the of chemistry vol. 258, 1983 in mechanisms underlying calcium homeostasis in ... ·...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 258, No. 2, Issue of January 25, pp. 731-741, 1983 Pnnted in U.S. A.

Mechanisms Underlying Calcium Homeostasis in Isolated Hepatocytes*

(Received for publication, August 11, 19821

Suresh K. Joseph, Kathleen E. Coll, Ronald H. Coopert, James S. Marks, and John R. Williamson@ From the Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

”

The steady state relationship between intra- and extramitochondrial free Ca2+ across the inner mitochondrial membrane has been investigated in isolated liver mitochondria. The extramitochondrial free Ca2+ concentration was essentially independent of the mitochondrial calcium content above 4 nmol/mg of protein. Below this value, a decrease in the mitochondrial calcium content was accompanied by a decrease in the extramitochondrial free Ca2+ concentration. The experimental data are compatible with a model in which the steady state distribution of calcium is described in terms of the kinetic parameters of the separate carriers catalyzing Ca2+ influx and efflux across the mitochondrial inner membrane. The corresponding relationship between cytosolic free Caz+ concentration and the amounts of calcium in the mitochondria and endoplasmic reticulum was investigated in isolated liver cells over a range of cellular Ca2+ contents by using a non- disruptive technique based on the selective release of calcium from mitochondrial and total cellular pools by addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone and A23181, respectively. A net increase in cell calcium from 1 to 5 nmol/mg dry weight, increased the cytosolic free Ca2+ concentration from 0.1 to about 0.3 p~ and increased the calcium contents of both mitochondria and endoplasmic reticulum. Above 5 nmol of calcium/mg cell dry weight, the endoplasmic reticulum calcium pool became filled, and further increases in calcium content were accounted for by increases of the mitochondrial pool but no further increase of the cytosolic free Ca” concentration. These studies and experiments with mixtures of isolated microsomes and mitochondria suggest that, in cells as normally isolated (containing 5 to 6 nmol of calcium/ mg dry weight), the endoplasmic reticulum is saturated with calcium and is unlikely to play a major role as an intracellular calcium buffer. The in situ mitochondrial calcium content is sufficiently high (approximately 16 nmol/mg of protein) for these organelles to buffer effectively the cytosolic free Ca2+ concentration at a value of about 0.3 pM. In addition, it may be concluded that intramitochondrial Ca2+-dependent enzymes will be exposed to saturating concentrations of free Ca2+.

A wide variety of cellular processes are thought to be regulated by changes in the cytosolic free Ca2+ concentration (1-5). In liver, a-adrenergic agonists and vasoactive peptides

* This work was supported by National Institutes of Health Grants AM-15120 and HL-14461. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by a Juvenile Diabetes Career Development Award. 8 To whom correspondence and reprint requests should be ad-

dressed.

appear to cause mobilization of intracellular calcium (6-9), which, through allosteric activation of phosphorylase kinase (10, ll), leads to increased glycogenolysis. However, the mechanisms which control the cytosolic free Ca2’ concentration and the distribution of intravesicular calcium within the cell are not fully understood. Further studies on the regulation of calcium homeostasis in the hepatocyte should therefore contribute towards determining how hormones may elicit changes of intracellular calcium concentration ( 3 ) .

Normally, the free Ca2+ in the cytosol of most cells is maintained between 50 and 200 n~ (9, 12-17). Attention has been focused for some time on the role of mitochondria in the regulation of intracellular free ea2+ concentration (1, 18, 19), but most of the early studies were performed using unphysi- ologically high extramitochondrial Ca’+ concentrations so that their relevance to the situation in the intact cell is difficult t o assess. Uptake and efflux of Ca2+ in mitochondria occur by different processes. Uptake of CaZ+ takes place by an electrophoretic uniport mechanism which is energetically driven by the electrogenic proton pump of the respiratory chain. This process is characterized by a high maximum velocity and an apparent K, for Ca2+ of 2 to 5 PM (20-22), which is increased further by the presence of Mg” (23) . With the discovery of a separate ruthenium red- insensitive Ca2+ efflux process in mitochondria, the potential physiological significance of Ca2+ cycling across the mitochondrial membrane at low external free Ca2+ concentrations has become apparent (20, 22, 24). The efflux carrier is electroneutral and in liver mitochondria catalyzes the exchange of Ca2+ for 2H’ (25), with an apparent K, for matrix free Ca2’ of approximately 10 ,UM (26) and a V,, of 4 to 5 nmol/min/mg of protein (24, 26). This efflux rate is much lower than the V,,, for ea2+ uptake (1, 21). By use of calcium electrodes to monitor the extramitochondrial free Ca2‘ concentration, it has been shown that liver mitochondria as normally prepared will either take up or release calcium in response to small additions of Ca2+ or EGTA‘ without a significant change of the steady state extramitochondrial free Ca” concentration (24,27). These studies were performed with mitochondria containing a relatively high range of intrarnitochondrial calcium contents (10 to 60 nmol/ mg of protein) so that the Ca2+ efflux carrier was operating close to V,,,. Under these conditions, the mitochondria effectively buffer the extramitochondrial free Caz+ concentration, suggesting that this organelle may be primarily responsible for stabilization of the cytosolic free Ca” in the intact cell (24).

On the other hand, Denton and colleagues (28, 29) from studies on the Ca2+ sensitivity of intramitochondrial dehydro- genases suggested that the physiological function of the Ca‘+ transporting systems in the inner mitochondrial membrane is

’ The abbreviations used are: EGTA, ethylene glycol bis(P-amino- ethyl ether)-N,N,N’,N’-tetraacetic acid; FCCP, carbonyl cyanide p - trifluoromethoxyphenylhydrazone; HEPES, 4-(2-hydroxyethyl)-l-pi- perazineethanesulfonic acid.

731

732 Calcium Homeostasis in Liver

to determine the intramitochondrial Ca2’ concentration and thereby regulate a variety of Ca2+-dependent enzymes. These authors have suggested that alternative Ca2+ transport systems in the plasma membrane (30) and the endoplasmic reticulum (31,32) could play the major role in maintenance of the cytosolic free Ca2+ concentration. Evidence that the endoplasmic reticulum may contribute to regulation of cytosolic free Ca2’ was provided by Becker et al. (33) who showed that addition of liver microsomes to isolated liver mitochondria in the presence of MgATP2- decreased the free Caz+ of the incubation medium from 0.5 to 0.2 p ~ .

