regulation and disorders of acid-base homeostasis...1430 section iii † regulation and disorders of...

Because of the large number of biological processes sensitive to changes in pH (see reviews [88, 117, 456]), the subject of acid-base homeostasis has attracted considerable attention over the past several decades. Until relatively recently, acid-base homeostasis, for both clinicians and basic scientists, has been synonymous with pH regulation in the two most easily accessed compartments, blood and cerebrospinal fl uid (CSF). The pH in these extracellular compartments (pHo) is cer-tainly important for organisms. For example, alterations in pHo may affect various extracellular biochemical reactions (e.g., hemostasis, complement fi xation) and infl uence bind-ing of various substances (e.g., hormones, metals, therapeu-tic agents) to plasma proteins or cell surface receptors. Moreover, certain ion channels (524, 555) as well as trans-porters that move solutes across cell membranes are sensitive to pHo changes. Nevertheless, the number of processes sen-sitive to changes of extracellular pH pales in comparison with the myriad processes sensitive to alterations in intracel-lular pH (pHi). Thus, pHi homeostasis should be a matter of central importance not only for individual cells, but also for the organism composed of these cells.

Although cellular metabolism can modulate pHi, the regulation of pHi is the province of membrane proteins that transfer acid-base equivalents across the plasma membrane. In addition, transporters that carry acid-base equivalents across organellar membranes can transiently modify pHi or can participate in the buffering of cytoplasmic H�.

Since the last edition of this book, the fi eld of pHi regula-tion has advanced on two broad fronts: (1) elucidating the complex mechanisms responsible for pHi regulation and (2) understanding the molecular mechanisms of pHi regulation.

ELUCIDATING THE COMPLEXITY OF pHi REGULATION

One of the themes that continue to emerge is that patterns of pHi regulation in particular cell types tend to be unique and complex. As a result, without previous knowledge of a cell’s physiology, it may be impossible to predict its response to a particular maneuver. For example, switching the extra-cellular buffer from a non-CO2/HCO3

� buffer to CO2/HCO3

� usually causes an abrupt fall in pHi, due to infl ux of the highly permeant CO2. On the other hand, some cell membranes show no evidence whatsoever of being perme-able to gases such as CO2 (573) or NH3 (289, 511, 573). In fact, some of the CO2 permeability of membranes may re-quire “gas” channels (126, 188, 392, 427). For cells with CO2-permeable membranes, the response to the initial CO2-induced acidifi cation may—depending on the cell type and initial pHi—be a pHi recovery that is totally absent (59), partial (591), complete (545), or even excessive (6, 49, 53, 75, 104, 151, 391, 431).

pHi regulation is not only diverse, it is also complex. Thus, a particular cell type may possess numerous plasma-membrane transporters that regulate pHi, each with its own unique properties. Since the last edition of this book, many pHi-regulating mechanisms have been functionally studied in greater detail. These mechanisms include Na�/HCO3

�

cotransporters.pHi regulation is also complex in that the Na�-H� ex-

changer and other acid-base transporters may be under the concerted control of growth factors (104, 199–202, 381) or other environmental infl uences, such as acidosis (428) or

Copyright © 2008, Elsevier Inc. All rights reserved.Seldin and Giebisch’s The Kidney

CHAPTER 51

Control of Intracellular pH

Mark O. Bevensee and Walter F. BoronUniversity of Alabama at Birmingham, Birmingham, Alabama, USAYale University School of Medicine, New Haven, Connecticut, USA

1429

Regulation and Disorders of Acid-Base Homeostasis

Ch51-P088488_1429-1480.indd 1429Ch51-P088488_1429-1480.indd 1429 7/31/07 10:30:10 AM7/31/07 10:30:10 AM

1430 SECTION III • Regulation and Disorders of Acid-Base Homeostasis

hypertonicity (397). A G-protein–coupled H� receptor has been described (345). In the proximal tubule, the basolateral concentration of [CO2] and [HCO3

�] per se—independent of basolateral pH—are powerful regulators of acid-base transport (618).

Given the complexity and diversity of patterns of pHi regulation, one must examine pHi physiology anew for each previously unexplored cell type.

UNDERSTANDING THE MOLECULAR MECHANISMS OF pHi REGULATION

Since the last edition of this book, the fi eld has seen major advances in the molecular biology of acid-base transporters. One of the advances has been in organizing the vast array of proteins responsible for transport. The Human Genome Organization (HUGO) created the SLC superfamily of solute carriers, which currently contains 43 families. Three of these contain members that pH physiologists have tradi-tionally regarded as pHi-regulatory proteins: the SLC4 family of HCO3

� transporters (618), the SLC9 family of Na�-H� exchangers (405), and the SLC26 family of multi-functional anion exchangers (384). Table 1 summarizes these and 21 other SLC families that contain members that transport acid-base equivalents. Over the past several years, investigators have cloned cDNAs encoding new members of many of these families and have identifi ed a myriad of splice variants of the SLC proteins listed in Table 1.

In addition to the SLC transport proteins, several other families of protein engage in acid-base transport, including vesicular H� pumps, P-type ATPases (e.g., the gastric H�-K� pump and the SERCA Ca2�-H� pump), Cl� channels that also mediate the fl ux of HCO3

�, H� channels, and aqua-porins that mediate the fl ux of CO2 or NH3.

Given the recent advances in our understanding of acid-base transport at the molecular level, the future holds great promise for (1) determining the molecular identity of trans-porters responsible for pHi regulation in a particular cell type, (2) studying transport function under well-defi ned conditions (e.g., transporters heterologously expressed in Xenopus oocytes or other cells), and (3) elucidating the structural basis of transporter function.

SCOPE OF THIS CHAPTER

In this chapter, we will fi rst consider the methodologies available for making pHi measurements. We will then exam-ine the factors that contribute to changes in pHi: the ther-modynamic forces acting on hydrogen ions (H�) and other charged acids/bases; the permeation of the cell membrane by electrically neutral acids and bases; the buffering power of the intracellular fl uid; and transporters that regulate pHi by moving H�, bicarbonate ions (HCO3

�) and/or other weak acid/bases across the plasma membrane. Because space does

not permit us to summarize the vast array of acid base trans-porters presented in Table 1, we will focus on transporters that play a major role in pHi regulation. Emerging from this discussion will be a model of pHi regulation. Finally, in light of this model, we will consider several factors that funda-mentally alter pHi regulation: alterations in pHo; tempera-ture changes; metabolic inhibitors and hypoxia; cell shrink-age; hormones, growth factors, and oncogenes.

METHODS FOR MEASURING pHi

Of the techniques available for measuring pHi, we will con-sider the four that are of greatest utility. These techniques differ from one another in terms of their theoretical founda-tions, the precise cellular properties that they measure, as well as in their accuracy and sensitivity.

pH-SENSITIVE MICROELECTRODES

In the mid 1990s, it was beginning to look like the use of pH microelectrodes might become a dying art, except for their restricted use for measuring pHi in large invertebrate cells.

TABLE 1 SLC Families with Members That Can Engage in Acid-Base Transport

SLC Family Number Function

1 High-affi nity glutamate transporters4 HCO3

� transporters5 Na/glucose cotransporters (which include Na/

monocarboxylate cotransporters)6 Na- and Cl-dependent neurotransmitter trans-

porters7 Cationic amino-acid transporters9 Na-H exchangers10 Na/bile-salt transporters11 H�-coupled metal-ion transporters13 Na/sulfate-carboxylate cotransporers15 H�/oligopeptide cotransporters16 H�/monocarboxylate cotransporters17 Type I Na/phosphate cotransporter and vesicular

glutamate transporters18 Vesicular amine transporters19 Folate/thiamine transporters20 Type III Na/phosphate cotransporters21 Organic-anion transporters22 Organic cation/anion transporters23 Na-dependent ascorbate transporters25 Mitochondrial carriers26 Multifunctional anion transporters27 Fatty-acid transporters32 Vesicular inhibitory-amino-acid (GABA) trans-

porters34 Type II Na/phosphate cotransporters36 H�/coupled amino-acid transporters42 Rh family (NH3 transport)


CHAPTER 51 • Control of Intracellular pH 1431

However, the emergence of the Xenopus oocyte expression system and the cloning of numerous acid-base transporters over the past decade have breathed new life into an old technology.

When a pH electrode and an indifferent reference elec-trode are placed in a solution, the voltage difference between the electrodes (EX) is linearly related to the solution pH (pHX):

pH pH (E E )F

RTln10X S X S= + − (Eq. 1)

Here, pHS is the pH of a standard solution, and ES is the voltage difference in this standard. F/(RT ln 10) is the theo-retical slope (�58 mV per pH-unit change at 22°C) of the line relating pH to voltage. The actual slope is determined empirically.

Although the pH sensor can be any of several materials (e.g., platinum–hydrogen, antimony, tungsten, or a liquid membrane), the most reliable remains pH-sensitive glass. Glass has the advantages of long lifetime, long-term stabil-ity, and insensitivity to cations other than H� (at physiolog-ical pH), redox reactions, and various gases. Several styles of glass pH-sensitive microelectrodes are available, including Hinke’s exposed-tip design (254), RC Thomas’ recessed-tip design (543), and RC Thomas’ eccentric design (488, 548). Glass electrodes are particularly well suited to measure pHi of relatively large cells (e.g., squid axons) or to measure pHo. However, because it is diffi cult to fabricate glass microelec-trodes that are small enough to be used in small cells, glass electrodes generally have been abandoned in favor of elec-trodes with liquid-membrane sensors, which are easier to make, but have a much shorter lifetime, are less stable and are sometimes sensitive to other parameters.

Ideally, the pH and reference microelectrodes must impale a single cell. An acceptable alternative may be to place them in two identical cells or in cells that are electri-cally coupled. Another solution is to use a double-barreled electrode (143, 359). Unfortunately, for each barrel, there is a trade-off between tip size and electrode performance. Thus, the larger the two barrels of a double-barreled elec-trode, the better the electrode performance, but the greater the cell damage caused by impalement. The performance of liquid-membrane electrodes can be improved by using a concentric design in which a saline-fi lled pipette is threaded into the column of liquid-membrane sensor, thereby reducing the overall longitudinal resistance be-tween the sensor at the electrode’s tip and the electrical contact in the electrode’s barrel (174, 406, 561, 562). Fedirko et al. (174) have described a simplifi ed approach for implementing this concentric design for H�- and Ca2�-selective microelectrodes, permitting rapid mea-surements of extracellular pH and Ca2� transients in rat hippocampal brain slices.

pH-sensitive electrodes, particularly those with glass sen-sors, remain the method of choice for monitoring pHi in

relatively large cells. Not only are these electrodes highly sensitive (�0.01 pH units), they are probably more precise and accurate than pH-sensitive dyes. Moreover, the micro-electrodes report pH and cell voltage simultaneously, and in real time. The “pHi

” that they report is almost certainly that of the bulk cytoplasm (i.e., the fl uid in direct contact with the plasma membrane), uncontaminated by the pH of or-ganelles. Disadvantages of using microelectrodes for mea-suring pHi include the time and skill required for making and using them. In addition, one can only use conventional microelectrodes on cells large enough to sustain the impale-ment. Although small mammalian cells generally cannot tolerate such impalement, pH-sensitive microelectrodes placed near the extracellular side of the plasma membrane of a mammalian cell can be used to measure H� fl uxes due to acid-base transporter activity. Using an elegant “self-referencing” pH microelectrode technique with Chinese hamster ovary (CHO) fi broblasts, Fuster et al. (194) mea-sured the continual extracellular H� gradient when a cell-attached patch pipette was repetitively positioned close to and away from an extracellular pH electrode. H� fl uxes due to altered Na-H exchanger activity were quantitated from changes in the extracellular H� gradient elicited by altering the cytoplasm via pipette perfusion.

DISTRIBUTION OF WEAK ACIDS AND BASES

Cell membranes are generally far more permeable to neutral molecules than to charged molecules of similar shape and size. Thus, if a cell is exposed to a monoprotic weak acid HA (HA →← H� � A�), then the neutral molecule rapidly enters the cell (Fig. 1). Assuming for the moment that A� cannot penetrate the membrane, the entry of HA continues until HA is in equilibrium across the cell membrane; that is, when the concentration of HA inside the cell ([HA]i) is the same as that outside ([HA]o). Because entering HA dissociates to H� and A�, the equilibration of HA across the membrane is necessarily accompanied by a fall in pHi. This principle underlies the pHi changes caused by neutral weak acids and bases, which we will discuss below in Effects of Weak Acids and Bases on pHi. Provided that the dissociation constant (K � [H�]� [A�]/[HA]) is the same both inside and out-side the cell, then, at equilibrium,

[H�]i � [A�]i � K � [HA]i

� K � [HA]o

� [H�]o � [A�]o (Eq. 2)

and

[H ][H ]

[A ][A ]

i

o

o

i

��

��

��

��= (Eq. 3)

Because the transmembrane distribution ratios of A� and H� are inversely related, we can use Eq. 3 to compute pHi.



The weak acid most commonly used to calculate pHi is 5,5-dimethyl-2,4-oxazolidinedione (DMO), used as 14C-DMO; benzoic acid has also been used (160, 321). Regard-less of the weak acid used, the amount of radioactivity in the intra- or extracellular fl uid is proportional to the total con-centration of the probe, [A�] � [HA]. Because it is impos-sible to measure [A�] directly, we must modify the foregoing equation to include the concentrations of the total probe:

[ ] [ ]

[ ] [ ]/ [ ]/ [

A HA

A HAK HK H

i i

o o

i−

−

+

+

+( )+( ) = +

+11 ]]o

(Eq. 4)

Equation 4 can then be solved for pHi:

pH pKA HA

A HAi

i i

o o

pH pi= ++( )+( )

−

−−log

[ ] [ ]

[ ] [ ]10 KK +( )⎡

⎣⎢⎢

⎤

⎦⎥⎥

1 (Eq. 5)

One can use a similar approach to compute pHi from the distribution of a permeant weak base, such as methylamine (67). Reviews by Waddell and Butler (570) and Roos and Boron (456) contain more detailed descriptions of the weak-acid/base method, including potential diffi culties.

The major advantages of the weak-acid/base method include its technical simplicity and applicability to even very small cells. The parameter actually measured is not the pH of the cytoplasm, but rather a volume-weighted mean pH of all intracellular compartments in which the weak acid or base is distributed. The practical sensitivity of this approach is 0.03 to 0.05 pH units—considerably less than the microelectrode technique. The major disadvantage of the weak-acid/base approach is that continuous pHi mea-surements are not possible.

pH-SENSITIVE DYES

Absorbance

Molecules with an absorbance, fl uorescence excitation and/or fl uorescence emission spectrum sensitive to pH may be convenient probes for measuring pHi. If one exposes a solu-

tion of a pH-sensitive dye to light, then dye molecules may absorb some of the light as electrons make the transition to a higher-energy state. From the intensities of incident light (Io) and the light transmitted through the solution (I), we can compute the absorbance (A):

AIIo= log (Eq. 6)

According to the Beer-Lambert law, A at a particular wavelength (A�) is proportional to both the length of the light’s path through the solution (l) and the concentration of the dye (C):

A CpH� ��= , l (Eq. 7)

where the proportionality constant �, pH is the dye’s extinction coeffi cient at a particular wavelength and pH. The wavelength dependence of �, pH defi nes the shape of the absorbance spec-trum, and the pH dependence of �, pH defi nes how this shape is affected by changes in pH. However, A depends not only upon pH, but also upon l and C. If we were to attempt to compute pH from absorbance data at a single wavelength, then we would need to know both l and C, two parameters that are in practice very diffi cult to ascertain in a live cell. Alternately, we could obtain the absorbance data at two wavelengths (�1 and �2), and compute the absorbance ratio (A�1/A�2):

AA

CC

pH

pH

pH

pH

�

�

�

�

�

�

�

�

�

�

1

2

1

2

1

2

= =,

,

,

,

ll (Eq. 8)

Thus, because the l and C terms cancel out, the absor-bance ratio in Eq. 8 depends only on pH. By choosing two wavelengths in such a way that ��1,pH/��2,pH varies consid-erably with pH, one can obtain sensitive pH measure-ments. Experimenters using fl uorescein derivatives typi-cally use the peak absorbance wavelength (∼510 nm), and the isosbestic wavelength (∼440 nm), where � is insensi-tive to pH changes. Monitoring dye absorbance at the isosbestic wavelength is attractive because one can determine the extent of dye loss during the experiment, assuming l is constant.

An advantage of absorbance for measuring pHi is that it tends to be extremely stable and sensitive. On the other hand, because absorbance is proportional to l and C, a rela-tively high intracellular dye concentration is required, even for thick preparations (e.g., renal tubules).

Fluorescence

After absorbing a photon, most molecules return to the ground state by gradually losing energy through a series of random collisions with other molecules (46). Some dyes, however, can lose a quantum of energy from an excited singlet state by emitting a photon (i.e., fl uorescing). The intensity of emitted fl uorescent light (Iemit) can be measured with a photomultiplier tube (e.g., see ref. [75]) or an intensifi ed

FIGURE 1 The distribution of a monoprotic weak acid across a cell membrane. When placed in the external solution, HA passively enters the cell, where it dissociates to form H� and A�. Once [HA]i � [HA]o, the equilibrium HA → H� � A� holds in both the intracellular and extracel-lular fl uids.



television camera, which provides imaging data (e.g., see ref. [412]). At most wavelengths, Iemit is sensitive to pH, but at all wavelengths, Iemit is sensitive to dye concentration as well as other parameters (e.g., position of cell in incident light beam). Therefore, one usually uses a ratio technique to generate a parameter more uniquely related to pH. With the fl uorescence-excitation ratio approach, commonly used with fl uorescein dyes such as bis-carboxyethyl-carboxyfl uorescein (BCECF), one alternately excites at two wavelengths while monitoring Iemit at one wavelength. With the fl uorescence-emission ratio approach, commonly used with some rhodamine dyes such as seminaphthorhodafl uor-1 (SNARF-1), one excites at one wavelength, while monitoring Iemit simultaneously at two wavelengths. By analogy with the absorbance ratio approach, one chooses three wavelengths to optimize the pH sensitivity of the ratio.

Fluorescence measurements offer the advantage of being extremely sensitive. Thus, it is possible to quantitate the fl uorescence from small amounts of dye in a portion of a single cell. In addition, one can use fl uorescence with two-dimensional imaging, confocal microscopy, and two-photon microscopy. Because fl uorescence is more sensitive than ab-sorbance to the environment of the dye molecule, fl uores-cence measurements are in principle more prone to artifact.

Calibration

SIMULTANEOUS MICROELECTRODE MEASUREMENTS

Because dyes interact with cytoplasmic components, the spectroscopic properties of an intracellular dye differ, some-times markedly, from those of the same dye examined in a cuvette. Therefore, intracellular dye calibration is essential. One calibration approach is to use a second, independent method for measuring pHi in the same cell or another cell under similar conditions. For example, simultaneous mea-surements with a pH-sensitive microelectrode have con-fi rmed that the absorbance indicator dimethylcarboxyfl uo-rescein in salamander proximal-tubule cells (107), and the fl uorescence indicator BCECF in leech glial astrocytes (395) yield reasonable values.

NIGERICIN APPROACH

The most popular approach has been to monitor intracel-lular absorbance or fl uorescence while using the high-[K�]o/nigericin technique (542) to clamp pHi to predetermined values. Nigericin is a carboxylic ionophore that exchanges K� (and to a lesser extent Na�) for H� across cell mem-branes. If one is successful in choosing [K�]o to match [K�]i, then pHi should equal pHo. Thus, by altering pHo, one can measure the dye’s spectral properties over a range of pHi values. A detailed calibration (i.e., spanning a wide pHi range) can be obtained for each experiment. Alternately, one can perform the detailed calibration on one set of cells, and routinely perform only a single-point calibration (75). Po-tential problems with the high-[K�]o/nigericin technique have been discussed in some detail (106).

In using nigericin-containing solutions for dye calibration, one must be careful to cleanse the perfusion system completely after each calibration procedure. Even trace amounts of nige-ricin can interfere with the assessment of pHi-regulating mechanisms, by mimicking a K-H exchanger and by increas-ing “background acid loading”1 through nigericin-mediated exchange of internal K� for external H� (49, 446).

NULL-POINT APPROACH

A novel calibration technique, originally proposed for microelectrodes by Szatkowski and Thomas (537), but ap-plied to dyes by Eisner et al. (168), involves sequentially ex-posing the cell to a permeant weak acid and weak base. As discussed in subsequent sections, the size of the pHi decrease caused by a weak acid increases predictably as the initial pHi rises, whereas the size of pHi increase elicited by a weak base decreases predictably as the initial pHi rises. It is thus possi-ble to compute the initial pHi from the magnitude of the change of the pHi indicator, assuming that the behavior of the weak acid and base is ideal and intracellular buffering power is fi xed over the observed pHi range. The application of this approach to fl uorescent dyes is straightforward when pHi is near the pK of the dye (i.e., when the pH dependence of fl uorescence is approximately linear). In principle, the ap-proach could be extended to more extreme pHi values, al-though the mathematics would be more complicated.

Eisner et al. (168) have extended the approach by intro-ducing an elegant null-point technique in which one ex-perimentally determines a combination of weak-acid and weak-base concentrations that produces no change in the measured fl uorescence. The null-point technique has the advantage of obviating the need for nigericin, but the disad-vantage is that one must expose the cell to many solutions before zeroing in on the null point. Another limitation is that the dye is calibrated at exactly one pHi. Nevertheless, the null-point approach can be particularly useful in assess-ing the validity of other dye-calibration procedures, particu-larly the high-[K�]o/nigericin technique (77–79).

Bleaching and Photodynamic Damage

Excessive illumination can photolyse (i.e., bleach) dye mol-ecules, causing a progressive decrease in the concentration of native dye. Among the photolysis products may be free radicals that react with cellular components and injure the cell (“photodynamic damage”). Excessive illumination can also cause a time-dependent shift in the apparent intracel-lular calibration curve of the dye (75), presumably due to bleaching-induced generation of dye-related products with spectral characteristics slightly different from those of the parent compound.

Bleaching can be particularly problematic during experi-ments on single cells, where the number of dye molecules

1 We defi ne “acid loading” as any process that causes pHi to fall. Examples in-clude the uptake of H�, the loss of HCO3

�, or the metabolic production of H�.



and thus the number of emitted photons is low. The prob-lem is that the limiting signal-to-noise (S/N) ratio is pro-portional to the square root of the number of photons measured. Although one can attempt to increase the number of emitted photons by increasing the intensity of the excita-tion light source, the greater intensity exacerbates the bleach-ing and photodynamic damage. These effects can be mini-mized by illuminating continuously with low-intensity light while integrating the signal over a suffi ciently long period or by limiting the duty cycle of high-intensity exciting light (e.g., illuminate for 200 milliseconds every 2 seconds; 75). Thus, one must sometimes trade off pHi resolution (i.e., S/N ratio) against time resolution. In imaging experiments, where a cell may be represented by thousands of picture elements (pixels), the photon emission rate per pixel is ex-ceedingly low. Thus, one must trade off pHi resolution, time resolution, and spatial resolution (i.e., the number of pixels that must be grouped to compute pHi values).

Two-photon excitation laser scanning microscopy is a rela-tively new imaging technique that has allowed investigators to reduce photobleaching of ion-sensitive fl uorescent dyes, even while acquiring high spatial and temporal resolution record-ings (153, 535). Multiphoton imaging technology is based on the general quantum principle that a molecule can exhibit fl uorescence after absorbing two photons simultaneously when excited by high-intensity light of twice the wavelength neces-sary for single-photon absorption. Because two-photon ab-sorption and subsequent fl uorescence rise very steeply with excitation intensity, lower intensity out-of-focus light does not generate appreciable fl uorescence. In addition, excitation at the higher wavelengths (and thus lower energies) reduces overall photobleaching of the dye. Thus, two-photon imaging can be used to perform high-resolution imaging without the draw-back of excessive photodynamic damage. As reviewed by Dunn and Young (165) and Zipfel et al. (620), two-photon microscopy has been particularly useful in examining intracel-lular Ca2� transients, especially in cellular microdomains such as dendritic spines. However, the technique has also been used with BCECF to measure intracellular pH in microdomains of the epidermis (244).

Choice of Dyes

BCECFThe most popular dye for fl uorescence measurements of

pHi is the fl uorescein derivative BCECF (449), which has four negatively charged carboxylate groups and a phenolic �OH moiety that is titrated by pH changes. The dye can be directly loaded into large cells (e.g., Xenopus oocytes) with an injection pipette or into small mammalian cells by diffusion from a patch pipette during whole-cell recordings (344, 553). However, BCECF is usually loaded into cells as an uncharged tetra-acetoxymethylester (AM) precursor that easily perme-ates most plasma membranes. Intracellular esterases hydro-lyze BCECF-AM to yield four or fi ve formaldehyde mole-cules for each charged BCECF molecule trapped inside the

cell. Dye loading can vary greatly among cell types—some take up large amounts of the precursor and convert it to BCECF very rapidly (i.e., ∼1 minute), whereas others may not load suffi ciently even after many minutes. Less conven-tional methods for dye loading, including scrape loading, os-motic lysis, and electroporation, are examined in more detail in Giuliano and Taylor (216). BCPCF, with carboxypropyl groups, is a derivative of BCECF that can be used as a dual-emission pH indicator (336).

OTHER DYES

Pyranine-based (215) and rhodamine-based dyes (557, 592) have been used for monitoring pHi. Rhodamine dyes such as SNARF-1 are excited and emit at longer wavelengths than the fl uorescein derivatives. An advantage of SNARF-1 is its absorbance and fl uorescence-excitation spectra are pH sen-sitive, as is its fl uorescence-emission spectrum (i.e., it can be used in the dual-emission mode). The pH sensitivity of its fl uorescence-emission spectrum makes SNARF-1 more useful than fl uorescein derivatives for confocal microscopy, in which one typically excites at only one wavelength. Dual-emission dyes also enable the user to sample more frequently and avoid the delay in alternating between two excitation wavelengths. SNARF-1 can be used simultaneously with fura-2 for moni-toring both pHi and [Ca2�]i (356). In general however, one should be cautious in using multiple ion–sensitive dyes simul-taneously to avoid quenching artifacts (357).

