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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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METHODS |
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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(
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RESULTS |
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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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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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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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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).
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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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