The present study was undertaken to investigate the relationship between steady state values of extramitochondrial free CaZ+ and the calcium content of liver mitochondria and to relate these findings to the possible role that mitochondria may have in regulating the cytosolic free Ca2+ concentration in the intact hepatocyte. A simple direct method has been developed for measuring the distribution of calcium between endoplasmic reticulum and mitochondria in intact hepatocytes. This has allowed the relationship between the cytosolic free Ca2+ concentration and the contents of calcium in the intracellular compartments to be determined over a wide range of total calcium contents of the hepatocyte. These findings together with further in vitro studies using mixtures of liver mitochondria and either hepatic microsomes or rabbit skeletal muscle sarcoplasmic reticulum vesicles, allow the conclusion to be drawn that at in uiuo levels of total hepatocyte calcium, the endoplasmic reticulum is almost fully saturated, while the mitochondrial calcium content is in the range to buffer effectively the cytosolic free Ca” at a value of about 0.3 p ~ . In addition, a model describing the relationship between intra- and extramitochondrial free Ca2+ concentrations has been developed which allows determination of the kinetic parameters of Ca2+ cycling from steady state measurements. The model also has predictive value in relation to hormonal effects on intracellular calcium homeostasis.

EXPERIMENTAL PROCEDURES’

RESULTS

The Relationship between Extramitochondrial Free Ca2+ Concentration and Mitochondrial Calcium Content-The uptake of small amounts of calcium (2 nmol/mg of protein) by isolated respiring mitochondria is illustrated in Fig. lA. The endogenous calcium content of the mitochondria used in this experiment was 12.5 nmol/mg of protein. As found by other workers (24, 27), the extramitochondrial free Ca2’ concentration maintained by the mitochondria in the steady state was relatively constant following addition of four aliquots of 2 nmol of Ca2+/mg of protein. However, when the same calcium additions were made to mitochondria which had been depleted of calcium to a level of 1.5 nmol/mg of protein, there was a progressive increase in the steady state extramitochondrial free Ca2+ concentration (Fig. l B ) , indicating that mitochondria buffered the extramitochondrial free Ca2+ concentration poorly when they were calcium-depleted.

In order to explore the relationship between intra- and extramitochondrial calcium in more detail, single additions of

Portions of this paper (including “Experimental Procedures,” Figs. 3-5, Table I, and the section entitled “Kinetic Model of Mito- chondrial Calcium Cycling”) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magni- fying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 82M-2183, cite the authors, and include a check for $5.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

calcium up to 12 nmol/mg of protein were added to mitochondria respiring in the presence of succinate and rotenone. The extramitochondrial calcium was measured when steady state conditions of Ca2+ cycling were attained, and the intramitochondrial calcium was subsequently determined by measuring the amount of calcium released after addition of the ionophore A23187. The results of a series of such experiments are shown in Fig. 2 - 4 . In the absence of added Mg2+ (control curue of Fig. ZS), the extramitochondrial free Ca” concentration was found to increase with the mitochondrial calcium content up to a value of about 4 nmol/mg of protein. Above this value, the Ca2+ concentration in the medium was relatively independent of the mitochondrial calcium content. Addition of 0.15 m~ Mg2+ markedly increased the steady state extramitochondrial free calcium at all mitochondrial calcium contents. At a value of 10 nmol of calcium/mg of protein, the extramitochondrial free Ca2+ concentration was about 0.25 ~ L M and was increased to about 0.8 p~ by the addition of Mg2’. Similar increases in the so-called “set point” for mitochondrial calcium buffering, caused by the addition of Mg“, have been observed previously (24, 27). Under conditions of low extramitochondrial free Ca2+, inhibition of Ca2+ influx by addition of Mg“ causes a displacement from the steady state because of an increase in the apparent K,,, for Ca2’ influx (50, 52). This

- 5

-z &INITIAL CALCIUM CONTENT=I25nrnol/mr3 -

FIG. 1. Effects of successive calcium additions on the extramitochondrial free CaZr concentration of isolated liver mitochondria. In A, the initial mitochondrial calcium content was 12.5 nmol/mg of protein, while in B, the initial mitochondrial calcium content was 1.5 nmol/mg of protein. The free Ca2+ concentrations were calculated from the measured absorbance changes of the Ca- arsenazo 111 complex at 675-685 nm.

0 2 4 6 8 1 0 1 2 0 2 4 6 8 10 12 CALCIUM CONTENT (nrnoVrng protein)

FIG. 2. Relationships between the extramitochondrial free CaZ+ concentration and the calcium content of liver mitochondria. In A, mitochondria (0.5 mg of protein/ml) were incubated in the absence of Mg2’ for control conditions or in the presence of 0.15 mM Mg2+ with and without 1 mM phosphate. In B , mitochondria (0.5 mg of protein/ml) were incubated in the absence of Mg2’ but with either 20 or 26 pmol of ruthenium red/mg of protein. The mitochondrial calcium content was measured by addition of 2 nmol of A23187/ mg of protein.

Calcium Homeostasis in Liver 733

results in a net efflux of Ca2+ from the mitochondria and an increased Ca2+ concentration in the incubation medium. A new steady state of Ca2+ cycling is attained but at a higher extramitochondrial free Ca2+ concentration.

Phosphate has also been shown to increase the steady state extramitochondrial free Ca2+ concentration (steady state [Ca2']0) maintained by isolated mitochondria in the presence of Mg2+ (53). In our hands, the relationship between intra- and extramitochondrial Ca2+ in the presence of Mg2' was not altered by the addition of 1 mM phosphate (Fig. 2A ). However, further experiments showed that the steady state [Ca2+]o at a mitochondrial calcium load of 7.4 nmol/mg of protein and a constant external free Mg2+ concentration of 0.15 m~ was decreased from 0.70 to 0.49 p~ by addition of up to 1.6 mM MgATPZ- (0.57 m~ free ATP4-), in general agreement with the observations of Becker (27). At present it is not known how MgATP2- (or ATP4-) interferes with the Mg2+ effect.

