Alanine uptake activates hepatocellular chloride channels

Steven D. Lidofsky and Richard M. Roman

Department of Medicine and The Liver Center, University of California, San Francisco, California 94143-0538; and Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Cells involved in the retrieval and metabolic conversion of amino acids undergo significant increases in size in response to amino acid uptake. The resultant adaptive responses to cell swelling are thought to include increases in membrane K+ and Cl- permeability through activation of volume-sensitive ion channels. This viewpoint is largely based on experimental models of hypotonic swelling, but few mammalian cells experience hypotonic challenge in vivo. Here we have examined volume regulatory responses in a physiological model of cell-swelling alanine uptake in immortalized hepatocytes. Alanine-induced cell swelling was followed by a decrease in cell volume that was temporally associated with an increase in membrane Cl- currents. These currents were dependent both on alanine concentration and Na+, suggesting that currents were stimulated by Na+-coupled alanine uptake. Cl- currents were outwardly rectifying, exhibited an anion permeability sequence of I- > Br- > Cl-, and were inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid, features similar to those reported for a widely distributed class of volume-sensitive anion channels evoked by experimental hypotonic stress. These findings suggest that volume-sensitive anion channels participate in adaptive responses to amino acid uptake and provide such channels with a new physiological context.

amino acids; cell volume; ion channels; liver

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

AMINO ACID UPTAKE presents a ubiquitous challenge to cell volume regulation. This is particularly true in hepatocytes, which extract nutrients absorbed from the intestine and provide metabolic substrates for the remainder of the organism. Hepatocytes respond to the osmotic challenges of amino acid uptake through adaptive mechanisms that serve to restore cell volume toward its resting state. Most information concerning hepatocellular volume regulation is derived from experiments involving hypotonic stress. Swelling of hepatocytes induced by hypotonic media is associated with increases in K+ and Cl- conductance, consistent with opening of K+ and Cl- channels, respectively (9, 14, 20, 21, 25). The ensuing efflux of electrolytes and water through these channels is thought to decrease hepatocellular volume toward basal values.

Although hypotonic challenge has been an excellent experimental model for the study of volume recovery after cell swelling, hepatocytes do not generally face hypotonic conditions in vivo. Rather, the major physiological stimulus for hepatocyte swelling is concentrative uptake of solutes such as amino acids (11). Studies at the whole organ level suggest that the volume regulatory responses to amino acid uptake resemble the responses to hypotonic challenge; that is, recovery from cell swelling is associated with a net loss of K+ and Cl- (6, 13). At the cellular level, the characteristics of these responses are less certain.

For example, Na+-coupled alanine uptake, which is known to produce hepatocyte swelling (3, 16), activates the Na+-K+ pump (4, 23), stimulates opening of K+ channels (1), and leads to a sustained membrane hyperpolarization (5, 24). Membrane hyperpolarization is thought to promote Cl- efflux (6, 24). However, it is not known whether Cl- efflux occurs passively through constitutive "leak" pathways or whether it occurs through regulated ion channels. We have therefore explored this question in further detail. Here we show in HTC cells, a model liver cell line (25), that alanine uptake produces cell swelling that is associated with an increase in membrane current, consistent with activation of Cl- channels. Moreover, the outward rectification, anion permeability, and pharmacological properties of this current resemble those of cell swelling-induced Cl- currents produced by hypotonic challenge. These results suggest that recruitment of volume-sensitive Cl- channels provides a potent mechanism for hepatocytes to respond to the osmotic stress of amino acid uptake.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell culture. HTC rat hepatoma cells were grown in minimal essential medium supplemented with 5% fetal calf serum and 2 mM glutamine, penicillin, and streptomycin. They were maintained at 37°C in a humidified 5% CO2 atmosphere and passaged weekly.

Measurement of cell volume. Mean cell volume (MCV) was determined in HTC cell suspensions by electronic cell sizing using a Coulter multisizer as previously described (25). Briefly, ~107 cells grown on polystyrene flasks were harvested with 0.05% trypsin, suspended in cell culture medium, and centrifuged for 1 min at 2,000 g. Cells were then resuspended in 3 ml of a standard bath solution that contained (in mM) 140 NaCl, 4 KCl, 1 KH2PO4, 2 MgCl2, 1 CaCl2, 10 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, pH 7.4). After a 45-min period of gentle agitation, a 500-µl aliquot of the cell suspension was added to 20 ml of standard solution.

MCV was determined from groups of 105 cells at intervals of 0 (basal), 1, 2, 3, 4, 5, 10, 15, and 20 min after addition of a concentrated aliquot of L-alanine to the final 20-ml suspension to achieve a final concentration of 5 mM. In selected experiments, isosmotic substitutions were made in which 140 mM N-methyl-D-glucamine-chloride (NMDG-Cl) replaced NaCl in the standard solution. Changes in cell size over time were expressed as relative volume by normalizing MCV to basal MCV, where relative volume has been reported as mean ± SE.

