A Possible Mechanism for the Insulin Stimulation of Alanine Uptake by Isolated Rat Hepatocytes

Comp. Biochem. Physiol. Vol. 116C, No. 3, pp. 245–253, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0742-8413/97/$17.00 PII S0742-8413(96)00199-5

A Possible Mechanism for the Insulin Stimulation of Alanine Uptake by Isolated Rat Hepatocytes Sawsan I. Kreydiyyeh,1 Muna M. El-Kasti, and Anwar B. Bikhazi2 Departments of 1Biology and 2 Physiology, American University of Beirut, Beirut, Lebanon

ABSTRACT. The mechanism underlying the insulin-induced stimulation of alanine uptake by isolated rat hepatocytes was studied by testing the involvement of the Na1-K1 pump and the Na1 -H1 exchanger. Insulin was found to inhibit the in vitro activity of the Na1-K1 ATPase in a liver homogenate. Ouabain, a specific inhibitor of the pump, enhanced alanine transport, as insulin did, and no additive effects were observed when it was added simultaneously with the hormone. When the membrane was depolarized by sodium removal from the incubation medium or by an increase in its K1 concentration, a stimulation of amino acid uptake was observed. Amiloride, an inhibitor of the Na1-H1 transporter enhanced also alanine uptake. The increase in transport observed when amiloride and insulin were added together was not significantly different from the one produced by amiloride alone. The stimulatory effect of the drug and the hormone disappeared, however, in presence of bicarbonate or when the membrane was hyperpolarized by potassium removal from the incubation medium. It was concluded that depolarization per se, increased alanine transport and that the effects of amiloride and insulin were mediated by intracellular acidification followed by membrane depolarization, which in turn activated the transporters. comp biochem physiol 116C;3:245–253, 1997.  1997 Elsevier Science Inc. KEY WORDS. Acidification, alanine, amiloride, depolarization, hepatocytes, Na1-H1 exchanger, Na1 -K1 ATPase, ouabain

INTRODUCTION Previous works have provided ample evidence for a stimulatory effect of insulin on the uptake of amino acids by rat hepatocytes (1–3). This effect was apparent only after a lag period of 30 to 120 min (2) and was attributed to an activation of the A transport system (2,4). The literature does not report, however, enough information about the mechanism of action of insulin, nor about the steps involved in its signal transduction. Some investigators ascribe some of the hormone effects, in the liver and other tissues, to an increase in the activity of the Na1-K1 ATPase (5–7), others to an induced change in the membrane potential (8), and still others to a stimulation of the Na1-H1 exchanger (9,10), which uses the sodium gradient established by the Na 1-K1 pump to extrude H1 ions from the cell and regulate intracellular pH (11,12). Since the neutral amino acids, and in particular alanine are cotransported with sodium across the hepatocyte membrane (13), the Na1 -K1 ATPase and the Na 1-H1 antiporter could be target sites of action of the hormone in its stimulation of amino acid uptake, and a change in membrane potential may be one of their manifestations. The aim of this work is to test this hypothesis, and to Address reprint requests to: Sawsan Kreydiyyeh, Department of Biology, Faculty of Arts and Sciences, American University of Beirut, Beirut, Lebanon. Fax 212 478 1995; E-mail: [email protected]

delineate the mechanism underlying the insulin-induced stimulation of alanine uptake by rat hepatocytes.

MATERIALS AND METHODS Isolation of Hepatocytes Liver cells were isolated from adult male Sprague-Dawley rats weighing 150–200 g by a modification of the enzymatic perfusion technique established by Ingebresten and Wagle (14). Throughout the experiment, the animals were handled in accordance with the Guide for Laboratory Animal Facilities and Care, U.S. Department of Health, Education, and Welfare. The rats were anesthetized by i.p. injection of pentobarbital (5 mg/100 g body weight), and the liver was perfused in situ, by cannulation of the portal vein, with 200 ml of Ca-free Krebs-Henseleit (K-H) buffer containing 1,000 U.S.P. units of heparin, at a flow rate of 30–40 ml/ min. Before the preparation was started, the abdominal vena cava was cut below the renal vein to allow for drainage. The K-H buffer had the following composition: NaCl: 94.8 mM; KCl: 4.74 mM; KH2PO4: 1.19 mM; MgSO4: 2.43 mM; NaHCO3: 23 mM; sodium pyruvate: 49.2 mM; sodium fumarate: 5.38 mM; sodium glutamate: 4.92 mM; D-glucose: 11.54 mM. At the end of this step, the liver was excised and perfused at a rate of 80 ml/min with a collagenase-containing (type CLS 2.325 U/mg, Worthington Biochemical Corporation, Freehold, NJ) K-H buffer (0.4 mg/ml) for 40

