From the Departments of Pediatrics and Medicine, Children's Hospital and the University of Colorado Health Sciences Center, Denver, Colorado 80262 and the § Department of Physiology and Biophysics, University of Alabama, Birmingham, Alabama 35294
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ABSTRACT |
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Regulated changes in cell volume represent a
signal that modulates a broad range of cell and organ functions. In HTC
hepatoma cells, increases in volume are coupled to membrane ion
permeability through a pathway involving (i) ATP efflux, (ii) autocrine
stimulation of P2 receptors, and (iii) increases in
anion permeability and Cl efflux, contributing to
recovery of volume toward basal values. Based on recent evidence that
cell volume increases also stimulate phosphoinositide kinases, the
purpose of these studies was to determine if phosphatidylinositol
3-kinase (PI 3-kinase) modulates these pathways. Exposure of cells to
hypotonic buffer (20 or 40% less NaCl) caused an initial increase in
cell volume and stimulated a rapid increase in ATP release. Subsequent
opening of Cl
channels was followed by recovery of cell
volume toward basal values, despite the continuous presence of
hypotonic buffer. Inhibition of PI 3-kinase with wortmannin
(Ki = 3 nM) significantly inhibited
both the rate of volume recovery and activation of Cl
currents; similar results were obtained with LY294002 (10 µM). Additionally, current activation was inhibited by
intracellular dialysis with antibodies specific for the 110-kDa
catalytic subunit of PI 3-kinase. Since release of ATP is a critical
element in the volume-regulatory pathway, the role of PI 3-kinase on
volume-stimulated ATP release was assessed. Both wortmannin and
LY294002 decreased basal and volume-stimulated ATP permeability but had
no effect on the current response to exogenous ATP (10 µM). These findings indicate that PI 3-kinase plays a
significant role in regulation of cell volume and suggest that the
effects are mediated in part through modulation of cellular ATP
release.
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INTRODUCTION |
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Polyphosphoinositides and their metabolites represent novel intracellular signaling molecules recently shown to mediate cellular responses to a number of hormones and growth factors (1-4). Activation of phosphatidylinositol (PI)1 3-kinase leads to phosphorylation of phosphatidylinositol at the D-3 position of the inositol ring, representing a distinct pathway of PI metabolism. The 3-phosphorylated lipids rapidly increase upon growth factor stimulation, suggesting that they may act as second messengers mediating PI 3-kinase signals (2, 5-7). However, the function and the targets of these lipid products are not fully known.
PI 3-kinase is a heterodimer composed of a 110-kilodalton catalytic peptide and an 85-kilodalton regulatory peptide, which are tightly associated (1, 8, 9). This protein has been purified from rat liver (10), and PI 3-kinase activity has been shown to increase in response to a number of hormonal and growth factor stimuli, including insulin, platelet-derived growth factor, insulin-like growth factor, epidermal growth factor, colony-stimulating factor, and hepatocyte growth factor (2, 11, 12). Although the physiologic role of PI 3-kinase and its lipid products has not been completely defined, it has been implicated in such diverse processes as cellular growth and transformation (12, 13), glucose uptake and transport (14-16), membrane ruffling (17, 18), actin rearrangement (14, 19), and vesicular trafficking (20-22).
Recently, physiologic increases in cell volume have also been shown to be a potent stimulus for PI 3-kinase activation (23). Regulation of cell volume is mandatory for maintenance of cellular integrity; in addition, the hydration state may represent a means of coupling changes in membrane transport to other organ level functions. In hepatocytes, for example, cell volume increases reproduce many of the metabolic effects of insulin, including stimulation of bile acid secretion, glycogen, and protein synthesis, and gene expression (24, 25). These and other observations have led to the concept that changes in cell volume per se may represent a signal regulating liver function (26, 27).
In model liver cells, increases in cell volume stimulate an adaptive
response that involves opening of membrane Cl channels
through an ATP-dependent mechanism (Fig.
