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Phosphatidylinositol 3-Kinase Contributes to Cell Volume Regulation through Effects on ATP Release*

Open AccessPublished:June 12, 1998DOI:https://doi.org/10.1074/jbc.273.24.14906
      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 (K i = 3 nm) significantly inhibited both the rate of volume recovery and activation of Clcurrents; 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.
      Polyphosphoinositides and their metabolites represent novel intracellular signaling molecules recently shown to mediate cellular responses to a number of hormones and growth factors (
      • Schliess F.
      • Schreiber R.
      • Haussinger D.
      ,
      • Kapeller R.
      • Cantley L.C.
      ,
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • Whitman M.
      • Downes C.P.
      • Keeler M.
      • Keller T.
      • Cantley L.C.
      ). Activation of phosphatidylinositol (PI)
      The abbreviations used are: PI 3-kinase, phosphatidylinositol 3-kinase; pF, picofarad(s); ALU, arbitrary light unit.
      1The abbreviations used are: PI 3-kinase, phosphatidylinositol 3-kinase; pF, picofarad(s); ALU, arbitrary light unit.
      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 (
      • Kapeller R.
      • Cantley L.C.
      ,
      • Toker A.
      • Cantley L.C.
      ,
      • Traynor-Kaplan A.E.
      • Harris A.L.
      • Thompson B.L.
      • Taylor P.
      • Sklar L.A.
      ,
      • Spiegel S.
      • Foster D.
      • Kolesnick R.
      ). 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 (
      • Schliess F.
      • Schreiber R.
      • Haussinger D.
      ,
      • Escobedo J.A.
      • Navankasattusas S.
      • Kavanaugh W.M.
      • Milfay D.
      • Fried V.A.
      • Williams L.T.
      ,
      • Hu P.
      • Mondino A.
      • Skolnik E.Y.
      • Schlessinger J.
      ). This protein has been purified from rat liver (
      • Carpenter C.L.
      • Duckworth B.C.
      • Auger K.R.
      • Cohen B.
      • Schaffhausen B.S.
      • Cantley L.C.
      ), 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 (
      • Kapeller R.
      • Cantley L.C.
      ,
      • Cantley L.C.
      • Auger K.R.
      • Carpenter C.L.
      • Duckworth B.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ,
      • Varticovski L.
      • Harrison-Findik D.
      • Keeler M.L.
      • Susa M.
      ). 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 (
      • Varticovski L.
      • Harrison-Findik D.
      • Keeler M.L.
      • Susa M.
      ,
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ), glucose uptake and transport (
      • Martin S.S.
      • Haruta T.
      • Morris A.J.
      • Klippel A.
      • Williams L.T.
      • Olefsky J.M.
      ,
      • Katagiri H.
      • Asano T.
      • Inukai K.
      • Ogihara T.
      • Ishihara H.
      • Shibasaki Y.
      • Murata T.
      • Terasaki J.
      • Kikuchi M.
      • Yazaki Y.
      • Oka Y.
      ,
      • DePaolo D.
      • Reusch J.
      • Carel K.
      • Bhuripanyo P.
      • Leitner J.W.
      • Draznin B.
      ), membrane ruffling (
      • Wennstrom S.
      • Siegbahn A.
      • Yokote K.
      • Arvidsson A.K.
      • Heldin C.H.
      • Mori S.
      • Claesson W.L.
      ,
      • Hawkins P.T.
      • Eguino A.
      • Qiu R.
      • Stokoe D.
      • Cooke F.T.
      • Walters R.
      • Wennstrom S.
      • Claesson-Welsh L.
      • Evans T.
      • Symons M.
      • Stephens L.
      ), actin rearrangement (
      • Martin S.S.
      • Haruta T.
      • Morris A.J.
      • Klippel A.
      • Williams L.T.
      • Olefsky J.M.
      ,
      • Walter R.J.
      • Hawkins P.
      • Cooke F.T.
      • Eguinoa C.
      • Stephens L.R.
      ), and vesicular trafficking (
      • Merlin D.
      • Guo X.
      • Martin K.
      • LaBoisse C.
      • Landis D.
      • Dubyak G.
      • Hopfer U.
      ,
      • Herman P.K.
      • Emr S.D.
      ,
      • Davidson H.W.
      ).
      Recently, physiologic increases in cell volume have also been shown to be a potent stimulus for PI 3-kinase activation (
      • Krause U.
      • Rider M.H.
      • Hue L.
      ). 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 (
      • Lang F.
      • Busch G.L.
      • Volkl H.
      • Haussinger D.
      ,
      • Al-Habori M.
      • Peak M.
      • Thomas T.H.
      • Agius L.
      ). These and other observations have led to the concept that changes in cell volume per se may represent a signal regulating liver function (
      • Haussinger D.
      • Lang F.
      • Gerok W.
      ,
      • Haussinger D.
      ).
      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 (
      • Schliess F.
      • Schreiber R.
      • Haussinger D.
      ,
      • Krause U.
      • Rider M.H.
      • Hue L.
      ,
      • Blommaart E.F.C.
      • Luiken J.J.F.P.
      • Blommaart P.J.E.
      • van Woerkom G.M.
      • Meijer A.J.
      ). 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.
      Figure thumbnail gr1
      Figure 1Autocrine signaling by ATP release contributes to cell volume regulation. A proposed model of Cl channel activation in HTC cells is shown, involving (i) ATP release stimulated by increases in cell volume, (ii) P2 receptor binding, and (iii) Cl channel opening. The resulting Cl efflux favors water loss and cell volume recovery.

