Role In Cell Volume Regulation Research Articles

Salt and osmosensing: role of cytoplasmic hydrogel.

Osmotic perturbations, occurring frequently under physiological and pathological conditions, alter cell size/volume and function. To protect cellular homeostasis, cell osmo- and volume-sensing mechanisms activate volume compensatory processes. The plasma membrane plays a prominent role in cell volume regulation by mediating the selective transport of extra- and intracellular osmolytes. The function of the membrane-enclosed cytoplasm in osmosensing and cell volume homeostasis is much less appreciated. We present current concepts and discuss evidence of cell volume sensors with emphasis on the hydrogel nature of the mammalian cytoplasm and its intrinsic osmosensitivity.

Disruption of NHE8 expression impairs Leydig cell function in the testes.

Multiple sodium/hydrogen exchanger (NHE) isoforms are expressed in the testes, and they play various roles in cell volume regulation, intracellular pH regulation, and fluid absorption. NHE8, the most recently characterized NHE family member, is detected in the Leydig cells in humans and mice in great abundance by immunohistochemistry in the current study. Male mice lacking NHE8 expression were infertile. Despite having intact male reproductive organs, male NHE8-/- mice have smaller testes and lacked spermatozoon in the seminiferous tubules and the epididymis. At the age of 39 wk, few spermogonia were seen in the testis in NHE8-/- mice. Although male NHE8-/- mice have normal serum levels of luteinizing hormone and follicle-stimulating hormone, serum testosterone level was significantly reduced. These mice have decreased expression of luteinizing hormone receptor in the testes. In NHE8 small-interfering RNA-transfected mouse Leydig cells (MLTC-1), silencing of NHE8 decreased the expression of luteinizing hormone receptor by ∼70%. Moreover, loss of NHE8 function in Leydig cells resulted in disorganized luteinizing hormone receptor membrane distribution. Therefore, male infertility in NHE8-/- mice is at least partially due to the disruption of luteinizing hormone receptor distribution and consequent low testosterone production, which leads to Sertoli cell dysfunction. Our work identified a novel role of NHE8 in male fertility through its effect on modifying luteinizing hormone receptor function.

ICln: a new regulator of non-erythroid 4.1R localisation and function.

To optimise the efficiency of cell machinery, cells can use the same protein (often called a hub protein) to participate in different cell functions by simply changing its target molecules. There are large data sets describing protein-protein interactions (“interactome”) but they frequently fail to consider the functional significance of the interactions themselves. We studied the interaction between two potential hub proteins, ICln and 4.1R (in the form of its two splicing variants 4.1R80 and 4.1R135), which are involved in such crucial cell functions as proliferation, RNA processing, cytoskeleton organisation and volume regulation. The sub-cellular localisation and role of native and chimeric 4.1R over-expressed proteins in human embryonic kidney (HEK) 293 cells were examined. ICln interacts with both 4.1R80 and 4.1R135 and its over-expression displaces 4.1R from the membrane regions, thus affecting 4.1R interaction with ß-actin. It was found that 4.1R80 and 4.1R135 are differently involved in regulating the swelling activated anion current (ICl,swell) upon hypotonic shock, a condition under which both isoforms are dislocated from the membrane region and thus contribute to ICl,swell current regulation. Both 4.1R isoforms are also differently involved in regulating cell morphology, and ICln counteracts their effects. The findings of this study confirm that 4.1R plays a role in cell volume regulation and cell morphology and indicate that ICln is a new negative regulator of 4.1R functions.