1). The resulting efflux of
Cl
favors water loss and recovery of cell volume toward
basal values. Interestingly, liver cell volume increases stimulate
parallel activation of multiple kinases, including PI 3-kinase,
tyrosine kinase, and mitogen-activated protein kinases (1, 23, 28). However, little is known regarding the cellular site(s) of action of
these kinases, and the cellular signals involved in
volume-dependent Cl
channel regulation in
liver have not been defined. Consequently, the purpose of these studies
was to assess the potential role of PI 3-kinase in recovery from cell
swelling and in cell volume-dependent changes in membrane
ion permeability.
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EXPERIMENTAL PROCEDURES |
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Cell Culture--
All studies were performed in HTC cells, a
model rat hepatoma cell line that expresses ion channels and purinergic
receptors similar to those found in primary rat hepatocytes. Increases
in HTC cell volume stimulated by uptake of alanine (29) or exposure to
hypotonic buffer (30, 31) are followed by ATP efflux, receptor activation, and opening of membrane Cl channels (30, 33).
Cells were passaged at weekly intervals and maintained in
HCO3
-containing minimal essential
medium (Life Technologies, Inc.) supplemented with 10%
heat-inactivated fetal bovine serum, L-glutamine (2 mM), penicillin (100 IU/ml), and streptomycin
(100 µg/ml) as described previously (34).
Measurement of Cl Currents--
Membrane
Cl
currents were measured using whole-cell patch clamp
techniques (35, 36). Cells on a coverslip were mounted in a chamber
(volume ~400 µl) and perfused at 4-5 ml/min with a standard
extracellular solution containing 140 mM NaCl, 4 mM KCl, 1 mM CaCl2, 2 mM
MgCl2, 1 mM
KH2PO4, 10 mM glucose, and 10 mM HEPES/NaOH (pH ~7.40). The standard intracellular
(pipette) solution for whole-cell recordings contained 130 mM
KCl, 10 mM NaCl, 2 mM MgCl2,
10 mM HEPES/KOH, 0.5 mM CaCl2 and 1 mM EGTA (pH 7.3), corresponding to a free
[Ca2+] of ~100 nM (37). Patch pipettes were
pulled from Corning 7052 glass and had a resistance of 3-10 megaohms.
Recordings were made with an Axopatch ID amplifier (Axon Instruments,
Foster City, CA) and were digitized (1 kHz) for storage on a computer
and analyzed using pCLAMP version 6.0 programs (Axon Instruments,
Burlingame, CA) as described previously (36, 38). Pipette voltages
refer to the bath. Current-voltage relations were measured between
120 and +100 mV in 20-mV increments (400-ms duration, 2 s
between test potentials). In the whole-cell configuration, pipette
voltage corresponds to the membrane potential, and upward
deflections of the current trace indicate outward membrane current.
Changes in membrane Cl
permeability were assessed at a
test potential of
80 mV (Ek) to minimize any
contribution of K+ currents (30).
Cell Size Measurements--
Mean cell volume was measured in
cell suspensions by electronic cell sizing (Coulter Multisizer,
Accucomp software version 1.19, Hialeah, FL) using an aperture of 100 µm. Cells in subconfluent culture were harvested with 0.05% trypsin,
suspended in cell culture medium, centrifuged for 1 min at ~1000 × g, resuspended in 3 ml of isotonic buffer, and incubated
with gentle agitation for 30-45 min. Aliquots (~500 µl) of cell
suspension were added to 20 ml of isotonic or hypotonic (40% less
NaCl) buffer. Measurements of ~20,000 cells at specified time points
after exposure to isotonic or hypotonic buffer were compared with basal
values (time 0). Changes in values are expressed as relative volume
normalized to the basal period. As a measure of volume recovery, the
percentage of regulatory volume decrease was calculated as (peak
relative volume at 3 min relative volume at measured time
point)/(peak relative volume
1) × 100. Experimental reagents
were added as indicated.