      DISCUSSION

      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 (
      • Meng X.J.
      • Weinman S.A.
      ). 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 (
      • Schliess F.
      • Schreiber R.
      • Haussinger D.
      ,
      • Krause U.
      • Rider M.H.
      • Hue L.
      ,
      • Lang F.
      • Busch G.L.
      • Volkl H.
      • Haussinger D.
      ,
      • Al-Habori M.
      • Peak M.
      • Thomas T.H.
      • Agius L.
      ). 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 (
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • Toker A.
      • Cantley L.C.
      ,
      • Spiegel S.
      • Foster D.
      • Kolesnick R.
      ). 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 (
      • Arcaro A.
      • Wymann M.P.
      ,
      • Vlahos C.J.
      • Matter W.F.
      • Brown R.F.
      • Traynor-Kaplan A.E.
      • Heyworth P.G.
      • Prossnitz E.R.
      • Ye R.D.
      • Marder P.
      • Schelm J.A.
      • Rothfuss K.J.
      • Serlin B.S.
      • Simpson P.J.
      ,
      • Okado T.
      • Sakuma L.
      • Fukui Y.
      • Hazeki O.
      • Ui M.
      ).
      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 (
      • Krause U.
      • Rider M.H.
      • Hue L.
      ). 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 (
      • Krause U.
      • Rider M.H.
      • Hue L.
      ,
      • Noe B.
      • Schliess F.
      • Wettstein M.
      • Heinricj S.
      • Haussinger D.
      ). 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 (
      • Folli F.
      • Alvaro D.
      • Gigliozzi A.
      • Bassotti C.
      • Kahn C.R.
      • Pontiroli A.E.
      • Capocaccia L.
      • Jezequel A.M.
      • Benedetti A.
      ).
      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 Clpermeability.
      These findings appear to be specific for PI 3-kinase. The inhibitory effects of wortmannin are detectable in low nanomolar concentrations (K i ∼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 (
      • Vlahos C.J.
      • Matter W.F.
      • Brown R.F.
      • Traynor-Kaplan A.E.
      • Heyworth P.G.
      • Prossnitz E.R.
      • Ye R.D.
      • Marder P.
      • Schelm J.A.
      • Rothfuss K.J.
      • Serlin B.S.
      • Simpson P.J.
      ,
      • Vlahos C.J.
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      ). 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 (
      • Nakanishi S.
      • Catt J.K.
      • Balla T.
      ,
      • Wong K.
      • Cantley L.C.
      ). 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 (
      • Roche S.
      • Koegl M.
      • Courtneidge S.A.
      ). 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 (
      • Roman R.M.
      • Wang Y.
      • Lidofsky S.D.
      • Feranchak A.P.
      • Lomri N.
      • Scharschmidt B.F.
      • Fitz J.G.
      ,
      • Wang Y.
      • Roman R.M.
      • Lidofsky S.D.
      • Fitz J.G.
      ). 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 (
      • Wang Y.
      • Roman R.M.
      • Lidofsky S.D.
      • Fitz J.G.
      ). 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 (
      • Harden T.K.
      • Boyer J.L.
      • Nicholas R.A.
      ). 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 (
      • Folli F.
      • Alvaro D.
      • Gigliozzi A.
      • Bassotti C.
      • Kahn C.R.
      • Pontiroli A.E.
      • Capocaccia L.
      • Jezequel A.M.
      • Benedetti A.
      ), 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 (
      • Schliess F.
      • Schreiber R.
      • Haussinger D.
      ,
      • Krause U.
      • Rider M.H.
      • Hue L.
      ,
      • Haussinger D.
      ). 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 (
      • Kapeller R.
      • Cantley L.C.
      ).
      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 Clcurrent 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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