Journal of Physiology (1997), 499.3, pp.763-771

Is resting state HCO3- secretion in frog gastric fundus mucosamediated by apical Cl--HCOO- exchange?

Rosa Caroppo *, Lucantonio Debellis *, Giovanna Valenti *, Seth Alper t,Eberhard Frdmter4 and Silvana Curci *§

*Istituto di Fisiologia Generale, Universitta di Bari, Via Amendola 165/A, 70126 Bari,Italy, t Molecular Medicine and Renal Units, Beth Israel Hospital, 330 Brookline Avenue,

Boston, MA 02215, USA and tZentrum der Physiologie, J W Goethe Universitdt,Theodor Stern-Kai 7, D-60590 Frankfurt/Main, Germany

1. We have tested the widely accepted hypothesis that resting-state bicarbonate secretion ofgastric fundus mucosa is mediated by Cl--HCO3- exchange in the apical membrane ofsurface epithelial cells (SECs). To this end, SECs of isolated fundus mucosa of Rana esculentawere punctured with double-barrelled microelectrodes to measure intracellular pH (pH1).

2. No significant pH1 changes were observed in response to changing luminal HC03- and/or Cl-concentrations. The change in pHi (ApHi) in response to luminal chloride substitutionaveraged 0 00 + 0T01 pH units (mean + S.E.M.; n = 48), and did not change after blockingputative basolateral acid/base transporters which could have masked the pHi response.

3. On the other hand, pHi responded readily and reversibly to luminal perfusion with eitherlow-pH (pH 2-5) solution (ApHi= -0-36 + 0'05; n =4; P < 0-01) or C02-free HCO3-Ringer solution (ApHi = +0-10 + 0-01; n = 29; P < 0001). These observations demonstratethat the solution change was effective and complete within 1 min and show that the apicalmembrane of SECs is permeable to C02.

4. The apical membrane of frog SECs could not be stained with an antibody against theC-terminal end of the mouse Cl--HCO3- exchanger isoform AE2, although this antibodyreadily stained the basolateral membrane of the oxyntopeptic cells (OCs).

5. In conclusion, the presence of a Cl--HCO3- exchanger in the apical membrane of SECs offrog gastric fundus mucosa in the resting state could not be confirmed, but other models ofHCOO- secretion cannot be fully excluded. Observations from electrical measurements,favouring a model of conductive HCOO- secretion, point to the OCs rather than the SECs as asite of origin of HCO3- secretion.

It is well known that the gastric fundus mucosa, in additionto secreting hydrochloric acid (HC1), is also able to secretebicarbonate (Flemstrbm, 1977; Garner & Flemstrbm, 1978).This alkaline secretion, together with the layer of mucusthat covers the epithelial surface, is thought to provide adefence mechanism against autodigestion. However, despiteits clinical importance and despite numerous in vivo and invitro studies on amphibian and mammalian gastric mucosae(Allen, Flemstrbm, Garner & Kivilaakso, 1993), it is stillunder debate which cells secrete HCO3- and how they do so.

Since the oxyntopeptic cells (OCs) in the gastric glands secreteHC1, it has been generally assumed that HCO3- secretionoriginates from the surface epithelial cells (SECs). Thisassumption seems to be supported by (1) the morphologicalsimilarity of fundus and antrum surface cells which also

secrete HCO3- (Flemstrom & Sachs, 1975; Flemstrbm, 1977),(2) the high content of carbonic anhydrase in SECs (O'Brien,Rosen, Trencis-Buck & Silen, 1977) and (3) their ability togenerate a more alkaline pH at the surface than in thelumen (micro-climate) (Allen & Garner, 1980; Takeuchi,Magee, Critchlow, Matthews & Silen, 1983). However, recentexperiments from our laboratory have indicated that, inRana esculenta, the OCs contribute to gastric HCO3-secretion, at least under carbachol stimulation (Debellis,Iacovelli, Frdmter & Curci, 1994).