Fig. 2B shows the effect of two suboptimal concentrations of ruthenium red, a noncompetitive inhibitor of calcium influx (46, 48), on the relationship between intra- and extramitochondrial calcium. Ruthenium red at concentrations of 20 and 26 pmol/mg of protein inhibited the initial rate of Ca2+ uptake by 83 and 94%, respectively, and caused related increases of the steady state [Ca2+]o at each level of mitochondrial calcium content.

The above results show that an increase of the apparent K, for Caz+ influx by M$* or a decrease of the Vmax increase the steady state [Ca".'], for a given mitochondrial calcium content. Consequently, it may be expected that the relationship between the intra- and extramitochondrial Ca2' concentrations under conditions of Ca2+ cycling can be defined by the kinetic parameters for Ca2+ influx and efflux on the separate transport systems. This has been verified by development of a kinetic model of mitochondrial Ca" cycling consistent with the experimental data, which is described in Miniprint (see Figs. 3- 5 and Table I). The lines drawn in Fig. 2 are theoretical curves generated by the model. For the control Mg"-free condition in Fig. 2 A , the following values for the kinetic parameters were used: 10 ~ L M free Ca2' for the K', for Ca2+ efflux together with the value of 7 X for the ratio of free/total Ca2+ in the mitochondrial matrix (26), 1.6 p~ for the K', for Ca2+ influx and 0.056 for the ratio of V', efflux/V',, influx (see Table I in Miniprint). The curve drawn through the points for the plus Mg2' condition was generated by increasing the K'm for Ca" influx to 4.5 pM, leaving the other kinetic parameters the same. As seen from Fig. 2, the fit of the theoretical curves to the data points is quite adequate.

The Role ofkfitochondria in the Maintenance of Cytosolic Free Ca2+ in Hepatocytes-The data in Fig. 2 suggest that isolated mitochondria can maintain a relatively constant Ca2+ concentration in the incubation medium provided that their Ca2+ content is more than 4 nmol/mg of protein. Any assess- ment of the ability of mitochondria to act as Ca2+ buffers in the intact cell requires information on the relationship between the mitochondrial Ca2+ content in situ and the cytosolic free Ca2+ concentration. In order to determine this relationship, hepatocytes as normally isolated were either partially depleted of calcium or were allowed to accumulate different amounts of calcium by a period of cold incubation in the presence of 10 m~ extracellular Ca2+ (38). At each level of calcium loading, the cytosolic free Ca2+ concentration was determined by the digitonin null point titration technique (9), while the total hepatocyte calcium content was measured by atomic absorption spectroscopy. The relationship between total cellular Ca'* content and cytosolic free Ca'+ is shown in Fig. 6A. Over the range of cellular Ca2+ contents from 1 to 16 nmol/mg cell dry weight, the cytosolic free ea2+ concentration

I

04 . . . . . , , I 0 4 8 12 16

TOTAL CELL CALCIUM lnmol/rng dry w t 1

FIG. 6. Variation of the cytosolic free Ca2+ concentration ( A ) and the phosphorylase activity (B) with the total cell calcium content of isolated hepatocytes. Rat liver cells (2 to 3 mg dry weight/ml) were incubated in modified Hanks' medium at 30 "C. The cytosolic free CaZ+ concentration was measured by the digitonin null point titration technique in Ca2+-!?ee and Mg"-free medium. The total cell calcium content was measured by atomic absorption spectroscopy. The total phosphorylase activity was 84 * 1.4 nmol/min/ mg cell dry weight.

increased from 0.1 to 0.3 p ~ , with a plateau value being reached at 4 to 5 nmol of calcium/mg cell dry weight. Fig. 6B shows the comparable relationship between the total cell calcium and the phosphorylase activity of isolated hepatocytes. Phosphorylase activity increased with cell calcium content up to about 5 nmol/mg dry weight, and thereafter re- mained constant. From Fig. 6B, a half-maximum increase of phosphorylase activity was obtained at a total cell calcium content of 0.7 nmol/mg dry weight, which from Fig. 6A corresponds to a cytosolic free Ca2+ concentration of 90 nM. This value presumably reflects the K, for binding of Ca2+ to the calmodulin component of liver phosphorylase kinase on the assumption that activation of phosphorylase kinase in this range is proportional to increased phosphorylase activity, The results of Fig. 6 show that the cytosolic free Ca2+ concentration is effectively buffered within a narrow range, which is independent of the total cellular calcium above 4 to 5 nmol/mg of cell dry weight.

The distribution of calcium between the major intracellular calcium pools contained in the mitochondria and endoplasmic reticulum of the hepatocyte was measured by a direct rapid method which does not require cell disruption. The principle of the method depends on measurement of the release of calcium selectively from the different intracellular calcium pools by addition of suitable Ca2*-releasing agents to the cell suspension. The mitochondrial uncoupling agent FCCP was used to release calcium from the mitochondria without affect- ing calcium release from the endoplasmic reticulum pool, as indicated by its lack of effect on isolated microsomes.3 On the other hand, the ionophore A23187 induces calcium release from all intravesicular calcium pools (54-56). Hence, the difference between A23187-releasable calcium and the uncou- pler-releasable calcium is considered to represent the content of the endoplasmic reticulum calcium pool. For the method to be valid, the cells must be of high quality and should be in a steady state with respect to cell calcium content during incubation with the arsenazo 111 buffer prior to addition of FCCP or A23187. Calcium uptake into mitochondria of damaged cells must be prevented, e.g. by preincubation of the cells with

J. S. Marks and J. R. Williamson, unpublished observations.


2 ,UM ruthenium red. Control experiments showed that the plasma membrane did not cause retention of released intracellular calcium, since addition of digitonin after either FCCP or A23187 gave no further increase in absorbance of the Ca- arsenazo I11 complex. Further control experiments showed that sequential additions of FCCP and A23187 caused the same total amount of calcium release from the intact hepatocytes as that obtained with A23187 alone.