Measurement of membrane currents. Membrane currents were measured in HTC cells using patch-clamp recording techniques in the whole cell configuration as previously described (17). Selected current segments were digitized and stored on computer for subsequent analysis with pClamp software. Cells were studied on tissue culture dishes (1-ml volume) ~24 h after plating. All measurements were performed at room temperature.

The standard bath solution was identical to that used for cell volume measurements described previously. In selected experiments, isosmotic substitutions were made in which 1) 140 mM NMDG-Cl replaced 140 mM NaCl, 2) 140 mM Na-gluconate and 4 mM K-gluconate replaced 140 mM NaCl and 4 mM KCl, respectively, 3) 140 mM NaI and 4 mM KI replaced 140 mM NaCl and 4 mM KCl, respectively, or 4) 140 mM NaBr and 4 mM KBr replaced 140 mM NaCl and 4 mM KCl, respectively.

The standard pipette solution contained (in mM) 10 NaCl, 130 KCl, 2 MgCl2, 0.5 CaCl2, 1 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 10 HEPES (pH 7.2). Under these conditions, using the standard bath solution, the current at 0 mV is near the reversal potential (Vrev) for Cl-, and the current at -80 mV is near the Vrev for K+ (7). In selected experiments, isosmotic substitutions were made in which 1) 10 mM NMDG-Cl replaced 10 mM NaCl or 2) 10 mM Na-gluconate and 130 mM K-gluconate replaced 10 mM NaCl and 130 mM KCl, respectively.

Whole cell currents were monitored with the membrane potential (Vm) held at -40 mV and transiently stepped to 0 and -80 mV (corresponding to K+ and Cl- currents, respectively) at 10-s intervals. Current-voltage relationships were obtained by measuring whole cell currents under conditions in which Vm was transiently stepped from -100 to +100 mV in 20-mV increments. This was performed under basal conditions and 7-10 min after the addition of a concentrated aliquot of alanine (5-50 µl) to the culture dish. In selected experiments, measurements were performed in the presence of the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)- benzoic acid (NPPB, Calbiochem).

In experiments involving anion substitution with Br- and I-, relative permeabilities were calculated from the Vrev of membrane currents, using the Goldman-Hodgkin-Katz equation with the simplifying assumption that Na+ permeability was negligible under these conditions
<IT>V</IT><SUB>rev</SUB> = <FR><NU><IT>RT</IT></NU><DE><IT>F</IT></DE></FR> ln <FENCE><FR><NU>Cl<SUB>i</SUB> + &agr;K<SUB>o</SUB> + &bgr;X<SUB>i</SUB></NU><DE>Cl<SUB>o</SUB> + &agr;K<SUB>i</SUB> + &bgr;X<SUB>o</SUB></DE></FR></FENCE>
where R is the gas constant, T is the absolute temperature, and F is the Faraday constant; Cli, Clo, Ki, Ko, Xi, and Xo refer to the intra- and extracellular concentrations of Cl-, K+, and permeant anion X-, respectively; and alpha  and beta  refer to the relative permeabilities PK/PCl and PX/PCl, respectively. Vrev was estimated from the linear portion of the current-voltage relationship (between -80 mV and +20 mV), obtained 7-10 min after alanine exposure. All results are reported as means ± SE.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Alanine uptake produces cell swelling. We first determined the effect of alanine on HTC cell volume. Under basal conditions, cell volume averaged 2,821 ± 60 µm3 (n = 20). Exposure to 5 mM alanine produced a prompt (~7%) increase in cell volume, peaking in 2 min. Cell volume subsequently decreased (Fig. 1). By 20 min after alanine exposure, the change in cell volume from basal values decreased to ~60% of its maximum. When the impermeant cation NMDG was substituted for Na+, basal cell volume was not significantly affected (2,854 ± 54 µm3, n = 20). However, alanine-induced cell swelling was abolished under these conditions (Fig. 1). These results suggest that the increases in cell volume were attributable to Na+-coupled alanine uptake.


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Fig. 1.   Effect of alanine on cell volume. Mean cell volume was measured in HTC cells as described in METHODS. Relative cell volume was determined, with respect to basal conditions, as a function of time after exposure to 5 mM alanine, where relative cell volume equals 1.0 under basal conditions. Measurements were performed in the presence or absence [N-methyl-D-glucamine (NMDG) substitution] of Na+ or in the presence of the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 10 µM). Each point corresponds to mean ± SE of 20 groups of cell suspensions.