ATP Bioluminescence Assay-- Cells were grown to confluence in a 35-mm dish, washed twice with phosphate-buffered saline, and incubated with Opti-MEM I reduced serum medium plus luciferase-luciferin reagent (2 mg/ml, lyophilized reagent; Sigma). The dish was placed on a platform, lowered into the recording chamber of a Turner model TD20/20 luminometer, and studied immediately in real time. Since background luminescence (cells and medium without luciferase-luciferin reagent) is less than 0.1 arbitrary light unit (ALU), ATP released from cells into the media catalyzes the luciferase-luciferin reaction. Bioluminescence was measured in continuous 15-s photon collection intervals. To induce cell volume increases, the extracellular buffer was diluted 20 or 40% as indicated by the addition of water. In control studies, an equal volume of isotonic buffer was added to assess possible ATP release due to mechanical stimulation (39). The small changes in bioluminescence associated with isotonic exposures were <10% of values associated with hypotonic exposure (data not shown).
Reagents--
Wortmannin (Sigma) and LY294002 (Calbiochem) were
used as PI 3-kinase inhibitors (40-42). For all studies with
wortmannin and LY294002, cells were preincubated with the respective
inhibitor for 10 min prior to hypotonic exposure. In separate patch
clamp studies, PI 3-kinase was inhibited by intracellular dialysis with a purified rabbit polyclonal antibody recognizing a sequence
corresponding to residues 1054-1068 of the 110-kilodalton
-catalytic subunit of PI 3-kinase (Upstate Biotechnology, Inc.)
(43). Polyclonal rabbit antibody to
-galactosidase was utilized as a
control antibody (5 Prime
3 Prime, Inc., Boulder, CO). ATP and
other reagents were obtained from Sigma.
Statistics-- Results are presented as the mean ± S.E., with n representing the number of cells for patch clamp studies and the number of culture plates or repetitions for other assays. Student's paired or unpaired t test was used to assess statistical significance as indicated, and p values < 0.05 were considered to be statistically significant.
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RESULTS |
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Inhibition of PI 3-Kinase Delays Cell Volume Recovery from Swelling-- Exposure of cells to hypotonic buffer (40% decrease in NaCl, ~205 mosM), caused a rapid initial increase in relative volume to 1.18 ± 0.01 (n = 5, p < 0.001) within 3 min. The increase was followed by gradual recovery toward basal values despite the continued exposure to hypotonic buffer (Fig. 2). To evaluate whether PI 3-kinase contributes to cell volume recovery, analogous studies were performed in the presence of wortmannin. Preincubation with wortmannin (50 nM) resulted in a greater initial volume increase to 1.24 ± 0.01 immediately after hypotonic exposure and caused significant inhibition of volume recovery at all subsequent time points. The relative volume of 1.14 ± 0.01 at 30 min in the presence of wortmannin significantly exceeded control values of 1.07 ± 0.01 (n = 5, p < 0.001, Fig. 2). These findings indicate that inhibition of PI 3-kinase impairs recovery from cell swelling.
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Effect of Kinase Inhibition on Cl Efflux--
In HTC
cells, cell volume recovery from swelling depends in part upon opening
of Cl
channels in the plasma membrane. To assess whether
PI 3-kinase contributes to channel opening, whole-cell currents were
measured under basal conditions and following cell volume increases
induced by hypotonic exposure.
ICl
was measured at a test
potential of
80 mV and values were reported as current density (pA/pF) to normalize for differences in cell size as recently described (30). Results are compared with control studies (basal and
swelling-induced ICl
) measured
on the same day to minimize any effects of day-to-day variability in
current amplitude.