Fig. 7A shows that the content of the endoplasmic reticulum calcium pool (A23187 minus FCCP-releasable calcium) increased with an increase of total cell calcium over the range from 1 to 5 nmol/mg dry weight, reaching a maximum calcium loading of 0.85 nmol/mg dry weight, with hepatocyte calcium contents above 5 nmol/mg dry weight. Separate experiments showed that the hepatocytes contained 90 mg of microsomal protein/g dry weight. Hence, from the data in Fig. 7A, it may be calculated that the maximum calcium loading capacity of endoplasmic reticulum in the hepatocyte is about 10 nmol/ mg of microsomal protein. This corresponds roughly to values reported in the literature for the maximum capacity of isolated hepatic microsomes to accumulate calcium (32,33,57), as well as our own data. Our failure to observe saturation of the so- called microsomal calcium pool with increasing hepatocyte calcium loads in previous experiments using rapid cell disruption techniques (9) suggests that there was a large unrecog- nized calcium contamination of the microsomal fraction from external sources.

Fig. 7B shows that the mitochondrial calcium pool (FCCP- releasable calcium) increased approximately linearly with an increase of total cell calcium, with no evidence of saturation. At a cell calcium content of 6 nmol/mg dry weight, which is similar to that obtained with normal freshly isolated hepato-

z

n 2 0

t 2 4 6 8 IO 12 14

TOTAL CELL CALCIUM hmol/rngdry wt)

TOTAL CELL CALCIUM(nmol/rngdrywt) FIG. 7. Distribution of intracellular calcium with an increase

of total cell calcium in isolated hepatocytes. Rat liver cells (2 to 3 mg dry weight/ml) were incubated in Ca2+-free and Mg*+-free Hanks' medium at 30 "C. A shows the estimated calcium content of the endoplasmic reticulum as measured by the difference between the amount of calcium released from intact hepatocytes by A23187 and that released by FCCP. B shows the estimated calcium content of the mitochondria as measured by the amount of calcium released from intact hepatocytes by FCCP.

cytes, mitochondria contained about 67% of the total cellular calcium (4.0 nmol/mg cell dry weight, equivalent to 16 nmol/ mg of mitochondrial protein), in agreement with results using the rapid cell disruption technique (9). At this calcium content, the mitochondria should be effective in buffering the cytosolic free Ca2+ concentration at a constant value (cf. Fig. 2).

In Fig. 8, the cytosolic free Ca2+ concentration has been related to the FCCP-releasable (mitochondrial) calcium pool of the hepatocytes. The relationship obtained is similar to that reported in Fig. 2 with isolated mitochondria and shows a relative independence of the cytosolic free Ca2+ concentration on the in situ mitochondrial calcium content a t values above about 6 nmol of calcium/mg of mitochondrial protein.

The amount of A23187-releasable calcium was consistently less than the total calcium content of the hepatocytes as measured by atomic absorption spectroscopy. This residual calcium was constant over the range of total cell calcium contents from 2 to 16 nmol/mg dry weight, and amounted to 0.89 * 0.02 nmol/mg dry weight (mean & S.E. of 23 observations). It was not further reduced by addition of 2 ,UM ruthenium red, which has been shown to displace membrane-bound calcium (58). Part of this residual calcium is probably contained in the mitochondria because of the high concentration of mitochondrial calcium binding sites. Assuming that the final free Ca" concentration of the mitochondrial matrix is 0.1 ,UM after addition of FCCP or A23187, this will correspond to a mitochondrial calcium content of 0.4 nmol/mg of cell dry weight, on the basis of a total/free ratio of mitochondrial calcium of 1 nmol/mg of PrOtein-pM (26) and a conversion factor of 0.25 mg of mitochondrial protein/mg, cell dry weight. The remainder (0.5 nmol/mg dry weight) is probably nonves- icular and could represent calcium bound in the cytosol (e.g. to intracellular proteins and inward facing phospholipid head groups of the plasma membrane). If so, then for a cytosolic free Ca2+ concentration of 0.3 p~ and a cytosolic water volume of 2.2 ml/g of cell dry weight (59), an upper limit for the ratio of free to bound calcium in the cytosol would be 1.3 X

The Role of Intracellular Non-mitochondrial ea2+ Trans- port Systems in Calcium Homeostasis-Since the data in Figs. 6 and 7 showed that the calcium content of the endoplasmic reticulum in the intact hepatocyte appeared to be closely related to the cytosolic free Ca2+ concentration in the range of total cell calcium contents below 5 nmol/mg dry weight, it was of interest to investigate the calcium handling properties of this system in more detail. Fig. 9 shows the results of a series of experiments designed to measure Ca2+ uptake by endoplasmic reticulum in situ. This was accom-

MITOCHONDRIAL CALCIUM (nrnol/rng protem)

0 8 16 24 32 40 48 56 - 5 1-1

FCCP RELEASABLE CALCIUM ( n r n o l h g dry wt )

FIG. 8. Relationship between the cytosolic free Ca2+ concentration and the in situ mitochondrial calcium content in intact hepatocytes. The data are taken from the results presented in Figs. 6A and 7B. The conversion of mitochondrial calcium content from units of nanomoles/mg cell dry weight to nanomoles/mg of mitochondrial protein is based on an estimated hepatocyte mitochondrial content of 250 mg of protein/g of cell dry weight.


w O Y 0 I 2 3 4 5 6

FREE COz* (pMI

FIG. 9. Calcium content of hepatocyte endoplasmic reticulum in situ as a function of steady state external free Ca" concentration. Calcium-depleted rat liver cells (6 to 8 mg dry weight/ml) were incubated in Ca2+-free modified Hanks' medium containing 50 p~ arsenazo 111, 2 PM ruthenium red, 200 p~ free Mg2+, 50 p~ MgATP2-, 2 mM creatine phosphate, and 100 pg/ml of creatine phosphokinase. Digitonin (20 pg/ml) was then added, followed by different amounts of calcium up to 20 nmol/ml. The final free Ca2+ concentration of the medium was measured after a steady state had been reached. The content of calcium in the endoplasmic reticulum was measured as A23187-releasable calcium, from which the small amount of initial FCCP-releasable calcium in the mitochondrial pool (0.2 to 0.3 nmol/mg of cell dry weight) was subtracted. The difference between the A23187-releasable and FCCP-releasable calcium of the intact hepatocytes used for these experiments showed that the initial calcium content of the endoplasmic reticulum was below 0.1 m o l / mg of cell dry weight.