MEASUREMENT OF PH IN ORGANELLES

Investigators have developed ingenious methods for targeting pH-sensitive probes to organelles. As reviewed by Maxfi eld and Yamashiro (360), pH-sensitive indicators can be targeted to the endocytic pathway following pinocytosis or receptor-mediated endocytosis. More re-cently, Kim et al. (292) have measured Golgi pH with either rhodamine- or fl uorescein-labeled subunit of verotoxin, which accumulates in the Golgi complex after receptor-mediated endocytosis and retrograde transport. Grinstein’s group has also measured pH in the endo-plasmic reticulum (291) and the trans-Golgi network (152) by creating chimeric proteins with organelle-specifi c retrieval signals and subsequently tagging them with pH-sensitive fl uorophores before internalization. A similar approach has been used to measure the pH of re-cycling endosomes (134, 541). The Machen group has examined pH regulation in the secretory pathway by tar-geting biotin-labeled pH probes to organelles expressing avidin-chimera proteins (596–598). Seksek et al. (492) have used a different technique to measure the trans-Golgi pH of fi broblasts. They injected the cells with 70-nm liposomes containing membrane-impermeable pH-sensitive fl uorophores; the liposomes then fused with the trans-Golgi. As described in the next section, investi-gators have also measured the pH of organelles by fusing pH-sensitive green fl uorescent protein (GFP) variants to protein markers for specifi c organelles.



GREEN-FLUORESCENT PROTEIN

Another fl uorophore that has become useful in the pH fi eld is GFP, which is a natural product of the jellyfi sh Aequo-rea victoria, and typically used to label proteins expressed in cells (108, 353). Several researchers have engineered GFP mutants such as pHluorins (366) that are sensitive to pH changes in the physiological range and can be targeted to the cytosol or organelles (159, 272, 309, 340, 346, 366, 388, 450). Compared with more traditional pH indicators, pH-sensitive GFP mutants are advantageous in displaying a low rate of photobleaching while remaining trapped inside of cells. pHluorins linked to markers of synaptic vesicles (syn-aptopHluorins) undergo marked changes in fl uorescence upon exocytosis at synaptic terminals and have therefore been used to characterize synaptic vesicle cycling associated with presynaptic activity (366, 474, 610). More recently, such vesicular cycling has been examined in transgenic mice ex-pressing synaptopHluorin (333). It is intriguing to speculate on the potential applicability of pH-sensitive GFPs to exam-ine cellular pH physiology. For example, one could perform in vivo pHi measurements on targeted cells that are induced by a specifi c promoter to express a pH-sensitive GFP.

Differential Dye Loading

Recall that some cells load more rapidly than others with dyes such as BCECF. One can actually exploit such differ-ential dye loading when working with mixed populations of cells. For example, intercalated cells in the cortical collecting tubule of the kidney incorporate BCECF-AM much more rapidly than the neighboring principle cells (484). In a somewhat different application, Chu et al. (121) found that mouse colonic crypt cells exclude SNARF, which then can be used to measure extracellular pH changes in the intact epithelium. BCECF generally leaks out of cells slowly be-cause the dye is negatively charged. However, a probenecid-sensitive organic-anion transporter can extrude BCECF from thyroid cells (210) and epithelial kidney and intestinal cells (13, 125). In an elegant study, Harris et al. (247) used BCECF to measure the pH of the lateral intracellular space (LIS) of MDCK cell monolayers by fi rst loading the cells with BCECF and subsequently allowing organic transport-ers to move the dye into the LIS.

BCECF generally stains the cytoplasm rather uni-formly, although in some cells the nuclear region is more intense than peripheral areas (439). Working on Ehrlich ascites tumor cells, Thomas et al. (542) found that 6-carboxyfl uorescein (when loaded into cells as the diacetate ester) is confi ned to the cytoplasm, but fl uorescein is dis-tributed in both the mitochondria and cytoplasm. In a more recent study, Slayman et al. (515) found that BCECF can accumulate in vacuoles of the fungus Neurospora when the cells are exposed to BCECF-AM.

One can evaluate a dye’s intracellular compartmentaliza-tion by monitoring the fl uorescence loss elicited by selec-tively permeabilizing different compartments with deter-

gents. For example, most of the BCECF loaded into rat hippocampal CA1 neurons appears to be in the cytoplasm because 0.01% saponin reduces the fl uorescence signal by ∼96% (48). Both absorbance- and fl uorescence-derived esti-mates of pHi can be infl uenced by dye in compartments other than the cytoplasm, depending upon each compart-ment’s volume, dye concentration and pK, and pH.

NUCLEAR MAGNETIC RESONANCE

Certain atomic nuclei, among them 31P and 19F, possess a quantum mechanical property termed “spin” and behave as tiny bar magnets with magnetic moments. When an atomic nucleus of this type is placed in an external magnetic fi eld, the magnetic moment precesses with a characteristic fre-quency about the axis of the applied fi eld. The nucleus can be excited to a high-energy state by irradiating it with an oscil-lating magnetic fi eld of the same frequency (i.e., resonance frequency) as the precession frequency. The resonance fre-quency depends not only on the identity of the atomic nu-cleus (e.g., 31P), but also on its chemical environment, which infl uences the strength of the magnetic fi eld at the nucleus. Thus, the resonance frequencies for 31P in HPO�

4 and H2PO�

4 are slightly different because of the different chemi-cal environments of the 31P. Because the exchange rate of 31P between individual HPO�

4 and H2PO�4 ions is very rapid,

nuclear magnetic resonance (NMR) detects only a single inorganic phosphate peak, the location of which depends on [H2PO�

4]/[HPO�4]. Because the dependence of this ratio on

pH is described by a modifi ed pH-titration curve (H2PO�4

# HPO�4 � H�; pK�a � ∼6.8), the position of the inorganic

phosphate peak is a good index of pHi.A major advantage of 31P-NMR is that, in addition to

providing nearly continuous measurements of pHi, it can also be used to monitor levels of a variety of phosphorus-containing compounds, such as ATP. The pHi value derived from NMR measurements is predominantly the pH of the cytoplasm as contaminated by inorganic-phosphate signals from other intracellular compartments, as well as the extra-cellular space. The mitochondrial inorganic phosphate peak can in some cases (503) be resolved from that of the cyto-plasmic peak. NMR measurements have been made on whole organs, whole small animals, and human limbs (196).

A common variation of the NMR approach for monitor-ing pHi has been the use of 19F-labeled probes having pK values in the physiological range (156). Similar to 31P-NMR, 19F-NMR involves observing a chemical shift of the 19F-labeled probe in response to the level of protonation of an attached acidic group. As discussed by Deutsch (157), NMR measurements with 19F-labeled probes are advantageous over 31P in that background signals are low and the fl uorinated probes are highly visible and sensitive to the environment. Some of the commonly used probes include fl uoroanilines, derivatives of fl uoroisobutyric acid, and fl uorinated pyridoxins.



These probes are generally introduced into cells as methyl esters, similar to the approach used for pH-sensitive dyes. Aside from cost and the need for technical expertise, the major disadvantage of NMR is its relatively low sensitivity. Thus, a considerable mass of 31P or 19F is required for detec-tion, precluding the use of the technique with single cells. For reviews, see refs. (196, 213, 402).

FORCES AFFECTING THE PASSIVE MOVEMENT OF H� AND OTHER CHARGED ACIDS AND BASES

Forces Affecting H�

Until the 1930s, it was generally assumed that hydrogen ions were in electrochemical equilibrium across the cell mem-brane, as defi ned by the Nernst equation:

VRTF

HH

mo

i

=+

+ln

[ ][ ]

(Eq. 9)

Here, Vm is the voltage difference across the plasma mem-brane (the unit is the volt), R is the universal gas content (8.31 J � °K�1 � equivalent�1), T is absolute temperature, F is Faraday’s constant (96,486 coulombs � equivalent�1), and the subscripts o and i refer to extracellular and intracel-lular, respectively. In terms of pH, the forgoing equation becomes

Vm � (0.0585 V) � (pHi � pHo) (Eq. 10)

assuming a temperature of 22°C. Thus, if H� were in equi-librium, then pHi would be one unit lower than pHo for each �58.5 mV of membrane potential. However, such a situation would present severe problems for the cell, inas-much as Vm changes would shift the equilibrium distribu-tion of H�, and thus alter pHi and pHi-sensitive processes. It was not until the mid 1930s that Fenn and colleagues (179, 180), in experiments on frog skeletal muscle, demon-strated that pHi is higher than expected for H� to be in electrochemical equilibrium. The authors, in effect, esti-mated Vm from the ratio [K�]o/[K�]i, and measured pHi using the weak-acid method.

Now both Vm and pHi can be measured directly, and for nearly all cells studied, pHi is well above the equilibrium pHi. In vertebrate skeletal muscle, for example, Vm is ∼�90 mV and pHi is ∼7.1 (456). Given a pHo of 7.4, Eq. 10 predicts an equilibrium pHi of ∼5.9, far lower than the actual pHi. Rather than calculating equilibrium pH, one could equally well compute equilibrium potential for H�, that is, the mem-brane voltage required for H� to be in equilibrium across the membrane (EH). In skeletal-muscle example introduced pre-viously (pHi � 7.1, pHo � 7.4), the EH computed from Eq. 9 or Eq. 10 is ∼�18 mV. Because the actual Vm (�90 mV) is more negative than EH, H� is drawn into the relatively negative cell, driven by an electrochemical gradient of 90 � 18 or 72 mV. Thus, there is a substantial electro-

chemical gradient (1.2 pH units or 72 mV) favoring the passive infl ux of H�. If the cell membrane were permeable to H�, then the resultant H� infl ux would represent a “chronic intracellular acid load” (i.e., an acid load imposed on the cell for an indefi nite period) that would tend to lower pHi.

In the previous discussion, we made no assumptions about the mechanism of the hypothetical infl ux of H�. It had gener-ally been thought that H� fl ux across the plasma membrane occurs via nonspecifi c pathways. As extensively reviewed by DeCoursey (145), voltage-activated H� currents were fi rst described in snail neurons (550) and have subsequently been characterized in numerous other cells, including epithelial cells (including kidney cells), connective tissue, skeletal muscle, lymphocytes, macrophages, granulocytes, and microglia. These channels become active during depolarizing pulses and con-duct outward H� currents in the direction predicted for pas-sive H� movement in highly depolarized cells (e.g., when the Vm of snail neurons is more positive than ∼�10 mV). The depolarization threshold for channel activation is lowered by increasing the pHo-to-pHi gradient.

Proton channels are sensitive to Cd2� and Zn2� and other polyvalent cations. In rat alveolar epithelial cells, the H� chan-nels are very selective for H�: The permeability of the channel to H� is approximately seven orders of magnitude greater than the permeability to TMA (115). Although unitary H� chan-nels have not been detected, noise analysis suggests that they are extremely small (4–10 fA) (89, 147). However, according to more recent analyses with improved S/N current measure-ments on inside-out patches of human eosinophils, the unitary conductance is larger: ∼40 fS at pHi 6.5 and 140 fS at pHi 5.5 (116). It is still diffi cult to distinguish these single-channel conductances from those estimated from electrogenic trans-porters and pumps (42, 89, 116, 146).

The role of this voltage-activated H� current has not been precisely defi ned. However, the current could serve as an elegant, although temporary, H�-extrusion mechanism that does not require the direct input of energy. A strong depolarization of the cell (e.g., during an action potential) would not only activate the H� conductance, but also shift Vm to values more positive than EH, thereby reversing the electrochemical H� gradient. Increasing the pHo-to-pHi gradient would also be expected to contribute to the activa-tion of the H� conductance. Murphy et al. (385) demon-strated that Zn2�-sensitive H� channels do contribute to the recovery of pHi from an NH4

�-induced acid load in rat alveolar epithelial cells exposed to both normal and high-K� solutions. Finally, two groups—Ramsey et al. (437) and Sasaki et al. (477)—have independently cloned a voltage-dependent, Zn2�-sensitive H� channel.

Forces Affecting Charged Weak Acids/Bases

In general, the passive fl uxes of ionic weak acids (e.g., NH4�)

and bases (e.g., HCO�3) also impose a chronic intracellular

acid load. As noted earlier (see Eq. 3), whenever a neutral weak acid (HA) is equilibrated across the cell membrane



([HA]i � [HA]o), the transmembrane distribution ratio for H� is the reciprocal of the distribution ratio for the anionic conjugate weak base (A-). Consider, for example, the CO2/HCO�

3 buffer system. If (1) [CO2]i equals [CO2]o, (2) CO2 is in equilibrium with H� and HCO�

3 both inside and out-side the cell, and (3) the equilibrium constant is the same inside and outside the cell, then

[ ][ ]

[ ][ ]

HH

HCOHCO

i

o

o

i

+

+

−

−= 3

3

(Eq. 11)

That is, the electrochemical gradient for HCO3� (and that

for any other monovalent anion that can be described by a similar equation) is equal to but opposite to the gradient for H�. Inasmuch as H� normally leaks into cells, HCO3

� and other anionic weak bases tend to leak out, also decreasing pHi. In crayfi sh muscle, the transmitter GABA opens Cl� channels that are also permeable to HCO3

� (280). The resul-tant HCO�

3 effl ux can reduce pHi by as much as 0.4. A similar GABAA-activated HCO3

� conductance is present in cells from turtle cerebellum (111) and rat hippocampus (112, 279). This GABAA-activated HCO�

3 conductance can be inhibited by antagonists of GABAA receptors such as picrotoxin. Other anionic weak bases that seem to penetrate at least some cell membranes include the DMO anion (287), formate (494), propionate (286), and salicylate (494).

We can make a similar analysis for cationic weak acids (HB� # H� � B). If the electrically neutral conjugate weak base (B) is equilibrated across the cell membrane (i.e., [B]i � [B]o), if the equilibrium HB� # H� � B holds on both sides of the membrane, and if the equilibrium constant is the same on both sides of the membrane, then

[ ][ ]

[ ][ ]

HH

HBHB

i

o

i

o

+

+

+

+= (Eq. 12)

That is, the electrochemical gradient for HB� is in the same direction as that for H�. Thus, similar to H�, cationic weak acids such as NH4

� tend to enter the cell and produce a chronic intracellular acid load.

Energetics of pHi Regulation

The preceding analysis shows that, under the conditions that normally prevail in most cells (e.g., pHi � 7.1, Vm � �60), the electrochemical gradients affecting H� and charged monovalent weak acids/bases (provided the neutral species equilibrates across the membrane) favor fl uxes that would lower pHi. These passive fl uxes are depicted for a model cell in Fig. 2. Note that the above thermodynamic analysis addresses only the net direction of these passive fl uxes, and does not address the rate of intracellular acid loading. How important for intracellular acid loading are these passive fl uxes? To our knowledge, this issue has yet to be considered in a comprehensive fashion, probably for two reasons. First, one would like to be able to block specifi c

individual pathways. Unfortunately, except for the examples of voltage-gated H� channels and GABAA-activated HCO�

3 conductances, no specifi c blockers of these pathways have been identifi ed. Second, one would like to study these path-ways in isolation. Unfortunately, cells may have multiple pathways for acid loading (e.g., cellular metabolism).

In predicting the passive fl ux of H� relative to that of monovalent acids/bases, one must consider factors other than the electrochemical gradients of the species involved. One such factor is concentration, which for H� may be several orders of magnitude lower than for ions such as HCO�

3 or inorganic phosphate. Another key factor is mem-brane permeability, which for H� may be several orders of magnitude higher than for other ions (144). However, as described by DeCoursey (145) there are many diffi culties associated with measuring membrane H� permeability.

The acid-loading mechanisms mentioned previously (i.e., passive fl uxes, carrier-mediated transport, metabolism) are “chronic” because they act continuously to lower pHi. Maintaining a normal pHi requires that acid loading be matched by a comparable—and continuous—extrusion of acid. By defi nition, this acid extrusion2 must be an active (i.e., energy-requiring) process. Acid extrusion can be ac-complished either by the active uptake of alkali (e.g., OH� or HCO�

3) and/or the active removal of acid (e.g., H�). As discussed in subsequent sections, a cell can also be subjected to an “acute intracellular acid load” (i.e., an acid load of defi ned and limited magnitude, imposed over a relatively

FIGURE 2 Factors affecting cellular H� balance. In a typical mamma-lian cell, membrane potential (Vm) might be �60 mV (inside negative) and extracellular pH (pHo), approximately 7.4. For H� to be in electrochemical equilibrium, pHi would have to be approximately 6.4. This is far lower than typical values, which range from 6.8 to 7.6, depending upon the cell type. Thus, H� tends to enter the cell passively (inward arrow). The same gradient that drives H� into the cell would also tend to drive HCO3

�out (outward arrow), thereby lowering pHi. The cell would also be acidifi ed by any trans-porters that mediate the effl ux of HCO3

� (curved arrow), and perhaps by the metabolic generation of acid (arrow). For pHi to be kept at its normal, rela-tively alkaline value, transporter(s) must actively remove acid from the cell or accumulate an alkali such as HCO3

�. Together, such active processes are re-ferred to as “acid extrusion.”

2 We defi ne “acid extrusion” as any process that causes pHi to rise. Examples include the effl ux of H� and the uptake of HCO3

�.



brief period of time). For example, injecting a cell with H� produces a sudden decrease in pHi. One of the key observa-tions in pHi physiology is that cells usually recover sponta-neously from such acid loads by extruding the acid load.

Some metabolic products, which are commonly thought of as acids, or are indeed acids, may actually have no net ef-fect on pHi. A classic example is CO2. In the steady state, the rate of cellular CO2 production equals the rate at which CO2 passively exits the cell, which equals the rate at which the CO2 leaves the organism. Thus, because [CO2] does not increase in the steady state, CO2 production under these conditions is “pHi silent.” Another example of a pHi-silent metabolic product, at least in some cells, is lactic acid. For cells with an H�/lactate cotransporter that normally func-tions to transport lactate out of cells, lactic-acid production would be pHi silent if the lactic-acid effl ux via the cotrans-porter kept pace with lactic-acid production.

EFFECTS OF WEAK ACIDS AND BASES ON pHi

Effects of CO2 and Other Neutral Weak Acids

In the section, Distribution of Weak Acids and Bases, for measuring pHi, we discussed the fl ux of neutral weak acids and bases across the cell membrane. In general, solutions containing the weak acid HA always contain the conjugate weak base A�. However, because membranes are usually far more permeable to HA than to A�, HA fl uxes generally have a greater effect on pHi.

FLUX OF THE NEUTRAL WEAK ACID

When a cell is exposed to a neutral weak acid, HA enters and dissociates into A� and H�, causing pHi to fall:

HA → A� � H� (Eq. 13)

This process continues until [HA]i equals [HA]o. Subse-quently, there should be no further change in pHi, provided there is no fl ux of A� and no change in the rates of acid-base transporter activity. Although CO2 is often regarded as a weak acid, it is not an acid at all. Only after reacting with H2O does CO2 yield the true weak acid, H2CO3, which then dissociates (as described previously for the idealized weak acid HA):

CO2 � H2O → H2CO3 → HCO�3 � H� (Eq. 14)

These two reactions can be combined into a single one with an overall apparent equilibrium constant (K′a � [HCO�

3][H�]/[CO2]). The enzyme carbonic anhydrase (CA) greatly accelerates the formation of HCO�

3 from CO2 by catalyzing the reaction:

CO OH HCOCA2 3+ ⎯ →⎯− −

(Eq. 15)

which generates H� by virtue of consuming OH�. The mechanism by which CA catalyzes this reaction is examined in more detail by Liljas et al. (334). In the absence of CA,

the reaction can also occur, particularly under alkaline con-ditions (i.e., pH � 8).

In the example of Fig. 3, a cell is successively exposed to solutions equilibrated with 1%, 2%, and 5% CO2 (constant [HCO�

3]o � 10 mM). Each time, CO2 produces a rapid and sustained fall of pHi. The period during the CO2 exposure in which pHi is relatively stable is termed the “plateau phase”. The magnitude of the CO2-induced acidifi cation is inversely related to the intracellular buffering power () (see following sections). The magnitude of the pHi decrease also increases with [CO2]o, as does the fraction of incoming CO2 that dissociates into HCO�

3 and H� (see Eq. 14). The de-gree of dissociation is governed by the relationship between pHi and pK′a, which, in logarithmic form, is the familiar Henderson-Hasselbalch equation:

pH pKHCOCO

i ai

i

= ′ +−

log[ ][ ]

3

2 (Eq. 16)

where pK′a is ∼6.1 at 37°C. Therefore, if pHi is 7.1, then 10 molecules of incoming CO2 dissociate into H� plus HCO�

3 for each molecule of CO2 that remains CO2. If the initial pHi is only 6.1, then this ratio falls to 1:1, and fewer incoming CO2 molecules dissociate. There are two practical consequences of this rule. First, the lower the initial pHi, the smaller the mag-nitude of the acidifi cation elicited by the subsequent exposure to the CO2. Second, as one raises [CO2]o by fi xed increments, the magnitude of the acidifi cation is not proportional to the successive [CO2]o increment. Thus, in Fig. 3, the pHi pro-duced by 2% CO2 is less than twice as large as that produced by 1% CO2, and that produced by 5% CO2 is substantially less than fi ve times that produced by 1% CO2.

Although investigators have long considered that all gases penetrate all membranes simply by dissolving in the membrane lipid, Waisbren et al. (573) demonstrated the fi rst

CO2:[HCO3

�]07.4

7.2

7.0

6.8

pHi

1%10 mM

2%10

5%10

30 min

1 2 3

FIGURE 3 Effect of CO2 on pHi. A giant barnacle muscle fi ber was exposed to CO2 at three different levels: 1% (pHo � 7.5), 2% (pHo � 7.2), and 5% (pHo � 6.8). After its entry into the cell, CO2 is rapidly hydrated to H2CO3, which in turn dissociates into H� and HCO3

�, causing pHi to fall. Removing CO2 reverses these processes. pHi was measured with an exposed-tip pH-sensitive microelectrode, of the design of Hinke. (Used with permission from Boron WF. Intracellular pH transients in giant bar-nacle muscle fi bers. Am J Physiol 1977;233:C61–C73.)



membrane with negligible permeability to a dissolved gas (i.e., CO2 and NH3). Moreover, Nakhoul et al. (392) and Cooper and Boron (126) demonstrated the fi rst gas channel, AQP1, which is permeable to CO2. Studies indicate that AQP1 is responsible for ∼60% of the CO2 permeability of the human erythrocyte (170). AmtB, the bacterial Rh ho-molog, functions as an NH3 channel (288, 615). Thus, at least two families of proteins can function as gas channels.

Weak acids such as acetic acid (389), lactic acid (504), and DMO (287) can also elicit intracellular acidifi cations, which have [HA]o and pHi dependencies similar to those described for CO2. CO2 is unusual, however, in that [CO2]o is easily controlled, independent of [HCO�

3]o, either by the experimenter (who can equilibrate solutions with a known CO2 mixture) or by the intact organism (which can alter its external respiration). Of course, the price one pays for vary-ing [CO2] at constant [HCO�

3] is that—at equilibrium—pH must vary as well, as predicted by Eq. 16. In the laboratory, it is possible to overcome this limitation by using out-of-equilibrium CO2/HCO�

3 solutions (see following sections) to change only one of the three parameters (pH, CO2, or HCO�

3) at a time. For nonvolatile weak acids, the experi-menter can only manipulate the total concentration of extra-cellular buffer ([TA]o � [HA]o � [A�]o) and pH. When [TA]o is fi xed, changes in pHo produce reciprocal alterations in [HA]o and [A�]o, so that the magnitude of the HA-induced pHi decrease is very pHo sensitive.

FLUX OF THE ANIONIC, CONJUGATE WEAK BASE

In the preceding discussion, we assumed that the move-ment of the uncharged weak acid HA was the only factor affecting pHi. In other words, we excluded the possibilities that: (1) the charged species A� can traverse the cell mem-brane and signifi cantly affect pHi and (2) acid-base trans-porters and metabolic processes that generate acid-base equivalents can alter the overall balance between acid extrusion/loading. In the absence of these complicating ef-fects, applying and withdrawing HA/A� would produce the pHi changes illustrated in Fig. 4B.

What would happen if A�, as well as HA, were able to cross the membrane passively? At the instant a cell is ex-posed to a solution containing HA and A�, the electro-chemical gradient for A� would be inward (assuming that the initial [A�]i were suffi ciently low). Initially then, both HA and A� would enter, the HA tending to lower pHi, and the A� tending to raise pHi. A signifi cant permeability of the membrane to A� would reduce the initial rate of the HA-induced acidifi cation. Eventually, however, the genera-tion of A� from incoming HA, as well as the infl ux of A�, would raise [A�]i to such a level that A� would tend to exit the cell passively, as described earlier. In addition, a trans-porter might move A� out of the cell; for example, a Cl-HCO3 exchanger normally moves HCO3

� out of the cell. Subsequently, the effl ux of A� would lower pHi, both during the initial HA-induced acidifi cation and during the later plateau phase, as illustrated in Fig. 4A. This A� effl ux rep-

resents what might be termed a “semi-chronic” acid load, inasmuch as the pHi decrease would continue until [A�]i was so low that A� was equilibrated across the membrane. Removing external HA/A� would elicit a rapid rise in pHi due to the effl ux of HA from the cell. Note, however, that pHi would rise to a value less than the initial pHi. The mag-nitude of the shortfall would be directly related to the net acid loading due to A� effl ux. The pHi changes illustrated in Fig. 4A, presumably due to permeability of the membrane to the A� form of a weak acid, have been observed in snail neurons exposed to salicylic acid (494).

Acid-extrusion mechanisms can also infl uence the pHi changes of a cell exposed to HA/A�. In practice, the ef-fects described in the following paragraphs are due to one or more acid-extruders that outstrip acid loaders. These transporters are described in more detail later, as are their effects on the dynamics of pHi regulation. We introduce these transporters here only to complete our consideration of pHi transients elicited by HA/A�. Consider a cell that is permeable to HA, but not to A�, and that has an acid-extrusion mechanism that is unaffected by A� (e.g., Na�-H� exchange). Applying HA/A� would acutely acid load the cell, due to the infl ux of HA, and subsequent forma-tion of A� and H�. However, an acid-extruder stimulated by the pHi decrease would remove this acute acid load and return pHi to exactly its starting value (Fig. 4C). Note that

HA and A�

Alkaline

pHi

Acid

A HA and A�B

HA and A�

Alkaline

pHi

Acid

C HA and A�D

Time

FIGURE 4 Hypothetical responses of a cell to the application of a neutral weak acid (HA) and its conjugate weak base (A�). In all cases, the initial effect is a rapid fall in pHi, due to the infl ux and partial dissociation of HA. The decrease in pHi halts once [HA]i � [HA]o. The subsequent course of pHi depends on the balance among (1) the passive effl ux of A� (which will lower pHi), (2) transporters (e.g., Cl�-HCO3

� exchanger) that lower pHi, and (3) transporters (e.g., Na�-H� exchanger) that raise pHi. A: Acid loading exceeds acid extrusion, and pHi falls during the plateau phase. When external HA/A� is removed, the recovery of pHi falls short of the initial value. B: Acid loading exactly balances acid extrusion, and pHi is stable during the plateau phase. When external HA/A� is removed, pHi returns to its initial value. C: Introducing HA/A� does not fundamentally alter acid loading or acid extrusion. pHi returns to exactly its initial level by the end of the plateau phase. When external HA/A� is removed, pHi over-shoots its initial value. D: Introducing HA/A� promotes acid extrusion more than acid loading. During the plateau phase, pHi recovers to a value that is higher than the initial pHi. When external HA/A� is removed, pHi overshoots its initial value by even more than in case C.



removing HA/A� causes pHi to overshoot its initial value by a substantial amount. The magnitude of the overshoot is directly related to the net amount of acid extruded dur-ing the HA/A� exposure. This pattern of pHi changes, including a complete recovery of pHi during the plateau phase, is observed in snail neurons acid loaded by exposure to CO2/HCO3

� (545). Other examples have been reported in which the plateau-phase pHi recovery is incomplete.