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Intracellular Dialysis with a Specific Antibody to PI 3-Kinase
Inhibits Volume-activated Cl Currents--
Both
wortmannin and LY294002 are thought to be selective inhibitors of PI
3-kinase, but the potential for inhibition of other kinases cannot be
fully excluded. Consequently, an alternative strategy was used to
assess the specificity of PI 3-kinase using intracellular dialysis with
antibodies to the 110-kDa catalytic subunit of PI 3-kinase. This
antibody has been shown to inhibit growth factor-stimulated PI 3-kinase
activity in cultured fibroblasts (43). For these studies, the
antibodies were delivered to the cell interior by inclusion in the
patch pipette. Intracellular dialysis with anti-PI 3-kinase antibody (5 µg/ml) completely inhibited Cl
currents in response to
hypotonic exposure with a maximal average current density of
2.9 ± 1.0 pA/pF. In contrast, currents during intracellular dialysis with
antibodies to
-galactosidase (5 µg/ml) were similar to controls
(
42.1 ± 5.6 pA/pF, n = 6, p < 0.001; Fig. 5).
These findings support a specific role of PI 3-kinase in
volume-stimulated Cl
channel activation.
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PI 3-Kinase Modulates Swelling-activated ATP Release--
As
depicted in Fig. 1, PI 3-kinase could potentially modulate current
activation and cell volume recovery through stimulation of ATP release,
modulation of P2 receptors, or coupling receptor binding to
channel opening. To assess the site of action of PI 3-kinase, two
strategies were utilized. First, the effect of wortmannin on the
current response to ATP was assessed. For these studies, cells in
hypotonic buffer were exposed to exogenous ATP in the presence of
wortmannin (50 nM). If PI 3-kinase modulates P2
receptors or couples receptor binding to Cl channel
opening, the presence of wortmannin would be expected to inhibit
ATP-dependent current activation. In the presence of wortmannin, hypotonic exposure failed to activate Cl
currents. However, the subsequent addition of ATP (10 µM)
to the perfusate resulted in instantaneous activation of
Cl
currents (representative trace, Fig. 3B),
increasing current density from
1.2 ± 0.4 pA/pF to
37.4 ± 13.1 pA/pF (n = 5, p < 0.05, Fig.
4A). Second, both removal of extracellular ATP and inhibition of PI 3-kinase with wortmannin delay cell volume recovery from swelling in Coulter counter studies. To assess whether these effects are related, additional studies were performed to assess the
rate of recovery from swelling (percentage of regulatory volume decrease) under control conditions in the presence of wortmannin to
inhibit PI 3-kinase and in the presence of wortmannin plus exogenous
ATP added to the extracellular bath. The presence of exogenous ATP (10 µM) bypassed the inhibitory effects of wortmannin and
restored volume recovery with values no different from controls at all
subsequent time points (n = 5 for each time point,
p < 0.01; Fig. 6). These
findings indicate that PI 3-kinase is likely to function more
proximally in the signaling pathway by modulating local ATP
concentrations outside of the cell.
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DISCUSSION |
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In hepatocytes and other epithelial cells, physiologic changes in cell volume are closely coupled to membrane ion permeability and directly modulate a broad range of cell and organ functions (44). For example, increases in cell volume produced by hypotonic exposure mimic many of the effects of insulin, stimulating protein and glycogen synthesis, bile flow, and exocytosis through selective effects on gene and protein expression (1, 23-25). The present studies indicate that PI 3-kinase may play a critical intermediary role in this process and suggest that the effects of PI 3-kinase are mediated in part through regulation of electrodiffusional movement of ATP across the plasma membrane.
PI 3-kinases catalyze phosphoinositol at the D-3 position of the inositol ring, leading to formation of at least three phosphoinositides that are presumed to function as intracellular second messengers (3, 5, 7). Tyrosine kinase-regulated PI 3-kinase is composed of a 110-kilodalton catalytic subunit that binds ATP, and its function is modified by interactions with a separate 85-kilodalton regulatory subunit. Both wortmannin and LY294002 effectively inhibit kinase activity (40-42).