plished by measuring Ca2+ uptake from the medium after addition of digitonin to hepatocytes incubated in the presence of ruthenium red to inhibit mitochondrial Ca2+ uptake and a creatine phosphokinase ATP-regenerating system. The extent of Ca2+ uptake by the endoplasmic reticulum depended on the final external free Ca2+ concentration up to 2 p~ and thereafter became saturated at a level of 1.1 nmol/mg of cell dry weight (Fig. 9). This value is similar to that obtained in the experiments shown in Fig. 7. Further experiments provided no evidence that the endoplasmic reticulum buffered the external free Ca2+ concentration. Thus, after addition of small amounts of EGTA to digitonized hepatocytes incubated as in Fig. 9 (sufficient to decrease the free Ca" to about 0.2 p ~ ) , CaL+ efflux could not be observed from the endoplasmic reticulum calcium pool even when it had become saturated with calcium. Control experiments showed that digitonin at the same concentration used in these experiments had no effect on Ca2+ uptake by isolated hepatic microsomes.

A question of fundamental importance for an understanding of intracellular calcium homeostasis in hepatocytes concerns the relationship between the cytosolic free Ca2+ concentration and the calcium sequestering activities of the endoplasmic reticulum and mitochondria. From the data in Figs. 6 and 7 with intact hepatocytes, it may be calculated that the endoplasmic reticulum calcium pool is half-filled a t a cytosolic free Ca2+ concentration of 0.17 p ~ . In contrast, the data in Fig. 9 show that when Ca2+ uptake into mitochondria is inhibited, the endoplasmic reticulum calcium pool of digitonin-treated hepatocytes is half-filed when the external free Ca" concentration is 0.8 p ~ . These results, in contrast to those of Becker et al. (33), suggest that the endoplasmic reticulum may have only a minor role in buffering the steady state cytosolic free Ca2+ concentration. Because of outstanding ambiguities between our data and those reported by Becker et al. (33), the problem has been reinvestigated by studying the calcium interactions between isolated rat liver mitochondria and either hepatic microsomes or rabbit skeletal muscle sarcoplasmic reticulum.

Fig. 10A illustrates the response of the mitochondria containing initially 8 nmol of calcium/mg of protein to small

pulses of either Ca2+ or EGTA. Under these conditions, the steady state extramitochondrial free Ca2+ concentration was effectively buffered at about 0.8 p~ after either Ca2+ or EGTA additions, as would be expected at this high mitochondrial calcium content (cf. Fig. 2 A ) . Fig. 10B shows that addition of 90 pg of rabbit muscle sarcoplasmic reticulum to the liver mitochondria incubated as above caused a rapid fall of the extramitochondrial free Ca2+ concentration from 0.75 to 0.5 PM followed by a slower rise back to the initial value. Control experiments showed that this effect was dependent on the presence of MgATP2- and was abolished by pretreatment of the sarcoplasmic reticulum vesicles with fluorescein 5"isothi- ocyanate, an inhibitor of the Ca2+-ATPase (60). Addition of sarcoplasmic reticulum thus had a similar effect on mitochondrial calcium cycling and the extramitochondrial free Ca8+ concentration as addition of EGTA. A further increase of the ratio of sarcoplasmic reticulum protein to mitochondrial protein caused the external free Ca2+ concentration to fall to lower values, which failed to return to the initial steady state level maintained by the mitochondria prior to addition of sarcoplasmic reticulum (Fig. lOC, together with data not shown). These results may be interpreted as indicating that uptake of Ca2+ by the sarcoplasmic reticulum causes a fall of the medium free Ca2+ concentration, thereby diminishing the rate of Ca2+ influx into mitochondria relative to efflux with consequent loss of calcium from the mitochondria to the medium. The vesicles will be expected to remove calcium from the medium until they become saturated or the mitochondria become depleted of calcium, at which point the free Ca'+ of the medium will reach a new steady state rate of Ca" cycling in accordance with the type of relationships shown in Fig. 2 A .

From the above interpretation of the data, it may be predicted that when calcium sequestering vesicles and mitochondria are mixed at a fixed ratio in the presence of MgATP2-, the difference between the initial and final steady state free Ca2+ concentration of the external medium will depend on the initial calcium content of the mitochondria. Fig. 11 shows the results of such an experiment in which mitochondria contain-

(c

FIG. 10. Effect of addition of rabbit muscle sarcoplasmic reticulum vesicles to rat liver mitochondria on the free Ca2+ concentration of the incubation medium. In A, B, and C, liver mitochondria (1.76 mg of protein) containing initially 8 nmol of calcium/mg of protein were incubated in 2.5 ml of Ca"-depleted medium containing 50 pM arsenazo 111, 0.2 mM Mg'+, 0.05 mM MgATP2-, 2 m~ creatine phosphate, and 100 pg/ml of creatine phosphokinase. Further additions were made at the arrows as shown in the figure.


I Mg A T P 2 - 0 8 I

MICROSOMES ALONE / A

MITOCHONDRIA [ I 74nmo1 Coz+/mq~

N 04 I + MICROSOMES 8

0 2

LL

0 "'I \ E

MITOCHONORIA I 4 OOnrnol Co2'/rnpI / + MICROSOMES

0 8 + MICROSOMES D

0 4

0 7-

5 10 15 Mlnutes

FIG. 11. Effect of addition of hepatic microsomes to liver mitochondria on the free Ca" concentration of incubation medium. Rat liver mitochondria (1.75 mg of protein/ml) containing different initial contents of calcium as noted in the figure were incubated at 30 "C in Ca2'-depleted medium containing 50 p~ arsenazo 111, 0.25 mM Mg'+, 2 mM creatine phosphate, and 100 pg/ml of creatine phosphokinase. Hepatic microsomes (0.9 mg of protein/ml) were preincubated with the mitochondria in traces B through I).