Regarding the cellular mechanism of HCO3- secretion inthe SECs of frog fundus mucosa, two different models havebeen proposed. Both are based on the operation of aCl--HCO3- exchanger, which is thought to reside in eitherthe apical (Flemstrbm, 1980) or the basolateral cell membrane

§ To whom correspondence should be addressed.

6028 763

R. Caroppo, L. Debellis, C. Valenti, S. Alper, E. Frimter and S. Curci

(Takeuchi, Merhav & Silen, 1982) of the SECs. Since theoperation of this exchanger has never been directlydemonstrated, we have now searched for its existence in theSECs of frog resting gastric fundus mucosa using electro-physiological, electrochemical and immunohistologicaltechniques.

METHODSThe experiments were performed on gastric fundus mucosa of Ranaesculenta in accordance with Italian Government guidelines foranimal experiments. The frogs were kept in an aquarium at roomtemperature and fed with earthworms until 3-7 days prior to theexperiment. The animals were killed by decapitation, followed bydestruction of the spinal cord and brain. The stomach was isolatedand the muscle layer and connective tissue removed by bluntdissection. The mucosa was then mounted horizontally betweentwo halves of a Lucite chamber (aperture, 0164 cm2) with themucosal side facing up. Both, the serosal and mucosal surfaces ofthe tissue were continuously superfused with oxygenated Ringersolution at room temperature (20-24 °C). Fluid exchange in themucosal compartment was achieved within seconds from a shock-free, remote-controlled, eight-way manifold.

The transepithelial potential difference (Vt) was measured with ahigh-impedance differential electrometer using two flowing-boundary calomel half-cells filled with 2-7 M KCl solution andconnected to each bath solution downstream of the tissue. Themucosal bath was connected to earth. Cell membrane potentials(Vm) and intracellular pH (pHi) were measured with double-barrelled microelectrodes (see below) using a model FD 223 dual-channel electrometer (WPI). All measurements were recorded on astrip-chart recorder (Kipp & Zonen, Delft, Holland).

The control Ringer solution had the following composition (mM):102-4 Nat, 410 K+t 1P8 Ca2+ 0-8 Mg2+, 91-4 Cl-, 17-8 HCO3-b 0-8S042-¾ 0-8 H2PO3- and 11 glucose. It was gassed with 5% C02 in 02and had a pH of 7-36. In experiments in which Cl- and/or HCO3-was reduced or removed from the bathing solutions, these ions werereplaced by equimolar amounts of gluconate. All chemicals were ofreagent grade and purchased from Farmitalia Carlo Erba (Milano,Italy), Sigma or Fluka.

Tissues were kept in the resting state by adding 0- 1 mm cimetidine(Sigma) to the serosal solution.

MicroelectrodesDouble-barrelled pH microelectrodes were constructed as describedby Kondo, Igarashi, Abe & Tada (1993). Briefly, two sections offilament-containing aluminum silicate glass tubing of differentdiameters (1-5 mm o.d. and 1-0 mm i.d. and 1-1mm o.d. and0-75 mm i.d.; obtained from Hilgenberg, Malsfeld, Germany) weretwisted together and then untwisted before they were pulled (tiplength ; 20 mm) in a Narishige PE2 vertical puller. Then, the backof the thin channel was closed and the thick channel was silanizedfor 180 s in dimethyl-dichloro-silane vapour (Serva, Heidelberg,Germany). After baking for 3 h at 140 °C, the thick channel wasbackfilled with a small amount of the H+ ligand cocktail containingtridodecylamine (Hydrogen Ionophore II, Cocktail A; Fluka) andits shaft was later filled with a buffer solution of pH 7-0. Thereference channel contained 0-5 M KCl. The slope of the electrodes(mean + S.E.M., n = 48) was 54-4 + 0-2 mV (pH unit)-' and theresistance was 279 + 46 GQ2 for the selective channel and 179 +

in place before and after the impalement by flushing the chamberwith NaCl solutions containing a mixture of KH2P04 and Na2HPO4to yield pH values between 6-8 and 7-8.

All measurements are quantified as mean values + S.E.M. of nindividual recordings on m tissues from which micropuncture datawere analysed. The significance of the observations was evaluatedby Student's t test for paired data.