S. I. Kreydiyyeh et al.

246

min by recirculation of the buffer. This was followed by a last 5 min perfusion step with the incubation buffer, but without recirculating the buffer. All perfusion steps were run at 37°C and the buffers used were gassed with 95% O2; 5% CO2 and their pH adjusted to 7.4. The liver was then transferred to a beaker, and the hepatocytes dispersed in 30 ml of incubation buffer. The cell suspension obtained was filtered through a 250 µm nylon-mesh and viability tests were routinely performed on every preparation using the trypan blue exclusion test at the beginning and at the end of the experiment. In all preparations used, the viability was above 90%, and did not decrease below 87% by the end of the experiment. The number of hepatocytes in the suspension was measured in a Coulter Counter Model A (Coulter Electronics, Hialeah, FL). Incubation Buffers Hepatocytes were incubated in five different types of buffers containing all 1 mM amino-oxyacetate as an inhibitor of alanine metabolism. 1. A phosphate buffer saline (PBS) containing 10 mM K 2HPO4, and 0.15 M NaCl. 2. A sodium-free buffer in which NaCl was replaced by choline chloride (K2HPO4: 10 mM, choline chloride: 0.15 M). 3. A high potassium buffer in which the concentration of K 2HPO4 was increased from 10 mM to 50 mM and NaCl was kept at 0.15 M. 4. A bicarbonate phosphate buffer (K2HPO4: 5 mM; KHCO3: 10 mM; NaCl: 0.15 M). 5. A potassium-free phosphate buffer in which K 2HPO4 was replaced by Na 2HPO4, and the sodium concentration kept constant (Na2HPO4; 10 mM; NaCl: 133 mM, choline chloride: 20 mM). All incubation buffers were gassed with 95% O2; 5% CO2 and their pH adjusted to 7.4.

Determination of Alanine Uptake Alanine uptake was determined by measuring the disappearance of the amino acid from the extracellular fluid. One ml aliquots of cell suspension were withdrawn at 10-min intervals starting from zero time and up to 1 hr. Immediately upon withdrawal they were centrifuged at 10,000 3 g for 30 sec to precipitate the cells, and 0.5 ml of supernatant were transferred to a scintillation vial and assayed for radioactivity. Alanine uptake was calculated as the difference in the amount of extracellular alanine present in the supernatant at zero time, and the one present at a specific time interval. Assessment of Water Movement For every treatment, and in every experiment, water movements were measured using [3H] inulin (1.07 Ci/mmol, Amersham International plc, Buckinghamshire, U.K.) as a marker of the extracellular space, and all alanine uptake data were corrected accordingly. IN VITRO ASSAY OF THE Na1 -K1 ATPase IN THE HEPATOCYTES HOMOGENATE. Hepatocytes, isolated from three livers, were suspended in Tris buffer (NaCl: 200 mM; MgCl2 ⋅ 6H2O: 5 mM; EGTA: 2mM; KCl: 5 mM; Tris-HCl: 200 mM, pH 7.4), and homogenized in a glass-teflon homogenizer at 2100 rpm (Arthur Thomas Scientific Apparatus, Philadelphia, PA). The in vitro activity of the Na1-K1 ATPase (the ouabain-sensitive component of the total ATPase activity) in the homogenate was assayed by measuring the amount of inorganic phosphate liberated by an aliquot of homogenate in presence of ATP at 37°C (15). Inorganic phosphate was determined by the method of Taussky and Shorr (16). Percent inhibition of the enzyme was calculated as follows:

%inhibition 5 {1 2 [Pi (Insulin) 2 Pi (Insulin 1 ouabain)/Pi (Control) 2Pi (Control 1 ouabain)]} 3 100.