0.1 m~ MgATP2-. Microsomal calcium uptake was initiated a t zero time by addition of

ing calcium contents from 1.74 to 6.25 nmol/mg of protein and hepatic microsomes were mixed in a protein ratio of 2:l. Trace A shows that addition of MgATP2- to microsomes alone (0.9 mg of protein/ml) decreased the free Ca2+ concentration in the medium to about 0.4 ,UM. This steady state value could be decreased further by addition of more microsomal protein or by small aliquots of EGTA, indicating the absence of an active Ca2+ efflux system. Traces B, C, and D in Fig. 11 show that upon addition of MgATP2- to activate the microsomal Ca2+- ATPase, the free Ca2+ concentration of the medium decreased over a period of 2 to 3 min to the same value of about 0.4 PM. However, the rate and extent of the subsequent increase of medium free Ca2+ concentration depended on the initial calcium content of the mitochondria, presumably reflecting the different rates of Ca2+ efflux. Thus, when the initial mitochondrial calcium content was 6.25 nmol/mg of protein, the final calcium content was still sufficient to buffer the medium free Ca2+ at about 0.8 PM. At lower initial mitochondrial calcium contents, the final steady state [Ca2+]o was correspondingly lower after addition of hepatic microsomes. Separate control experiments showed that addition of 100 ,UM MgATP" in the presence of 250 free Mg2+ to mitochondria alone had no effect on the extramitochondrial free Ca2+ concentration.

Direct evidence of a transfer of calcium from the mitochondria to hepatic microsomes was obtained in separate experiments similar to those shown in Fig. 11. The total calcium of the mixed pools contained in the mitochondria and microsomes was measured by addition of A23187. The contents of a duplicate cuvette were centrifuged for 30 s at 12,000 X g in an Eppendorf centrifuge to remove the mitochondria, and the calcium content of the hepatic microsomes in the supernatant was measured similarly by addition of A23187. With mitochondria containing initially 1 nmol of calcium/mg of protein, a total of 1.3 nmol of calcium was lost from the mitochondrial pool over a 10-min period, 1.1 nmol of calcium was removed from the medium, and the microsomes gained 2.4 nmol of calcium. With mitochondria containing initially 5 nmol of

calcium/mg of protein, mitochondria lost 2.9 nmol of calcium, 0.2 nmol of calcium was removed from the medium, and the microsomes gained 3.0 nmol of calcium. Saturation of the microsomes with calcium under these conditions was ascer- tained by their failure to remove further calcium from the medium in the presence of 2 p~ ruthenium red.

Qualitatively, these data support the observations of Becker et al. (331, although there are important quantitative differ- ences. These relate to the considerably higher mitochondrial calcium content required for buffering the extramitochondrial free Ca2+ and the slower time course observed in their study. The present studies, with in vitro mixtures of mitochondria and microsomes, indicate that in the presence of MgATP2-, microsomes will continue to sequester Ca2+ until they become saturated. At equilibrium, the extramitochondrial free Ca2+ concentration is determined solely by the kinetics of Ca2+ cycling across the mitochondrial membrane. The effect of the microsomes in the above experiments is to regulate the external free Ca2+ indirectly by causing a redistribution of calcium from the mitochondria to the microsomes.

DISCUSSION

One of the main purposes of the present study was to define the relationships between the cystolic free Ca2+ concentration in the hepatocyte and the calcium transport systems of the intracellular organelles, with the goal of increasing our understanding of the regulation of intracellular calcium homeostasis. Studies with isolated liver mitochondria reported in this paper provide quantitative information concerning variation of the extramitochondrial free Ca2+ concentration with the total calcium content of the mitochondria, which previous studies (26) have shown is linearly related to the intramitochondrial free Ca2+ concentration. The relationship between the extra- and intramitochondrial free Ca2+ concentration is a dynamic one as implied qualitatively by earlier studies (24, 27). The quantitative relationship can be described by a simple kinetic model of Ca2+ cycling via the electrophoretic Ca2+ uniport and the electroneutral Ca"/2H+ antiport. The importance of these studies is that they define the ranges where external steady state free Ca2+ concentration (steady state [Ca2'],) is either dependent upon or essentially independent of the mitochondrial calcium content. With isolated mitochondria, the dependent region is up to about 4 nmol of calcium/mg of protein (Fig. 2), while with mitochondria in situ, a somewhat higher value is obtained (Fig. 8). The actual value of the steady state [Ca2+Io for a certain mitochondrial calcium content is variable, being dependent on ionic or other factors that may affect the kinetic constants of the Ca'+ transport systems. These possible variables have not been exhaustively investigated in the present study, but it is perti- nent that the kinetic model of Ca2+ cycling predicts that the steady state [Ca2+Io is much more sensitive to changes of the apparent K, of the Ca2+ uniporter than to changes of the other kinetic parameters. Thus, Mg2+, which increases the apparent K,,, of the Ca2+ uniporter (23, 50, 52) , caused a 2 to %fold increase of the steady state [Ca2+lo (Fig. 2 and Refs. 24 and 27). On the other hand, phosphate depletion of mitochondria, which under some conditions increases the activity of the Ca2+ efflus carrier, has only a small effect on the steady state [Ca2+Io (61). The effects of Mg2+ on the steady state [&*+lo are potentially important for extrapolation of the data to physiological conditions since the hepatocyte cytosolic free Mg2+ concentration is 0.5 to 1.0 m~ (9, 62, 63). However, MgATP2- counteracts the Mg2+ effect, and the net effect of 1 m~ free M$+ plus 3 mM MgATP2- on the mitochondrial steady state [Ca2+lo has been shown to be similar to that


obtained in the absence of both free Mg2+ and MgATP2- (27). This is borne out by the fact that the plateau value of the steady state [Ca2+]o with isolated mitochondria incubated in Mg2+-free medium is similar to that of the hepatocyte cytosolic free ea2+ concentration (Fig. 6 and Ref. 9). A half-maximum cytosolic free Ca2+ concentration was obtained at a total cell calcium content of 1.6 nmol/mg dry weight (Fig. 6), which corresponds to a mitochondrial calcium content of approximately 2 nmol/mg of mitochondrial protein. This is only slightly higher than the value of 1.4 nmol/mg of protein obtained from the data of Fig. 2 with isolated mitochondria having a calcium content corresponding to a half-maximal extramitochondrial free Ca" concentration. These data strongly suggest that in the intact cell, the cytosolic free Ca2+ concentration is in a steady state with respect to the mitochondrial calcium content over a wide range of cellular calcium contents.