As illustrated in Fig. 4D, exposing a cell to HA/A� may cause the pHi during the plateau phase to rise to a value even greater than the initial pHi. For example, if the cell had an acid extruder that is stimulated either by HA or A�, then the infl ux of HA would initially acidify the cell. However, the stimulated acid extruder would not merely return pHi to its initial value, as in Fig. 4C, but increase pHi beyond the initial value, as in Fig. 4D. We can provide two such exam-ples in which A� stimulates acid extrusion and thereby elicits pHi changes similar to those in Fig. 4D: (1) exposing renal mesangial cells to CO2/HCO�

3 stimulates a Na�-driven HCO�

3 uptake mechanism (75, 76), and (2) adding acetic acid/acetate to the lumen of a rabbit S3 proximal tu-bule stimulates a Na�/acetate cotransporter (389, 393). HA can also stimulate acid extrusion. In the rabbit proximal tu-bule, CO2 per se appears to stimulate Na-H exchange and H� pumping (391).

EFFECTS OF NH3 AND OTHER NEUTRAL WEAK BASES

Solutions containing the neutral weak base B also contain its conjugate weak acid HB�. Because membranes are usually far more permeable to B than to HB�, B fl uxes tend to have a greater effect on pHi.

Flux of Neutral Weak Base

When a cell is exposed to a neutral weak base, B enters and associates with H� to form HB�, causing pHi to rise:

B � H� → HB� (Eq. 17)

This process continues until [B]i equals [B]o. Subsequently, there should be no further change in pHi, provided there is no fl ux of HB� and no change in the rates of acid-base transporter activity. Removing external B reverses the reac-tion as written in Eq. 17, and B passively diffuses out of the cell. pHi should then return to exactly its initial level. An example of the effect of the weak base NH3 is shown in Fig. 5. Applying 20 mM total ammonium (i.e., [NH3]o � [NH�

4]o) causes pHi fi rst to rise, and then stabilize (segment ab, pulse 1). Removing the NH3/NH4

� elicits a fall in pHi to a value somewhat lower than the initial one (compare points a and c). The reason for this small pHi undershoot will become clear in the following sections. As discussed above for HA-induced acidifi cations, the magni-tude of the NH3-induced alkalinization depends on intra-

cellular buffering power, [NH3]o, and the degree to which entering NH3 is protonated. The last is governed by the relation:

pH pKNHNH

i ai

i

= ′ ++

log[ ][ ]

3

4

(Eq. 18)

where pK′a (∼9.2 at 22°C) is the acid dissociation constant. Thus, at the initial pHi of 7.3 (point a), approximately 99.4% of entering NH3 is protonated to form NH4

�, whereas at a pHi of 7.8 (point b), only approximately 98% is protonated. The dependence of the protonation of NH3 on the difference (pK′a � pHi) has two consequences, analogous to the two discussed earlier for CO2. First, the higher the initial pHi, the smaller the magnitude of the alkalinization elicited by the subsequent exposure to the NH4

�. Second, as one raises [NH3]o by fi xed incre-ments, the magnitude of the alkalinization with each suc-cessive [NH3]o increment does not increase in proportion. Thus, in Fig. 5, the alkalinization produced by 10 mM total NH4

� is less than twice as great as that produced by 5 mM total NH4

�.In Fig. 5, the magnitude of the NH4

�-induced alkalini-zation actually depends more on [NH3]o than on [total NH4

�]o. Thus, the NH4�-induced alkalinizations will be

identical for solutions in which [total NH4�] and pHo are

varied reciprocally so as to keep [NH3]o constant (56). Neutral weak bases such as lidocaine (50), procaine (50), and methylamine (67) also elicit intracellular alkaliniza-tions that have [B]o and pHi dependencies similar to those described for NH3.

7.8

7.6

7.4

7.2

pHi

20mMNH4Cl

10mM 5mM

30 min

1 2 3

7.0

2mM 1mM

4 5

b

a

c

pHo � 7.70

FIGURE 5 Effects of brief exposures to NH3 on pHi. A giant barnacle muscle fi ber was exposed to NH3 at fi ve different levels of [NH3] � [NH4

�]: 20, 10, 5, 2, and 1 mM. Because pHo was 7.70 throughout, [NH3]o was approximately 312, 156, 78, 31, and 16 �M, respectively, in the fi ve pulses. After its entry into the cell, NH3 rapidly combines with H� to form NH4

�. The resultant rise in pHi continues until [NH3]i � [NH3]o. Remov-ing external NH3/NH4

� reverses these processes. Between NH3/NH4�

pulses, the external solution was buffered with 5-mM HEPES to pH 7.5. pHi was measured with an exposed-tip pH-sensitive microelectrode, of the design of Hinke. (Used with permission from Boron WF. Intracellular pH transients in giant barnacle muscle fi bers. Am J Physiol 1977;233:C61–C73.)



As noted previously, the bacterial protein AmtB, an Rh homologue, functions as a gas channel permeable to NH3 (288, 386, 615). In addition, aquaporins-1, -4, -5, and -8 (387, 475) are permeable to NH3.

Flux of Cationic, Conjugate Weak Acid

When one exposes a cell to a weak base, the dominant effect on pHi generally refl ects the infl ux of the highly permeant neutral weak base (e.g., NH3). However, the fl ux of the conjugate weak acid (e.g., NH4

�) may produce pHi decreases that are substantial or even dominant. The ef-fects on pHi of applying and withdrawing NH3/NH4

� can be separated into four steps. When a cell is exposed to NH3/NH4

�, the rapid infl ux of NH3 leads to an initial rise in pHi (Fig. 6A). However, this alkalinization is actually blunted by the simultaneous, although smaller, infl ux of NH4

�. The most general mechanism of NH4� infl ux is the

passive diffusion of NH4� (61), although the carrier-

mediated transport of NH4� into cells can be substantial.

For example, NH4� can enter cells by substituting for K�

on the Na�-K� pump (7) and/or the Na�-K�-Cl� co-transporter (289). Although most of the entering NH4

� remains NH4

�, a small fraction (governed by the differ-ences between pK′a and pHi) dissociates to form NH3 and H�. When external NH3/NH4

� is removed, the NH4� that

previously entered, but failed to dissociate, now dissociates into NH3 (which rapidly leaves the cell) and H� (which is trapped within the cell). As a consequence, the fi nal pHi (point c in Fig. 5) is slightly lower than the initial value (point a).

The acidifying effect of NH4� is much more evident when

the NH3/NH4� exposure lasts beyond the initial infl ux

(Fig. 6A) and equilibration of NH3 (Fig. 6B). During an ex-tended plateau phase, the continuing net infl ux of NH4

� pro-duces a plateau-phase acidifi cation, as illustrated in Fig. 6C. In cells with a Cl�-HCO�

3 exchanger (e.g., sheep cardiac Pur-kinje fi bers), this plateau-phase acidifi cation may be aug-mented by effl ux of HCO�

3 in exchange for Cl� (566). When the external NH3/NH4

� is eventually withdrawn, the pHi un-dershoot is greatly exaggerated (Fig. 6D). The magnitude of the undershoot refl ects the previous infl ux of NH4

�. The longer the exposure to NH3/NH4

�, the more extensive the plateau-phase acidifi cation, and the larger the undershoot. This under-shoot represents an acute intracellular acid load. Indeed, the NH4

�-prepulse technique is widely used for acid loading cells in studies of pHi regulation (61).

If the NH4� electrochemical gradient is reversed during

the plateau phase, in a cell for which the dominant mecha-nism of NH4

� entry is a passive fl ux, then pHi rises during the plateau phase and the pHi undershoot is converted to a shortfall (56).

FIGURE 6 The NH4�-prepulse technique. A: When a cell is fi rst exposed to a solution

containing NH4� and NH3, there is a rapid increase in pHi, due to the infl ux of NH3. This rise

in pHi is blunted if NH4� can also enter the cell. B: Eventually, [NH3]i rises to the point where

[NH3]i transiently equals [NH3]o. Here, NH4� is in equilibrium with NH3 and H�, both inside

and outside the cell. pHi is transiently stable. C: During the plateau phase, the entry of NH4�

leads to the formation of intracellular NH3, which then exits the cell. This sets up a shuttling system for carrying H� into the cell. The NH4

� may enter via a simple conductive pathway or be transported by a carrier such as the Na�-K� pump or Na�/K�/Cl� cotransporter. Other acid-loading processes (e.g., Cl�-HCO3

� exchange or metabolism) could also contribute to the plateau-phase acidifi cation. D: Upon removing external NH3/NH4

�, most of the intracellular NH4

� gives up its H� and exits the cell as NH3. Because NH4� had accumulated in the cell

during the plateau (either because of NH4� uptake per se or other acid-loading processes), pHi

undershoots its initial value and the cell is acid loaded.



INTRACELLULAR BUFFERING

Role of Buffering in pHi Regulation

In the broadest sense, a pH buffer is any system that moder-ates the effects of an acid or alkali load by reversibly consum-ing or releasing H�, respectively. As we shall see, buffering in the intracellular fl uid (e.g., the cytosol) is considerably more complex than in the blood and extracellular fl uids. One can determine the buffering power () of the extra- or intracel-lular fl uid by applying an acute acid or alkali load, and mea-suring the resultant change in pH. is defi ned as the amount of strong base (or strong acid) required to raise (or lower) pH by a given amount. Strictly speaking, the defi nition is given in differential notation. However, if the amount of base added ( , given in mM) and the resultant pH increase ( pH) are suffi ciently small, then is approximated by:

=

BpH

(Eq. 19)

This defi nition was originally proposed in slightly different form by Koppel and Spiro in 1914 (311, 455) and later modifi ed to its present state independently by Michaelis (365) and Van Slyke (564). Note that is always a positive number; for a strong acid, and pH are both negative. is usually reported in the unit mM per pH unit.

Among the several methods available for applying acid and alkali loads to the cytoplasm, the most direct is by injec-tion of acid (363, 545) or alkali into the cell with a micropi-pette. In all cells that regulate their pHi, such an acute acid load produces an abrupt fall in pHi, followed by a recovery towards its initial value (Fig. 7). The extent of the abrupt fall

in pHi (segment ab) is determined by the amount of acid injected, the intracellular buffering power, and the ability of pHi-regulatory mechanisms to blunt the pHi decrease. As discussed in later sections, these pHi-regulatory mechanisms can actively extrude acid from the cell and thereby return pHi to its initial value (b–f ). Figure 7 shows a model ex-periment in which these pHi-regulatory mechanisms are allowed to function. If we were to calculate blindly, then we would see that the apparent depends critically on the time of the pHi measurement following the acid load. For example, the apparent is relatively low at point b and rises to infi nity at point f, where pHi is zero.

An approach for avoiding this ambiguity is to defi ne in-tracellular buffering power (i) under conditions in which we completely block pHi regulation. In this case, pHi would fol-low the trajectory indicated by ag in Fig. 7, the observed pHi at “infi nite” time would be relatively large, and the computed would be smaller than any of the possibilities described previously. There are three justifi cations for exclud-ing acid-base transport processes from the defi nition of i:

1. As we have just seen, excluding cellular transport pro-cesses makes the calculated i time independent (pro-vided we allow enough time for pHi to fall).

2. As we shall see later, transport of acid and/or base can generally be distinguished from other buffering mecha-nisms by applying transport inhibitors or performing ion substitutions in the extracellular fl uid.

3. Generally, these transport processes are not fully revers-ible. The buffering mechanisms included in our defi ni-tion of i fall into three categories: (1) weak acids and bases (i.e., chemical buffers), (2) biochemical reactions consuming or releasing H�, and (3) transport of acids or bases across organellar membranes. These mechanisms, which will be discussed in more detail later, can be justi-fi ably grouped because there is presently no practical basis for distinguishing among them. Furthermore, they proceed very rapidly compared to the time currently re-quired to measure pHi.

Buffering mechanisms abate pHi changes produced by acute acid and alkali loads that otherwise could damage the cell. However, these mechanisms cannot prevent a change in pHi, only reduce its magnitude. Furthermore, buffering mechanisms cannot restore pHi to its initial value following an acid or alkali load. Such recoveries are brought about by transport of acid and/or base across the cell membrane. As an illustration, consider the response of the cell to an acute intracellular acid load, as in Fig. 7. Cellular buffers respond very rapidly, typically consuming more than 99.99% of the applied acid and limiting the initial fall in pHi (ag). Active transport mechanisms respond more slowly, extruding acid from the cell and returning pHi toward normal (b–f ). During the transport-mediated pHi recovery, H� previ-ously taken up by the buffering mechanisms is now released and extruded from the cell. By the time pHi returns to its initial level (f ), the entire acid load has been extruded from

FIGURE 7 Determination of intracellular buffering power (i) in an intact cell from the pHi change produced by an acute acid load. i is defi ned as the amount of strong acid injected (mmole/l of cytoplasm) divided by the negative of the resultant fall in pHi. Injecting acid, however, causes a bipha-sic change in pHi: a rapid fall (segment ab), followed by a slower recovery (bcdef ). The latter is due to extrusion of acid from the cell by one or more active transport processes. The true i must be computed from the pHi change ( pHag) that would have occurred had all acid extrusion been blocked (segment ag). If the pHi instead is computed from the pHi at points b, c, d, e, or f, the computed i will be, at best, artifactually high (in the case of pHab) and, at worst, will approach infi nity (in the case of pHaf � 0). If the acid load is applied by exposing the cell to CO2, then the active transport of HCO3

� into the cell can sometimes cause the pHi at point f to be greater than the pHi at point a. In this case, pHaf is less than 0, and the computed i will be negative.



the cell, and cellular buffering mechanisms have been re-turned to their pre-acid-loading state (a). Buffering mecha-nisms play no role in the steady state; in this case, acid loading exactly balances acid extrusion, and the state of cellular buffers is unchanged.

MECHANISMS OF INTRACELLULAR BUFFERING

The total intracellular buffering power (T) is the sum of chemical (i.e., weak-acid/base equilibrium), biochemical, and organellar buffering powers.

Chemical Buffering

A weak base (B) of valence n reacts with H� to produce its conjugate weak acid (HB) of valence n � 1:

Bn � H� → HBn�1 (Eq. 20)

This equilibrium is described by the relation:

′ =+

+K a

n

n

B HHB

[ ][ ]1

(Eq. 21)

where K′a is the apparent acid dissociation constant. This relation can also be expressed in logarithmic form, where-upon it takes the form of Eq. 18. Starting with the defi nition of (Eq. 19) and the statement of chemical equilibrium (Eq. 21), one can express as a function of [H�], K′a, and total buffer concentration (311, 365).

= ′′ +

+

+

2 32

. [ ]( [ ])

[ ]K H

K HTBa

a

(Eq. 22)

where 2.3 approximates ln(10), and [TB] is the concentra-tion of total buffer (i.e., [TB] � [Bn] � [HBn�1]). By ob-taining the derivative of with respect to [H�] (i.e., d/d[H�]) and setting this equal to zero, one can show that is maximal when [H�] equals K′a (311). This maximal buff-ering power (max) is simply:

max � 0.58 [TB] (Eq. 23)

Although the pH of maximal buffering power is in general unique to the buffer, all buffers have the same molar buffer-ing power (max/[TB]), and same dependence of on the difference pH � pK′a. If more than one buffer is present, then T is simply the sum of the individual buffering pow-ers, each computed from Eq. 22.

The foregoing equation describing was derived by tak-ing the partial derivative of [n] with respect to pH at a fi xed [TB]. That is, the analysis applies only to buffers in a closed system, in which [TB] is constant. Closed-system buffers include a solution of a nonvolatile buffer (e.g., inorganic phosphate) in a beaker, a solution of a volatile buffer (e.g., CO2/HCO�

3) in a capped syringe, and an impermeant buffer

(e.g., a protein) in the cytoplasm of a cell. In such a closed system, for CO2/HCO�

3 would be rather low at normal pHi values, because pHi (∼7.0) is far higher than the pK′a of 6.1. However, cells are generally not a closed system with respect to CO2/HCO�

3. Rather, CO2 usually freely exchanges with the extracellular fl uid (ECF) so that the total intracel-lular CO2 (i.e., [CO2]i � [HCO�

3]i) can change appreciably during intracellular acid-base disturbances. For example, when H� is added to cytoplasm containing CO2/HCO�

3, H� combines with HCO�

3 according to the reactions:

H� � HCO�3 → H2CO3 → H2O � CO2 (Eq. 24)

The cytoplasmic CO2 so formed is lost to the ECF. Thus, it is [CO2]i that remains constant, while [HCO�

3]i and [total CO2]i both fall. Because there is no buildup of CO2 within the cell, the extent of the reaction in Eq. 24 is limited only by the availability of HCO�

3. The amount of H� absorbed (i.e., buffered) by the CO2/HCO�

3 system exactly equals the amount of HCO�

3 that disappears. That is,

CO HCOpH

23=−

[ ] (Eq. 25)

where CO2 is the CO2 buffering power. When this equa-

tion, in differential form, is combined with the Henderson-Hasselbalch equation, it can be shown that

CO2 � 2.3 [HCO�3] (Eq. 26)

when [CO2] is held constant. At very high pH values, when the equilibrium H� � CO�

3 # HCO�3 cannot be ignored,

the term 4.6 [CO�3] must be added to the right-hand side of

Eq. 26. This equation describes the buffering power of CO2/HCO�

3 in an open system, such as a solution in a beaker equilibrated with gaseous CO2. Because most cell mem-branes are highly permeable to CO2, this gas rapidly equili-brates across membranes and stabilizes [CO2]i, provided the ECF behaves as an infi nite reservoir for CO2. Thus, an iso-lated cell in vitro behaves as an open system for CO2/HCO�

3. Because the intact organism has mechanisms (e.g., alveolar ventilation in higher vertebrates) for stabilizing [CO2] in the ECF, CO2/HCO�

3 behaves as an open-system buffer in vivo in the extra- and intracellular spaces.

In an open system, the CO2/HCO�3 buffer pair generally

makes a substantial contribution to T. For example, at a pHi of 7.1, the buffering power of all non-CO2 buffers in rat renal mesangial cells is only about 10 mM (75), whereas the computed CO2 is nearly 29 mM when the cell is equili-brated with 5% CO2. Thus, CO2 accounts for nearly 75% of the T of 39 mM/pH unit.

It is essential to distinguish clearly between open- and closed-system buffers of a cell. The cell is a closed system for intracellular buffers that do not readily cross the cell mem-brane (e.g., inorganic phosphate and the imidazole groups of proteins). Thus, these buffers are infl uenced by the attributes of the cell (e.g., volume, temperature, and metabolic state), and not those of the extracellular fl uid. These are termed



“intrinsic buffers” (56). Biochemical and organellar buffer-ing mechanisms (vide infra) are also intrinsic, and the total intrinsic buffering power (I) is the sum of biochemical, organellar, and intrinsic chemical buffering powers.

The cell is an open system when one member of the buf-fer pair readily crosses the cell membrane. The buffering power of such a buffer pair is very sensitive to extracellular conditions. As noted earlier, CO2 is proportional to [HCO�

3]i, which in turn is completely determined by pHi and the extracellular PCO2 (assuming that CO2 equilibrates across the cell membrane). The cell also behaves as an open system for buffers other than CO2/HCO�

3. These are gen-erally conjugate pairs of which one member is a small, neutral molecule. Examples include NH3/NH4

� and acetic acid/acetate. Regardless of whether the charged species is a cation or an anion, the intracellular buffering power of an open-system buffer pair is always 2.3 � [charged species]i, provided the neutral species is equilibrated across the cell membrane. Thus, the intracellular buffering power of such a buffer pair is sensitive not only to the total amount of the buffer in the ECF (i.e., [TB]o), but also to pHo. As with CO2/HCO�

3, these other open-system buffers can some-times be the dominant component of T. For example, I in squid axons is ∼9 mM (61). When these axons are exposed to a pHo�7.7 NH3/NH4

� solution containing only 10 mM total ammonium, [NH4

�]i rises to 10 mM. The computed NH

3 is 23 mM, nearly 70% of T. Because the buffering power of such open-system buffers is so sensitive to factors external to the cell (e.g., pHo), these buffers are termed “extrinsic” (56). The total chemical buffering power of a cell is the sum of the buffering powers of the individual conju-gate acid/base buffer pairs, be they intrinsic (closed system) or extrinsic (open system).

Biochemical Buffering

Because certain biochemical reactions consume or release H� and are pHi sensitive, they can act as H� buffers (456). Examples include hydrolysis of ATP (which releases H�) and hydrolysis of phosphocreatine (which consumes H�). During the Cori cycle, the liver converts lactic acid into glucose, whereas skeletal muscle breaks down the glucose to produce ATP and more lactic acid. Also, H� buffering can arise when reactions induce a change in the pK′a of an ioniz-able group. A classic example is the buffering reactions of oxygenated versus deoxygenated hemoglobin.

Well-studied examples of biochemical buffering are reac-tions involving intermediary metabolites in rat brain (183). Cells that are acutely acid loaded with an increase of PCO2

in the ECF respond by reducing intracellular levels of several acid metabolic intermediates (i.e., the acids of lactate, pyru-vate, citrate, �-ketoglutarate, malate, glutamate, and aspar-tate), while raising those of glucose and glucose-6-phosphate. These observations are consistent with the hypothesis that reducing pHi inhibits a step in the glycolytic pathway, possibly the phosphofructokinase reaction. Indeed, mouse

muscle phosphofructokinase is markedly sensitive to pH changes (556); reducing in vitro pH from 7.2 to 7.1 produces more than a 90% inhibition. Rat brain cells respond in the opposite fashion to acute intracellular alkali loads. Increases in pHi induced by lowering the PCO2

in the ECF lead to increased levels of lactate and pyruvate (348). Thus, the bio-chemical machinery of these cells seems to respond appropri-ately to pHi changes, consuming H� in response to intracel-lular acid loads and releasing H� in response to alkali loads. The extent to which such biochemical reactions contribute to overall buffering power can be computed from changes in steady-state metabolite levels, provided that such changes are reciprocally linked to the production or consumption of neu-tral and/or readily diffusible molecules. For example, the consumption of one citrate molecule removes three H�, whereas malate removes two, and pyruvate, one. From data on rat brain (508), the buffering power of biochemical reactions amounts to about half that provided by all non-CO2 physicochemical buffers. Biochemical buffering power can be defi ned and quantitated in a manner analogous to chemical (vide supra) and organellar (vide infra) buffering powers (see Eq. 19). The biochemical buffering reaction can be written as:

Rn � q H� # Pn�q (Eq. 27)

where R is the reactant, P is the product, n is the valence, and q is the H� stoichiometry (q � 0). The biochemical buffering power is thus:

=⋅

[ ]R qpH

n

(Eq. 28)

As noted previously, biochemical and organellar (vide infra) buffering mechanisms, in addition to closed-system chemi-cal buffers, comprise the intrinsic buffers.

Organellar Buffering

H�-transport systems have been identifi ed or implicated in many intracellular organelles (456, 459), including mito-chondria (83, 181, 369–372, 513, 514), lysosomes (401), sarcoplasmic reticulum (398), Golgi network (152, 292), endosomes (134, 237, 253, 394, 563), chromaffi n granules (240, 401), and zymogen granules (22). In addition, three of the cloned Na-H exchangers (NHE6, -7, and -9) are found in organellar membranes (see Chapter 54) where they may contribute to organellar pH regulation (for reviews of ve-sicular H� pumps, see [185, 217]). It would be reasonable to expect decreases in cytoplasmic pH to stimulate H� uptake and/or inhibit H� extrusion by at least some organelles and increases in pHi to produce the opposite effects. Such buff-ering would lead to a net transfer of H� into these organ-elles following intracellular acid loads and a net movement of H� into the cytoplasm following alkali loads. A transfer of acid or base across organellar membranes would consti-tute a de facto buffer mechanism for the bulk intracellular



fl uid. Although the extent to which such hypothetical or-ganellar buffering mechanisms contribute to T is not estab-lished, it can be inferred from published data that changes in extraorganellar pH can produce at least small changes in the intraorganellar pH of mitochondria (503), lysosomes (401), and sarcoplasmic reticulum vesicles (398). Organellar buff-ering power can be defi ned and quantitated in a manner analogous to that for physicochemical and biochemical buff-ering (see Eq. 19). If the organellar buffering reaction is written:

Organelle() � H� # organelle(H�) (Eq. 29)

where the parentheses refer to organellar contents. Organel-lar buffering power is thus:

=

[ ()]organellepHi

(Eq. 30)

Organellar buffers, along with the biochemical and closed-system chemical buffers, comprise I.

MEASUREMENT OF INTRACELLULAR BUFFERING POWER

Titration of Cell Homogenates

The easiest method for estimating i is to homogenize a tissue sample and titrate the homogenate. The slope of the pH titration curve is the buffering power of the homoge-nate. However, some disadvantages limit the accuracy of this technique. First, homogenization-induced changes in metabolism must be prevented, for example by quick-freezing the sample before homogenization (193), and then performing the titration at low temperature (193) or in the presence of an inhibitor such as fl uoride (508). However, with metabolism blocked, it is unlikely that the contribu-tion of biochemical buffering can be accurately assessed. In addition, reducing temperature generally increases pK’a values of chemical buffers, and thus modifi es the relation-ship between and pH. Finally, homogenization may dis-rupt cellular organelles and thus obscure the organellar buffering contribution. It might be possible to assess the organellar contribution to the buffering of the cytoplasm by using new techniques to monitor pH changes inside organ-elles (see previous section).