Incubation Procedures Hepatocytes were incubated in 100 ml incubation buffer (104 cells/ml) at 37°C in water-jacketed beakers, and stirred all through with a glass stirrer rotated by a 120 rpm motor to prevent cell agglutination. When the effect of insulin, ouabain, or amiloride (Sigma Chemical Co, St Louis, MO) was studied, they were added to the different incubation buffers at a respective concentration of 0.1 µM, 4 mM, and 1 mM. L-[UL-14C] alanine (159.1 mCi/mmol, Sigma Chemical Co, St Louis, MO) was added at 114 nM. In all experiments, the hepatocytes were dispersed after the addition of all agents, and just before the beginning of the incubation period. The experimental treatments and their respective controls were run on the same hepatocyte preparation and were always replicated together.

Protein was determined by the method of Lowry et al. (17). Statistical Analysis The data were analyzed by a one-way ANOVA and whenever statistical significances were detected, the Student’s t-test was applied. P , 0.05 was considered significant. RESULTS Effect of Insulin and Ouabain When Added to PBS Insulin (0.1 µM) increased significantly and up to 60 min alanine transport (Fig. 1). The enhancement in the amino acid uptake observed with ouabain (4 mM) was even more prominent (Fig. 1) and was significant at all time intervals.

Mechanism of Action of Insulin in Hepatocytes

247

FIG. 1. Effect of insulin and ouabain on alanine uptake by hepatocytes. The cells were incubated in PBS containing insulin, ouabain, or both. Alanine uptake was measured at 10-min intervals.

When both insulin and ouabain were added simultaneously, the values obtained for alanine transport were significantly higher, at all time intervals, than the control values, but were not significantly different from the ones obtained with ouabain alone. Effect of Sodium Removal The uptake of alanine by hepatocytes was increased upon removal of sodium from the incubation medium (Fig. 2). This increase was significant at all time intervals and was comparable to the one observed with insulin alone. Effect of High Potassium An increase in K 1 concentration of the extracellular fluid (from 20 mM to 100 mM) led to an increase in the transport of alanine, and the values obtained at all time intervals were significantly higher than their corresponding control values (PBS) (Fig. 3). Effect of Insulin and Amiloride When Added to PBS At all time intervals, alanine transport was significantly increased by insulin and amiloride when added to a PBS buffer

(Fig. 4). When both were added simultaneously, the values obtained were significantly higher than their corresponding control values (PBS) but were not different from the ones observed with amiloride alone (Fig. 4). Effect of Insulin and Amiloride in Presence of Bicarbonate The addition of bicarbonate to the extracellular fluid did not affect alanine uptake (Fig. 5) and the values obtained were not significantly different from their corresponding controls (PBS). However, the stimulatory effect of insulin and amiloride disappeared in presence of bicarbonate, and although the values obtained in the simultaneous presence of amiloride and bicarbonate were higher than the ones observed with bicarbonate alone, yet they were not significantly different from each other (Fig. 5). Effect of Insulin and Amiloride in the Absence of Potassium Potassium removal from the incubation medium did not have any effect on alanine transport (Fig. 6), and the values obtained in the presence and absence of potassium were not significantly different from each other. In the absence of

248

S. I. Kreydiyyeh et al.

FIG. 2. Effect of insulin and sodium removal from the extracellular fluid on alanine uptake by hepatocytes. Cells were incu-

bated in PBS or in a Na1-free buffer in presence or absence of insulin. Alanine uptake was measured at 10-min intervals.

FIG. 3. Effect of a high-potassium concentration in the incubation medium on alanine uptake by hepatocytes. Cells were incubated in PBS, or in a high K1 buffer. Alanine uptake was measured at 10-min intervals.

Mechanism of Action of Insulin in Hepatocytes

249

FIG. 4. Effect of insulin and amiloride on alanine uptake by hepatocytes. The cells were incubated in PBS in presence of insulin, or amiloride, or both, and alanine uptake was measured at 10-min intervals.

potassium, however, the stimulatory effect of insulin and amiloride was not observed, and the data obtained with insulin or with amiloride, were not significantly different from their corresponding controls (potassium-free buffer) (Fig. 6).

did not exert any inhibitory effect on the amount of inorganic phosphate liberated and the values obtained with insulin alone and with insulin and ouabain were not significantly different from each other (2.332 vs. 2.341).