An evaluation of calcium homeostasis in the intact cell requires a knowledge of the mitochondrial calcium content in situ and the effect of the Ca2+ transporting activities of other intracellular systems on the cytosolic free Ca2+. Results presented in this paper and in previous reports (9,56) show that between 65 and 80% of the total cell calcium is contained in the mitochondria. A tabulation of liver calcium contents given by Borle (4), which includes values for slices, perfused liver, and hepatocytes, provides values ranging from 1.7 to 13.8 nmol/mg dry weight. In our hands, liver cells as normally prepared and suspended at 37 "C in medium with 1.25 mM Ca2+ contained 5.9 f 1.3 nmol of calcium/mg dry weight (mean f S.E. of 15 cell preparations), after a brief Ca2+-free wash. At this cell calci-Jm content, the mitochondria contain at least 16 nmol/mg of protein (Fig. 7B). This finding has several important implications. First, on the basis of the mitochondrial calcium activity coefficient reported previously (26), mitochondria in a normal liver cell will have a free Ca2+ concentration in the matrix of about 16 PM, which is more than 1 order of magnitude above the range of free Ca2+ concentrations required to activate mitochondrial Ca2+-sensitive enzymes (28, 29). Consequently, there are little grounds, at least in liver, to indicate that these enzymes, namely pyruvate dehydrogenase phosphatase, NAD-linked isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase, are regulated by changes of mitochondrial free Ca2+ in the physiological range (26, 64 ). Second, at this level of mitochondrial matrix free Ca", the velocity of Ca2+ efflux will be relatively independent of changes of the mitochondrial calcium content, and the extramitochondrial free Ca2+ concentration should be well buffered. The data relating the cytosolic free CaZ+ concentration to the hepatocyte calcium content (Fig. 6) show that when total cell calcium is above 4 nmol/mg cell dry weight, the cytosolic free Ca2+ concentration is approaching its maximum value of 0.3 PM. Thus, when the cell is in a steady state with respect to calcium homeostasis, a fall of total cell calcium content of about 30% from the normal physiological value of 6 nmol/mg cell dry weight will have little effect on the cytosolic free Ca2+ concentration, while an increase of total cell calcium will likewise have no effect.

In contrast to the role of mitochondria, there is no evidence that endoplasmic reticulum in situ or hepatic microsomes in vitro are capable of buffering the external free Ca2+ concentration. Endoplasmic reticulum vesicles have a CaZ+-ATPase transport system (31-33,57), which, although much less active than rabbit skeletal muscle sarcoplasmic reticulum, is able to decrease the extravesicular free Ca2+ concentration to below 0.1 PM. The V',,, obtained with isolated hepatic microsomes at 22 "C was 1.75 nmol/mg of protein.min, compared with the value of about 6 nmol/mg of microsomal protein. min at

30 "C obtained with endoplasmic reticulum in situ using experimental conditions similar to those of Fig. 9 (data not shown). The apparent K,,, value for free Ca2+ of in situ microsomal Ca2+ uptake was 2.8 PM, which is 1 order of magnitude higher than the lowest value reported for isolated hepatic microsomes (57). No evidence for an interaction with calmodulin or trifluoperazine could be obtained with either system: and the discrepancy between the apparent K, values for Ca2+ uptake remains to be resolved. The maximum calcium sequestering activity of endoplasmic reticulum is very small compared with mitochondria, amounting to about 1 nmol/mg of cell dry weight (Figs. 7 and 9), which is reached at a total cell calcium content of about 5 nmol/mg dry weight. An unregulated, unidirectional carrier, transporting calcium into the lumen of the endoplasmic reticulum would be expected to sequester Ca2+ from the cytosolic calcium pool until complete saturation is attained. The experimental data, in contrast, indicate that intermediate stages of loading of the endoplasmic reticulum are possible under steady state conditions (Fig. 7A ) . This fiiding suggests that the endoplasmic reticulum must have a Ca2+ efflux pathway. At present, there is no direct evidence for such an efflux mechanism in endoplasmic reticulum either in situ or in vitro. The important conclusion is reached from these studies that the endoplasmic reticulum calcium pool is largely filled in liver cells containing a physiological calcium content and that this organelle appears to have a negligible role in buffering the cytosolic free Ca2+ concentration.

A further and more difficult question is what additional factors contribute to the regulation of the cytosolic free Ca2+ concentration in the liver cell in its physiological environment. A 5000-fold concentration gradient of Ca2+ is normally present across the plasma membrane of the hepatocyte. Little is known of the mechanisms of Ca2+ entry into liver cells, but possible processes include a Ca2'/H+ or Ca2+/Na+ antiporter, a channel regulated by protein phosphorylation or simple passive diffusion (65). A small but finite permeability of the plasma membrane to extracellular Ca2+ necessitates the presence of an active Caz+ extrusion mechanism for achievement of calcium homeostasis under physiological conditions. This is clearly altered with cold exposure of hepatocytes in the presence of Ca2+ since the cells gain calcium under these conditions but subsequently lose it slowly when incubated at elevated temperatures in a Ca2+-depleted medium. Recently, a high affinity Ca2'-ATPase has been identified in liver plasma membranes which has been shown to be regulated by low molecular weight proteins functioning as both activators (30) and inhibitors (66), although the physiological significance of regulation by these proteins is presently unclear. Ultimately, the balance between rates of Ca2+ influx and efflux across the plasma membrane will determine directly the total calcium content of the cell and indirectly the cytosolic free Ca2+ concentration.