Microinjection

The most straightforward approach for estimating i in an intact cell is to microinject a known amount of acid or base into a cell and monitor the resultant change in pHi. The microinjection can be achieved by iontophoresis (545) or pressure injection (363). With the iontophoresis technique, two electrodes, one fi lled with acid or base and the other with a neutral salt, are placed in a cell, and electric current is

passed between them. From the amount and duration of current, and from an in vitro calibration of current versus ejected acid/base, one can compute how much acid or base entered the cell. The pressure-injection method (363) also requires that the cell be impaled with two micropipettes, one for pressure-injecting the Cl� salt of an acid (e.g., HCl), and the other for monitoring intracellular Cl� activity (ai

Cl). From the rise in ai

Cl and the Cl� activity coeffi cient, one can calculate the amount of HCl injected per unit volume of cytoplasm. This technique enables one to compute the total i (i.e., sum of physicochemical, biochemical, and organellar buffering) of the cell. For small neurons incubated in a CO2-free solution, the iontophoresis and pressure-injection meth-ods give i values of 10.8 mM/pH unit (545) and 10.3 mM/pH unit (363), respectively. Disadvantages of this direct in vitro titration approach include the necessity of us-ing cells that (1) are large enough to withstand the micro-electrode impalements and (2) have a suffi ciently compact geometry to permit rapid equilibration of the injected acid or base throughout the cell.

Weak-Acid and Base Methods

An approach that, at least in principle, is applicable to cells of all shapes and sizes, is measurement of the pHi change produced by exposing the cell to a neutral weak acid HA (HA # H� � A�), or weak base B (HB� # H� � B). As discussed earlier, exposing a cell to a neutral weak acid leads to a decrease in pHi as HA enters the cell and partially dis-sociates into H� and A�. The entry and dissociation halt once [HA]i � [HA]o. The extent of the dissociation de-pends on the weak acid’s pK′a, as well as the initial pHi. Virtually all of the released H� is consumed by intracellular buffers (Fig. 8A); the remaining fraction is responsible for the accompanying pHi decrease. Although HA/A� is a buf-fer, it does not participate in buffering the acid load imposed by the intracellular dissociation of HA. Similarly, a B/BH� buffer system can not buffer changes in pHi caused by the entry (or exit) of B. Because the dissociation of HA leads to the formation of one A� for each H�, the magnitude of the intracellular acid load is [A�]i. This amount of added acid is the additive inverse of the amount of base added ( B) in the preceding defi nition of :

ii

i

i

BpH

ApH

= = − −

[ ] (Eq. 31)

Note that i is the non-HA/A� buffering power of the cell. It is the sum of chemical, biochemical, and organellar buffering. pHi is directly measured, and [A�]i is calcu-lated from fi nal and initial values of pHi and [HA]o. In the simplest case, the initial [A�]i is zero, and fi nal [A�]i is given by:

[ ] [ ]A HAipH pKi a− − ′= ⋅10 (Eq. 32)



assuming [HA] � [HA]i � [HA]o. A commonly used weak acid for measuring i is CO2, for which:

ii

i

i

BpH

HCOpH

= = − −

[ ]3 (Eq. 33)

i is the non-CO2/HCO3� or intrinsic buffering power, i

I. Once i

I is known, the total intracellular buffering power (i

T) can be obtained by computing the open-system CO2 buffering power:

iT

iI

iCO2= + (Eq. 34)

where iCO2, the CO2 buffering power, is given by 2.3 �

[HCO�3]i, as outlined previously.

There are as many as three major drawbacks of using the weak-acid method to measure . First, [HA]i may not exactly equal [HA]o. In rapidly metabolizing cells for in-stance, [CO2]i may exceed [CO2]o. Second, the pHi de-crease elicited by the entry of HA may stimulate acid-extrusion mechanisms (i.e., pHi-regulating systems described in subsequent sections). Thus, the actual pHi may be smaller than if all acid-base transport had been blocked (Fig. 7). By underestimating pHi, one will over-estimate i. This error should in principle be eliminated by blocking the pHi-regulating systems (56). One can also minimize the error by extrapolating the pHi-versus–time curve back to a point at which acid extrusion is judged to have had no effect on pHi (8, 47, 49, 113). For example, in the hypothetical experiment of Fig. 7, one could extrapo-late the pHi recovery (i.e., bcdef ) back to the time of the acid load. A third potential drawback of the weak-acid method is that the weak acid could indirectly alter pHi by modifying cellular metabolism. Thus, it is critical that the weak acid affect pHi only by entering and dissociating into H� and A�. Otherwise, any other effect on pHi will lead to an error in the calculation of i.

The intrinsic intracellular buffering power can also be determined by exposing a cell to a neutral weak base, B (Fig. 8B). As described in the preceding section, exposing a cell to B leads to a pHi increase as most of the entering B combines with H� to form HB�. The entry of B and the alkali loading of the cell continues until [B]i � [B]o. Almost all of the entering H� that is converted to HB� is derived from cellular buffers. The minute amount that comes from the pool of free H� is responsible for the rise in pHi. Thus, the change in [HB�]i is equivalent to the amount of strong base added to the intracellular fl uid:

ii

i

i

BpH

HBpH

= =+

[ ] (Eq. 35)

In the simplest case, the initial [HB�]i is zero, and the fi nal [HB�]i is calculated from the statement of chemical equilib-rium:

[ ] [ ]HB BipK pHa i+ ′ −= ⋅10 (Eq. 36)

assuming that [B] � [B]i � [B]o. The most commonly used weak base is NH3:

ii

i

i

BpH

NHpH

= =+

[ ]4 (Eq. 37)

[NH4�]i is determined in a manner analogous to that outlined

earlier for [HCO�3]i. Note that i in this case is the non-

NH3/NH4� buffering power (i.e., the buffering power of all

buffers other than NH3/NH4�). If the NH3 titration is per-

formed in the absence of CO2/HCO�3, then the measured i

is iI. If CO2/HCO�

3 is present, then the measured i is iI

� iCO2 � i

T. An approach commonly used with NH4� is to

compute from the pHi decrease caused by decreasing or withdrawing external NH4

�. During such experiments, one usually blocks acid-extrusion mechanisms (e.g., by removing external Na�) that would likely counteract the acidifi cation. By reducing [NH4

�]o in a stepwise fashion (Fig. 9A), one can compute i as a function of pHi (Fig. 9B), as has been done in mesangial cells (75) and gastric parietal cells (590). An alternate approach one can use with NH4

� is to compute i from the pHi increase that accompanies stepwise increases of external NH4

� (49).

ACID-BASE TRANSPORT SYSTEMS

Acid-base transporters can be divided into two groups: acid loaders and acid extruders. Acid loaders move H� into or base equivalents (e.g., OH� or HCO3

�) out of cells and gen-erally contribute to the recovery of pHi from acute intracel-lular alkali loads. In contrast, acid extruders move H� out of or base equivalents into cells and generally contribute to the recovery of pHi from acute intracellular acid loads. In the remainder of this section, we discuss major acid-base trans-porters in each of these two categories. For each transporter,

FIGURE 8 Measurement of intracellular buffering power by exposure to a neutral weak acid or base. A: When a cell is exposed to a neutral weak acid (HA), HA enters the cell, where some of it dissociates into H� and A�. Virtually all of this H� is taken up by buffers (M; valence � n) that are titrated to their conjugate weak acids (HM; valence � n � 1). Thus, for each A� formed, almost exactly one H� is consumed by non-HA/A� buf-fers. � � [A�]i / pHi. B: When a cell is exposed to a weak base, B, the base enters the cell, where some of it combines with H� to form HB�. Virtually all of this H� is derived from buffers (HM; valence � n � 1) that are titrated to their conjugate weak bases (HM; valence � n). Thus, for each HB� formed, almost exactly one H� is released by non-B/HB� buf-fers. � [HB�]i / pHi.



we will examine at least the following three issues: (1) general function and molecular identity, (2) energetics and role in pHi regulation, and (3) effects of pharmacological agents. Since the third edition of this book was published, there have been considerable advances in the molecular identifi cation and characterization of many acid-base transporters. For a discussion of the molecular biology of the Na-H exchangers (see Chapter 54), Na�-coupled HCO�

3 transporters (see Chapter 52), and anion exchangers (see Chapter 53), please consult the relevant chapters in this book.

ACID-LOADING MECHANISMS

As noted previously, passive fl uxes of H�, monovalent cat-ionic acids (e.g., NH4

�), and monovalent anionic bases (e.g., HCO�

3) are governed by the electrochemical gradient for the ion in question, and normally tend to acidify the cell. In

principle, such passive transport processes could be ex-ploited for protecting the cell against alkaline loads. Never-theless, such passive processes generally do not appear to have a substantial impact on pHi. A notable exception dis-cussed above is the GABAA-activated Cl� channel at the crayfi sh neuromuscular junction (280) and in cells from turtle cerebellum (111) and rat hippocampus (112, 279). This Cl� channel can conduct HCO�

3 and thus mediate a substantial HCO�

3 effl ux that lowers pHi. For the most part, however, major acid-loading transport pathways are HCO�

3 transporters— the most common ones being Cl�-HCO�

3 exchange and Na�/HCO�

3 cotransport.

Cl�-HCO3� and Cl�-OH� Exchangers

GENERAL FUNCTION AND MOLECULAR IDENTITY

Found in a wide variety of animal-cell membranes, Cl�-HCO�

3 exchangers normally couple the infl ux of Cl� to the effl ux of HCO�

3 (Fig. 10A). Na�-independent Cl�-HCO�3

exchangers (to distinguish them from the Na�-dependent Cl�-HCO�

3 exchangers discussed later) are thought to serve two major functions in nonepithelial cells: regulation of in-tracellular pH (pHi), and regulation of intracellular [Cl�]. The erythrocyte exchanger is known as the “band 3 protein” because of its position on sodium dodecyl sulfate (SDS)–polyacrylamide gels (91). Band 3 has also been termed

7.2

6.8

6.4

6.0

pHi

7.6

0 Na�

a

b

c d

e

f

g

NH 4�: 20 10 5 2.5 1 0.5

hi

k

j2 min 07018802

7.6

10

0Buf

ferin

g po

wer

(m

M/p

H)

6.1 6.4 7.0

pHi

7.3

15

5

6.7

FIGURE 9 The dependence of intrinsic intracellular buffering power on pHi. A: The effect on pHi of stepwise reductions in [NH4

�]o in the absence of CO2/HCO3

�. The cell was fi rst exposed to a solution containing 20 mM. Na� then was removed to block acid-extruding mechanisms. The subsequent reduc-tion in [NH4

�]o from 20 to 10 to 5 to 2.5 to 1 to 0.5 to 0 mM produced discrete acid loads. This experiment was performed on a single renal mesangial cell, the pHi of which was measured using bis-carboxyethyl-carboxyfl uorescein and a microscope-based fl uorometry system. B: Computation of i. The acid load imposed by each stepwise reduction in [NH4

�]o was computed from the change in [NH4

�]i. The latter was calculated from [NH4�]o, pHo and pHi. The mean

buffering power in the pHi range covered by the stepwise fall in pHi was com-puted as i � [NH4

�]i/ pHi. (Used with permission from Boyarsky G, Ganz MB, Sterzel B, Boron WF. pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO3-. Am J Physiol 1988;255: C844-C856.)

FIGURE 10 Acid-loading mechanisms. A: Cl�-HCO3� exchanger.

B: Cl�-organic anion exchanger. C: The 1:3 electrogenic Na�/HCO3� co-

transporter. The electrogenic Na�/HCO3� cotransporter present in the ba-

solateral membranes of epithelial cells has a stoichiometry of three HCO3�

equivalents for each Na�. Three alternative models are shown. D: Electro-neutral K�/HCO3

� cotransporter. E: Ca2�-H� pump. F: Glutamate trans-porter. G: Peptide transporter. H: Divalent-cation transporter.

A

B



“AE1” because it was the fi rst “anion exchanger” to be cloned and sequenced (310). As described in greater detail in Chap-ter 53, the AE gene subfamily comprises three related genes AE1, AE2, and AE3. The AE gene subfamily is a branch of the bicarbonate-transporter (SLC4) family that includes the Na�-coupled HCO�

3 transporter subfamily (see Chapter 52). Several members of the SLC26 family can also mediate Cl�-HCO�

3 exchange (384).

INVOLVEMENT OF CL� AND HCO3

�/OH�: ENERGETICS

Red-cell AE1 mediates electroneutral homo- or hetero-exchange of monovalent anions with a 1:1 stoichiometry, exhibiting a substrate preference of Cl� � HCO�

3 � NO3 � Br � F � I � divalent oxyanions (31, 274). Sulfate is co-transported with H� (367). Monovalent anion exchange can be reasonably well modeled by a ping-pong kinetic scheme, thought to refl ect the alternation of an anion binding site between one side and the other of the membrane (90, 192, 306, 443). Because the Cl�: HCO�

3 stoichiometry is 1:1, the transporter is electroneutral. Thus, the direction of net trans-port is determined by the sum of the chemical gradients for Cl� (i.e., [Cl�]i/[ Cl�]o) and HCO�

3 (i.e., [HCO�3]i/

[HCO�3]o). Because the inward Cl� gradient is generally

greater than the inward HCO�3 gradient in cells other than

erythrocytes, the former dominates, driving HCO�3 out of

the cell. The transporter is easily reversed, however, by invert-ing the sum of the Cl� and HCO�

3 gradients. Indeed, a clas-sic pHi assay for Cl�-HCO�

3 exchange activity involves re-moving external Cl�: this evokes a rapid pHi increase (105, 565). In some cell types, the transporter can also be reversed by lowering [HCO�

3]i, as would occur during severe intracel-lular acid loads. The exchanger operating in reverse then becomes an acid extruder, moving HCO�

3 into the cell and contributing to the pHi recovery from the acid load.

Some Cl�-HCO�3 exchangers have a very high affi nity for

HCO�3 and transport can take place with low levels of HCO�

3 (�100 �M) in a nominally HCO�

3-free solution. For exam-ple, removing external Cl� from the solution bathing rat os-teoclasts can elicit a dramatic 4,4�-diisothiocyanatostilbene- 2,2� disulfonate (DIDS)–sensitive pHi increase, even in the nominal absence of CO2/HCO�

3 (507). However, vigorously bubbling the solutions with 100% N2 gas greatly reduces the pHi increase.

Sun et al. (534) reported the presence of a Cl�-OH� ex-changer (or H�/Cl� cotransporter), which operates in paral-lel with a “conventional” Cl�-HCO�

3 exchanger, in guinea-pig ventricular myocytes. The putative Cl�-OH� exchanger is Cl� dependent; it is activated by low pHo (534) and high pHi (329). The evidence that it is a Cl�-OH� exchanger, rather than a Cl�-HCO�

3 exchanger is that it functions as an acid loader in the presence or absence of CO2/HCO�

3, and is insensitive to DIDS (534). Activating the exchanger by low-ering pHo is unaffected by a CO2-free, 100% N2 atmosphere (328), thereby ruling out the involvement of the small levels of HCO�

3 present in air-equilibrated solutions.

Based on experiments on rat-brain synaptosomes, Martínez-Zaguilán et al. (354) have concluded that a H�/Cl� cotransporter is responsible for (1) the pHi increase elicited by removing extracellular Cl�, and (2) the faster pHi recovery from an acid load in the absence of extracellular Cl�. However, it is generally diffi cult to distinguish H� transport in one direction from OH� and/or HCO�

3 trans-port in the opposite direction.

DEPENDENCE ON pHi

By analogy to acid extruders, which are often stimulated by decreases in pHi, one might expect acid loaders such as Cl�-HCO�

3 exchange to be stimulated by increases in pHi. Indeed, based on DIDS-sensitive 36Cl� fl uxes in Vero and L-cells (404), pHi increases elicited by returning Cl� to Cl�-depleted mesangial cells (76), and rates of pHi change as well as 36Cl� fl uxes in lymphocytes (358), Cl�-HCO�

3 exchange activity in these cells is low but measurable at pHi values as low as 6.5, increases to somewhat higher levels in the physiological pHi range, and then rises very steeply as pHi increases above �7.6. Caution is in order concerning the two methods used to study the pHi sensitivity of Cl�-HCO�

3 exchange. First, isotopic fl uxes refl ect only unidirec-tional movements, and thus cannot distinguish Cl�-Cl� exchange from Cl�-HCO�

3 exchange unless both infl uxes and effl uxes are determined. Second, fl uxes derived from rates of pHi change depend heavily on computed values of CO2, which (like the computed fl uxes) rise steeply with in-creasing values of pHi.

In more recent expression studies, the widely distributed AE2 polypeptides also appear to exhibit greater activity at more alkaline pHi values (263, 275, 327). The pH sensor is located somewhere within the C-terminal transmembrane domain (527, 612), and its apparent pK′a is modulated by residues within the N-terminal cytoplasmic domain (317, 527, 528). Multiple histidines within the transmembrane domain contribute to AE2 activity, as well as pHi and pHo sensitivities (529).

EFFECTS OF PHARMACOLOGICAL AGENTS

The nonerythroid Cl�-HCO�3 exchanger is blocked by

stilbene derivatives such as DIDS and 4-acetamido-4′-diisothiocyanatostilbene-2,2′ disulfonate (SITS) (105, 403), as well as the noncompetitive inhibitor nifl umic acid (132, 307). Some of the more potent AE inhibitors include oxonol dyes at the nanomolar level (14, 305, 308, 434).

Cl�-Organic Anion Exchangers

In addition to the transporters mediating a direct ex-change of Cl� for HCO�

3, as described previously, some transporters exchange Cl� for organic anions that are the conjugate bases of weak acids (Fig. 10B). Although members of the AE family of Cl�-HCO�

3 exchangers can exchange organic anions for Cl� (132a, 274a), investigators have described other Cl�-base exchange



activities that cannot be attributed to AE family mem-bers. Since the last edition of this book, investigators have identified 10 SLC26 family members that trans-port monovalent and divalent anions, including sulfate, formate, and oxalate, in addition to chloride, bicarbon-ate, and hydroxyl ions. SLC26 proteins exhibit a range of ion specificities and many individual proteins exhibit functional versatility by transporting at least three types of anions (384). These proteins likely contribute to the recycling of organic anions associated with NaCl reab-sorption in the proximal tubule. A detailed examination of these gene families is presented in Chapter 53.

Electrogenic Na�/HCO3� Cotransporters


Present in the basolateral membrane of several epithelia, including the renal proximal tubule, is an electrogenic Na�/HCO�

3 cotransporter (59) that mediates the isodirectional fl ux of one Na� and—in the simplest model—three HCO�

3 ions (523). In epithelia, the cotransporter normally mediates a net effl ux of Na� and HCO�

3 (see following sections), and therefore functions as an acid loader (Fig. 10C). In the early proximal tubule, Na�/HCO�

3 cotransport appears to be the major route of HCO�

3 effl ux across the basolateral mem-brane. The transporter thus plays a key role in HCO�

3 reab-sorption, and probably makes an important contribution to Na� reabsorption as well.

Our understanding of Na/bicarbonate cotransporters (NBCs), and other bicarbonate transporters has expanded immensely over the past 10 years with the molecular identi-fi cation of these proteins. As described in Chapter 52, 10 SLC4 genes encode members of the family of bicarbonate transporters including electrogenic and electroneutral NBCs, the Na-driven Cl-HCO3 exchanger (NDCBE), and the Na�-independent anion exchangers (AEs). At the amino-acid level, the transporters share considerable homology to one another, and are therefore predicted to have similar membrane topologies.

INVOLVEMENT OF NA� AND HCO3�: ENERGETICS

In the salamander proximal tubule, the cotransporter has an absolute requirement for Na� and HCO�

3 (59). The co-transporter in rabbit proximal convoluted tubules can still function even in the absence of added HCO�

3 (312), probably due to intracellular formation of HCO�

3 from metabolically-derived CO2. Removing external HCO�

3 elic-its a rapid fall in pHi and [Na�]i, and a near-instantaneous depolarization of the basolateral membrane. A similar pat-tern of changes is caused by removing external Na�. All ef-fects are fully reversible. The DIDS-sensitive depolarization elicited by removing Na�, which makes it appear as though Na� is moving as an anion, is practically diagnostic of elec-trogenic Na�/HCO�

3 cotransport.A 1:3 stoichiometry is accounted for by any of the

schemes outlined in Fig. 10C (341). The model in which

Na� exits together with one CO�3 and one HCO�

3 (Fig. 10C, model B) is supported by the observation that SO�

3 stimulates Na� uptake by basolateral membrane vesi-cles (519), presumably by interacting at a CO�

3 site. How-ever, more recent studies indicate that NBCe1-A—at least as expressed in Xenopus oocytes—does not interact with SO�

3 or HSO�3 (224, 225). Grichtchenko and Boron (222)

performed a more direct assessment of CO�3 transport by

using extracellular pH electrodes to measure NBCe1-induced changes in surface pH (pHs) of Xenopus oocytes expressing both the transporter and extracellular-facing car-bonic anhydrase (CAIV). Depolarization-induced activa-tion of the NBC elicited an expected decrease in pHs as base equivalents were transported into the cell. However, apply-ing a CA inhibitor caused the magnitude of the pHs de-crease to increase rather than to diminish. The larger re-sponse is consistent with the hypothesis that NBCe1 mediates CO�

3 transport and that the role of CAIV is to consume H� generated as the reaction HCO�

3 → CO�3 �

H� replenishes CO�3 near the cell surface.

The direction of net cotransport is determined by the com-bined transmembrane gradients for Na�, HCO�

3, and voltage. If one focuses on Na�, then the epithelial cotransporter acts as an auxiliary Na� pump. As far as its classifi cation as a primary, secondary, or tertiary active Na� transporter is concerned, the cotransporter occupies an unusual niche. The Na�-K� pump directly or indirectly establishes the membrane voltage. This pump also indirectly establishes the Na� gradient that ener-gizes the Na�-H� exchanger, which raises pHi and thereby establishes the HCO�

3 gradient. These voltage and HCO�3

gradients, directly or indirectly established by the Na�-K� pump, drive the epithelial Na�/HCO�

3 cotransporter in the direction of net Na� effl ux. Thus, this Na� extrusion can be viewed as a secondary/tertiary active transport process.


Similar to Cl�-HCO�3 exchangers, Na�/HCO�

3 cotrans-porters are inhibited by stilbene derivatives such as DIDS (59, 454). Cloned NBCe1 expressed in oocytes can also be inhibited by benzamil (162), the nonsteroidal anti-infl ammatory drug tenidap (162, 342), nifl umic acid (337), and diBAC oxonol dyes (337).

Electroneutral K�/HCO3� Cotransporter

General Function and Molecular IdentityA K�/HCO�

3 cotransporter (Fig. 10D), presumably elec-troneutral, has been documented in squid giant axons, where it normally mediates the effl ux of one K� and one HCO�

3 ion (256). Because the transporter moves HCO�

3 out of the axon, it functions as an acid loader. A similar transporter has been reported in cells of the medullary thick ascending limb of rat kidney (331, 580) and may exist in cultured cortical astro-cytes of rat (M.O.B. and W.F.B., unpublished data). In mammalian glia, K�/HCO�

3 cotransport may contribute to the pHi decrease following an acute intracellular alkali load.



INVOLVEMENT OF K� AND HCO3�: ENERGETICS

In the squid giant axon dialyzed with a fl uid containing K�, but lacking Na� and Cl�, the cotransporter moves K� and HCO�

3 out of the axon, eliciting a pHi decrease (256). The base effl ux is reduced by removing K� from the dialysis fl uid. Also, the base effl ux is reduced by elevating extracel-lular K� and HCO�

3, thereby eliminating the gradient favor-ing K�/HCO�

3 effl ux. Under the appropriate conditions, the K�/HCO�

3 cotransporter can also operate in the forward direction, moving HCO�

3 into the squid giant axon (142, 256). Exposing axons dialyzed with a fl uid devoid of K�, Na�, and Cl� to an artifi cial seawater containing K� and CO2/HCO�

3 elicits a pHi decrease (due to CO2 entry), fol-lowed by a pHi increase (due to K�/HCO�

3 infl ux). The pHi

increase requires the presence of both extracellular K� and HCO�

3. The HCO�3 fl uxes mediated by the K�/HCO�

3 co-transporter can be enhanced by using out-of-equilibrium CO2/HCO�

3 solutions to introduce HCO�3 exclusively to

either the inside or outside of the axon (142, 614).As mentioned previously, two independent groups have

also reported K�-coupled HCO�3 transport in the medullary

thick ascending limb (mTAL) from tubule suspensions (331) or perfused tubules (580). In both preparations, the K�-coupled HCO�

3 transport did not require the presence of Na� or Cl� and could be inhibited by DIDS. In the perfused-tubule study (580), luminal DIDS increased tran-sepithelial HCO�

3 reabsorption—a fi nding consistent with DIDS-sensitive K�-coupled HCO�

3 transport opposing HCO�

3 reabsorption.


While the transporter in the mTAL is sensitive to stilbene derivatives such as DIDS, the K�/HCO�

3 cotransporter in the squid giant axon is not. However, quaternary ammonium ions introduced into the cell inhibit inwardly directed cotransport (142). The potency of inhibition increases with increasing alkyl chain length of the quaternary ammonium ion. For example, the apparent Ki for phenylpropyltetraethylammonium or PP-TEA (�91 �M) is �850-fold less than the apparent Ki for tetraethylammonium or TEA (�78 mM).

Ca2�-2H� Exchange Pump


The P-type ATPase family includes not only the Na-K and H-K pumps, but also the Ca2� pumps, of which there are two types—the sarcoplasmic (endoplasmic) reticulum Ca2�-ATPase (SERCA) and the plasma membrane Ca2�-ATPase (PMCA). These Ca2� pumps, along with Na�/Ca2� exchangers, play a pivotal role in returning [Ca2�]i to low levels following stimuli-induced infl ux through Ca2� chan-nels in the plasma membrane or release from intracellular stores. SERCAs pump Ca2� back into intracellular stores, whereas PMCAs pump Ca2� out of the cell across the plasma membrane. In both cases, the pumps exchange Ca2� for H�. Thus, from the standpoint of pHi, the PMCAs,

which take up H� as they extrude Ca2� across the plasma membrane, function as acid loaders (Fig. 10E).

Four PMCAs (PMCA1-4) have been cloned and charac-terized (see reviews by 100, 233, 375, 599). The cDNAs encode proteins with �1200 amino acids. Based on hy-dropathy analysis, the PMCAs have 10 transmembrane �-helices (TM 1–10) with both the N and C termini being cytoplasmic. The ATPase catalytic domain lies between TM4 and TM5. Based on localization studies (see review [232]), PMCA1 and PMCA4 are expressed ubiquitously. In contrast, PMCA2 and PMCA3 are found predominantly in the nervous system. mRNA encoding all four isoforms have been found in kidney (102, 349). However, PMCA levels of mRNA and protein are highest in the distal tubule, particu-larly in the distal convoluted tubule (55, 350).