Effect of Insulin on the In Vitro Activity of the Na1-K1 ATPase in the Hepatocyte Homogenate

DISCUSSION Effect of Insulin, Ouabain, Sodium Removal and High Potassium

Insulin increased the amount of inorganic phosphate liberated from 2.16 to 2.332 µmol/hr/mg protein (Table 1). Because many other phosphatases are present in the homogenate in addition to the Na1 -K1 ATPase, the effect of the hormone on the ATPase alone was studied using ouabain as a specific inhibitor of the enzyme. Ouabain at 2 mM inhibited the Na1-K1 pump maximally and was used at such a concentration in the enzymatic assay. When applied to the homogenate, the drug decreased the amount of inorganic phosphate liberated from 2.160 to 2.080 µmol/hr/mg protein (Table 1). In presence of insulin, however, ouabain

The literature reports a stimulatory effect of insulin on amino acid uptake (1–3) by rat hepatocytes, which needs 30 to 120 min to manifest itself and which is mediated by activation of the A transport system (2–4). In this work, however, the effect of the hormone appeared from the first 10 min, and was maintained up to 1 hr. Since alanine is cotransported with sodium (13) across the hepatocyte membrane, and since some researchers have reported a stimulation of the Na1-K1 pump by insulin (5–7), the insulin-induced stimulation in alanine uptake was thought to be due to an activation of the Na 1-K1 ATPase, which would in-

S. I. Kreydiyyeh et al.

250

FIG. 5. Effect of insulin and

amiloride in presence of bicarbonate, on alanine uptake by hepatocytes. Cells were incubated in PBS or in a HCO32 containing buffer in presence of insulin or amiloride. Alanine uptake was measured at 10-min intervals.

crease the sodium gradient needed for the active transport of alanine. To test this hypothesis, ouabain was applied as a specific inhibitor of the enzyme, and the results obtained were opposite to the expectations. Ouabain, instead of inhibiting alanine uptake, stimulated it, as did insulin. No additive effects were observed when both were added together, inferring that both are acting through the same mechanism. The inhibitory effect of the hormone on the Na1-K1 ATPase was confirmed by the in vitro enzymatic assay, which demonstrated a 100% inhibition of the pump by insulin. This effect could be exerted through activation of some intermediate phosphatases or kinases present in the homogenate. Since the dissipation of the sodium gradient increases alanine uptake, then the presence of sodium in the extracellular fluid does not seem crucial for the transport process. In fact Heinz and coworkers (18) have demonstrated the persistence of concentrative amino acid uptake in the absence of a sodium gradient. In this work also, when sodium was omitted from the incubation medium, an increase in alanine transport was observed, which was not enhanced fur-

ther upon addition of insulin. This suggests that insulin and sodium removal affect probably a common pathway that leads eventually to a stimulation of alanine uptake. Both the inhibition of the Na1-K1 pump and sodium removal lead to a depolarization of the hepatocyte membrane. To check whether depolarization per se is responsible for the increase in alanine transport, the membrane was depolarized by raising extracellular potassium from 20 mM to 100 mM. In this case also, a significant enhancement of the amino acid uptake was observed (Fig. 4). Effect of Amiloride Amiloride, a specific inhibitor of the Na 1-H1 exchanger, increased significantly alanine transport across the hepatocyte membrane. This antiporter exchanges extracellular Na1 for intracellular H1 . When it is inhibited, H1ions accumulate inside the cell and lead to cellular acidification (12). Fitz et al. (19) have demonstrated that intracellular acidosis, induced by a variety of maneuvers, results consistently in membrane depolarization. Depolarization per se, was shown

Mechanism of Action of Insulin in Hepatocytes

251

FIG. 6. Effect of insulin and

amiloride on alanine uptake by hepatocytes in the absence of potassium. Hepatocytes were incubated in a K1-free buffer, in presence of insulin or amiloride. Alanine uptake was measured at 10-min intervals.