Tyrosine 284 phosphorylation is required for ClC-3 chloride channel activation in vascular smooth muscle cells

The ClC-3 chloride channel (and current, ICl,ClC-3) plays an important role in cell volume regulation, proliferation, and apoptosis in vascular smooth muscle cells, and is a potential target for prevention of vascular remodelling and stroke. However, modulation of ICl,ClC-3 by intercellular signalling is not fully understood. Although it has been suggested that tyrosine phosphorylation is required for ICl,ClC-3 activation, the potential tyrosine residues in the ClC-3 protein are not clear. In the present study, the critical tyrosine residues in ClC-3 protein were investigated. Site-specific mutagenesis, immunoprecipitation, patch clamp, and Cl(-) transport imaging techniques were employed. We found that activation of ICl,ClC-3 was associated with tyrosine phosphorylation of the ClC-3 protein. Three potential tyrosine residues, Y284, Y572, and Y631, were mutated to phenylalanine, and only mutation, at Y284 within a consensus Src-phosphorylation site, completely blocked ICl,ClC-3. Phosphomimetic mutation Y284D increased the Cl(-) current and Cl(-) efflux mediated by ClC-3. The Y284F mutation completely abolished the protective effect of ClC-3 on apoptosis, whereas the Y284D mutation potentiated it. There was an interaction between Src kinase and ClC-3 protein, and the Y284D mutation abrogated the inhibitory effect of SU6656, a Src family kinase inhibitor, on ClC-3 Cl(-) current. Tyrosine 284 phosphorylation in the ClC-3 channel targeted by Src kinase is an important molecular mechanism for ClC-3 channel activation.

High-throughput fluorescent-based NKCC functional assay in adherent epithelial cells.

BackgroundThe kidney-specific NKCC cotransporter isoform NKCC2 is involved in the Na+ reabsorption in the Thich Ascending Limb (TAL) cells and in the regulation of body fluid volume. In contrast, the isoform NKCC1 represents the major pathway for Cl- entry in endothelial cells, playing a crucial role in cell volume regulation and vascular tone. Importantly, both NKCC isoforms are involved in the regulation of blood pressure and represent important potential drug targets for the treatment of hypertension.ResultsTaking advantage of an existing Thallium (Tl+)-based kit, we set up a Tl+ influx-based fluorescent assay, that can accurately and rapidly measure NKCC transporter activity in adherent epithelial cells using the high-throughput Flex station device. We assessed the feasibility of this assay in the renal epithelial LLC-PK1 cells stably transfected with a previously characterized chimeric NKCC2 construct (c-NKCC2). We demonstrated that the assay is highly reproducible, offers high temporal resolution of NKCC-mediated ion flux profiles and, importantly, being a continuous assay, it offers improved sensitivity over previous endpoint NKCC functional assays.ConclusionsSo far the screening of NKCC transporters activity has been done by 86Rb+ influx assays. Indeed, a fluorescence-based high-throughput screening method for testing NKCC inhibitors would be extremely useful in the development and characterization of new anti-hypertensive drugs.

Uncoupling of K+ and Cl− transport across the cell membrane in the process of regulatory volume decrease

It is accepted that K+ and Cl− flows are coupled tightly in regulatory volume decrease (RVD). However, using self referencing microelectrodes, we proved that K+ and Cl− transport mainly by channels in RVD was uncoupled in nasopharyngeal carcinoma CNE-2Z cells, with the transient K+ efflux activated earlier and sustained Cl− efflux activated later. Hypotonic challenges decreased intracellular pH (pHi), and activated a proton pump-dependent H+ efflux, resulting in a decline of extracellular pH (pHo). Modest decreases of pHo inhibited the volume-activated K+ outflow and RVD, but not the Cl− outflow, while inhibition of H+ efflux or increase of pHo buffer ability promoted K+ efflux and RVD. The results suggest that the temporal dynamics of K+ channel activities is different from that of Cl− channels in RVD, due to differential sensitivity of K+ and Cl− channels to pHo. H+ efflux may play important roles in cell volume regulation, and may be a therapeutic target for human nasopharyngeal carcinoma.