In liver cells, the lipid products of PI 3-kinase are not present under basal conditions. However, exposure to insulin or increases in cell volume lead to rapid kinase activation (23). Inhibition of PI 3-kinase by wortmannin or LY294002 prevents the increases in glycogen synthase activity, acetyl-CoA carboxylase, and bile acid excretion normally caused by cell volume increases, suggesting that PI 3-kinase activation represents one of the signals coupling changes in cell volume to cell metabolism and transport (23, 45). Moreover, results in different models have implicated PI 3-kinase as a modulator of vesicular trafficking, cytoskeletal organization, and bile formation, processes also directly influenced by physiologic changes in cell volume (46).
In these studies of HTC cells, observations using a variety of
techniques support a broader role for activation of PI 3-kinase as an
early and important step coordinating changes in cell volume and
membrane Cl permeability. Inhibition of PI 3-kinase
significantly impairs cell volume recovery after hypotonic exposure and
uncouples cell volume from changes in membrane Cl
permeability.
These findings appear to be specific for PI 3-kinase. The inhibitory effects of wortmannin are detectable in low nanomolar concentrations (Ki ~3 nM) and in individual cells are partially reversible. Moreover, similar inhibitory effects are caused by LY294002, a structurally unrelated compound that also inhibits the ATP binding activity of p110 (41, 47). Despite the potency of these compounds, it is acknowledged that inhibitors can have unanticipated effects on other signaling pathways as well; wortmannin, for example, has recently been shown to inhibit a separate PI 4-kinase in higher concentrations (48, 49). Consequently, an alternative strategy was utilized to inhibit PI 3-kinase by intracellular dialysis with antibodies that bind selectively to the p110 catalytic subunit. These antibodies have previously been shown to block growth factor-stimulated PI 3-kinase effects in cultured fibroblasts (43). When antibodies were allowed to equilibrate with the cell interior by inclusion in the patch pipette, current activation following hypotonic exposure was inhibited. Antibodies unrelated to PI 3-kinase had no effect. These findings are likely to reflect specific inhibition of PI 3-kinase activity through antibody binding to a critical functional site on the cell interior.
Several observations indicate that the effects of PI 3-kinase are
mediated in part through modulation of cellular ATP release. Previous
studies of HTC cells indicate that increases in cell volume lead to
electrodiffusional release of ATP. The localized increase in
extracellular ATP is thought to activate P2 receptors in
the plasma membrane coupled to Cl channels, and the
resulting Cl
efflux contributes to restoration of cell
volume toward basal values (31, 33). In individual cells, exposure to
exogenous ATP (added to the bathing solution) bypasses the inhibitory
effect of wortmannin on swelling-induced current activation; and in
cell suspensions, supplemental ATP partially reverses the inhibitory effect of wortmannin on cell volume recovery from swelling. Thus, wortmannin is not likely to modulate volume regulatory responses through inhibition of P2 receptors or blockade of membrane
Cl
channels.
To assess whether PI 3-kinase is functioning at a more proximal site in the signaling cascade, the effect of cell volume on release of ATP from cells into the supernatant was assessed by a sensitive and specific luminometric assay. This approach has a number of advantages over electrophysiologic methods that are based on detection of currents carried by high (100 mM) concentrations of ATP (33). Specifically, luminometry studies are performed using intact cells maintained in conventional media under conditions where signaling mechanisms are intact, minimizing the potential adverse effects of intracellular dialysis with unphysiologic solutions. In addition, the marked increase in sensitivity of this assay permits detection of ATP in the absence of nucleotidase inhibitors or other agents that might modify ATP availability.
Under basal conditions in isotonic buffer, low levels of ATP were always detectable in supernatant media. Increases in cell volume caused a rapid increase in ATP release, and the magnitude of the response was proportional to the transmembrane osmolar gradient. ATP release was not related to apparent cytotoxicity, since the same maneuvers had no effect on trypan blue exclusion, propidium iodide staining, or lactate dehydrogenase release (data not shown). Moreover, ATP release was inhibited by PI 3-kinase inhibitors, supporting a specific process mediated by signaling events. In the presence of wortmannin or LY294002, both basal release and the response to hypotonic exposure were significantly diminished. The most direct interpretation is that cell volume-dependent activation of PI 3-kinase is necessary for increases in membrane ATP permeability.