ImmunocytochemistryFrogs were killed as described above and the stomach removed,sliced and fixed overnight at 4°C, by immersion in a fixativecontaining 2% paraformaldehyde, 10 mm sodium periodate, and75 mm lysine (PLP). The mucosae were then washed (3 times) withphosphate-buffered saline (PBS; 0-9% NaCl in 10 mm phosphatebuffer, pH 7-4). Tissue blocks were infiltrated with 30% sucrose

overnight, mounted in embedding medium (Miles, Elkhart, IN,USA), frozen in liquid nitrogen, and sectioned at 4 am thickness ona Reichert Frigocut cryostat. Sections were placed on Superfrost/Plus Microscope Slides (Fisher Scientific, Pittsburg, PA, USA), keptin PBS for O min, pre-incubated for 15 min with 1% bovineserum albumin in PBS, and then incubated at room temperature for2 h with anti-mouse AE2 affinity-purified antibodies, diluted asindicated in the figure legends. Sections were washed twice for5 min in PBS containing 2-7% NaCl (high-salt PBS), and twice innormal PBS. The sections were then incubated for 60 min withfluorescein-conjugated goat anti-rabbit IgG (10 ug mlF in PBS;Calbiochem) followed by washing twice for 5 min in high-salt PBS,and twice in normal PBS. Sections were mounted in 50% glycerolin 0-2 M Tris-HCl (pH 8-0) containing 2-5% n-propyl gallate toretard quenching of the fluorescence. Sections were examined andphotographed with a Nikon FXA photomicroscope equipped forepifluorescence and photographed using Kodak TMAX 400 filmpush-processed to 1600 ASA.

RESULTSEffect of changes in luminal H0O3- concentration onelectrical parameters and pHiA basic experiment to test for the presence of a HCOJ-transport mechanism (conductance or coupled transporter)is to measure the effect of changes in luminal HC03-concentration on the apical cell membrane potentialdifference (Vm) and on intracellular pH (pHi). As shown inFig. 1A, intermittently decreasing luminal HCO3concentration at constant Pco2 (36 mmHg) by a factor of 10(from 18 to 2 mM) had virtually no effect. In ten experimentson seven mucosae in the resting state (0-1 mm cimetidine),pHi increased by +0-03 + 0-01 pH units, which was notsignificant (n.s.), while Vt slightly increased from-17-2 + 2-1 (luminal surface negative) to -17-9 + 2-3 mV(A Vt = -0-7 + 0-3 mV; P= 0-05) and Vm slightly decreasedfrom -31-7 + 3-1 to -30-6 + 3-3 mV (AVm = +1 1 +0-4 mV; P < 0-02). These changes do not readily indicate thepresence or absence of a specific HCO3- transport mechanism.

To test whether the high luminal Cl- concentration interferedwith the operation of a ClF-HCO3 exchanger, as was

previously noticed in studies on renal proximal tubule (Kondo& Frdmter, 1990), we repeated the experiment at a reduced

22 M.Q for the reference channel. All microelectrodes were calibrated

764 J: Physiol.499.3

luminal Cl- concentration (from 91-8 to 9 mm) (Fig. IB).

Gastric HCO3- secretion and surface cells

However, ApH, in response to a change in HC03-concentration was still only of borderline significance(ApH1 = -003 + 0-01 pH units, n = 8, m= 4; P= 005)while the changes in Vt and Vm were highly significant. Vtincreased from -46-7 + 2-3 to -49-1 + 2-4 mV(A Vt = -2-4 + 03 mV; P < 0O001) and Vm decreased from-7-7 + 2-0 to -5-1 + 2-2 mV (A Vm = +2-6 + 05 mV;P <0O001). These potential changes would be compatiblewith the presence of an apical HC03- conductance pathway.