Rapid Aquaporin Translocation Regulates Cellular Water Flow

The control of cellular water flow is mediated by the aquaporin (AQP) family of membrane proteins. The structural features of the family and the mechanism of selective water passage through the AQP pore are established, but there remains a gap in our knowledge of how water transport is regulated. Two broad possibilities exist. One is controlling the passage of water through the AQP pore, but this only has been observed as a phenomenon in some plant and microbial AQPs. An alternative is controlling the number of AQPs in the cell membrane. Here, we describe a novel pathway in mammalian cells whereby a hypotonic stimulus directly induces intracellular calcium elevations through transient receptor potential channels, which trigger AQP1 translocation. This translocation, which has a direct role in cell volume regulation, occurs within 30 s and is dependent on calmodulin activation and phosphorylation of AQP1 at two threonine residues by protein kinase C. This direct mechanism provides a rationale for the changes in water transport that are required in response to constantly changing local cellular water availability. Moreover, because calcium is a pluripotent and ubiquitous second messenger in biological systems, the discovery of its role in the regulation of AQP translocation has ramifications for diverse physiological and pathophysiological processes, as well as providing an explanation for the rapid regulation of water flow that is necessary for cell homeostasis.

Anoctamins

Endogenous Ca(2+)-activated Cl(-) channels (CaCC) demonstrate biophysical and pharmacological properties that are well represented in cells overexpressing anoctamin 1 (Ano 1, TMEM16A), a protein that has been identified recently as CaCC. Proteins of the anoctamin family (anoctamin 1-10, TMEM16A-K) are widely expressed. The number of reports demonstrating their physiological and clinical relevance is quickly rising. Anoctamins gain additional interest through their potential role in cell volume regulation and malignancy. Available data suggest that Ano 1 forms stable dimers and probably liaise with accessory proteins such as calmodulin or other anoctamins. In order to understand how anoctamins produce Ca(2+)-activated Cl(-) currents, it will be necessary to obtain better insight into their molecular structure, interactions with partner proteins, and mode of activation.

Na-K-Cl cotransporter-1 in the mechanism of cell swelling in cultured astrocytes after fluid percussion injury

Brain edema and associated increased intracranial pressure are major consequences of traumatic brain injury (TBI). An important early component of the edema associated with TBI is astrocyte swelling (cytotoxic edema). Mechanisms for such swelling, however, are poorly understood. Ion channels/transporters/exchangers play a major role in cell volume regulation, and a disturbance in one or more of these systems may result in cell swelling. To examine potential mechanisms in TBI-mediated brain edema, we employed a fluid percussion model of in vitro barotrauma and examined the role of the ion transporter Na(+)-K(+)-2Cl(-)-cotransporter 1 (NKCC1) in trauma-induced astrocyte swelling as this transporter has been strongly implicated in the mechanism of cell swelling in various neurological conditions. Cultures exposed to trauma (3, 4, 5 atm pressure) caused a significant increase in NKCC1 activity (21%, 42%, 110%, respectively) at 3 h. At 5 atm pressure, trauma significantly increased NKCC1 activity at 1 h and it remained increased for up to 3 h. Trauma also increased the phosphorylation (activation) of NKCC1 at 1 and 3 h. Inhibition of MAPKs and oxidative/nitrosative stress diminished the trauma-induced NKCC1 phosphorylation as well as its activity. Bumetanide, an inhibitor of NKCC1, significantly reduced the trauma-induced astrocyte swelling (61%). Silencing NKCC1 with siRNA led to a reduction in trauma-induced NKCC1 activity as well as in cell swelling. These findings demonstrate the critical involvement of NKCC1 in the astrocyte swelling following in vitro trauma, and suggest that blocking NKCC1 activity may represent a useful therapeutic strategy for the cytotoxic brain edema associated with the early phase of TBI.

ClC-3 chloride channel prevents apoptosis induced by hydrogen peroxide in basilar artery smooth muscle cells through mitochondria dependent pathway