INVOLVEMENT OF CA2�: ENERGETICS

It has been known for some time that the plasma-membrane Ca2� pump in erythrocytes exchanges Ca2� for H� (101, 396). Schwiening et al. (487) have used Ca2�- and pH-sensitive microelectrodes to demonstrate that the plasma membrane Ca2� pump in snail neurons also exchanges extra-cellular H� for intracellular Ca2�. In this preparation, activa-tion of the Ca2� pump (generated by increasing intracellular Ca2� with depolarization or iontophoretic Ca2� injection) elicits an increase in extracellular Ca2�, and a simultaneous increase in extracellular pH. Both the increase in extracellular Ca2� and pH can be inhibited by the Ca2�-pump inhibitor vanadate. A similar Ca2� pump, present in hippocampal neurons (554), appears responsible for causing an increase in the extracellular pH of the hippocampal slice elicited by stimuli-induced neuronal activity (408, 409, 518).

Glutamate Transporters


In the mammalian central nervous system (CNS), the predominant excitatory amino acids glutamate and aspar-tate are transported from the extracellular space back into neurons and glial cells by ion-coupled glutamate transport-ers in the SLC1 family (282). Since 1992, cDNAs encoding several of these electrogenic glutamate transporters have been cloned and characterized (27, 28, 135, 136, 172, 281, 284, 421, 531). These transporters are predominantly ex-pressed in the nervous system, but also in certain epithelial cells (135, 453, 490). Because the nomenclature in this fi eld is not uniform, we will follow the suggestion of Amara (28) and refer to them as excitatory amino acid transporters (EAATs). The L-glutamate/L-aspartate trans-porter (GLAST1 or EAAT1) and the GLT1 (or EAAT2) transporter are mainly found in glia such as astrocytes and Bergmann glia. In contrast, the excitatory amino acid car-rier 1 (EAAC1 or EAAT3) and EAAT4 are found pre-dominantly in neurons. EAAT3 is also expressed in liver and is also present in small intestine and kidney, where it mediates the uptake of glutamate and aspartate across the



apical membrane (250). Of the more recently cloned gluta-mate transporters, EAAT4 is expressed in cerebellum, whereas EAAT5 is predominantly expressed in retina.

INVOLVEMENT OF GLUTAMATE/ASPARTATE, NA�, AND K�: ENERGETICS

Using the results from 22Na� and [14C]glutamate uptake studies and pHi measurements on Xenopus oocytes express-ing EAAT3, Kanai et al. (283) concluded that the protein transports one glutamate, two Na� and one H� into the cell, in exchange for one K�, which moves out of the cell (Fig. 10F). This stoichiometry, which predicts a net move-ment of one positive charge into the cell, agrees with that proposed for the glutamate transporter in salamander reti-nal glia based on kinetic analysis (70). Klöckner et al. (304) suggested that EAAT1, when expressed in Xenopus oocytes, has a stoichiometry of one glutamate and three Na� into the cell, in exchange for one K� out of the cell, without transport of an acid-base equivalent. However, in experi-ments on EAAT3-expressing oocytes, adding glutamate or aspartate to the extracellular fl uid causes a steep fall in pHi (283), consistent with the infl ux of H� or the effl ux of alkali.

Peptide and Divalent-Cation Transporters

Although most solute exchangers and cotransporters in ani-mal cells are Na�-driven, some are H�-coupled (Figs. 10G and 10H). The absorption of oligopeptides and peptide-based antibiotics by intestinal and proximal-tubule epithelial cells is mediated by two members of the SLC15 family, H�/oligopeptide transporters PepT1 (SLC15A1) and PepT2 (SLC15A2) (see review [137, 197]). PepT1 and PepT2 transport the same di- and tripeptides, but with dif-ferent affi nities/capacities (137). PepT1 exhibits low-affi nity, high-capacity transport, whereas PepT2 exhibits the opposite: high-affi nity, low-capacity transport. Both trans-porters are expressed in kidney (among other tissues), with PepT1 found in the S1 segment of the proximal tubule and PepT2 found in the S3 segment (498, 516). More recently identifi ed members of the peptide transporter family include PTH1 (SLC15A4) and PTH2 (SLC15A3), both of which can transport histidine as well as di- and tripeptides in a H�-coupled manner (137).

The cDNA encoding PepT1 has 707 amino acids with 12 putative membrane-spanning segments (175). In oocytes expressing PepT1, the net transport of dipeptides occurs with a proton: dipeptide stoichiometry of 1:1 (525). For negatively charged dipeptides, one H� binds to the cation site of the transporter, whereas another H� neutralizes the dipeptide on the peptide site. The net transport stoichiom-etry of negatively charged dipeptides is therefore one proton for one neutralized dipeptide. Adding extracellular glycyl-glycine, glycyl-leucine, or glycyl-lysine (dipeptides) to oo-cytes expressing PepT1 causes a steep fall in pHi (283, 525), consistent with the H� infl ux.

Gunshin et al. (235) cloned the cDNA encoding a mam-malian divalent-cation transporter (DCT-1)—now called the divalent metal transporter (DMT)—that mediates intes-tinal nonheme Fe2� absorption. DMT contains 561 amino acids with 12 putative membrane-spanning domains. By Northern blotting, DMT is ubiquitously expressed; heavily so in the duodenum. DMT is electrogenic, and cotransports any one of a group of metals (e.g., Fe2�, Zn2�, Mn2�, Co2�, Cd2�, Cu2�, Ni2�, Pb2�) together with one H�. By analogy with the EAATs and PepT1, adding Fe2� to the extracellular fl uid of an oocyte expressing DMT leads to a rapid fall in pHi, consistent with the infl ux of H�.

ACID-EXTRUSION MECHANISMS

As discussed earlier, acid extrusion, that is, the movement of H� (or protonated monovalent weak acids) out of cells, or the movement of OH� or HCO�

3 (or monovalent weak bases) into cells, generally occurs against a steep energy gra-dient. Based on their source of energy, acid-extrusion mech-anisms can be divided into three groups: primary, secondary, and tertiary active transporters (23). Primary active trans-port mechanisms, also called pumps, derive their energy from electron transport (e.g., the electron transport chain that drives H� out of mitochondria) or, as with the trans-porters such as the H� pump considered in the following sections, from the hydrolysis of ATP. Secondary active transport systems (e.g., Na�-H� exchange) derive their en-ergy from the electrochemical gradient of an ion (e.g., Na�), a gradient that is in turn established by a primary active transporter (e.g., Na�-K� pump). Finally, tertiary active transporters (e.g., the monocarboxylate system) are driven by an ion gradient established by a secondary active trans-porter.

From the point of view of feedback control, the effective-ness of an acid extruder would be enhanced greatly if de-creases in pHi increased transport. The most productive ap-proach for studying the pHi dependence of acid extruders in intact cells has been to load cells with acid acutely, as sche-matized in Fig. 11A. The acid load can be applied by pre-pulsing with NH4

� (61), microinjecting H� (363), dialyzing with a low-pH solution (467), or reducing pHo in the tem-porary presence of nigericin (228). In the model experiment of Fig. 11A, the pHi decrease produced by the acid load (seg-ment ab) is followed by a rapid recovery to the initial pHi (bcd). In the most general case, this pHi recovery is mediated by one or more acid extruders, but may be opposed by one or more acid loaders. Identifying the component of pHi recov-ery that is mediated by a particular transporter requires that the transporter be specifi cally, instantaneously and com-pletely inhibited. Such inhibitors exist for two of the acid extruders that we will discuss, the Na�-driven Cl�-HCO�

3 exchanger and the Na�-H� exchanger. Figure 11A illustrates three possible outcomes (indicated by broken curves follow-ing the arrow) of applying such an agent during a pHi



recovery due at least in part to a mechanism sensitive to the blocker. First, applying the blocker may slow but not halt the pHi recovery. This result indicates that a second, blocker-insensitive acid extruder also contributes to the pHi recovery. Second, the blocker may completely eliminate the pHi recov-ery, indicating that the entire recovery is due to a blocker-sensitive mechanism(s). However, complete blockade by the blocker does not rule out the possibility that the blocker-sensitive acid-extrusion process is opposed by a less potent blocker-sensitive acid-loading process. Third, applying the blocker may not only halt the pHi recovery, but may also unmask a slow pHi decline. This result implies that the blocker-sensitive acid-extrusion process is opposed by at least one blocker-insensitive acid loader.

In any of these three eventualities, the difference in rates of pHi change (dpHi/dt) immediately before and immedi-ately after applying the blocker at point c in Fig. 11A is the component of the pHi recovery—at the pHi value prevailing at point c—that is due to blocker-sensitive transporters. In

order to convert this blocker-sensitive dpHi/dt to a net acid extrusion rate ( JE), we must multiply this dpHi/dt by the total intracellular buffering power (T) prevailing at the pHi of the blocker application, as well as the inverse of the surface-to-volume ratio (�):

JE � (T/�) � (dpHi/dt)Blocker-sensitive (Eq. 38)

Because acid-base transporters are generally sensitive to changes in pHi, this analysis must be repeated over a range of pHi values to produce a plot of the pHi dependence of the blocker-sensitive fl ux(es).

Rather than repeating the experiment in Fig. 11A many times, each with a different pHi at the instant we apply the blocker (i.e., point c), we could use a more practical approach, which requires only three types of experiments. First, we monitor the pHi recovery—in the absence of blocker—over the entire pHi range indicated by bcd in Fig. 11B. At regular pHi intervals during this recovery, we compute the “gross” dpHi/dt, from which we compute the gross JE (Fig. 11C).

FIGURE 11 Determining the pHi dependence of an acid-base transporter. The pHi dependence of the Na�-driven Cl�-HCO3

� exchanger. A: Hypothetical experiment illustrating how an inhibitor (e.g., DIDS) can be used to ascertain the component of a pHi recovery from an acid load that is mediated by an acid extruder such as the Na�-driven Cl�-HCO3

� exchanger. The blocker is applied at the arrow (point c). The broken curves illustrate three possible effects of the blocker on the pHi recovery. B: Another hypothetical experiment illustrating a different approach for determining the pHi dependence of an acid extruder during the pHi recovery from an acid load. The blocker is applied at the time of the acid load or after the pHi recovery (dotted curves). C: The pHi dependence of “gross” acid extrusion (gross JE) is calculated from the pHi recovery in the absence of blocker (bcd). The pHi dependence of “blocker-unmasked” acid loading (blocker-unmasked JL) is calculated from the pHi recovery in the presence of blocker (be) or the pHi decrease elicited by applying the blocker after the pHi recovery (de). D: The pHi dependence of “blocker-sensitive” acid extrusion (blocker-sensitive JE) is calculated by subtracting the plot of blocker-unmasked JL from the plot of gross JE.



Second, we monitor the pHi recovery in an experiment in which the blocker is present during the entire pHi recovery. If the blocker-sensitive transporter is the only acid extruder present in the cell, then pHi will not recover at all, or may even decline. On the other hand, if other acid extruders are present (as is more often the case), pHi will recover slowly and incompletely, as in be. At regular pHi intervals during this abbreviated recovery, we compute the “blocker-insensitive” dpHi/dt, from which we compute the blocker-insensitive JE (Fig. 11C).

Third, we allow pHi to recover fully (bcd), and then add the blocker. If, during the steady state indicated by point d, one or more acid loaders oppose the blocker-sensitive acid extruder, as is typically the case, then applying the blocker will cause pHi to drift downward (de). The initial rate of pHi decline at point d refl ects the rate of acid loading ( JL) at the pHi at d. At regular pHi intervals during this pHi decline, we compute the “blocker-unmasked” dpHi/dt, from which we compute the blocker-unmasked JL (Fig. 11C).

Finally, at each pHi value, we subtract the blocker-unmasked JL from the blocker-insensitive JE in Fig. 11D. The result is the pHi dependence of the “blocker-sensitive” JE (i.e., the fl ux mediated by the transporter of interest). This approach was fi rst used by Boron et al. (65) to deter-mine the pHi dependence of the Na�-driven Cl�-HCO�

3 exchanger of barnacle muscle, and then by Boyarsky et al. (74) to determine the pHi dependence of the Na�-H� exchanger of renal mesangial cells. Others extended this approach to study the pHi dependence of Na�-H� ex-change in IEC-6 colonic cells (512), UMR-106 osteoblas-tic cells (236), osteoclasts (439), rabbit S3 proximal tubules (113), 3T3 fi broblasts transformed with the c-H-ras onco-gene (285), Chinese hamster lung fi broblasts (Yoon M.O.B. and W.F.B., unpublished data), rat hippocampal astrocytes (49) and shrunken mesangial cells (44). Finally, the approach outlined in Figs. 11B, 11C, and 11D also has been used to determine the pHi dependence of the Na�-driven Cl�-HCO�

3 exchanger in NIH 3T3 cells trans-formed with the c-H-ras oncogene (285), and the pHi dependence of the H� pump in rabbit S3 proximal tubules (113) and osteoclasts (439).

H� Pumps


An electrogenic vacuolar-type H� pump (Fig. 12A, left) is present in the distal nephron of the kidney (185), as well as in macrophages and neutrophils (491, 536), os-teoclasts (110), tumor cells (355), and goblet cells of the insect midgut (593). Similar H� pumps are present in clathrin-coated vesicles and other organellar membranes (185, 186).

The V-type H� pump is composed of two domains, V0 and V1. The V0 domain, involved in translocating H� across the membrane, is a 260-kD complex of at least fi ve different subunits (a, d, c, c�, c �), ranging from 17 to 100 kD. The H�

pump inhibitor DCCD binds to subunit c. The V1 domain, involved in ATP hydrolysis, is a 570-kD complex of at least eight different subunits (A–H), ranging from 14 to 70 kD. The catalytic nucleotide binding sites are located on subunit A. Also, the sulfhydryl reagent N-ethylmaleimide inhibits the pump by binding to subunit A. A more complete review of the structure and function of the V0 and V1 subunits of the pump is presented in refs. (187, 526). Finally, similar vacuolar H� pumps have been examined in Neurospora and plant cells (72, 73, 619).

Immunohistological studies with antibodies raised against various subunits of the H� pump have revealed expression of the pump throughout the nephron on both apical and basolateral membranes (571). In the cortical collecting tu-bule, the H� pump is localized to the apical membrane of � intercalated cells (which secrete acid) and the basolateral membranes of intercalated cells (which secrete alkali) (84, 85, 509). Vacuolar H� pumps are also present at the apical membrane of the proximal tubule (84, 85, 509).

ENERGETICS

The extrusion of H� by this primary active transporter is not coupled directly to the movement of other ions that might balance electrical charge. Rather, this pump is electro-genic. Because the H� pump is driven by the hydrolysis of ATP, which provides a substantial amount of energy, it is capable of generating a large H� gradient across the cell

FIGURE 12 Acid-extrusion mechanisms. A: Primary active transport mechanisms include the electrogenic H� pump and the electroneutral K�-H� pump. B: Secondary active transporters include the Na�-driven Cl�-HCO3

� exchanger (four models of which are shown), the Na�-H� ex-changer, the 1:2 electrogenic Na�/HCO3

� cotransporter, and the 1:1 electroneutral Na�/HCO3

� cotransporter. C: The tertiary active transport system consists of a Na�/monocarboxylate cotransporter that brings the monocarboxylate (X�) into the cell and a H�/monocarboxylate cotrans-porter that uses the energy stored in the X� gradient to drive H� out of the cell. In renal proximal tubules, the Na�/monocarboxylate cotransporter is confi ned to the luminal membrane.



membrane. For example, if the voltage across the membrane were zero, then the H� pump could establish a pH differ-ence of about three pH units. On the other hand, when compared with other H� transporters, H� pumps have a relatively low transport rate. Thus, the H� pump is ex-pressed in epithelia needing a high-gradient/low-capacity H� transporter. A H� pump contributes to pHi regulation in a number of cells, including macrophages and neutrophils (536), osteoclasts (439), renal cells (493, 572), glial cells (411, 420), and corneal epithelial cells (599).

K�-H� Exchange Pumps


As discussed earlier, K�-H� exchange pumps (Fig. 12A, right) are members of the P-type ATPase family. Both the gastric and nongastric H�-K� pumps are ATP-driven trans-porters that exchange intracellular H� for extracellular K�. However, the gastric and nongastric pumps differ in their molecular biology, localization, functional properties, and pharmacological sensitivities (see reviews [270, 510]). Simi-lar to the Na�-K� pump, the H�-K� pumps have a catalytic � subunit and a subunit. The cDNAs encoding the two subunits of the gastric H�-K� pump (502, 551) are similar to the corresponding subunits of the Na�-K� pump. The cloning of the cDNAs encoding the � subunit of nongastric H�-K� pumps (133, 176, 271, 374) reveals that they are �65% homologous to the � subunits of the Na� pump and the gastric H�-K� pumps.

Based on localization studies, in some species, both the gastric and nongastric forms of the H�-K� pump are present in the collecting duct of the kidney (see reviews [270, 510]). Although both forms are localized to the re-nal collecting duct, they are thought to play only a small role in the overall K� reabsorptive ability of the kidney. In fact, the nongastric form of the H�-K� pump may only be important during pathophysiological conditions, such as K� depletion.

INVOLVEMENT OF K�: ENERGETICS

In using the energy from ATP hydrolysis, H�-K� pumps can establish extremely large pH gradients. For instance, gastric parietal cells (433) can produce gastric secretions containing as much as 140 mM HCl (i.e., pH �0.7). In parietal cells of rabbit gastric glands, applying omeprazole (which blocks H�-K� pumps) reduces pHi by nearly 0.1 in glands stimulated with histamine and IBMX, though not in resting glands. This result implies that the H�-K� pump contributes to the maintenance of pHi in stimulated parietal cells.

Functional studies of nongastric H�-K� pumps have been performed in vitro using expression systems such as the Xenopus oocyte and HEK-293 cells. In such expression sys-tems, the nongastric pumps exchange extracellular K� for intracellular H�. However, the pumps may also possess the ability to transport Na� in place of H� (see review, 270).

Na�-Driven Cl�-HCO3� Exchanger


A secondary active transport system that exchanges exter-nal Na� and HCO�

3 for internal Cl� and H� (or its equiva-lent, vide infra) was fi rst identifi ed in invertebrate cells such as squid axons (68, 468) and snail neurons (547). Indeed, the work on the squid axon was the fi rst evidence for the dy-namic regulation of pHi (61). This transporter makes an important contribution to pHi regulation in mammalian neurons (486). Not certain is the precise identity of the trans-ported acid-base equivalents, inasmuch as pHi measurements cannot clearly distinguish inward movements of HCO�

3, CO�

3 or NaCO�3 from one another, or from outward move-

ments of H�. All four schemes outlined in Fig. 12B (top left) can account for the data. For simplicity, we shall refer to the transporter as the Na�-driven Cl�-HCO�

3 exchanger.As mentioned earlier and described in greater detail in

Chapter 52, investigators have cloned the cDNA encoding the Na-driven Cl-HCO3 exchanger (NDCBE or SLC4A8) and have also examined the transport properties of the pro-tein (223). When expressed in Xenopus oocytes, human NDCBE exhibits all the functional hallmarks of the endog-enously expressed transporter. For instance, stimulation of NDCBE activity in oocytes causes an increase in pHi that (1) requires external Na� and HCO�

3, (2) can be inhibited by stilbenes, and (3) correlates with a decrease in intracel-lular Cl� (223). Using the surface-pH measurement tech-nique described previously for examining CO�

3 transport by NBCe1, Grichtchenko and Boron (221) have provided evi-dence that NDCBE can also transport CO�

3.

DEPENDENCE ON pHi AND CALCULATION OF ACID EXTRUSION RATES

Using the approach outlined in Fig. 11B through 11D, Boron et al. (65) separately determined the pHi dependence of the Na�-driven Cl�-HCO�

3 exchanger of barnacle muscle at pHo values of 6.8, 7.4, 8.0, and 8.6 (at a fi xed [CO2] of 0.4%). As shown in Fig. 13, the fl ux is highest at low pHi values, and gradually falls toward zero at a pHi of �7.4, the threshold for activating the exchanger. This work was the fi rst to demon-strate threshold behavior for an acid-extruder; similar work followed on the Na�-H� exchanger (see following sections). The pHi sensitivity of acid extrusion in Fig. 13 is probably not a Michaelis-Menten type of dependence on intracellular H�, which would translate into a sigmoid (rather than a linear) acid-extrusion rate versus pHi relationship. The pHi sensitivity may refl ect an allosteric site for intracellular H� (vide infra).

It is important to recognize that, for two reasons, an analysis of the type shown in Fig. 13 is far more valuable than a simple plot of the pHi recovery rate (dpHi/dt) versus pHi. First, because the analysis includes buffering power, the result is a fl ux rather than merely a rate of change in pHi. This con-sideration is important when buffering power changes ap-preciably over the pHi range in question, or if a transporter is to be compared under conditions when buffering power is



expected to vary. Second, the analysis takes into account pro-cesses other than the transporter in question; these other processes may contribute to the pHi recovery (or lack thereof!). Failing to make this correction can lead to a serious over- or underestimation of the fl ux due to the transporter of interest.

DEPENDENCE ON pHO

Although the Na�-driven Cl�-HCO�3 exchange rate is

stimulated at low values of pHi, it is apparently inhibited at low values of pHo. For example, both in barnacle muscle and squid giant axons, decreases in pHo—produced by lowering [HCO�

3]o at a fi xed PCO2—cause JE to decrease (64, 66). However, at least for the squid axon, this apparent inhibition at low pHo may be only illusory: when pHo is decreased at a fi xed [NaCO�

3]o, the inhibition disappears (64). Thus, at least in squid axons, the transporter per se may be relatively pHo insensitive. Most methods of producing acidosis may inhibit Na�-driven HCO�

3 transport because they simulta-neously lower [NaCO�

3]o.

INVOLVEMENT OF NA�, CL�, AND HCO3�

The Na�-driven Cl�-HCO�3 exchanger has an absolute

requirement for external Na� (545), external HCO�3 (60) or

a related species, and internal Cl� (467). Removing any one of these three ions from the appropriate side of the mem-brane completely blocks acid extrusion. Moreover, Na� and Cl� are actually transported along with acid equivalents (467, 545). The observed stoichiometry is one Na� entering the cell, one Cl� exiting, and two acid equivalents neutral-ized intracellularly. This stoichiometry is consistent with all

four models presented in Fig. 12B (top left). The prediction that Na�-driven Cl�-HCO�

3 exchange should be electro-neutral is supported by the observations that transport nei-ther alters (56, 544) nor is altered by Vm (68).

ENERGETICS

The models of Fig. 12B (top left) make equivalent ener-getic predictions, based upon which physiological gradients for Na�, Cl�, and HCO�

3 should support Na�-driven Cl�-HCO�

3 exchange with no external source of energy (456). The major predicted energy source is the steep Na� gradi-ent, established by the Na�-K� pump. This model of a secondary active transporter, driven only by ion gradients, is supported by the observation that transport can be reversed by reversing gradients for Na� and/or HCO�

3 (65, 469, 546). Curiously, however, the transporter in squid axons requires ATP; not as an energy source, but probably for an essential phosphorylation event or as an essential cofactor (62).

THE pHi THRESHOLD

In barnacle muscle, the activity of the Na�-driven Cl�-HCO�

3 exchanger falls toward zero as pHi approaches a pHi threshold of �7.4 (see Fig. 13). This reduction in transport does to drive pHi to �8.0. Threshold behavior is thus prob-ably due to an allosteric effect of H� on the transporter or an essen not refl ect the unavailability of energy, however, which is suffi cient tial activator. This hypothesis is supported by the observation that the barnacle-muscle Na�-driven Cl�-HCO�

3 exchanger, in addition to mediating net acid extrusion, also mediates what appears to be Na�-Na� (469) and Cl�-Cl� (69) exchange. Like acid extrusion, these ancillary transporter activities are also stimulated by reducing pHi below the threshold.

SENSITIVITY TO PHARMACOLOGICAL AGENTS

The Na�-driven Cl�-HCO�3 exchanger is totally and ir-

reversibly blocked by the stilbene derivatives SITS and DIDS. The site of permanent action is apparently a free amino group on the transporter that reacts with the isothio-cyano moiety of the stilbene to form an N,N-disubstituted thiourea (Edman reaction). However, even stilbene deriva-tives lacking an isothiocyano group can inhibit the trans-porter, although the inhibition is often less than complete and is reversible. Thus, 4,4�-dinitro-2,2�-stilbene disulfo-nate (DNDS), at a concentration of 1 mM, reversibly inhib-its the squid-axon transporter by approximately 80% (63). By analogy to the erythrocyte Cl�-HCO�

3 exchanger, the Na�-driven Cl�-HCO�

3 exchanger probably forms a revers-ible, ionic complex with each of the aforementioned stil-benes. This interaction presumably involves the negatively charged sulfonate groups on the stilbene and cationic moi-eties on the transporter. The stilbenes with amino-reactive groups (i.e., SITS and DIDS) subsequently form a covalent bond that eventually blocks transport irreversibly. Interesting, the inhibition of squid-axon Na�-driven Cl�-HCO�

3 ex-changer by DNDS is apparently competitive with Na� as

FIGURE 13 The pHi dependence of the Na�-driven Cl�-HCO3� ex-

changer from giant barnacle muscle fi bers. The rate of pHi recovery from an acid load (NH4

� prepulse) was determined over a range of pHi values, multiplied by the sum of intrinsic and HCO3

� buffering powers (both of which vary with pHi), and corrected for an acidifi cation unmasked by applying SITS. (Used with permission from Boron WF, McCormick WC, Roos A. pH regulation in barnacle muscle fi bers: dependence on intracel-lular and extracellular pH. Am J Physiol 1979;237:C185-C193).



well as with HCO�3; this result is quantitatively consistent

with the hypothesis that DNDS actually competes with the NaCO�

3 ion pair for binding (63).Two nonstilbene amino-reactive agents also inhibit Na�-

driven Cl�-HCO�3 exchange (56). The inhibition by p-

isothiocyanatobenzenesulfonate is irreversible, as is its reaction with free amino groups. The inhibition by pyridoxal phosphate is reversible, as is its reaction with free amines. Na�-driven Cl�-HCO�

3 exchange is also reversibly inhibited by relatively high levels (�0.5 mM) of furosemide (68, 69).