These findings are of interest in light of recent evidence that diverse cellular processes are directly regulated by ATP release, metabolism, and binding. Indeed, more than 10 purinergic receptors responding to different nucleotides have been defined by pharmacologic and molecular techniques (32). Regulation of nucleotide release by changes in cell hydration may provide one mechanism for autocrine/paracrine signaling coupling changes in cell volume and other cell and organ functions. If so, several points merit further investigation. First, it is notable that PI 3-kinase inhibitors failed to completely suppress basal or swelling-induced ATP release and did not completely prevent recovery from cell swelling (Fig. 2). These observations imply that additional PI 3-kinase-independent mechanisms are operative as well. Given the important role for PI 3-kinase in regulation of endocytic and transcytotic pathways (46), it is attractive to speculate that ATP release may involve two separate pools of transporters, including transporters in the plasma membrane and those in submembrane vesicles. By interference with vesicle trafficking and cytoskeletal organization, wortmannin could decrease cellular ATP release by preventing insertion of new transporters from submembrane vesicles. Thus, wortmannin would be expected to delay but not eliminate the adaptive responses to cell volume increases. While the present studies do not address these possibilities directly, the findings are consistent with emerging observations in other models.
Second, the initial events that couple cell volume increases to activation of PI 3-kinase remain to be identified. Indeed, identifying the proximal signal(s) mediating volume-dependent cellular processes represents a critical focus for many laboratories, and cell volume is known to cause rapid activation of multiple kinases as well as sustained biochemical and genetic effects (1, 23, 27). Since PI 3-kinase utilizes membrane constituents as a substrate, it is possible that changes in the substrate availability or presentation associated with volume may contribute to kinase activity. However, alternative signals including other kinases or stretch-activated ion channels must be considered as well. It is notable, for example, that tyrosine kinases and other G-protein-coupled receptors have also been shown to regulate PI 3-kinase (2).
Third, there are quantitative differences in the time course and/or
magnitude of the wortmannin-sensitive parameters involved in cell
volume recovery. Wortmannin completely inhibits Cl
current activation in isolated cells, but only partially inhibits ATP
release and cell volume recovery. These differences may be related in
part to the different experimental techniques used. For example,
dialysis of the intracellular space during whole-cell recordings is
likely to alter signal transduction and may prevent actual cell volume
recovery since the volume of the pipette solution is orders of
magnitude greater than the volume of individual cells. While it will be
important to address the relationship between ATP release and volume
recovery in a more quantitative manner, the inhibitory effect of
wortmannin on ATP release, current activation, and volume recovery,
measured using different experimental approaches, supports an important
role for PI 3-kinase in cell volume regulation. Therefore, PI 3-kinase
may be an early and essential signal coupling changes in cell volume to
membrane Cl
permeability through effects on cellular ATP
release. Given the tissue-specific expression of multiple P2 receptor
subtypes, further definition of the mechanisms linking cell volume and
ATP release represents an attractive and previously unrecognized target
for modulation of the diverse cellular processes regulated by PI
3-kinase.
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FOOTNOTES |
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* These studies were supported by Cystic Fibrosis Clinical Fellowship Grant 2533599 (to A. P. F.) and National Institutes of Health Grants DK-43278 and DK-46082 (to J. G. F.).
To whom correspondence should be addressed: Campus Box B-158, Room
6412, University of Colorado Health Sciences Center, 4200 East 9th
Ave., Denver, CO 80262. Tel.: 303-315-2537; Fax: 303-315-5711; E-mail:
drew.feranchak{at}UCHSC.edu.
1 The abbreviations used are: PI 3-kinase, phosphatidylinositol 3-kinase; pF, picofarad(s); ALU, arbitrary light unit.
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REFERENCES |
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