ClC-3 Cl(-) channel plays an important role in cell volume regulation and cell cycle. In vascular smooth muscle cells, we have found that ClC-3 was involved in ET-1 induced cell proliferation. The present study was designed to further investigate the role of ClC-3 Cl(-) channel in H(2)O(2)-induced apoptosis and its underlying mechanisms in rat basilar arterial smooth muscle cell (BASMCs). By using ClC-3 cDNA and small interference RNA (siRNA) transfection strategy, it was found that overexpression of ClC-3 significantly decreased the apoptotic rate of H(2)O(2)-treated BASMCs and increased the cell viability, whereas silencing of ClC-3 with siRNA produced opposite effects and increased the apoptotic rate. ClC-3 overexpression decreased cytochrome C release and caspase-3 activation, and increased both the stability of mitochondrial membrane potential and the ratio of Bcl-2/Bax, whereas silencing of ClC-3 produced opposite effect. Furthermore, we demonstrated that overexpression of ClC-3 attenuated, whereas silencing of ClC-3 facilitated, the degradation of LaminA, one of the structural matrix proteins, in BASMCs. Our data suggest that ClC-3 Cl(-) channel can modulate H(2)O(2)-induced apoptosis in BASMCs via the intrinsic, mitochondrial pathway.

Caveolin-3 regulates the Volume-Regulated Anion Channel in Mouse Ventricular Cells

Caveolae are small invaginated microdomains located with a variety of signal transduction molecules on the plasma membrane. Recent reports showed that the knockout mice of a muscle-specific protein caveolin-3 (Cav-3 KO), a principal component of the caveolae in heart, displayed an enlargement of ventricular cells. Volume-regulated outwardly rectifying anion channel (VRAC) is activated by membrane stretch, and play a significant role in cell volume regulation in cardiac cells. However, it is unknown the properties of VRAC in the enlarged cardiac cells from Cav-3 KO mice. In this study, we examined that VRAC current and the cell volume regulation in freshly isolated single ventricular cells from Cav-3 KO mice (Hagiwara et al. 2000). Whole-cell current recording showed that the density of VRAC current induced by extracellular hypotonic solution (HYPO) is markedly reduced in the cells from Cav-3 KO mice, compared to that from wild-type (WT) mice. Video-image analysis revealed that the degree of HYPO-induced cell swelling in Cav-3 KO mice is significantly bigger than that in WT mice, and the regulatory volume decrease, which was seen in WT cells after osmotic swelling, is almost lost in cells from Cav-3 KO mice. This result is in parallel with the VRAC inhibition. In contrast, acidic extracellular pH-activated chloride current and extracellular UTP-activated CFTR current were affected less by the deficiency of caveoline-3. The attenuated VRAC current was restored by intracellular application of a VRAC modulator, phosphatidylinositol 3,4,5-trisphosphate (PIP3). These findings suggested that the attenuation of cardiac VRAC current is due to the PIP3 depletion in Cav-3 KO mice.

The C-terminus of ICln is Natively Disordered but Displays Local Structural Preformation

ICln is a vital, ubiquitously expressed protein with roles in cell volume regulation, angiogenesis, cell morphology, activation of platelets and RNA processing. In previous work we have determined the 3D structure of the N-terminus of ICln (residues 1-159), which folds into a PH-like domain followed by an unstructured region (residues H134 – Q159) containing protein-protein interaction sites. Here we present sequence-specific resonance assignments of the C-terminus (residues Q159 – H235) of ICln by NMR, and show that this region of the protein is intrinsically unstructured. By applying ¹³Cα- ¹³Cβ secondary chemical shifts to detect possible preferences for secondary structure elements we show that the C-terminus of ICln adopts a preferred α-helical organization between residues E170 and E187, and exists preferentially in extended conformations (β-strands) between residues D161 to Y168 and E217 to T223.

Angiotensin-(1–7) attenuates hyposmolarity-induced ANP secretion via the Na +–K + pump