Na�-H� Exchangers


Na�-H� exchangers (Fig. 12B, bottom left) have been identifi ed in the plasma membranes of a wide variety of vertebrate cells (for reviews, see [24, 131, 191, 230, 255, 397, 574]). At least two functional classes of Na�-H� exchangers have been identifi ed. The fi rst, which is apparently the most widespread version, are the Na�-H� exchangers of nonepi-thelial cells (7, 103, 226, 228, 378, 379, 413, 458) as well as the basolateral membranes of many epithelial cells (52, 107). This distribution is characteristic of what have been re-garded as “housekeeping” transporters (e.g., Na�-K� pump and Na�-Ca2� exchanger), which are inhibited by relatively low concentrations of fi ve-amino–substituted amiloride an-alogs, (e.g., ethylisopropyl amiloride [EIPA]). The second class of exchangers is the one characteristically located at the apical membrane of epithelia such as the renal proximal tu-bule (16, 266, 294, 318, 393, 478, 485). It is inhibited by only relative high concentrations of the aforementioned amiloride analogs. These two pharmacologically distinct Na�-H� exchangers have been identifi ed on the apical and basolateral membranes of LLC-PK1 cells (239). A third pharmacological class of Na�-H� exchanger is found in rat astrocytes (80) and hippocampal neurons (36, 47, 436, 486). These cells have an Na�-driven acid extruder that functions in the nominal absence of HCO�

3 and is not inhibited even by high levels of amiloride and/or amiloride analogs.

A cDNA encoding a Na�-H� exchanger (NHE1) was fi rst cloned by Sardet et al. (476), using an expression strategy outlined in Chapter 54. Nine additional NHE isoforms (NHE2 to NHE10) have been identifi ed, all members of the SLC9 family. The sequence comparisons, tissue distributions, and functions of the various NHE isoforms are examined in Chapter 54. In the remainder of this section, we will examine the physiology of Na�-H� exchange.

DEPENDENCE ON pHi

The fi rst convincing evidence that decreases in pHi stimulate Na�-H� exchange came from experiments on membrane vesicles, in which low pHi stimulates amiloride-sensitive Na� effl ux (25). This result, analogous to the Cl�-infl ux and Na�-effl ux data on the Na�-driven Cl�-HCO�

3 exchanger, extends the allosteric-H�-site hypothesis to the Na�-H� exchanger.

One can also determine the pHi dependence of the Na�-H� exchanger in experiments on intact cells. However, an ex-amination of the literature describing the pHi dependence of Na�-H� exchangers reveals that many investigators assume that the Na�-H� exchanger is the only factor contributing to the recovery of pHi from an acute acid load. In support of this conclusion, they point out that the recovery occurs in the nominal absence of HCO�

3 and is inhibited by blockers of Na�-H� exchanger. However, there is a serious fl aw in this line of reasoning. It is true that, after a cell containing a Na�-H� exchanger is acutely acid loaded, there is a more-or-less exponential pHi recovery (e.g., see bcd in Fig. 11A) that may be prevented by pretreating with amiloride analogs. However, this result only proves that an intact Na�-H� exchanger is required for the pHi recovery. Such experiments do not address the issue of how background acid-loading processes infl uence the rate of the pHi recovery, and thus cannot—by them-selves—provide the pHi sensitivity of the Na�-H� exchanger.

An analysis of the pHi sensitivity of Na�-H� exchange in intact cells requires that one rule out or at least account for background acid-loading processes that oppose the action of the exchanger, as discussed above in connection with Fig. 11. In experiments on isolated hepatocytes in the nominal ab-sence of HCO�

3 (82, 530), applying amiloride has no effect on steady-state pHi. This implies that both Na�-H� ex-change and background acid loading are inactive at this pH of �7. During the recovery from an acid load, amiloride analogs block the pHi recovery, but do not unmask a back-ground acid-loading process. Thus, in these cells, the product of (T/�) and the gross pHi recovery rate is the true net Na�-H� exchange rate, which varies with pHi in a nearly linear fashion (Fig. 14A).

In many other cells, however, applying an amiloride ana-log causes a sizeable fall in steady-state pHi (322, 379, 380), as indicated by de in Fig. 11B, implying that both acid-loading and Na�-H� exchange were active at the original steady-state pHi. A similar EIPA-induced pHi decrease oc-curs in renal mesangial cells. The pHi dependence of the true Na�-H� exchange rate thus can be computed only after correcting the gross pHi recovery rate for EIPA-insensitive acid extrusion and EIPA-unmasked acid loading, as dis-cussed in connection with Figs. 11B, 11C, and 11D. The result for the mesangial cell is the curve shown in Fig. 14B, which indicates that the Na�-H� exchanger is substantially less pHi sensitive in the physiological range than under con-ditions of severe acid loading (74).

DEPENDENCE ON pHO

The Na�-H� exchanger is inhibited by decreases in pHo. In intact cells, lowering pHo decreases the rate at which pHi recovers from an acid load (58, 376). In crayfi sh neurons, the pHi recovery halts completely at the pHo at which the en-ergy required to move one H� out of the cell equals that released by moving one Na� (376). In brush-border mem-brane vesicles from the renal cortex, the pHo dependence of Na�-H� exchange is described by a simple pH titration



curve with a pK′a of 7.45 (26). Based on kinetic analyses, external H�, Na�, Li�, and amiloride all compete for a com-mon site on the exchanger (for review, see [24]).

INVOLVEMENT OF NA�

Acid extrusion by the Na�-H� exchanger in intact cells is blocked by removing extracellular Na� (7, 58, 376). Further-more, in the presence of extracellular Na�, acid extrusion is accompanied by a rise in intracellular Na� activity (7, 58, 376) that is blocked when acid extrusion is blocked by low-ering pHo (58). Reported values of the apparent KM for ex-

ternal Na� vary over a fairly wide range, between �5 and �40 mM at a pHo of 7.4 (107, 273, 295, 296, 447, 578). Accordingly, external Na� nearly saturates the transporter at a normal pHo. The KM values tend to be inversely related to pHo, consistent with competition between external Na� and H� (for review, see [24]). In membrane-vesicle studies, the exchanger can transport Li� in place of Na� (294). Indeed, Li� transport by the exchanger is a key element of the “proton-suicide” technique used to kill cells with a func-tional Na�-H� exchanger (426).


Amiloride (Fig. 15A) is a K�-sparing diuretic that inhib-its apical ENaC Na� channels in tight epithelia; Ki values are in the range 0.1 to 1 �M (18, 38). However, at substan-tially higher concentrations, amiloride also inhibits certain Na�-H� exchangers (Ki: 3 to 1000 �M; 39, 351) and Na�-Ca2� exchange (Ki: 300 to 1100 �M) (277, 481). The inhi-bition of Na�-H� exchange by amiloride has generally been found to be purely competitive with respect to external Na� (295, 413, 448, 568), although both mixed-type (267) and noncompetitive inhibition (382) have been reported. Be-cause of the competition with Na�, millimolar levels of amiloride generally are necessary to inhibit Na�-H� ex-change at physiological Na� concentrations (7, 58, 376, 448). Structure-function relationships, which have been re-viewed in some detail (38, 39, 190), indicate that different regions of the amiloride molecule alter the molecule’s

FIGURE 14 Two patterns of pHi dependence of the Na�-H� ex-changer. A: The Na�-H� exchanger of rat hepatocytes exhibits true pHi threshold behavior. Although applying EIPA completely blocks the recov-ery of pHi from an acid load, EIPA has no effect on pHi after the pHi re-covery is completed. Thus, the exchanger is inactive in the normal steady state. The experiments were performed on single freshly disaggregated he-patocytes in the nominal absence of CO2/HCO3

�. pHi was measured using BCECF and a microscope-based fl uorometry system. (Used with permis-sion from Boyarsky G, Rosenthal N, Barrett E, Boron WF. Effect of dia-betes on Na�-H� exchange by single isolated hepatocytes. Am J Physiol 1991; 260:C167-C175.) B: The Na�-H� exchanger in rat mesangial cells is active in the steady state. Applying EIPA in the steady state unmasks a sizeable acidifi cation, indicating that the exchanger is normally active. The experiments were performed on single cells (passage 2–5) in the nominal absence of CO2/HCO3

�. (From ref. 74)

FIGURE 15 Amiloride and two analogues. A: Amiloride inhibits the Na� channel in the luminal membrane of many tight epithelia at micro-molar levels. It inhibits Na�-H� exchange at approximately millimolar concentrations, and inhibits Na�-Ca2� exchange at even higher concen-trations. B: Benzamil is even more active than amiloride against the Na� channel, but has almost no effect on the Na�-H� exchanger. C: Ethyliso-propyl amiloride can be up to 200 times more potent than amiloride against the Na�-H� exchanger, but is almost without effect on the Na� channel.

A

B

A

B

C



affi nity for the apical Na� channel, the Na�-Ca2� exchanger, or the Na�-H� exchanger. Substitutions at the terminal guanidine nitrogen of amiloride, such as the introduction of the benzyl group that produces benzamil (Fig. 15B), can result in compounds with substantially higher affi nity for the Na� channel, but extremely low affi nity for the Na�-H� exchanger. Conversely, alkyl substitutions at the fi ve-amino position, such as the introduction of ethyl and isopropyl groups that produce EIPA (Fig. 15C), result in compounds with greatly diminished affi nity for the apical Na� channel, but substantially increased affi nity for the Na�-H� ex-changer. Replacement of the Cl� at position 6 with Br� or I� produces compounds with increased affi nity for the Na�-H� exchanger, but decreased affi nity for the Na� channel and Na�-Ca2� exchanger. HOE compounds such as HOE694 and HOE642 (Cariporide) are relatively new Na�-H� exchange inhibitors that have different affi nities for several NHE isoforms, with NHE1 being the most sen-sitive. Similar to amiloride, the base structure of HOE compounds is guanidinium. However, unlike amiloride and its sister compounds, HOE compounds have an attached benzene ring.

As summarized previously, the inhibition produced even by fi ve-amino–substituted derivatives of amiloride (e.g., EIPA), can range from powerful to modest to nil. Furthermore, in rat osteoclasts (439), rat CA1 neurons (47), and rat astrocytes (81) (M.O.B., Frey, and W.F.B., unpublished data), 50 �M EIPA not only fails to inhibit Na�-H� exchange, it elicits a marked increase in steady-state pHi. The mechanism responsible for the EIPA-in-duced increase in pHi is presently unknown.

Electroneutral and Electrogenic Na�/HCO3

� Cotransporters

As discussed earlier, an electrogenic Na�/HCO�3 cotrans-

porter with a Na�: HCO�3 stoichiometry of 1:3 is present on

the basolateral membrane of proximal tubule cells of the kidney, where it mediates Na� and HCO�

3 reabsorption, and therefore functions as an acid loader. A member of the SLC4 family, NBCe1 (SLC4A4) is responsible for HCO�

3 transport in the proximal tubule. A second transporter, NBCe2 (SLC4A5), which is expressed at high levels in he-patocytes, can also mediate electrogenic Na/HCO�

3 cotrans-port with an apparent stoichiometry of 1:3 (479). However, both NBCe1 (362, 489) and NBCe2 (569) can also operate with a stoichiometry of 1:2, in which case they move Na� and HCO�

3 into cells, and therefore function as acid extrud-ers. An electrogenic Na�/HCO�

3 cotransporter with a Na�: HCO�

3 stoichiometry of 1:2 (Fig. 12B, top right) has been documented in both invertebrate and mammalian glial cells (see reviews [148, 150, 361, 457]).

An electroneutral Na�/HCO�3 cotransporter with a Na�:

HCO�3 stoichiometry of 1:1 (Fig. 12B, bottom right) has

been described in sheep Purkinje fi bers (138), guinea pig myocytes (184) and vascular smooth-muscle cells (1). Two

SLC4 members, NBCn1 (120, 430) and apparently also NCBE (Musa-Aziz, Parker, and W.F.B., unpublished data) mediate electroneutral Na/HCO�

3 cotransport. The molecu-lar biology of electrogenic and electroneutral NBCs is pre-sented in more detail in Chapter 52.

Monocarboxylate Transporters

GENERAL FUNCTION AND MOLECULAR IDENTITY OF MCTS

Na�-independent transport of monocarboxylates such as lactate occurs by a process of cotransport with H� (or exchange with OH�) across the plasma membranes of most cell types (see review [424]). The responsible trans-porters are members of the SLC16 family, which includes 14 members (241). The two best characterized monocar-boxylate transporters are MCT1 (SLC16A1) (204) and MCT2 (SLC16A7) (203). However, only MCT1-MCT4 exhibit H� coupled monocarboxylate transport. In fact, most of the other family members have yet to be function-ally characterized. The sequences of the encoded proteins are predicted to have 12 transmembrane (TM) segments, with a large intracellular loop between TM6 and TM7 and greatest conservation in the predicted transmembrane re-gions. Although both MCT1 and MCT2 have broad tis-sue distributions, in the kidney, MCT1 is expressed on the basolateral membrane of proximal tubule cells, and MCT2 is expressed on the basolateral membrane of medullary col-lecting duct cells (203).

SUBSTRATE SPECIFICITY AND ENERGETICS OF THE MCTS

MCT isoforms appear to have different affi nities for the same substrates. For example, both MCT1 and MCT2 transport lactate and pyruvate (203), but MCT2 has a much higher apparent affi nity for pyruvate (335).

In all cells examined, H�/X� cotransport appears to be electroneutral (155, 424, 504). Because MCT-mediated transport of monocarboxylates involves cotransport with H�, the net driving force depends on the gradients of both monocarboxylate and H� across the plasma membrane. Depending on the direction of the net driving force, MCTs can mediate either the infl ux or effl ux of monocar-boxylates. As described in the following sections, MCT1 on the basolateral membrane of renal proximal-tubule cells acts in conjunction with an apical Na�/monocarboxylate (Na�/X�) cotransporter to facilitate transepithelial lactate reabsorption.

EFFECTS OF PHARMACOLOGICAL AGENTS ON MCTS

MCT1 and MCT2, but not MCT3 and MCT4, are inhibited substantially by �-cyano-4-hydroxycinnamate (CHC) (203, 241, 606). However, because CHC has yet to be widely tested on other classes of transporters, it is not clear whether this compound is specifi c for MCTs.



THE NA�/MONOCARBOXYLATE COTRANSPORTER

The Na�/X� cotransporter is rather nonspecifi c, capable of transporting d- and l-lactate, pyruvate, acetate, and other monocarboxylates. The cotransporter is found in apical membranes of both the proximal tubule and small intestine (504, 595). The Na�/X� cotransporter appears to be electro-genic (moving more Na� than X�) in mammals (32, 364, 473, 563), but electroneutral in amphibians (504). Investiga-tors have found that one member of the SLC5 family of “Na/glucose cotransporters,” namely SLC5A8, encodes an electrogenic sodium-coupled monocarboxylate transporter (SMCT) (122, 220, 373, 422). In addition, zebrafi sh SLC5A12 encodes an electroneutral SMCT (422). As ex-pected, the transporters are relatively nonselective for mono-carboxylates, although substrate affi nities can vary among the transporters.

INTERACTION OF NA�/X� AND H�/X� COTRANSPORTERS

Some proximal-tubule cells possess both a Na�/X� co-transporter at the apical membrane and an MCT at the baso-lateral membrane (Fig. 12C). Acting in concert, these two monocarboxylate transporters can extrude substantial amounts of acid while reabsorbing monocarboxylates (389, 504).

The fi rst step of the acid-extruding process is the coupled entry of Na� and X� across the apical membrane, an ex-ample of secondary active transport. Because the pK�a gov-erning the dissociation of monocarboxylic acids (HX #H� � X�) is generally far below pHi, very little of the entering X� can combine with H� to form HX, and thus Na�/X� cotransport by itself has very little effect on pHi. In the sec-ond step, X� entering via luminal Na�/X� cotransport rap-idly exits across the basolateral membrane via MCT—along with H�. In this system, basolateral H�/X� cotransport is an example of tertiary active transport because the extrusion of H� is driven by an X� gradient that is, in turn, established by a secondary active transporter.

The above system of monocarboxylate transport is ex-pected to have effects on transepithelial transport and pHi regulation:

1. Na� reabsorption. Following the coupled uptake of Na� and X� across the apical membrane, the extrusion of Na� across the basolateral membrane by the Na�-K� pump contributes to transepithelial Na� reabsorption. Indeed, acetate promotes volume reabsorption (480).

2. X� reabsorption. Following the coupled uptake of Na� and X� across the apical membrane, H� and X- exit across the basolateral membrane. The net effect is the transepithelial reabsorption of monocarboxylate from lumen to blood.

3. Luminal H� secretion. When acetate is added to the lu-men of the rabbit S3 proximal tubule, pHi rises mark-edly, probably due to the apical uptake of X� via the Na�/X� cotransporter, followed by the apical effl ux of HX, presumably by nonionic diffusion or an MCT-like

transporter. The net effect is apical H� secretion and lu-minal acidifi cation (208).

4. Extrusion of H� across basolateral membrane. H� and X� exit across the basolateral membrane represents a net extrusion of acid that causes a substantial rise in pHi (389, 504). As far as pHi regulation is concerned, the monocarboxylate transporters masquerade as an amiloride-insensitive/monocarboxylate-dependent “Na�-H� exchanger.”

THE REGULATION OF pHi

In the preceding sections, we have discussed a wide range of factors that infl uence pHi. In this and the following section, we will examine how these factors interact to produce transient changes in pHi and, fi nally, to establish a steady-state pHi.

Fundamental Law of pHi Regulation

What determines the stability of pHi? It is intuitively obvi-ous that pHi will remain constant as long as the rate of acid extrusion (including uptake of alkali) out of the cytoplasm (JE) is equal to the rate of acid loading (including effl ux of alkali) into the cytoplasm (JL).3 pHi will rise if JE exceeds JL, and pHi will fall if JL exceeds JE. The rate of pHi change is proportional to the difference between JE and JL, propor-tional to the surface-to-volume ratio (�), but inversely pro-portional to buffering power:

d

dJ J

pHt

i = −( )�

E L (Eq. 39)

Equation 39 makes good intuitive sense. If JE � JL, then dpHi/dt � 0, and the cell is in a steady state with respect to pHi. Should JE exceed JL, pHi must increase because dpHi/dt will be greater than zero. Furthermore, the rate of this alka-linization is expected to increase as the difference between JE and JL increases. If JE and JL are unequal, then the rate of pHi change must be proportional to the surface-to-volume ratio (�). Thus, pHi transients tend to be faster in smaller cells. Finally, the rate of pHi change must be strongly infl u-enced by . If were infi nite, then pHi would be fi xed, re-gardless of JE and JL. Conversely, if approached zero, then even a small difference between JE and JL would cause pHi to change very rapidly.

3 We defi ne acid-extrusion and acid-loading rates as fl uxes ( J ), with the units of moles per unit area of cell surface, per unit time. In practice, such a defi nition is practical for calculating values from experiments only if the cell has a very simple geometry (e.g., cylinder in the case of an axon). For most mam-malian cells, which have complicated geometries, investigators express acid-loading and acid-extrusion rates in what we would term “pseuofl uxes” (φ) (45), with the units of moles per unit volume of cytoplasm, per unit time. In the case of a pseudofl ux, the surface-to-volume ratio is folded into the φ value. One must use caution in dealing with φ values, which can change with cell swelling or shrinkage, even without a change in the true underlying acid-base “fl ux.”



Factors That Infl uence the Steady-State pHi

A GRAPHICAL REPRESENTATION OF EQ. 39Figure 16 is a plot of JE and JL as a function of pHi for a

hypothetical cell. The shape of the JE curve is modeled after that reported for the Na�-H� exchanger of cultured mesan-gial cells (74), whereas the shape of the JL curve is modeled after that reported for background acid loading in renal mesangial cells (74), or Cl�-HCO�

3 exchange in several cells (76, 404). It is clear from Eq. 39 that pHi can be in a steady state (i.e., dpHi/dt � 0) only if JE and JL are equal. Thus, in Fig. 16, the steady-state pHi is determined by the intersection of the JE and JL curves. By using the graphical model in Fig. 16, one can evaluate how steady-state pHi is altered by either acid-base disturbances, which we will discuss below, or by other factors such as hormones, mitogens, and oncogenes. Some examples will be presented in the following paragraphs, from which the following three key concepts will emerge.

BUFFERING POWER DOES NOT INFLUENCE STEADY-STATE pHi

Buffering is important for cells because it reduces the magnitude of the pHi excursions that result from acute (i.e., one-time-only) acid and alkali loads. However, buffering does not prevent pHi changes; it only minimizes them. Moreover, after an acute acid or alkali load, buffering cannot return pHi to normal. As we will see in the next paragraph, it is the acid-extruding mechanisms that are responsible for returning pHi toward normal. According to Eq. 39, only infl uences the rate of pHi recovery from such an acid load.

ACUTE ACID OR ALKALI LOADS DO NOT AFFECT STEADY-STATE pHi

Consider the consequences of an acute acid load (e.g., an injection of H� or an infl ux of a weak acid that leads to the intracellular generation of H�). The vast majority of H� in-troduced into the cell is neutralized by buffers (the small amount of unbuffered H� is responsible for the pHi de-

crease). pHi can return to normal only when the entire acid load—both the additional free H� and the added H� that is reversibly neutralized by buffers—is extruded from the cell. During such a pHi recovery, acid extruders remove free H� from the cytoplasm, and buffers then partially replenish these extruded protons. Thus, during the pHi recovery, the added protons move from the buffers to the cytoplasm, from where the acid-extruders translocate them to the outside of the cell. If there has been no fundamental change in the properties of the acid loaders and acid extruders, then pHi will return pre-cisely to its initial value. Thus, by themselves, acute acid and alkali loads produce only transient changes in pHi.

ONLY FUNDAMENTAL CHANGES IN ACID-EXTRUDING AND ACID-LOADING PROCESSES CAN AFFECT STEADY-STATE pHi

Consider the consequences of modifying the pHi depen-dence of an acid-extruding process in such a way that the in-tersection of the JE and JL curves in Fig. 16 is altered. Such a change in transporter kinetics would produce a shift in steady-state pHi. Note that, whereas a change in the JE and/or JL curves is required for a shift in steady-state pHi, the JE and JL curves can shift without a change in steady-state pHi. For ex-ample, if there were appropriate offsetting changes in both curves, then it would be possible for the steady-state pHi to remain unaltered, even though JE and JL were modifi ed.

Examples of Intracellular Acid-Base Disturbances

ACUTE INTRACELLULAR ACID LOAD

Figure 17A illustrates the time course of pHi in a hypo-thetical experiment in which we acutely acid load a cell (seg-ment ab) and monitor the subsequent pHi recovery (bcd). Figure 17B is reproduced Fig. 16 and shows the pHi depen-dencies of JE and JL, as well as JE and JL values at different times during segment abcd in Fig. 17A. As described previously, the intersection of the JE and JL curves at point a in Fig. 17B de-termines the initial steady-state pHi, where dpHi/dt � 0.

We could regard the acute acid load as a square-wave increase in JL. The product of the width (i.e., duration) of the square wave, the height of the square wave, and the surface-to-volume ratio is the amount of acid we are loading into the cell. Dividing this amount by yields the magni-tude of the pHi decrease, which is represented in Fig. 17A as an instantaneous fall in pHi (ab). This imposed decrease in pHi causes JE to rise (b on the JE curve) and JL to fall (b on the JL curve). According to Eq. 39, because JE now exceeds JL, pHi must increase. However, as the pHi recovery proceeds (bcd), JE gradually falls and JL rises. Eventually, JE and JL come into balance at point d, which is identical to point a. Thus, as long as an acute intracellular acid load does not produce fundamental changes in the kinetics of acid extrusion or acid loading, the effect on pHi is only transitory. Stated differently, if the pHi dependencies of JE and JL in Fig. 17B are unchanged, then steady-state pHi also is un-

FIGURE 16 Dependence of acid-extrusion rate ( JE) and acid-loading rate (JL) on pHi for a hypothetical cell. The shapes of the curves are similar to those describing the Na�-H� exchanger and background acid-loading process in renal mesangial cells.



changed. In principle, the precise shape of the pHi recovery (bcd) could be obtained by integrating Eq. 39. An acute al-kaline load will have the opposite effects on pHi, and can be analyzed in an analogous way.

CHRONIC INHIBITION OF ACID EXTRUSION

Imagine that our hypothetical cell, initially in a steady state described by point a in Fig. 17C and Fig. 17D, is treated with suffi cient HOE694 (an inhibitor of Na�-H� exchange) to

reduce JE to approximately JE one eighth of its original level at all pHi values in Fig. 17D. Thus, immediately after this reduc-tion in Na�-H� exchange rate, JL is unchanged (point b on the JL curve), but JE is greatly reduced (point b on the JE curve). According to Eq. 39, because JL now exceeds JE, pHi must slowly fall (bcd in Fig. 17C). However, as pHi declines, JE rises and JL falls (e.g., point c in Fig. 17D). Eventually, JE and JL come into balance at point d, where pHi now stabilizes. If does not depend steeply on pHi, then the rate of pHi decline during bcd will be greatest at point b, where the differ-ence (JL � JE) is greatest. Thus, a chronic inhibition of acid extrusion causes steady-state pHi to fall.

CHRONIC INHIBITION OF ACID LOADING

Imagine that our hypothetical cell, initially in a steady state described by point a in Fig. 17E and Fig. 17F, is treated with suffi cient DIDS (an inhibitor of Cl�-HCO�

3 exchange) to reduce JL to approximately one fourth of its original value over the entire pHi range indicated in Fig. 17F. In Fig. 17F, the reduced acid loading is repre-sented by shifting the JL-pHi curve downward. Thus, im-mediately after this inhibition of Cl�-HCO�

3 exchange, JE is unchanged (point b on the JE curve), but JL is substan-tially reduced (point b on the JL curve). According to Eq. 39, because JL is now much less than the unchanged JE, pHi must slowly rise (bcd in Fig. 17E). However, this gradual increase of pHi causes JE to fall and JL to rise (e.g., point c in Fig. 17F). Eventually, JE and JL come into bal-ance at point d, where pHi stabilizes. Thus, a chronic inhi-bition of acid loading causes steady-state pHi to rise.

FACTORS INFLUENCING pHi

Interaction of pHi and pHo

It is well established that changes in pHo can affect pHi, and also that changes in pHi can be accompanied by changes in pHo (444, 549). Such interactions between intra- and extra-cellular acid-base metabolism are almost certainly mediated by changes in acid-base transport across the cell membrane. We have already discussed several such transport processes, including (1) nonionic diffusion of weak acids and bases (including metabolites), (2) passive fl ux of charged weak acids and bases through channels, and (3) acid-base move-ment via a variety of transporters. In the remainder of this section, we will examine how an alteration on one side of the cell membrane can alter transport and thus produce a pH change on the other side.