Prior studies have suggested that activation of Cl- channels is important in hepatocellular volume recovery from hypotonic swelling (9, 20, 25). We therefore examined the effect of Cl- channel blockade on volume recovery from alanine-induced cell swelling. Although exposure of HTC cells to the Cl- channel blocker NPPB (10 µM) did not affect the increase in cell volume induced by alanine, NPPB completely inhibited cell volume recovery (Fig. 1). These data support a role for Cl- channel activation in volume recovery from cell swelling induced by alanine uptake.

Alanine uptake stimulates membrane currents. To determine if Cl- channels are activated in response to alanine uptake, we examined the effect of alanine exposure on membrane currents. Under basal conditions, whole cell currents were small in magnitude (Fig. 2). Exposure to 5 mM alanine was associated with the development of inward membrane currents. This occurred within 3-5 min after alanine exposure and reached steady state between 7 and 10 min. The current-voltage relationships for basal and steady-state currents are shown in Fig. 2C. Steady-state currents were outwardly rectifying, a property similar to that described for swelling-induced currents in HTC cells produced by hypotonic challenge (25). Currents reversed polarity near 0 mV, suggesting that the currents were predominantly carried by Cl- rather than by K+ (Table 1). Moreover, steady-state currents increased in a dose-dependent fashion with alanine concentration (Fig. 3). These results suggested that alanine uptake stimulated Cl- currents.


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Fig. 2.   Effect of alanine on membrane currents. Whole cell currents were measured in HTC cells as described in METHODS. A: time course of membrane currents after alanine exposure. Currents were measured with membrane potential (Vm) held at -40 mV and transiently stepped to 0 mV and -80 mV, corresponding to upward and downward deflections, respectively. Arrow indicates time at which cell under study was exposed to 5 mM alanine. B: higher resolution recordings of currents under basal conditions and 8 min after exposure to alanine. Here Vm was transiently stepped from -100 to +100 mV in 20-mV increments. C: current-voltage relationship for basal conditions and 7-10 min after alanine exposure. Data correspond to means ± SE of 6 cells each.

                              
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Table 1.   Anion selectivity of alanine-stimulated membrane currents


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Fig. 3.   Relationship between current magnitude and alanine concentration. Membrane currents at -80 mV (reversal potential for K+) were measured 7-10 min after alanine exposure, and absolute values of currents were normalized to cell capacitance in picofarads (pF). Each point corresponds to mean ± SE of 6 cells.

Alanine-stimulated currents are sodium and chloride dependent and blocked by NPPB. If alanine stimulated membrane currents as a result of Na+-coupled alanine uptake and subsequent Cl- channel opening, it would be expected 1) that such currents would be abolished by removal of either Na+ or Cl- and 2) that currents would be inhibited by the Cl- channel blocker NPPB. To test the first set of predictions, two sets of ion substitutions were made. First, replacement of Na+ by the impermeant cation NMDG markedly inhibited alanine-stimulated membrane currents (Fig. 4). Second, replacement of Cl- by the impermeant anion gluconate also inhibited alanine-stimulated membrane currents (Fig. 4). In addition, alanine-stimulated membrane currents were significantly inhibited in the presence of 10 µM NPPB (Fig. 4). The Na+ and Cl- dependence of alanine-stimulated membrane currents and their pharmacological inhibition by NPPB are consistent with the opening of Cl- channels in response to Na+-coupled alanine uptake.


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Fig. 4.   Alanine-stimulated currents are dependent on Na+ and Cl- and blocked by NPPB. Currents at -80 mV were measured 7-10 min after exposure to 5 mM alanine, and absolute values of currents were normalized to cell capacitance. Experiments were performed in presence of Na+ and Cl- (control), NMDG-Cl (Na+ free), Na- and K-gluconate (Cl- free), and in presence of Cl- channel blocker NPPB (10 µM) as described in METHODS. Data correspond to means ± SE of 6 cells for each condition.

Anion selectivity of alanine-stimulated membrane currents. Cl- currents activated by hepatocellular swelling evoked by hypotonic challenge exhibit an anion permeability sequence of I- > Br- > Cl- (20). If alanine-stimulated currents were activated in response to cell swelling, it would be anticipated that they would display a similar permeability sequence. We therefore evaluated the selectivity of alanine-stimulated currents. In these experiments, anion substitutions were made in which either I- or Br- replaced Cl-, and the effects of these maneuvers on alanine-stimulated membrane currents were observed. Each of these substitutions shifted Vrev in the direction expected for anion-selective currents (Table 1). The anion selectivity of these currents was I- > Br- > Cl- (Table 1).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Previous work has demonstrated that concentrative uptake of amino acids such as alanine produces hepatocyte swelling, hyperpolarizes the membrane (through activation of the Na+-K+ pump and K+ channels), and stimulates Cl- efflux (1, 3-5, 23, 24). Although hepatocytes exhibit a high resting membrane permeability to Cl- (8), our results suggest that alanine-stimulated Cl- efflux is not simply constitutive. Rather, our data indicate that regulatable Cl- channels appear to be involved as well. Efflux of Cl- through the latter is likely to amplify the cellular response to alanine-induced cell swelling to restore hepatocyte volume toward basal levels. Furthermore, our results suggest that Cl- channel activation is a consequence of cell swelling. This interpretation is based on several findings.