Effect of luminal Cl- substitution on electricalparameters and pHiAn alternative approach to test for the presence of aCl--HC03- exchanger is to replace luminal Cl- and to seewhether the cells take up HC03- and alkalinize. Thisexperiment was performed in a total of forty-eightmeasurements on thirty tissues, always in the resting state.However, as shown in Fig. 2, upon substitution of luminalCl- by gluconate no significant alkalinization developed(ApHi = 0 00 + 0-01 pH units, n.s.) while Vt simultaneouslyincreased from -21-4 + 1'1 to -401 + 1P6 mV (P < 0O001)and Vm decreased from -29-5 + 1P3 to -12-3 + 1P7 mV(P < 0O001), indicating that the solution change must havereached the mucosal surface within the exposure time of

A

Vt 2 minL0 mV

-20

-40

3 min. Figure 3 shows the frequency distribution of allindividual ApHi values, referred to their mean pre- andpost-experimental pHi values. Inspite of the large scatter itcan be seen that the distribution is bell-shaped with thepeak exactly at ApHi = 0.00 pH units. Before acceptingthis as evidence against an apical C1--HCO3- exchanger wetried to validate the measurements as follows. Onepossibility was that effective pHi regulatory mechanisms inthe basolateral cell membrane (a Na+-H+ exchanger and/ora Cl--HC3- exchanger, or a Na+-(HCO3-)n cotransporter)might have blunted the pH, response of an apicalCl--HC03- exchanger.

To test for this possibility we repeated some experiments inthe presence of serosal dimethyl-amiloride (DMA, 10-5 M)and 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid(DIDS, 2 x 10-4 M), which are known to block the above-mentioned acid/base transporters. However, the pHiresponse to luminal Cl- removal was not affected. ApHi was0-01 + 002 in control and 0-01 + 0-02 in the presence ofserosal DMA and DIDS (n = 7, m = 5). This argues againstthe possibility that pHi buffering mechanisms masked thepHi response to luminal substitution of C1-. In view of theseasonal dependence of gastric mucosal HC03- secretion

B

--20 mV

F-40L 60

D E~~~~~~~~~~~1

Vm -20 mV

llEEEE - 40pH- -60

2mMHCO3

Vm

pH1

7-4j

Irn L0 mV-20

IiuI^III - -40iyvymrI"T, ---

12mM HCO3|

l 9 mmCl- Il

Figure 1. Effect of luminal HCO3- reduction on transepithelial potential (Vt), mucosal membranepotential (Vm) and intracellular pH (pHj) of surface epithelial cells (SECs)Luminal HC03- was reduced from 18 to 2 mm during the periods indicated by the boxes. A, in normalRinger solution (Cl- concentration 91-8 mM); B, in low-Cl Ringer solution (9 mm Cl-) for the periodindicated by the box. Superimposed voltage pulses indicate response to transepithelial current pulses of50 /zA cm-2 and 1 s duration.

7-8 jI

Ifill imiiuil II 1111illilliliIIIIIIIIIIIIII07-A 11117 lifffl -v ---- Il I...1-

J Physiol. 499.3 765

2 minVt

9 I 6

R. Caroppo, L. Debellis, C. Valenti, S. Alper, E. Fr6mter and S. Curci

v,

2 m2 min

--10 mV

- -30

L-50

-_70

pHi

| Cl-free

(Curci, Debellis, Caroppo & Fromter, 199compared experiments performed in springwith those performed in winter, but no differesults was observed. In addition, the followiiwere performed to exclude specific technical pro

Effect of luminal C02 removal on pHiTo verify that our microelectrodes did faithfull;and were indeed able to detect pHi changes durilin some of the above experiments we intermitteluminal PCO2 by equilibrating the apical H(solution with pure 02. This should alkaliniprovided the cell membrane was C02 pei

12

10

8

n 6

2

0

Figure 2. Effect of luminal Cl- removal in SECs--10 mV Details as in Fig. 1.