EFFECTS OF THE “CLASSIC” CHANGES IN EXTRACELLULAR ACID-BASE STATUS ON pHi

One can defi ne at least six simple disturbances of extracel-lular acid-base status, each of which has a unique affect on pHi. Four of these are the “classic” extracellular disturbances: metabolic acidosis and alkalosis and respiratory acidosis and

FIGURE 17 Response of a hypothetical cell to three insults to intracel-lular acid-base homeostasis. A, B: An acute intracellular acid load produces no fundamental change in the kinetics of acid extrusion and acid loading. Initially, pHi is described by point a in panel A, and JE and JL balance each other, as described by point a in panel B. The acid load, which causes pHi to fall to point b (A) also causes JE to rise and JL to fall, as described by the b points (B). Because JE now exceeds JL, pHi recovers (segment bcd in A). During this recovery, JE gradually decreases and JL gradually increases until they reach their original values at point d (B). C, D: Inhibiting acid extru-sion is assumed to reduce JE by 75% over the entire pHi range. The original JE-pHi relationship is given by the broken curve. Because JL exceeds JE im-mediately after the insult (point b in panel D), pHi declines (C). As pHi declines (bcd in panel C), however, JE slowly increases and JL decreases until they once again come into balance at point d (D), which describes the new steady state. E, F: Inhibiting acid loading is assumed to reduce JL by 75% over the entire pHi range. The original JL-pHi relationship is given by the broken curve. Because JE exceeds JL immediately after the insult (point b in panel F), pHi rises (E). As pHi rises (bcd in panel E), JL slowly increases and JE decreases until they once again come into balance at point d (F), which describes the new steady state.



alkalosis. The other two extracellular acid-base disturbances, isohydric hyper- and hypocapnia, are unusual in that they produce alterations in pHi with no change in pHo.

Metabolic Acidosis A sudden extracellular acidifi cation produced by a reduction of [HCO�

3]o at a fi xed PCO2 is ex-pected to alter pHi only by mechanisms that we shall de-scribe as “chronic.” The reductions in pHo and/or [HCO�

3]o are expected to inhibit acid-extrusion processes, but stimu-late acid-loading processes. Such kinetic changes could be produced by alterations in any rate-determining parameter, such a pHo, [Na�]o, [Na�]i, binding constants, or the density of transporters. The net effect is expected to be a gradual but sustained decline in pHi, similar to that schematized in Fig. 17C. The modeling of this problem is similar to that described in Fig. 17D, except that not only would JE de-crease, but JL would increase as well. The time course of the pHi decline depends on and on the pHi dependencies of JE and JL. The actual value of the new steady-state pHi would, of course, be independent of .

The predictions of the foregoing analysis have been con-fi rmed experimentally. Measurements of steady-state pHi with DMO indicate that metabolic acidosis lowers pHi in mammalian skeletal and cardiac muscle (3, 456). The time course of this intracellular acidifi cation has also been moni-tored with pH-sensitive microelectrodes in several verte-brate cells (8, 59, 149, 169).

Metabolic Alkalosis A sudden extracellular alkalinization produced by raising [HCO�

3]o at a fi xed PCO2 is expected to have chronic effects on acid extrusion, acid loading, and pHi that are opposite to those described above for metabolic acido-sis. Metabolic alkalosis generally produces a gradual but sus-tained pHi increase, similar to that schematized in Fig. 17E. The modeling of this problem would be similar to that of Fig. 17F, except that, not only would JE increase, but JL would decrease as well. Steady-state pHi measurements with DMO in mammalian muscle (3, 251) have confi rmed the foregoing prediction that pHi should rise during metabolic acidosis.

Respiratory Acidosis A sudden extracellular acidifi cation produced by an increase in PCO2 is expected to alter pHi by both acute and chronic mechanisms. In the laboratory, it is possible to produce a respiratory acidosis in which [HCO�

3]o is constant. However, in vivo, respiratory acidosis is always accompanied by a rise in [HCO�

3]o. Regardless of the effect on [HCO�

3]o, the “acute” effect of respiratory acidosis is produced by the infl ux of CO2, which elicits a rapid fall in pHi. This represents an acute intracellular acid load from which the cell would fully recover if the kinetics of acid-extruding and acid-loading mechanisms were not funda-mentally altered by the accompanying fall in pHo. The ex-pected “chronic” effects of respiratory acidosis are qualitatively the same as for metabolic acidosis: an inhibition of JE and a stimulation of JL. These chronic effects will tend to lower steady-state pHi, as in metabolic acidosis. Indeed, pHi de-terminations by the DMO technique (3, 456) show that respiratory acidosis does reduce steady-state pHi. In princi-ple, the time course of the pHi decrease could be predicted

from our discussion of CO2-induced acidifi cations (the acute effect) and from Eq. 39 (the chronic effect). Thus, , initial pHi, initial and fi nal PCO2, and the kinetics of acid extrusion and acid loading would all infl uence the time course of acidifi cation. All these parameters except would also infl uence the fi nal steady-state pHi. After the acute ef-fect (i.e., the rapid CO2-induced pHi fall), respiratory acido-sis could then produce (1) a slower but sustained pHi decline (chronic effect), (2) no further pHi change (i.e., immediate stabilization), or (3) a pHi recovery that is partial, complete or—at least in principle—even exaggerated. Which of the three steady-state outcomes we observe depends upon the relationship between JE and JL.

Respiratory Alkalosis The pHi changes produced by re-spiratory alkalosis, and the mechanisms effecting these changes, are opposite those produced by respiratory acidosis. The effect on acid extrusion is noteworthy because the im-posed rise in pHo is expected to stimulate acid extrusion, whereas the resultant increase in pHi (caused by the effl ux of CO2) is expected in inhibit or even totally block acid extru-sion. Thus, the expected time course of pHi after its rapid initial rise could be (1) a further, slower increase to a new steady state; (2) no change; or (3) a decline. Studies with pH-sensitive microelectrodes have demonstrated the fi rst pattern in mouse skeletal muscle (8), and steady-state pHi measurements have confi rmed that respiratory alkalosis causes a sustained elevation of pHi (3, 456).

Isohydric Hypercapnia Raising PCO2 at constant pHo (i.e., raising PCO2 and [HCO�

3]o proportionally) produces a rapid fall in pHi (61, 543), due to the infl ux of CO2, just as does respiratory acidosis (vide supra). However, for at least two reasons, the long-term infl uences on pHi can be very different from those produced by respiratory acidosis. First, because pHo is fi xed, one expects no immediate inhibition of acid extrusion or stimulation of acid loading. Thus, the cell should be better able to resist the acute acid load caused by the infl ux of CO2. Second, because [HCO�

3]o is higher in isohydric hypercapnia than respiratory acidosis, acid loading due to the passive and carrier-mediated effl ux of HCO�

3 should be lower than during respiratory acidosis. Third, the aforementioned increase in [HCO�

3]o could also stimulate HCO�

3-dependent acid-extrusion (e.g., Na�-driven Cl�-HCO�

3 exchange). According to recent data, raising [CO2] per se (i.e., in the nominal absence of HCO�

3) can stimulate H� extrusion from proximal-tubule cells (618). Thus, al-though isohydric hypercapnia is expected to elicit the same initial, CO2-induced fall in pHi as respiratory acidosis, the fi nal steady-state pHi should be higher, as has been con-fi rmed on mammalian skeletal muscle (3).

The time course of pHi during isohydric hypercapnia depends on the four previously mentioned factors (i.e., , initial pHi, initial and fi nal PCO2, and the kinetics of acid extrusion and acid loading). In salamander proximal-tubule cells, pHi does not recover from the initial fall, due to the very high HCO�

3 effl ux across the basolateral membrane, via NBC (59). In cells with lesser degrees of HCO�

3-dependent



acid loading, pHi makes a partial or even complete recovery (57, 456, 591). Finally, in cells with dominant HCO�

3-dependent acid-extrusion mechanisms, such as smooth muscle (6) and renal mesangial cells (75)¸ isohydric hyper-capnia increases steady-state pHi. In both dissociated CA1 neurons (486, 517) and cultured astrocytes (49) from the rat hippocampus, the degree of this pHi increase becomes less and less at progressively higher initial values of pHi. The pHi increase produced by isohydric hypercapnia in the rabbit proximal tubule (390) may refl ect stimulation of apical Na�-H� exchange and H� pumping (113, 114), likely involving sensors for basolateral CO2 and HCO�

3 (618).Isohydric Hypocapnia Lowering PCO2 at constant pHo

(i.e., lowering PCO2 and [HCO�3]o proportionally) produces

an initial rise in pHi due to CO2 effl ux, as in respiratory al-kalosis. The long-term infl uences on pHi are the opposite of those described previously for isohydric hypercapnia. Thus, the fi nal steady-state pHi is expected to be somewhat lower in isohydric hypocapnia than in respiratory alkalosis, as con-fi rmed for mammalian skeletal muscle (4). The time course of changes in pHi during isohydric hypocapnia has been monitored in several cells, especially during the transition between normal PCO2/[ HCO�

3] to nominally CO2/HCO�3-

free conditions. As expected, the reduction in PCO2 at con-stant pHo causes an abrupt increase in pHi (due to CO2 ef-fl ux), followed by a partial return of pHi toward normal (probably due to inhibition of acid extrusion and stimulation of acid loading at high pHi). In mesangial cells, the switch from 10% CO2/50 mM HCO�

3 (pHo � 7.4) to 5% CO2/25 mM HCO�

3 (pHo � 7.4) causes an abrupt increase in pHi that represents an acute intracellular alkali load. The subsequent recovery of pHi, due primarily to Cl�-HCO�

3 exchange, can then be examined at physiological levels of extracellular CO2 and HCO�

3 (200). A similar approach has been used by Ou-yang et al. (407) to examine Cl�-HCO�

3 exchanger activity in neurons cultured from the rat cortex.

USE OF OUT-OF-EQUILIBRIUM CO2/HCO3� TO MAKE

ISOLATED CHANGES IN EXTRACELLULAR [CO2], [HCO3

�], AND pH ONE AT A TIME

In 1995, Zhao et al. (614) introduced a rapid-mixing technique that allows one to create out-of-equilibrium (OOE) CO2/HCO�

3 solutions with virtually any combina-tion of [CO2], [HCO�

3], and pH in the pathophysiological pH range. The approach is to mix the outputs of two syringes—each containing a different combination of equil-ibrated [CO2], [HCO�

3], and pH—and then rapidly (�200 ms) deliver the newly mixed solution to the cells, while continuously sweeping away the solution after it has contacted the cells.

SENSORS FOR EXTRACELLULAR [CO2], [HCO3

�], AND pHZhou et al. (618) used the powerful OOE approach (see

previous section) to alter, systematically, the basolateral (BL) [CO2], [HCO�

3], and pH—one at time—in experiments on

isolated, perfused rabbit proximal tubules. They found that increasing [CO2]BL—while holding [HCO�

3]BL and pHBL fi xed—caused an increase in the rate of HCO�

3 reabsorption (measured during a period of �20 minutes). This is the ap-propriate response to the “respiratory” part of acute respira-tory acidosis. Conversely, increasing [HCO�

3]BL—while holding [CO2]BL and pHBL fi xed—caused the rate of HCO�

3 reabsorption to decrease. This is the appropriate response to the “metabolic” part of acute metabolic acidosis. Finally, they found that increasing pHBL—while holding [CO2]BL and [HCO�

3]BL fi xed—had no effect on the rate of HCO�3 reab-

sorption. In other words, at least during acute stresses, the proximal tubule is incapable of responding to changes in pHBL per se. Rather, the tubule responds to changes in the concentrations of the two major buffers, CO2 and HCO�

3. These data are consistent with the hypothesis that the proximal tubule has some sort of sensor or sensors that de-tect extracellular levels of CO2 and extracellular HCO�

3. The response to increased [CO2]BL is blocked by nanomolar levels of compounds believed to be specifi c for the ErbB family of receptor tyrosine kinases, but not by an inhibitor of Src kinases (617).

Although proximal tubule cells apparently do not re-spond to pHBL changes per se, Ludwig et al. (345) have identifi ed two G-protein–coupled receptors (GPCRs) that do respond to changes in extracellular pH. The fi rst is the ovarian cancer GPCR (OGR1). Lowering pHo from 7.8 to 6.8 converts OGR1 from its fully inactivated to its fully activated state, which causes an increase in inositol phos-phate formation. According to additional work by Tomura et al. (552), OGR1 can lead to an increase in [cAMP]i via phospholipase C and the cyclo-oxygenase pathway. OGR1 is highly expressed in osteoblasts, lung, intestine, and kidney. The second pH-sensitive GPCR is GPR4, which also ap-pears to signal through cAMP.

In addition to these GPCRs, several ion channels are pH sensitive. Thus, it is possible that changes in pHo could sig-nal via changes in membrane potential. The fi rst such chan-nel to be identifi ed was the Na�-permeable channel ASIC (575). This channel is activated by large (�1 pH unit) and rapid pHo decreases (19). pH also modulates 2P-domain K� channels (TASK, TALK, TREK, and TWIK). TASKs are acidosis inhibited, whereas TALKs are alkalosis activated (330). TASK-1 (pK �7.3) is very sensitive to pHo variations (166) and TASK-3 has a pK of �6.6 (109, 435).

EFFECTS OF INTRACELLULAR pH CHANGES ON pHO

The pH of the bulk extracellular fl uid (pHECF) is closely regulated by the respiratory and renal systems. Neverthe-less, the movement of acids and bases across cell mem-branes can modify pHECF. For example, in the central nervous system, where the neurons and glial cells are in close apposition to one another, acid-base transport across the cell membranes can profoundly alter pHo (see reviews, 117–119, 438). Acid-base movement across cell mem-branes can result in parallel or reciprocal changes in pHi



and pHo. Parallel decreases in pHi and pHo can occur when an increased rate of lactic acid production (which lowers pHi) leads to the subsequent effl ux of lactic acid via H�/Lac� cotransport (which lowers pHo). The effl ux of lactic acid minimizes the fall in pHi. Reciprocal increases in pHi and decreases in pHo occur in gallbladder epithelial cells due to Na�-H� exchange (444), and in depolarized snail neurons due to a voltage-activated conductance pathway (549). In both of these examples, stabilization of pHi oc-curs at the expense of pHo homeostasis.

ROLE OF THE EXTRACELLULAR FLUID IN pHi REGULATION

It is well established that changes in pHo will elicit recip-rocal changes in pHi. A general rule of thumb: the magni-tude of a pHi change will be approximately one third the magnitude of the pHo change. However, pHi/ pHo can vary considerably among cells, being as high as 0.7 in mes-enteric vascular smooth muscle (29), carotid-body glomus cell (86), and CNS neurons (71). As noted previously, extra-cellular metabolic acidosis causes a sustained fall in pHi. The overall effect of this acidosis on acid-base transport is there-fore a net transfer of acid from the ECF to the ICF of all accessible cells, causing pHo to increase somewhat toward normal, but at the expense of causing pHi to decrease throughout the organism. One interpretation of these recip-rocal changes in pHi and pHo is that cells act as a pH buffer for the ECF, preventing large fl uctuations in pHo. For ex-ample, “healthy” cells taking up H� minimize the fall in pHo due to the release of lactic acid by a subpopulation of hy-poxic cells. The partial uptake of this acid from the ECF by the healthy cells may thus be viewed as a mechanism for distributing the acid insult among all cells accessible to the ECF until such time that the renal and respiratory systems can correct the acid-base disturbance.

In the steady state in most animals, there is a net transfer of metabolically generated acid from cells to the ECF, and from the ECF to the urine, as depicted in Fig. 18. In the kidney, tubules from both the proximal nephron (16, 58, 59, 318) and distal nephron (493, 585) are composed of cells that regulate pHi much as would nonepithelial cells: They respond to intracellular acid loads by extruding acid. How-ever, these cells are unique in that HCO�

3 effl ux, which provides the major intracellular acid load, is limited to the cells’ basolateral (or blood side) membrane (9, 15, 17, 59, 319, 607). On the other hand, acid extruders (e.g., the Na�-H� exchanger and H� pump) are generally most active at the luminal membrane (16, 268, 393). Thus, the acid ex-truded into the ECF from nonrenal cells enters renal tubule cells via basolateral HCO�

3 effl ux, lowering renal tubule pHi. Presumably stimulated by this pHi decrease, luminal acid extruders move the acid into the tubule lumen. Thus, the regulation of pHi by certain renal tubule cells ultimately leads to acid excretion by the kidney. The renal tubule cells therefore provide the fi nal link between pHi and pHo: pHi regulation by renal tubule cells is responsible for pHo regula-

tion, which in turn makes possible pHi regulation by the body’s other cells.

Temperature Changes

Decreasing the temperature generally produces a pH in-crease in the blood and intracellular fl uid (440, 456). In principle, a temperature change could alter pHi in at least three ways: (1) by modifying the pK�a of pH buffers, (2) by modifying the pK�a of enzymes or transporters involved in acid-base homeostasis, or (3) by changing the activation energy for an enzymatic reaction or transport process.

EFFECT OF TEMPERATURE CHANGES ON THE PK�a of BuffersThe integrated form of the van’t Hoff equation predicts

that, in a closed system (i.e., for a fi xed total amount of buf-fer), a buffer’s pK should vary inversely with temperature:

pK pKH

Rln101T

1T

1 21 2

� � � ⎡

⎣⎢⎤⎦⎥ (Eq. 40)

where H is the heat of ionization, R is the universal gas constant, T1 and T2 are two absolute temperatures, and the subscripts 1 and 2 refer to pK�a values at the corresponding two temperatures. If a solution contains only one buffer, then a temperature change will produce parallel shifts in pK�a and pH. In the case of a solution containing several buffers (e.g., intracellular fl uid), the temperature-induced pH shift depends on the sum of the individual pK shifts, each weighted by the buffer’s relative contribution to total buffering (87).

Reeves and Malan (441) have analyzed the pHi changes produced by temperature shifts in frog muscle in vivo. Over the same temperature range, the in vivo pHi values in these

FIGURE 18 Schematic representation of factors affecting H� balance in the single cell and whole organism. A nonepithelial cell is acid loaded by metabolic production of acid, by HCO3

� effl ux, and by H� infl ux. pHi is maintained by a mechanism that extrudes acid from the cell into the extra-cellular fl uid (ECF) and blood. This acid effectively gains entry into certain renal tubule cells by the effl ux of HCO3

� across the renal tubule basolateral membrane. This HCO3

� effl ux, in turn, acid loads the renal tubule cell and stimulates extrusion of acid across the luminal membrane and into the urine. Thus, the necessity of pHi regulation by these renal tubule cells brings about pHi regulation of the blood, which in turn keeps the extracel-lular pH high enough for nonepithelial cells to regulate their pHi.



experiments are approximated by the in vitro pH values of a mixture of imidazole, phosphate, and CO2/HCO�

3, all at physiological levels. This frog-muscle data seems consistent with the idea that temperature changes can shift steady-state pHi by shifting pK�a of buffers, and thus releasing or con-suming H�. However, this interpretation is inconsistent with the general principle that only alterations in the properties of acid-base transporters can change steady-state pHi. Consider the example of raising temperature, which would lower the pK�a values of intracellular buffers, thereby releasing H�. This represents an acute intracellular acid load, no different in principle from the acid load produced by the injection of H�. If the only effect of the temperature increase were to lower pK�a values of buffers, then pHi would abruptly de-crease and subsequently recover to its initial value over the course of a few minutes. The only lasting effect would be subtle changes in the pHi profi le of . Thus, although there can be little doubt that temperature changes elicit a rapid release or consumption of H�, we must look to other mech-anisms to account for changes in steady-state pHi.

EFFECT OF TEMPERATURE CHANGES ON ENZYMES AND TRANSPORTERS

The effects of temperature changes on acid-base trans-porters and other enzymes affecting pHi are not well docu-mented. The effects are most likely of two sorts: (1) A temperature-induced shift in the pK�a of an ionizable group on an enzyme or transport protein could affect the kinetics of the reaction or transport process, or (2) raising the tem-perature speeds reactions by increasing the energy of the reactants. As far as pK�a shifts are concerned, a change in temperature would be expected to alter the ionization state of titratable groups on proteins, with effects being greatest for groups with pK�a values similar to the prevailing pH. For instance, a rise in temperature would lower the pK�a of an amino group of a protein, thereby reducing the fraction of molecules with a positive charge at that position. Conversely, the fraction of molecules with a negatively charged carboxyl group would rise. Such alterations in ionization could lead to conformational changes that could have major effects on protein function.

As far as direct effects of temperature changes on reac-tion rates are concerned, enzymatic reaction rates as well as passive and active fl uxes of ions all are expected to fall with decreasing temperature, although to varying degrees. In mouse soleus muscle, reducing the temperature from 37°C to 28°C causes the acid-extrusion rate to fall by 65% (7). Similarly, in cultured astrocytes from rat hippocam-pus, reducing the temperature from 37°C to room tem-perature causes the acid-extrusion rate elicited by expos-ing the cells to CO2/HCO�

3 to fall by �40% (M.O.B. and W.F.B., unpublished data). In a study on cultured fetal rat hippocampal neurons, Baxter and Church (36) have pre-sented evidence that the Na�-driven Cl�-HCO�

3 ex-changer contributes to the maintenance of steady-state pHi at 18–22°C, but not at 37°C. In these neurons, in-

creasing temperature may activate the Na�-H� exchanger more than the Na�-driven Cl�-HCO�

3 exchanger. In ad-dition to cellular changes brought about by temperature shifts, whole-body physiological changes (e.g., hormone levels, PCO2, plasma [HCO�

3]) may also infl uence pHi.

METABOLIC INHIBITORS AND ANOXIA/HYPOXIA

Metabolic inhibitors, anoxia/hypoxia, and ischemia gen-erally produce slow decreases in pHi. Metabolic inhibitors have been examined most closely in isolated nerve and muscle preparations using pH-sensitive microelectrodes. Both azide and 2,4-dinitrophenol (DNP) produce two-stage decreases in pHi (61, 543): an abrupt fall followed by a slower one. The abrupt pHi decrease is probably due to the infl ux and dissociation of the protonated weak-acid form of the inhibitor, whereas the slower pHi decrease may be caused by a net increase in the production of acidic metabo-lites, confounded perhaps by a decrease in the rate of acid extrusion. Squid axons that are exposed to hydrocyanic acid (HCN #H� � CN�; pK�a � �10) display only a slow decrease in pHi (61), the origin of which is likely the same as those of the slow phase of the azide and DNP-induced acidifi cations. The failure of HCN to produce a rapid pHi decrease in squid axons is due to its high pK�a: at physiolog-ical pHi, only a small fraction of incoming HCN dissociates into CN- and H�.

Ischemia and anoxia/hypoxia can also elicit decreases in pHi due to the lactic acid production that results from in-creased glycolysis in the absence of oxidative phosphoryla-tion (252, 604). For example, using 31P-NMR to measure the pHi of beating rat hearts, Gadian et al. (196) observed that ischemia produced a gradual fall in pHi from 7.05 to 6.2 in 13 minutes. However, in hearts previously depleted of glycogen, the pHi fall triggered by a subsequent episode of ischemia is reduced by �50% (206). Similarly, preincubating the hearts in 2-deoxyglucose, which inhibits phosphorylase b and thus glycogen breakdown, reduces the ischemia-induced pHi decrease by �25%. Although anoxia/hypoxia might be expected to decrease pHi by interfering with active-transport processes that extrude acid, the effects of anoxia/hypoxia appear to be more complex and are likely to depend on several factors including the cell type, the ex-perimental preparation (e.g., cells in situ vs cells in culture), duration of anoxia/hypoxia, and the presence versus absence of CO2/HCO�

3 as well as other parameters that infl uence the relative activity of various acid-base transporters. For example, in the isolated turtle heart, anoxia inhibits a DIDS-sensitive, Na�- and HCO�

3-dependent mechanism by �50% (499). In contrast, acute anoxia appears to stimu-late a stilbene-sensitive, electrogenic Na�-coupled HCO�

3 transporter in rat hippocampal neurons (603). Anoxia/ischemia or subsequent reoxygenation has also been shown to stimulate Na�-H� exchange activity in mammalian neu-rons and astrocytes (43, 158, 276, 301, 497, 602), and such stimulation can involve kinases such as ERK1/2 and protein kinases A and C (301, 497, 602).



Effects of Cell Shrinkage on pHi

EFFECT OF HYPERTONIC SOLUTIONS ON NA�-H� AND CL�-HCO3

� EXCHANGE

In several cell types, including erythrocytes (92, 93, 314, 416, 417, 505, 506), lymphocytes (226), mesangial cells (44), glioma cells (273, 500), and barnacle muscle (140, 141), shrinking a cell in a hypertonic medium stimulates the exchange of external Na� for internal H�. In at least Amphiuma red blood cells (RBCs), shrinkage in hypertonic solutions also stimulates the exchange of external Cl� for internal HCO�

3. Because the H� extruded from the cells is derived almost exclusively from buffers, and because the HCO�

3 is derived from the freely diffusible CO2 and H2O, these movements of H� and HCO�

3 are “osmotically silent.” Thus, the net effect of Na�-H� and Cl�-HCO�

3 exchange is an uptake of NaCl—along with osmotically obligated H2O—and a recovery of cell volume termed a “volume-regulatory increase” (VRI). As discussed in the following paragraphs, shrinkage appears to activate the Na�-H� ex-changer directly. However, the stimulation of the Cl�-HCO�

3 exchanger appears to be indirect, refl ected by the rise in pHi caused by the activated Na�-H� exchanger (264).

Interestingly, in lymphocytes, the shrinkage-induced ac-tivation of the Na�-H� exchanger is observed only after a volume-regulatory decrease that follows a previous cell swelling (226). Regulation of cell volume per se is discussed in Chapter 6. For other reviews of cell-volume regulation, see refs. (95, 229, 230, 243, 255, 313, 325, 326).

MECHANISM BY WHICH SHRINKAGE/HYPERTONICITY AFFECTS NA�-H� EXCHANGE

We do not know how cells sense shrinkage. However, we are beginning to understand more about the signal-transduction processes involved in the acute and chronic re-sponses to shrinkage. Hypertonicity can trigger the rapid de-phosphorylation phosphatidylinositol-5-kinase type I (PIP5KI), which activates the enzyme and increases the rate of PIP2 synthesis (601). On a longer time scale, kinases in the Ste20 family, with the subsequent activation of the MAPK pathway, are critical for the chronic response to hypertonic shock in yeast (259, 302) and mammalian cells (495).