TABLE 1. Effect of ouabain and insulin on the in vitro activ-

ity of the Na1-K1 ATPase in hepatocyte homogenates Treatment

N

Control Ouabain Insulin Insulin 1 ouabain

10 10 10 10

conclusive evidence, that both the drug and the hormone exert their effect through the same mechanism.

mmol Pi/hr/mg protein 2.160 2.080 2.332 2.341

6 0.020a 6 0.006b 6 0.011c 6 0.013c

Values are means 6 SEM. N is the number of replicates. a,b,c Values within a column not sharing a common superscript are significantly different at P , 0.05 as determined by Student’s t-test.

to enhance alanine uptake. The stimulatory effect of amiloride could thus be attributed, to an eventual depolarization of the cell membrane. Since the simultaneous addition of insulin and amiloride did not lead to any further enhancement in alanine uptake as compared to amiloride alone, it was speculated, without

Effect of Bicarbonate Gleeson et al. (20) reported that in hepatocytes, the recovery from an acute acid load is inhibited by amiloride only in the absence, but not in the presence of bicarbonate. It was suggested that a Na1 -HCO32 transporter situated in the basolateral membrane and a HCO32-Cl2 exchanger present on the apical side, are responsible, in addition to the Na1 H1 exchanger, for intracellular pH regulation (21). Accordingly, if the enhancement in alanine uptake observed with amiloride is ascribed to membrane depolarization that results from intracellular acidification, then the addition of bicarbonate to the incubation medium should correct the decrease in pH induced by the drug and, consequently, prevent cell depolarization and alanine uptake stimulation. In fact, amiloride did not cause any significant enhancement

S. I. Kreydiyyeh et al.

252

in alanine uptake in the presence of bicarbonate, confirming thus the hypothesis. The stimulatory effect of insulin disappeared also in presence of bicarbonate, suggesting that it is mediated through intracellular acidification or inhibition of the Na1-H1 exchanger. The hormone was shown, in fact, to inhibit the Na1-K1 pump, and dissipate consequently the sodium gradient needed for the activity of the exchanger. Insulin seems to act by inducing indirectly acidification through an inhibition of the Na1-H1 transporter followed by depolarization. This disagrees with the findings of Jakubowski and Jacob (9) who reported a stimulation of the Na1 -H1 transporter by insulin. Effect of Potassium Removal Potassium was removed from the incubation medium in order to induce membrane hyperpolarization and to see whether this would antagonize the effect of amiloride and insulin. In fact, the drug and the hormone did not stimulate alanine uptake in absence of potassium, and the values obtained were not significantly different from their corresponding controls (Fig. 6). This confirms the hypothesis that the effect of insulin and amiloride is mediated through a depolarization of the hepatocyte membrane. In conclusion, the following mechanism underlying the stimulatory effect of insulin on alanine uptake could be proposed: insulin inhibits the Na 1-K1 ATPase through an effect on some intracellular kinases and/or phosphatases, and dissipates the sodium gradient that drives the Na1 -H1 exchanger. This leads consequently to intracellular acidification followed by membrane depolarization. The depolarization in turn activates the alanine transporters, probably by inducing a change in their conformation. This effect of the hormone on membrane potential has also been reported by Friedman and Dambach (8) who observed a blocking of the glucagon-induced hyperpolarization of the hepatocyte membranes by insulin. The present findings can be reconciled with the known dependency of alanine transport on the sodium gradient, by assuming that two types of alanine carriers are present in the hepatocyte membrane. One type is activated by sodium binding (sodium-dependent), and the other by membrane depolarization (voltage-dependent). The dissipation of the sodium gradient by ouabain addition or removal of extracellular sodium, inhibits the Na1H1 and Na 1-HCO32 transporters and leads to a decrease in intracellular pH that can be corrected in presence but not in the absence of bicarbonate from the extracellular fluid, as demonstrated by Gleeson et al. (20). Since in presence of bicarbonate the pH is adjusted, no membrane depolarization will occur, and an inhibition in alanine uptake will be observed. In the present study, the cells were not able to recover from the intracellular acidity induced by ouabain or sodium

removal because bicarbonate was not present in the incubation media. As a consequence of the bicarbonate absence, the intracellular acidity was maintined, [and even enhanced by the increased activity of the HCO 32-Cl2antiporter (22)], the membrane depolarized, and the voltage-dependent alanine carriers activated, leading thus to a higher alanine uptake. This work was supported by a grant from the National Lebanese Council for Scientific Research.