--30

- -50- 7.7

- 7.5

- 7.3

4), we also depicted in Fig. 4, following C02 removal pH, always roseand summer markedly. The mean ApH, was +0O10 + 0O01 pH units,rence in the (n = 29, m = 20; P < 0O001).ng two tests Effect of strong luminal acidification on pHi and onblems. electrical parameters

Although the above-reported Vt (and Vm) response toy record pH, luminal substitution of Cl- indicates that the solutionng punctures, changes were effective and eventually must have reached,ntly changed even the OCs (see Discussion), access to the apical membrane503- Ringer of the SECs could have been unduly retarded or evenze the cells, prevented by a protective mucus layer covering these cells.rmeable. As Mucolytic pretreatment, i.e. superfusion of the luminal

Figure 3. Frequency distribution of the SEC pH1response to luminal Cl- removal (ApHi)Data from 48 different cells and 30 mucosae, n number ofobservations.

/I I

-0-195 -0-135 -0-075 -0-015 0-045 0-105 0-165ApHi

766 J Physiol.499.3

Gastric HCO3- secretion and surface cells

A0 mV

-20

-40

-7-6

-7-4

7-2

|C2 free

B8-0 -

7-8 -

7-6 -pH, -

7-4 -

7-2 -

7-0 - Control C02 free Control

-7-0

Figure 4. Effect of luminal C02 removal in SECsA, trace recordings of a single cell; details as in Fig. 1. B, pH, changes in 29 different cells, 20 mucosae.

surface for 30 min with Ringer solution containing 5 U ml-'papain and 5 mM L-cysteine (Flemstrom, Hiillgren,Nylander, Engstrand & Allen, 1995), did not change theresults, although a consistent reduction of the mucus layerwas observed (data not shown). We therefore tested theresponsiveness of SEC pH by changing the luminalperfusate pH without involving the readily diffusible C02.In these experiments the luminal fluid compartment wasperfused with Ringer solution titrated to pH 2f5. As shownin Fig. 5, exposure to low pH resulted in a fast andreversible cell acidification (ApHi = -036 + 0 05 pH units,n =4, m= 2 ; P < 001) which was accompanied by asignificant decrease in Vm (from -31P0 + 7 0 to -14-1 +7 0 mV; P < 0 02), but Vt was only little affected (it rosefrom -26 5 + 5-1 to -27-3 + 3-5 mV; n.s.). In addition, thevoltage divider ratio (which measures the ratio of apical to

basolateral cell membrane resistance) was not significantlyaltered; it fell from 1P7 + 0-26 to 1P4 + 0-16 (n.s.). Since thecell acidification reached its peak in less than 1 min (averageresponse time was 54 + 8-8 s), we conclude that the accessof the SECs to ion concentration changes in the luminalperfusate was not severely restricted by a mucus layer in ourexperiments.

Histochemical experimentsAs an alternative test for an apical Cl--HCO3 exchanger inthe SECs, we performed histochemical experiments with anaffinity purified rabbit antibody against the C-terminalaminoacids Nr 1224-1237 of the mouse exchanger isoformAE2.

Figures 6 and 7 show the immunolocalization of AE2 isoformin frozen sections of frog stomach. As can be seen in Fig. 6,

Vt-0 mV

Figure 5. Effect of low luminal pH (pH 2 5) in SECsDetails as in Fig. 1.

pH;

2 min. . Jr . W - ] 1|X1|§ mns s_ ^~I

--40

767J Phy8iol.499.3

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R. Caroppo, L. Debellis, G. Valenti, S. Alper, E. Frdmter and S. Curci

staining is restricted to the gastric glands cells (OCs) and isabsent from cells of the surface mucosa (SECs). Figure 7Ashows that in OCs immunostaining is localized to the baso-lateral cell membrane as expected (Stuart-Tilley, Sardet,Pouyssegur, Schwartz, Brown & Alper, 1994), while in theSECs (Fig. 7B) neither the apical nor the basolateralmembrane is stained.

This result suggests that the AE2 isoform is not expressedin the SECs, at least not in an amount sufficient to bedetected by immunofluorescence studies. However, wecannot exclude the possibility that different isoforms mightbe present in the membrane of the SECs.