Calcium homeostasis must be regarded as a dynamic state in which Ca2+ flux is balanced across at least three membranes, i.e. the plasma membrane, the endoplasmic reticulum membrane, and the mitochondrial membrane. The relative distribution of the total intracellular calcium between the cytosol, the reticular system, and the mitochondria is expected to be determined by the kinetic constants of the various Ca2+-transporting systems, the amount of Ca2+-binding ligands in the various compartments, and their affinity for Ca" under the prevailing local ionic conditions. Liver is characterized by having a high ratio of mitochondria to smooth endoplasmic reticulum, and while mitochondria have a very high capacity

R. H. Cooper and J. S. Marks, unpublished observations.


to accumulate calcium, the calcium pool of the endoplasmic reticulum is very limited. Thus, in liver, the cytosolic free Ca2' concentration remains in a steady state with respect to Ca'+ cycling across the mitochondrial membrane at different cellular calcium levels. The situation in kidney appears to be similar to that in liver (4, 67). However, experiments using a variety of techniques to measure intracellular calcium distribution with intact or permeable plasma membranes in pre- synaptic nerve terminals (68-71), squid axon (72-74), smooth muscle (75), myocytes (76, 77), and frog skeletal muscle (78) have concluded that over the physiological range of cytosolic free Ca2+ concentrations, the mitochondrial calcium content is low compared with that of the reticular system and that mitochondria in these tissues have little or no role in regulating the cytosolic Ca2+ concentration. Unlike liver, these organs have a low ratio of mitochondria to reticular system, and the latter has a much greater Ca2'-binding capacity than liver or kidney endoplasmic reticulum. Consequently, it is unlikely in these tissues that mitochondria buffer the cytosolic free Ca2+. The cytosolic free Ca2+ concentration in nerve tissue appears to be below 0.1 p~ (13, 73), but it remains to be determined whether the low value in the resting state is in a steady state with respect to Ca2+ cycling across the mitochondrial membrane.

When calcium homeostasis is disturbed by a hormonal or electrical signal to the cell, the steady state of CaT+ flux is perturbed by regulation of one or more kinetic parameters of the Ca2+ transport systems. An increase of the cytosolic free Ca" concentration may be achieved by an increased influx of Ca2+ across the plasma membrane or by a release of bound calcium from intracellular organelles, while subsequent return to the resting state may be caused by an increased rate of Ca2+ efflux from the cell or removal of calcium by intracellular organelles. Clearly, which Ca"-transporting systems are most important in re-establishing calcium homeostasis after a per- turbation will be determined by their relative kinetic constants. In heart, for instance, there is little evidence that mitochondria are important in the regulation of beat-to-beat cytosolic calcium transients (79). A similar conclusion has been reached with respect to nerve tissue (69, 73), skeletal muscle (78), and smooth muscle (75). A number of hormones have been shown to elevate transiently the cytosolic free Ca2' concentration and to result in the net efflux of calcium from the liver (6-9). The relative roles of the endoplasmic reticulum, mitochondria, and plasma membrane in regulating the cytosolic free Ca2' concentration in liver during stimulation by a-adrenergic hormones and vasopressin is currently under further investigation in this laboratory.

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Assuming that the efflux carrier obeys Michaelis-Yenten kinetics 1261 the rate of ea2+ efflux ( v 2 ) 1 s given by equation 2 where X2 and V2 are the apparent Km and Vmax Of the efflux carrier and [Ca2+l1 is the intrmitoehondrial free Ce.2' concentration.

for ICaZtl,, to give equation 3, which describes the Steady state relationship In the Steady state VI = v 2 and equatlons 1 and 2 can be rearranged and solved

between Intra- and extramitochondrlal free Ca2+ Concentrations in terms of 3 independent kinetic pnrameters. K1, X2 end 9 (defloed BE the ratio V2/V,).

by equation 3 is Shown in Fig. 2A where the theoretlcnl c u ~ v e s generated at fixed v&luee of the kinetic parameters 2re s h o w along with the experimental data points (see main body of text].

An illustratioo of the relltionshlp between ICa2+li and [Ca2+lo predicfed

to give the linear relationship shorn in equation 4. Equation 3 IS of t h e form of a recraogular hyperhola and can be rearranged

Semce of inhibitor should be linear with the values of the Y-intemept (a ) and According to equation 5 a plot of 1/[Ca2+]: against 1/[Ca2+Ii in the pre-

the X-intercept lbl being related to the inhibitor concentration by equations 6 and 7.

1 1 1

K12e (1 + Ill/Ki) K12 a"."- (61

Flg. 3 Show9 a double reciprocal p l o t BCcordLn. to equation 5 or the deta points shorn In Pip. 2 at 0 . 20 . and 26 p o l ruthenium redjmg protein. AS predicted a linear relationship betmen 1/[Ca2'l: and l/lCa2+Ii was observed in the presence or absence of the inhibitor. which supports the validity of the aS6umPt- i ons made i n derivinx equation 5 The effect of added inhibitor was to decrease both the Slope and intercepts of the line. The X and Y intercept va lues . obtained fT0m l inear resression analpsis of the data points. have been replotted accordinp- to equltions 6 and 7 (Fig. 4 ) The term 1/11 + ljKil, for a "on-

canpetitive inhibitor is given by the rate of Ca2+ uptake ~n the presence of inhibitor divided by the rate of Uptake in the absence of inhihifor (491 The intercept replot8 we?e also found to be linear

I IO04 100- N

-64 -0:2 0 0:2 0.4 0.6 0.8 1.0 1.2 . . . .

1

Calcium Homeostasis in Liver

TABLE I

Sumnary of kinetic psPmeter8 obtained frDm

the steady State distribution of Ca2+ B C ~ D S ~

the mitochondrial inner membrane

Kinetic parameter Steady state metnod Initial rate methodsh lderlved from Fig. 4A & B)

2.8-4.7 uY(23,46 ,47 ,SO)

9.7 "Y

5 nmol/min/mg (24. 26) 250 nmol/min/mg ( S O )

0 . 0 2

'Mean of values obtained from Flg. 4A L B of Y = 0.06 and 0.08, respectively.

'Klnetlc parmeters determined with succinate (t rotenone) as substrate. 0 10 20 K'm efflux (rM)

I

04 , , , , , ! I 0 1 2 3 4 5 0 0.05 0. I

K'm Influx ( F M ) Vmax efflux /Vmax Influx

Flg. 5 The effect of varxatlon of lndlYldUsl kinetic Darameters on

drhal) Ca2' Of 25 "mol. as calculated from equatlon 10. When the Steady State ICa2+l,,, at a total (intra- and extramltochon

f l x e d , the va lues of the klnetic parameters were K1 = 2 . 0 uM. K2 = 10 UU and o s 0.056. Other conStants were set at the following values vo = 2 5 mi V I = 0.0011 m l , I F 40 L U , Kd =

30 U M . F = 7 X 10-4,

the of chemistry vol. 258, 1983 in mechanisms underlying calcium homeostasis in ... ·...

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