References 1. McGivan, J.D.; Ramsell, J.C.; Lacey, J.H. Stimulation of alanine transport and metabolism by dibutyryl cAMP in the hepatocytes from fed rats. Biochim. Biophys. Acta 644:295–304; 1981. 2. Le Cam, A.; Freychet, P. Effect of insulin on amino acid transport in isolated rat hepatocytes. Diabetologia 15:117–123; 1978. 3. Kletzien, R.F.; Pariza, M.W.; Becker, J.E.; Potter, V.R. Induction of amino acid transport in primary cultures of adult rat liver parenchymal cells by insulin. J. Biol. Chem. 251:3014 – 3020;1976. 4. Shotwell, M.A.; Kilberg, M.S.; Oxender, D.L. The regulation of neutral amino acid transport in mammalian cells. Biochim. Biophys. Acta 737:267–284;1983. 5. Fehlmann, M.; Freychet, P. Insulin and glucagon stimulation of (Na1-K1) ATPase transport activity in isolated rat hepatocytes. J. Biol. Chem. 256:7449–7453;1981. 6. McGill, D.L.; Guidotti, G. Insulin stimulates both the α1 and the α2 isoforms of the rat adipocyte (Na1-K1)ATPase. J. Biol. Chem. 266:15824–15831;1991. 7. Weil, E.; Sasson, S.; Gutman, Y. Mechanism of insulininduced activation of Na1 -K1ATPase in isolated rat soleus muscle. Am. J. Physiol 261:C224–C230;1991. 8. Friedmann, N.; Dambach, G. Antagonistic effect of insulin on glucagon-evoked hyperpolarization. Biochim. Biophys. Acta 596:180–185;1980. 9. Jakubowski, J.; Jakob, A. Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na1 transport mediated by (Na1-K1)ATPase or Na1 /H1 exchanger of rat liver plasma membrane vesicles. Eur. J. Biochem. 193:541–549;1990. 10. Lynch, C.J.; Wilson, P.B.; Blackmore, P.F.; Exton, J.H. The hormone-sensitive Na1 pump: evidence for regulation by diacylglycerol and tumor promoters. J. Biol. Chem. 261:14551– 14556;1986. 11. Henderson, R.M.; Graf, J.; Boyer, J.L. Na-H exchange regulates intracellular pH in isolated rat hepatocytes couplets. Am. J. Physiol. 252:G109–G113;1987. 12. Renner, E.L.; Lake, J.R.; Persico, M.; Scharchmidt, B.F. Na1H1 exchange activity in rat hepatocytes: role in regulation of intracellular pH. Am. J. Physiol. 256:G44–G52;1989. 13. Oxender, D.L.; Christensen, H.N. Evidence for two types of mediation of neutral amino acid transport in Erlich cells. J. Biol. Chem. 238:3686–3699;1963. 14. Ingebresten, W.R.; Wagle, S.R. A rapid method for the isolation of large quantities of rat liver parenchymal cells with high anabolic rates. Biochem. Biophys. Res. Commun. 47:403– 410;1972. 15. Kreydiyyeh, S.I.; Baydoun, E.A.; Churukian, Z. Tea extract inhibits intestinal absorption of glucose and sodium in rats. Comp. Biochem. Physiol. 108C:359–365;1994. 16. Taussky, H.H.; Shorr, E. Microcolorimetric method for deter-

Mechanism of Action of Insulin in Hepatocytes

mination of inorganic phosphorous. J. Biol. Chem. 202:675– 685;1953. 17. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265–275;1951. 18. Heinz, A.; Jackson, J.W.; Richey, B.E.; Sachs, G.; Schafer, J.A. Amino acid active transport and stimulation by substrates in the absence of a Na1 electrochemical potential gradient. J. Membr. Biol. 62: 149–160;1981. 19. Fitz, J.G.; Trouillot, T.E.; Scharschmidt, B.F. Effect of pH on membrane potential and K1 conductance in cultured rat hepatocytes. Am. J. Physiol. 257:G961–G968;1989.

253

20. Gleeson, D.; Smith, N.D.; Boyer, J.L. Bicarbonate-dependent and -independent intracellular pH regulatory mechanisms in rat hepatocytes. Evidence for Na1 -HCO32 . J. Clin. Invest. 84: 312–321;1989. 21. Weintraub, W.H.; Machen, I.E. pH regulation in hepatoma cells: Roles for Na1-H1 exchange, Cl2 -HCO32 cotransport. Am J. Physiol. 257:G317–337;1989. 22. Meier, P.J.; Knickelbein, R.; Moseley, R.H.; Dobbins, J.W.; Boyer, L. Evidence for carrier-mediated chloride/bicarbonate exchange in canalicular rat liver plasma membrane vesicles. J. Clin. Invest. 75:1256 –1263;1985.