DISCUSSIONThe present experiments show that the Cl--HCO3exchanger, postulated to reside in the apical membrane ofSECs and to mediate resting HCO3- secretion in fundusgastric mucosa (Flemstr6m, 1980), cannot be detected bysensitive and controlled measurements. This conclusion isbased on present observations of the absence of detectablechange in pH, of SECs in response to changes in gastricluminal concentrations of HCO3- and Cl- (this work) and onour previous observation that SEC Cl- concentration doesnot change in response to replacement of luminal Cl- bygluconate (Curci, Schettino & Fromter, 1986).

The validity of the negative results obtained with pH-sensitive microelectrodes was substantiated by measurementsof reasonably rapid pHi changes in response to independentvariation of CO2 concentration and pH in perfusatesolutions. The absence of limiting diffusion barriers acrossthe luminal surface was further demonstrated by alteration ofVt by luminal Cl- replacement, which probably reflectsaltered Vm in OCs (see below).

A point of potential concern was the relatively large scatterin the pHi measurements, which might reflect the presenceof two different acid/base transport mechanisms indifferent cells, one alkalinizing and the other acidifying inresponse to luminal Cl- substitution. This interpretation isunlikely, however, in view of the near-Gaussian distributionof the data. Instead, we assume that small sudden shifts inthe pHi records most probably reflect mechanical disturbanceduring solution change and small slow drifts reflectdifferences in CO2 equilibration of the perfusion solutions.Thus we conclude that apical Cl--HCO3- exchange is eitherabsent from resting SECs or present at an extremely lowlevel.

This conclusion is strengthened by our immunohistochemicalobservations which showed that an antibody which clearlydetected AE2 in the basolateral membrane of the OCs failedto detect immunostaining of SECs (Fig. 7). Moreover, an

Figure 6. Immunolocalization of the AE2 isoformImmunocytochemical detection of AE2 in frozen section of frog gastric mucosa by anti-1224-1237 (affinitypurified antibodies 1: 2000 dilution) showing that staining is restricted to cells of the deep gastric glands.Scale bar = 20 ,um.

768 J Physiol.499.3

Gastric HCO3- secretion and surface cells

antibody specific for the cardiac isoform of AE3 present inthe apical membrane of certain epithelial cells (Alper,Stuart-Tilley, Yannoukakos & Brown, 1995) similarly failedto produce consistent immunostaining of SECs in Ranacatesbeiana and Xenopus laevis, though vascular and muralsmooth myocytes stained brightly in the same sections(S. Alper, unpublished results). Thus, any quiescent anionexchanger that might be present in SECs was not recognizedby antibodies that cross-react with amphibian anionexchanger polypeptides.

A hint towards which transport mechanisms might beresponsible for HC03- secretion may be obtained from theelectrical measurements during luminal HC03- substitution.These measurements, which are less prone to artifacts(particularly with regard to Vt), show a small hyperpolar-ization of the tissue (increase in Vt), and a small decrease inVm, which in one series of experiments was even highly

significant. Such potential changes would be expected todevelop if a HC03- conductance was present in the apicalcell membrane and such a conductance could also mediateHC03- secretion. However, this conductance, if present,cannot be located in the apical membrane of the SECs(which also do not respond with a change in pH1) but mustbe located in the apical membrane of the OCs for thefollowing reasons.

We know from a great number of measurements that theOCs in the glands form the main conductance pathwayacross gastric mucosa, amounting to at least 90% of totaltransepithelial conductance (Kottra, Jacovelli, Caroppo,Curci, Bakos & Fromter, 1996), while the SECs constituteonly a low-conductance pathway operating in parallel withthe OC pathway. In such a parallel array of conductances,any electrical signal that originates at the apical membraneof SECs will be strongly attenuated and will be difficult to

Figure 7. Immunolocalization of the AE2 isoformImmunocytochemical localization of AE2 (detected with anti-1224-1237, affinity purified antibodies,1: 2000 dilution) in frozen sections of frog stomach at higher magnification. Scale bar = 10 jum. A, AE2expression in the basolateral membrane of the oxyntopeptic cells; B, AE2-negative surface epithelial cells.

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detect in measurements of Vt. This situation is exemplifiedin the present experiments with luminal perfusion of low-pH solution when Vm changed drastically, but Vt showedonly a very small response (Fig. 5). By contrast, anyelectrical signal originating at the apical membrane of theOCs will be strongly reflected in Vt and will also besuperimposed onto all cell potential measurements on SECsthrough the circular current loop effect.