The alteration in osmolarity challenges cell volume regulation, a vital element for cell survival. Hyposmolarity causes an increase in cell volume. Recently, it has been reported that the renin–angiotensin system (RAS) plays a role in cell volume regulation. We investigated the effect of angiotensin-(1–7) [Ang-(1–7)] on hyposmolarity-induced atrial natriuretic peptide (ANP) secretion in normal and diabetic (DM) rat atria and modulation of the effect of Ang-(1–7) by the Na +–K + pump. Using isolated control rat atria, we observed that perfusion of hyposmotic solution into the atria increased ANP secretion. When Ang-(1–7) [0.1 μM or 1 μM] was perfused in a hyposmolar solution, it decreased the hyposmolarity-induced ANP secretion in a dose-dependent manner. This effect of Ang-(1–7) could be mediated by the Na +–K + pump, since ouabain, an Na +–K + pump inhibitor, significantly decreased the effect of Ang-(1–7) on hyposmolarity-induced ANP secretion. In contrast, N ω Nitro- l-arginine methyl ester hydrochloride ( l-NAME) did not modify the effect of Ang-(1–7) on the hyposmolarity-induced ANP secretion. Interestingly, the ANP secretion was increased robustly by the perfusion of the hyposmolar solution in the DM atria, as compared to the control atria. However, the inhibitory effect of Ang-(1–7) on the hyposmolarity-induced ANP secretion was not observed in the DM atria. In the DM atria, atrial contractility was significantly increased. Taken together, we concluded that Ang-(1–7) attenuated hyposmolarity-induced ANP secretion via the Na +–K + pump and a lack of Ang-(1–7) response in DM atria may partly relate to change in Na +–K + pump activity.

Kcnmb4−/− mice have decreased tubular dilation under high urinary flow

Our laboratory has shown that with volume expansion, flow through the tubules increased by 10‐fold as Na and water reabsorption was inhibited. How the renal tubules accommodate the very large increases in tubular flow has never been answered. We propose that during high tubular flow, tubular cross‐sectional area (CA) is increased by reducing the size of the cells lining the nephron. The large‐conductance, calcium‐activated K channels (BK) can play a role in cell volume regulation. Furthermore, BK are expressed in several nephron segments including the thick ascending limb (TAL), distal convoluted tubule (DCT), connecting tubule (CNT), and cortical collecting duct (CCD). We placed wild type (WT) and BK‐β4−/− (Kcnmb4−/−) mice on a normal (0.6% K) or K‐adapted (5% K or KA) diet for 10 days and measured tubular CA of the proximal tubule (PCT), TAL, CNT, and CCD. Immunohistochemical analysis with segment specific antibodies revealed that KA mice had significantly increased CA of all tubular segments compared to phenotype matched controls on a normal diet. However, the CA of the TAL, CNT, and CCD of the KA Kcnmb4−/− were significantly less than that of the KA WT mice (TAL: 174.4 vs 203.3; CNT: 278.1 vs 444.0; CCD: 901.6 vs 1401.0 μm2) indicating an inability of the Kcnmb4−/− tubules to fully dilate. No edema was present in the KA kidneys. These results reveal that increased flow leads to tubular dilation by a BK‐β4 dependent mechanism. Supported by NIH/NIDDK grant R01DK71014 (SCS).

Ammonia and proinflammatory cytokines modify expression of genes coding for astrocytic proteins implicated in brain edema in acute liver failure

There is evidence to suggest that, in acute liver failure (ALF), brain ammonia and proinflammatory cytokines may act synergistically to cause brain edema and its complications (intracranial hypertension, brain herniation). However, the molecular mechanisms involved remain to be established. In order to address this issue, semi-quantitative RT-PCR was used to measure the expression of genes coding for astrocytic proteins with an established role in cell volume regulation in cerebral cortical astrocytes exposed to toxic agents previously identified in experimental and clinical ALF. Such agents include ammonia, the proinflammatory cytokine interleukin-1beta (IL-1beta) and combinations of the two. Exposure of cultured astrocytes to recombinant IL-1beta (but not ammonia) resulted in increased expression of aquaporin-4 (AQP-4). Both ammonia and proinflammatory mediators led to decreased expression of glial fibrillary acidic protein (GFAP), a cytoskeletal protein, but these effects were not additive. On the other hand, heme oxygenase-1 (HO-1) and inducible nitric oxide synthase (iNOS) expression were significantly increased by exposure to both ammonia and proinflammatory mediators and although modest, these effects were additive suggestive of a synergistic mechanism. These findings suggest that worsening of brain edema and its complications in ALF due to proinflammatory mechanisms may result from exacerbation of oxidative stress-related mechanisms rather than upregulation of AQP-4 or decreases in expression of the astrocytic structural protein GFAP.