How does acute cell shrinkage affect one of the key end-points of the signal-transduction cascade, namely, the Na�-H� exchanger? Although hyperosmolarity does not alter NHE1 phosphorylation (231), the phosphorylation of other proteins is likely to be involved. For example, in work on primary rat astrocytes (501) and confl uent C6 glioma cells (500), Shrode et al. have discovered that shrinkage appears to stimulate Na�-H� exchange through phosphorylation of myosin light chain. Kinases such as tyrosine kinases, MAPKs, and Janus kinase 2 ( Jak2) also appear to be involved (11, 207, 214, 315). For example, the general tyrosine kinase in-hibitor genistein inhibits tyrosine kinase–mediated protein phosphorylation and hypertonic-induced stimulation of NHE1 in polymorphonuclear leukocytes (315).

According to results from an intriguing structure–function study involving chimeras of NHE1 (stimulated by shrinkage) and NHE2 (not stimulated by shrinkage), the extracellular loop between TM domains 1 and 2 of NHE2 is suffi cient to inhibit shrinkage-induced activation of the exchanger (533).

How does acute shrinkage mechanistically alter the Na�-H� exchanger? Dunham and colleagues (163, 164) have reported that cell shrinkage stimulates Na�-H� exchange activity in dog RBCs by reducing the transporter’s affi nity for external Na� at an extracellular inhibitory site.

Other insights into the events linking shrinkage to the acute stimulation of Na�-H� exchange come from work on dog RBCs, lymphocytes, and/or giant-barnacle muscle fi -bers. First, although Na�-H� exchange appears inactive in these cells under physiological conditions, it can be stimu-lated by either a decrease in pHi (139, 226), a decrease in cell volume (141, 226, 416), or an increase in [Li�]i (139, 415). Second, the shrinkage-induced activation of Na�-H� ex-change requires Cl� (414, 416), specifi cally intracellular Cl� (141, 258). In rat mesangial cells as well, the shrinkage-induced increase in pHi is inhibited by preincubating the cells in a Cl�-free solution for a minimum of �15 minutes (and presumably decreasing [Cl�]i considerably) (44). Third, in barnacle muscle fi bers, the shrinkage-induced activation of Na�-H� exchange is inhibited by GDPS (140), sug-gesting involvement of a G protein in the signal transduc-tion system. Consistent with this last observation, Na�-H� exchange is stimulated by either GTPS, AlF3, or cholera toxin (CTX) (258). However, the CTX effect is not via a classic Gs pathway, inasmuch as neither cAMP nor cAMP analogues stimulate Na�-H� exchange. It should be empha-sized that these data do not prove that CTX stimulates the Na�-H� exchanger by the same signal transduction path-way as does shrinkage. Finally, in barnacle muscle fi bers, the Cl� requirement in the shrinkage-induced activation of the exchanger is at or before activation of the heterotrimeric G protein (258).

K�-H� EXCHANGE

Cala has observed that an exchange of internal K� for ex-ternal H� contributes to the volume-regulatory decrease (VRD) that occurs after swelling Amphiuma RBCs (93). This exchange is inhibited in the nominal absence of Ca2�, and is stimulated by applying the Ca2� ionophore A23187, suggest-ing that increases in [Ca2�]i may activate K�-H� exchange. Two observations are of special interest. First, increased [Ca2�]i failed to stimulate K�-H� exchange when Na�-H� exchange was stimulated by shrinkage. Second, if the cells were shrunk (so that Na�-H� exchange should have been stimulated) and then pretreated with amiloride (to block Na�-H� exchange), then swelling failed to activate K�-H� exchange. However, amiloride did not inhibit swelling-activate K�-H� exchange when the drug was applied for the fi rst time to swollen cells. These data led Cala to suggest that Na�-H� and K�-H� exchange may be mediated by the same



entity (94). K�-H� exchange can also be activated by milli-molar concentrations of N-ethylmaleimide (NEM) (5).

Hormones, Chronic Stress, Growth Factors, and Oncogenes

HORMONES

A number of hormones modulate transepithelial acid-base transport in various nephron segments (for examples, see [205, 265, 339, 410]). In addition, hormones modulate specifi c acid-base transporters in a variety of cells, including those from the kidney. The Na�-H� exchanger is the trans-porter that has most often been the subject of these hormone studies—probably refl ecting the transporter’s popularity rather than its unique sensitivity to hormonal control. In the following paragraphs, we will review some of the hormones that can alter the activity of acid-base transporters, particu-larly those in the kidney.

Glucocorticoids Elevations in plasma levels of glucocorti-coids in response to metabolic acidosis increase acid excretion in the kidney by stimulating Na�-H� exchange, thereby in-creasing H� secretion and HCO�

3 reabsorption. In studies on brush-border membrane vesicles, Na�-H� exchange rates are higher in vesicles obtained from adrenalectomized (adx) animals treated with the glucocorticoid dexamethazone than in adx animals treated with the mineralocorticoid aldoste-rone or adx animals receiving no supplementation (189, 299, 472). In proximal-tubule cells isolated from the kidney, Bidet et al. (51) have shown that even a 1-hour treatment with dexamethazone can enhance Na�-H� exchange. The dexa-methazone increases the Vmax of the transporter, and stimu-lates new protein synthesis. Glucocorticoids exert their effect predominantly on the Na�-H� exchange isoform NHE3. For example, in ileal brush-border membranes from animals injected with methylprednisolone, the glucocorticoid stimu-lates the activity and mRNA levels of NHE3, but not of NHE2 or NHE1 (609). In opossum kidney (OKP) cells, glucocorticoids increases both Na�-H� exchange activity (35) and NHE3 mRNA levels (34). Hydrocortisone raises both the activity and protein level of NHE3 in OKP cells subjected to acidosis (20). More recent data are consistent with a nongenomic component to glucocorticoid-induced activation of NHE3 that involves both the stimulation of protein kinase SGK1 and the presence of the Na�-H� ex-changer regulatory factor NHERF2 (608). Glucocorticoids can also enhance the stimulatory effect of insulin on NHE3 activity and expression in OKP cells (195, 303).

Glucocorticoids appear to stimulate Na�/HCO�3 co-

transport as well. For example, removing hydrocortisone from the medium of cultured proximal tubule cells causes a decrease in Na�/HCO�

3 activity, whereas returning the hy-drocortisone or adding dexamethasone increases the activity of the transporter (466). In addition, both NBCe1 activity and mRNA levels are increased 80% to 90% in proximal tubules from rats 4 days after subcutaneous injection with glucocorticoids (12).

Aldosterone can have both nongenomic (short term) and genomic effects (long term) on Na�-H� exchange. For ex-ample, in renal and nonrenal cells, applying aldosterone elicits a stimulation of the Na�-H� exchanger after a maxi-mum delay of 20 minutes (400, 581–584), probably due to an alkali shift in the set point of the transporter (127). How-ever, a 30-minute exposure of aldosterone to frog early distal tubule also stimulates Na�-H� exchange activity, but due to increased expression of the transporter. In this frog-tubule preparation, the stimulation can be inhibited by transcrip-tion or translation inhibitors (128). In a nongenomic fash-ion, aldosterone also stimulates NHE3 activity in the med-ullary thick ascending limb of the kidney (218, 219) through an ERK-dependent pathway (579). As presented in greater detail in Chapter 54, aldosterone also stimulates the V-type H� pump in the distal tubule.

Catecholamines The catecholamines norepinephrine and dopamine infl uence acid-base transporter activity in the kidney. Norepinephrine has been proposed to stimulate Na�-H� exchanger activity in the proximal tubule (211, 399), probably through activation of �1A- and/or �1B-adrenergic receptors (338). However, in working on the Ambystoma proximal tubule, Abdulnour-Nakhoul et al. (2) found that norepinephrine has no effect on Na�-H� ex-changer activity, but rather, elicits an increase in pHi by in-hibiting NBC-mediated HCO�

3 effl ux.Regarding dopamine, it is well established that this cat-

echolamine inhibits Na�-H� exchanger activity in the brush-border membrane of the proximal tubule (177, 178, 212, 269, 496, 594). Such inhibition is mediated through cAMP/PKA-dependent and -independent events (177, 178, 594) and can involve endocytotic removal of NHE3 trans-porters from the apical membrane (262). Working on iso-lated proximal tubules from rabbit, Kunimi et al. (316) have demonstrated that dopamine also inhibits Na�/HCO�

3 co-transporter activity, although such inhibition was not ob-served in tubules from hypertensive rats. Dopamine has also been shown to inhibit Cl�-HCO�

3 exchanger activity in im-mortalized proximal tubule cells from normotensive, but not hypertensive rats (418).

Other agents that can infl uence acid-base activity in the kidney include adenosine, which stimulates Na�/HCO�

3 cotransporter activity in the proximal tubule (539), as well as cholinergic agonists that stimulate the cotransporter in proximal-tubule cells (451, 461).

Thyroid Hormones The effect of thyroid hormone levels on Na�-H� exchange activity has been examined in brush-border vesicles obtained from the renal cortex of hypo-, eu-, and hyperthyroid rats (297, 298, 300, 472). Compared with vesicles derived from euthyroid animals, those derived from hypothyroid animals had only half the normal Na�-H� ex-change activity, whereas those derived from hyperthyroid animals had twice the normal exchanger activity. In both cases, the altered Na�-H� exchange activity is due to an increase in the Vmax of the transporter, rather than a decrease in the apparent KM for Na�. The effect of thyroid hormones



triiodo-l-thyronine (T3) and l-thyroxine (T4) on Na�-H� exchange has also been examined in cultured cells. In opos-sum kidney cells, both T3 and T4 stimulate amiloride-sensitive 22Na� uptake (605), probably by increasing the transcription of NHE3 (97).

Parathyroid Hormone, Angiotensin II, and Cyclic AMP In the renal proximal tubule, HCO�

3 reabsorption is inhibited by PTH (154, 234, 265), which increases intracellular levels of cAMP. At the level of brush-border membrane vesicles, PTH and cAMP analogues both reduce Na�-H� exchange activity (278), whereas parathyroidectomy of the donor ani-mal increases Na�-H� exchange activity (123). At the cel-lular level, PTH and cAMP both inhibit Na�-H� exchange in a renal epithelial cell lines (383, 423) and in rat medullary thick ascending limb (mTAL) tubule suspensions (54). Based on work by Weinman and colleagues (586), the in-hibitory effect of cAMP in the proximal tubule requires the presence of the Na�-H� exchange regulatory factory NHERF1, which is a PDZ adaptor protein that can bind to the carboxy terminus of NHE3 (588, 589). The outcome of PTH stimulation is probably a shift in the pHi sensitivity of Na�-H� exchange (368), likely associated with PTH-induced phosphorylation of NHE3 (124, 173). PTH can also have a delayed secondary effect of stimulating internal-ization of NHE3 from apical membranes (124, 173, 613).

PTH and cAMP can also inhibit HCO�3 transporters.

Treatments that raise [cAMP]i inhibit apical Cl�-HCO�3

exchange in the Necturus gallbladder epithelium (445) and in renal epithelial cells (245). Similarly PTH appears to inhibit Na�/HCO�

3 cotransport activity in rabbit renal basolateral membranes via G proteins and a calmodulin-dependent protein kinase (462). Increases in cAMP and activated Ca2�-dependent protein kinases also inhibit Na�/HCO�

3 cotransport in these basolateral membranes (460). As described previously for cAMP-mediated inhibition of NHE3, NHERF1 is also involved in cAMP-mediated inhi-bition of NBCe1, although there is no direct association between the two proteins (41, 587).

Low doses of angiotensin (Ang) II (e.g., 10�9 M) stimu-late Na�-H� exchange and/or Na�/HCO�

3 cotransport in the proximal tubule (129, 167, 209, 246, 248, 261, 339, 339, 442, 471, 616), distal tubule (33, 332, 577), and cortical collecting duct (482). On the other hand, higher doses of Ang II (e.g., 10�6 M) inhibit these transporters (37, 129, 261, 616). Long-term effects of Ang II include regulation of NBCe1 expression in the proximal tubule. For example, expression levels of NBCe1—but not NHE3—in the kidney increase when rats are infused with Ang II, but de-crease when infused with the AT1 receptor blocker cande-sartan (560). Regarding the signal-transduction pathway, in renal cortical basolateral membrane vesicles (464) and OKP cells (98), Ang II stimulation of the exchanger involves G-protein activation. Ang II stimulation of Na�/HCO�

3 cotransporter activity in OKP cells involves activation of Src family tyrosine kinases (SFKs) and the classic MAPK pathway (452).

In isolated perfused superfi cial S1 segments of the rab-bit proximal tubule, Ang II stimulates Na�-H� exchange and Na�/HCO�

3 cotransport (209), as judged from rates of pHi change measured at relatively low pHi values. Interest-ingly, Ang II has very little effect on steady-state pHi. Thus, if Ang II indeed stimulates both transporters in the physiological pHi range, then the combined increase in activities of the acid extruder and acid loader serves to in-crease transepithelial HCO�

3 reabsorption without altering pHi. Ang II can also stimulate the Na�-H� exchangers on the apical (419) and basolateral (37) sides of macula densa cells.

Ang II can also stimulate other acid-base transporters. For example, in cat papillary muscles of the ventricular myo-cardium, Ang II stimulates Cl�-HCO�

3 exchange, via activation of a PKC-dependent pathway (96). In the kidney, Ang II has been shown to stimulate the H� pump in iso-lated proximal tubule cells by colchicine-sensitive apical membrane insertion (572).

CHRONIC STRESSES

Chronic Metabolic Acidosis and Alkalosis Chronic meta-bolic acidosis typically increases Na�-H� exchange activity in the kidney, whereas chronic metabolic alkalosis has the opposite effect. For example, brush-border membrane vesi-cles derived from animals with chronic metabolic acidosis have increased Na�-H� exchange activity (123), due to an increase in the apparent Vmax, rather than to a decrease in the KM for Na� (293, 472). Increases in the activity of Na�-H� exchange have been observed in other preparations, such as membrane vesicles of rabbit kidney proximal tubules previ-ously incubated in a low-pH solution (521), and cells from the medullary thick ascending limb (mTAL) of rats sub-jected to metabolic acidosis (323). In the mTAL, such acti-vation is paralleled by increases in NHE3 mRNA and pro-tein levels (323), but not NHE1 mRNA levels (324). Working with cultured proximal-tubule cells, Alpern et al. (260) found that chronic metabolic and respiratory acidosis increases Na�-H� exchange activity via new protein synthe-sis. This effect, as well as increased expression of NHE3 mRNA, is blocked by overexpressing Csk (600), a natural inhibitor of Src kinases. Recall that in acute experiments (see previous sections) on isolated perfused proximal tu-bules, CO2 appears to signal through a receptor tyrosine kinase, not Src.

Chronic metabolic acidosis/alkalosis can also elicit changes in the activity of other acid-base transporters. For example, the increase in apical Na�-H� exchange activity with metabolic acidosis is paralleled by an increase in baso-lateral Na�/HCO�

3 cotransporter activity (10, 428, 521). More recently, Kwon et al. (320) used semiquantitative im-munoblotting and immunohistochemistry to examine the effect of chronic metabolic acidosis on the expression of three Na�-coupled HCO�

3 transporters in rat kidney. The acidosis increased NBCn1 abundance in the medullary thick ascending limb and another NBCn1 variant in intercalated



cells, but had no effect on NBCe1 abundance in the proxi-mal tubule.

Metabolic acidosis also causes an increase in the activity of apical H� pumps in the kidney (522, 558, 559), presum-ably contributing to the increase in HCO�

3 reabsorption. In fact, chronic acidosis stimulates the redistribution of H� pumps to the apical surface of collecting-duct intercalated cells, whereas chronic alkalosis stimulates the redistribution to the basolateral surface (470). Finally, Sabolic et al. (470) observed that expression levels of Cl�-HCO�

3 exchange in-crease in the cortical collecting duct (CCD) of rats subjected to chronic metabolic acidosis, but decrease in animals sub-jected to chronic metabolic alkalosis. Also, working on the CCD, Tsuruoka and Schwartz (558) found that chronic metabolic acidosis decreases apical Cl�-base exchange of the -intercalated cells, but increases basolateral Cl�-base ex-change of the �-intercalated cells.

Chronic Hypercapnia Na�-H� exchange activity is in-creased in renal brush-border vesicles derived from animals that were chronically hypercapnic (611). Chronic respiratory acidosis also increases levels of band 3 mRNA in the kidney (540). Na�-H� exchange activity is increased in renal brush-border vesicles derived from animals that were chronically hypercapnic (611). Chronic respiratory acidosis also in-creases levels of band 3 mRNA in the kidney (540). Work-ing on either opossum kidney cells or cultures of proximal-tubule cells, Arruda and colleagues have found that 10% CO2 (i.e., respiratory acidosis) has a biphasic stimulatory effect on NBCe1 activity that initially (within 5 minutes) involves activation of Src family kinases and phosphati-dylinositol 3 kinase and increased membrane insertion of NBCe1 (40, 171, 463, 465). Later (24 hours), these authors observe increases in mRNA and protein levels. In isolated perfused proximal tubules, an inhibitor of Src kinases has no effect on the acute stimulation of HCO�

3 reabsorption by increased levels of basolateral CO2 (617).

Renal Hypertrophy, High-Protein Diet, and Chronic Potassium Depletion Renal hypertrophy can increase the activity of several acid-base transporters (99). Brush-border membrane vesicles derived from the remnant kidney follow-ing a uninephrectomy exhibit increased Na�-H� exchange activity (249, 491). Interestingly, the denervation associated with the uninephrectomy may be the predominant stimulus. Indeed, the hypertrophy-induced activation of Na�-H� ex-change elicited by the uninephrectomy can be mimicked by contralateral denervation (347). Nevertheless, hypertrophy of renal proximal-tubules cell in vitro does lead to an in-crease of Na�-H� exchange activity (182). In microperfu-sion studies of rat proximal tubule, Preisig and Alpern (429) demonstrated that the hyperfi ltration preceding a decrease in renal mass increases the activities of Na�-H� exchange and Na�/HCO�

3 cotransport.Rats fed a 40% protein diet, rather than a 6% protein diet,

also yield cortical brush-border membrane vesicles with enhanced Na�-H� exchange activity, and the effects of uni-nephrectomy and high-protein diet are additive (249, 491).

Finally, K� depletion can also increase the function and expression of acid-base transporters. For example, vesicles derived from K�-depleted rats exhibit enhanced Na�-H� exchange and Na�/HCO�

3 cotransport activity due to in-creases in apparent Vmax values, with no changes in the ap-parent KM values for Na� (520). Also, in functional and antibody studies, dietary K� depletion enhances the H� pump in rat distal nephron (30). As reviewed by Silver and Soleimani (510), K� depletion also increases function and expression of K�-H� pumps in the kidney, particularly the colonic form of the K�-H� pump. K� depletion appears to enhance overall HCO�

3 reabsorption in the renal tubule by increasing NBCe1 expression and activity in the proximal tubule, as well as eliciting NBCe1 mRNA expression and/or transporter activity in the mTAL and inner medullary collecting duct (21).

GROWTH FACTORS

Classical Model of Growth-Factor Effects on pHi A po-tential role for pHi in mitogenesis is suggested by the ob-servation that growth-factor–induced proliferation of fi -broblasts (425) and mesangial cells (202) is blocked at relatively low pHi values, and rises sharply as pHi enters the physiological range. Indeed, there are numerous ex-amples in which adding a mitogen to quiescent cells, in the nominal absence of CO2/HCO�

3, causes a sustained in-crease in pHi (199, 227, 380, 432, 458, 483, 532, 538, 567). Because the mitogen-induced pHi increase is prevented by removing Na� (227, 380) or pretreating with amiloride or an amiloride analogue (227, 322, 380), many investigators had deduced that the increase in pHi is due to stimulation of Na�-H� exchange (for more examples, see review [377]). Furthermore, in fi broblast mutants lacking Na�-H� exchange, mitogens fail to elicit proliferation, or to alkalinize the cells (425, 426). Wang et al. (576) have re-ported that a P19 embryonal carcinoma cell line lacking Na�-H� exchange has a reduced ability to growth and dif-ferentiate. The ability to differentiate can be resurrected by reintroducing the exchanger into the cells. These data led to an attractive hypothesis: (1) in quiescent cells, pHi is too low to sustain proliferation; (2) mitogens, in addition to affecting other cellular functions, stimulate Na�-H� ex-change; (3) this stimulation of Na�-H� exchange elevates pHi to a range that is permissive for proliferation. Thus, according to this model, pHi would play a central role in proliferation.

Effect of Mitogens on pHi in the Presence of CO2/HCO3

� One of the fi rst hints that the mitogen–pHi model described previously might be incomplete was the observa-tion that A431 cells incubated in the presence of CO2/HCO�

3 do not alkalinize in response to FCS plus EGF (104). Mesangial cells (199), fi broblasts (53), and NIH 3T3 cells (538) also fail to alkalinize. In fact, in mesangial cells, all 13 mitogens or other agents that increased steady-state pHi in the nominal absence of CO2/HCO�

3 caused pHi to decrease in the presence of this physiological buffer



(202). In all four of the aforementioned cell types, the steady-state pHi in the presence of CO2/HCO�

3 is substan-tially higher than that observed in the absence of CO2/HCO�

3, with or without mitogen. Thus, it seems that, at least for some cells, the pHi prevailing in the presence of CO2/HCO�

3 is high enough to put pHi in a range permis-sive for proliferation, and that pHi remains in this permis-sive range even after a mitogen-induced acidifi cation. Al-though the pHi increase elicited by mitogens in the absence of CO2/HCO�

3 is real, it appears that a change in pHi is not an intrinsic part of the mitogenic response.

Transporters Affected by Mitogens in the Presence of CO2/HCO3

� The effect of growth factors on the activity of acid-base transporters of cells incubated in the presence of CO2/HCO�

3 has been examined in some preparations. In renal mesangial cells, application of arginine vasopressin (AVP) (198, 200) or epidermal growth factor (EGF) (198) “activates” all three acid-base transporters known to be present: the Na�-H� exchanger, the Na�-driven Cl�-HCO�

3 exchanger and the Cl�-HCO�3 exchanger. The two

acid-extrusion mechanisms, assayed at the single pHi of 6.6, were stimulated by �100%. The acid loader, assayed at the single pHi of 7.7, was stimulated to an even greater extent, �140%. AVP also stimulates both Na�-H� and Cl�-HCO�

3 exchange in the A10 vascular-smooth-muscle cell line (290). It should be emphasized that none of the data discussed thus far address the issue of whether in fact the growth factors stimulated the transporters in the physiological pHi range (i.e., approximately 7.1–7.3 for these cells).

Growth factors can also have time-dependent effects on pHi and acid-base transporters. For example, in working on mesangial cells, Ganz et al. (198) observed that both AVP and EGF, which are thought to act through different signal transduction pathways, have similar effects on the time courses of the three aforementioned transporters. Both growth factors elicit an immediate (i.e., within 10 minutes) increase in the activities of all three transporters (assayed at the single pHi values indicated previously). The immediate response is followed within an hour by a fall in the activities of all three transporters, even though the activities remain substantially above control levels. Finally, there is a transient dip in the activities of all three transporters at times corre-sponding to the period of the maximal rate of increase in cell number (�35–45 hours for AVP and �12–20 hours for EGF). Growth factors can have “early” and “late” effects on Na�-H� exchange in vascular smooth muscle cells (see re-view [343]). The late effect can include increased expression of the exchanger.

Effect of Growth Factors on the pHi Dependence of the Na�-H� Exchanger In the past, many investigators believed that the pHi dependence of the Na�-H� exchanger is linear, and that the exchanger is inactive at the physiological (i.e., “threshold”) pHi. Investigators also believed that the mitogen-induced increase in pHi that is observed in the absence of CO2/HCO�

3 exclusively refl ects stimulation of

Na�-H� exchange in the physiological pHi range. Accord-ing to this view, mitogens cause an alkaline shift and/or an increase in the steepness of the Na�-H� exchange activity versus pHi relationship, without having other effects on acid extrusion and/or acid loading. Indeed, careful analyses of the pHi dependence of Na�-H� exchanger activity in intact hepatocytes indicates that Na�-H� exchange activity can vary linearly with pHi (82, 530) and that a mitogen can shift this line in the alkaline direction (530). However, as dis-cussed previously (pHi dependence of Na�-H� exchange), it is clear that the approach often used to reach similar conclu-sions in other cells (i.e., comparison of pHi recovery rates with and without amiloride at a single low pHi) is seriously fl awed.

Applying the approach used to generate the pHi depen-dence of Na�-H� exchange activity in Fig. 11A or Fig. 11B leads to the conclusion that AVP has a complex pHi- and time-dependent effect on Na�-H� exchange in mesangial cells. At short times (�8 minutes) after apply-ing AVP, the pHi dependence of Na�-H� exchange activ-ity (similar to that shown in Fig. 14A) becomes linear in such a way that the transporter is unaffected at pHi values below 6.7, but inhibited at higher pHi values. At longer times (�14 minutes), the new linearized plot is shifted to more alkaline values. In the new “stimulated” steady state, the Na�-H� exchanger is more active at pHi values below �6.9, but less active at higher pHi values. How can AVP therefore raise pHi in the absence of CO2/HCO�

3, if the Na�-H� exchanger is inhibited in the physiological pHi range? The answer seems to be that AVP has an even greater inhibitory effect on background acid-loading pro-cesses. Thus, AVP causes pHi to rise in mesangial cells in the absence of CO2/HCO�

3 not because the growth factor stimulates Na�-H� exchange, but because it inhibits Na�-H� exchange to a lesser extent than it inhibits back-ground acid loading.

Oncogenes

Hagag et al. (238) found that microinjecting the v-H-ras p21 gene product into mouse NIH 3T3 cells causes a rapid and sustained pHi increase that is inhibited by amiloride or low [Na�]o. Thus, a functional Na�-H� exchanger is re-quired for the ras-induced pHi increase, although these data do not prove that ras actually stimulates the exchanger. Dop-pler et al. (160) took a different approach, transfecting NIH 3T3 cells with the v-mos or Ha-ras oncogene under the control of the MMTV-LTR promoter. They found that expression of either oncogene, upon addition of a glucocor-ticoid, elicited an increase in pHi as well as progression into the S phase of the cell cycle. Expression of the proto-oncogene of Ha-ras (i.e., the normal cell product) had no affect on pHi and was only weakly mitogenic. The pHi in-crease associated with expression of the Ha-ras oncogene was blocked by dimethylamiloride (352), demonstrating a requirement for a functional Na�-H� exchanger. In NIH



3T3 cells transformed with the c-H-ras oncogene, Kaplan and Boron (285) found that a higher steady-state pHi —compared to that of the nontransformed cells—was due to an alkali shift of �0.7 pH unit in the pHi dependencies of both the Na�-H� exchanger and the Na�-driven Cl�-HCO�

3 exchanger.

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