The latter situation is exemplified in the present experimentswith luminal perfusion of Cl-free solution (Fig. 2). The largesecondary change in Vt (hyperpolarization) and the almost aslarge secondary change in Vm (depolarization) indicate thatthese potential changes (Vt and Vm) originate in the apicalmembrane of the OCs. This interpretation is supported bythe slow onset (and parallel development) of both potentialchanges which appears to reflect the delayed access of thenew solution to the surface of OCs inside the glands. Thefast initial changes of Vt and Vm in Fig. 2 indicate liquidjunction potentials that develop between the low Cl-solution suddenly flooding the mucosal surface, and thestagnant Ringer-like solution which persists initially in thegland lumen, and is only gradually being replaced bydiffusion. From these observations we conclude that theapical membrane of the OCs, but not that of the SECs,contains a significant Cl- conductance which is operativeeven in the resting state. Analogous observations withHCO3- suggest that rheogenic HC03- secretion should alsooriginate from OCs, possibly via the same anion conductance.

Summarizing the above discussion, we conclude that our datado not support a model according to which HCO3- secretion ismediated via a C1F-HCO3- exchanger in the apicalmembrane of SECs. This conclusion agrees with previousobservations (Seidler, Carter, Ito & Silen, 1989; Kraniak,Koyanagi & Fromm, 1995) on isolated non-polarized SECsof rabbit stomach. Instead, our observations would becompatible with a conductive bicarbonate permeationmechanism located in the OCs rather than in the SECs. Thelatter conclusion supports a model concept which we havepublished recently (Debellis et al. 1994).

In addition, two further points need to be considered. Thefirst concerns the observation made following luminalperfusion of a low-pH solution. In these experiments thecells acidified rapidly by up to 0-36 pH units, upon whichpH, recovered partially (in the presence of low-pH solution)in all experiments and eventually returned to its controlvalue, when perfusion with control solution was resumed.This observation, which resembles observations in Necturusantrum mucosa (Kiviluoto, Mustonen, Salo & Kivilaakso,1995), is surprising, at least with regard to the extent of thepH, change observed and the extent of the cell membranedepolarization associated with acidification. It suggests thatpHi regulatory mechanisms, which are probably located inthe basolateral membrane of the SECs, are not strongenough, at least in the isolated mounted mucosa, to keeppHi constant under the influence of luminal pH values such

as those prevailing under physiological conditions. Thereason for the collapsing cell membrane potential is notknown. However, since the voltage divider ratio remainedvirtually constant, we can exclude the development of anunspecific membrane leak. Instead we hypothesize that thelow pHi inhibited apical (and probably basolateral) K+channels in the SECs.

The second point concerns the observation of cellalkalinization during perfusion with CO2-free solutions. Intwo recent papers Boron and collaborators (Boron, Waisbren,Modlin & Geibel, 1994; Waisbren, Geibel, Modlin & Boron,1994) have reported that the apical membrane of rabbitgastric gland cells, in contrast to virtually all other cellmembranes, is practically impermeable to the lipid solublegasses CO2 and NH3. These authors have postulated thatthis impermeability was essential for the defence of gastricmucosal epithelium against acid damage (autodigestion).This point is of interest in the context of the presentexperiments, since our measurements on SECs, which facethe same problem as the OCs of defending their integrityagainst acid intrusion, show unequivocally that the apicalmembrane of these cells (at least in frog stomach) is readilypermeable to CO2.

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AcknowledgementsThe authors wish to thank Dr B. Landolfi for assistance in some ofthe experiments. This study was supported by Ministerodell'Universita e della Ricerca Scientifica e Tecnologica (ProgettoNazionale 40% 1994-95) and by Consiglio Nazionale delle Ricerche(grant no. 94.2630.04).

Author's email addressS. Curci: curci@bioserver.uniba.it

Received 12 August 1996; accepted 10 December 1996.

J Physiol.499.3 771