First, exposure to alanine rapidly produced HTC cell swelling that was followed by recovery of cell volume toward its resting state. Alanine-induced cell swelling was Na+-dependent, suggesting that it was attributable to Na+-coupled solute uptake. Although the degree of cell swelling evoked by alanine was less than that seen with hypotonic challenge, the time course of cell volume recovery was similar (25). This is compatible with the concept that volume regulatory mechanisms in response to alanine uptake and hypotonicity share common features.

Second, in parallel studies, alanine increased membrane currents in HTC cells. The magnitude of the currents increased as a function of alanine concentration, and the currents were Na+-dependent and thus consistent with current stimulation by Na+-coupled alanine uptake. In addition, the ablation of alanine-induced membrane currents by Cl- removal or exposure to the Cl- channel blocker NPPB suggested that these currents resulted from opening of Cl- channels. Taken together, these data imply that alanine uptake stimulated Cl- currents.

Third, the onset of alanine-induced Cl- currents was delayed with respect to the onset of cell swelling, and the time course of the increase in membrane currents paralleled that of cell volume recovery. These findings suggest that alanine-induced cell swelling preceded the increase in Cl- currents and could thus serve as a stimulus for current stimulation. Moreover, they imply that opening of Cl- channels is associated with cell volume recovery. This is supported by the observation that the Cl- channel blocker NPPB inhibited volume recovery from alanine-induced cell swelling.

Fourth, the time course and properties of alanine-induced Cl- currents resembled those of Cl- currents activated by cell swelling in response to hypotonic stress (25). Like volume-sensitive anion channels, alanine-stimulated Cl- currents were outwardly rectifying (20, 25), they were blocked by NPPB (20), and they exhibited an anion permeability sequence of I- > Br- > Cl- (20). Collectively, our data support a model in which 1) Na+-coupled alanine uptake produces cell swelling, 2) cell swelling stimulates volume-sensitive anion channels, and 3) Cl- efflux through such channels contributes to cell volume recovery.

Na+-coupled amino acid transport is widespread among mammalian tissues (19), and it would thus be expected to represent an osmotic challenge to cell types other than hepatocytes. Indeed, alanine-induced cell swelling has been documented in both kidney and intestine, where recovery from cell swelling is associated with increases in membrane permeability to K+ and anions (2, 18). This is analogous to the cellular response to hypotonic challenge. Although it is well known that hypotonic challenge increases membrane Cl- permeability through volume-sensitive anion channels in many cell types (22), the mechanisms involving amino acid-induced increases in membrane Cl- permeability have had limited study. Single channel data in Ehrlich ascites cells indicate that glycine exposure results in opening of 23 pS Cl- channels with conductances similar to those stimulated by hypotonic challenge, suggesting that the mechanism by which amino acids activate Cl- channels is through cell swelling (15). The results reported here suggest that this phenomenon may be a general one. Moreover, they provide a new context for volume-sensitive anion channels, because most mammalian cells are not subjected to hypotonic challenge in vivo.

Activation of hepatocellular Cl- channels by alanine may have implications for physiological processes beyond that of liver cell volume homeostasis. For example, one intriguing possibility is in regulation of bile flow. It is well known that bile flow increases with eating. This may be related in part to dietary absorption of amino acids, which are carried to hepatocytes via the portal vein. In support of this concept, experimental infusion of physiological (mM) concentrations of alanine and other amino acids into the portal vein increases both cell volume and bile output (10, 12). The present study provides a potential basis for this observation at the cellular level, because stimulation of Cl- channels could promote fluid secretion into the bile canaliculus. Thus activation of Cl- channels by amino acids may not simply represent a mechanism for control of liver cell volume but may also serve to couple nutritional signals to stimulation of bile flow. By extension, such channels may also coordinate the regulation of organ-specific functions by amino acid uptake in other cell types.

    ACKNOWLEDGEMENTS

This work was supported in part by grants from the American Diabetes Association, the National Institute of Diabetes and Digestive and Kidney Diseases (DK-47849), and the University of California, San Francisco, Huntington Research Fund.

    FOOTNOTES

Address for reprint requests: S. D. Lidofsky, GI Division, Burgess Building, MCHV Campus, Univ. of Vermont, Burlington, VT 05401.

Received 2 April 1997; accepted in final form 23 June 1997.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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AJP Gastroint Liver Physiol 273(4):G849-G853
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