Sites of Regulated Phosphorylation that Control K-Cl Cotransporter Activity

Modulation of intracellular chloride concentration ([Cl(-)](i)) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl(-) exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl(-)](I); however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3's intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl(-)](i) and have implications for control of cell volume and neuronal function.

Cell volume control in phospholemman (PLM) knockout mice: do cardiac myocytes demonstrate a regulatory volume decrease and is this influenced by deletion of PLM?

In addition to modulatory actions on Na+-K+-ATPase, phospholemman (PLM) has been proposed to play a role in cell volume regulation. Overexpression of PLM induces ionic conductances, with 'PLM channels' exhibiting selectivity for taurine. Osmotic challenge of host cells overexpressing PLM increases taurine efflux and augments the cellular regulatory volume decrease (RVD) response, though a link between PLM and cell volume regulation has not been studied in the heart. We recently reported a depressed cardiac contractile function in PLM knockout mice in vivo, which was exacerbated in crystalloid-perfused isolated hearts, indicating that these hearts were osmotically challenged. To address this, the present study investigated the role of PLM in osmoregulation in the heart. Isolated PLM wild-type and knockout hearts were perfused with a crystalloid buffer supplemented with mannitol in a bid to prevent perfusate-induced cell swelling and maintain function. Accordingly, and in contrast to wild-type control hearts, contractile function was improved in PLM knockout hearts with 30 mM mannitol. To investigate further, isolated PLM wild-type and knockout cardiomyocytes were subjected to increasing hyposmotic challenges. Initial validation studies showed the IonOptix video edge-detection system to be a simple and accurate 'real-time' method for tracking cell width as a marker of cell size. Myocytes swelled equally in both genotypes, indicating that PLM, when expressed at physiological levels in cardiomyocytes, is not essential to limit water accumulation in response to a hyposmotic challenge. Interestingly, freshly isolated adult cardiomyocytes consistently failed to mount RVDs in response to cell swelling, adding to conflicting reports in the literature. A proposed perturbation of the RVD response as a result of the cell isolation process was not restored, however, with short-term culture in either adult or neonatal cardiomyocytes.

The Regulation of Volume-Regulated Outwardly Rectifying Anion Channels by Membrane Phosphatidylinositides in Mouse Ventricular Cells

Volume-regulated outwardly rectifying anion channel (VRAC) plays an important role in cell-volume regulation. We examined the effect of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PIP2) on the VRAC current activated by hypotonic solution, in mouse ventricular cells. The VRAC current was inhibited strongly by intracellular application of LY294002 (a PI3 kinase inhibitor) or anti-PIP3 antibody (PIP3-Ab), and less strongly by anti-PIP2 antibody (PIP2-Ab). Intracellular application of PIP3 or PIP2 influenced neither the basal background current in isotonic solution nor the VRAC current in hypotonic solution. However, PIP3, but not PIP2, restored the VRAC current suppressed by LY294002 or PIP2-Ab. These results suggest that that PI3K-mediated PIP3 production is essential to activate the VRAC current. Furthermore, we found that an α1-adrenergic receptor (α1R) agonist, phenylephrine (PE), inhibited the VRAC current. This inhibition didn't occur in the presence of prazosin, an α1R antagonist, or when the cells were dialyzed with anti-Gq/11 antibody. U-73122, a PLC inhibitor, prevented the PE-induced inhibition of VRAC current, whereas several PKC inhibitors were without effect. Since PE unaffected the VRAC current in cells dialyzed with PIP2, PE-induced inhibition of the VRAC current may be related to PIP2 depletion. In addition, the reduction of VRAC current was also found in cells from STZ-induced insulin-deficient diabetic mice. In these cells, the attenuated VRAC current was restored by incubating the cells with insulin or by dialyzing the cells with PIP3. PIP2 loading could not restore the current. These findings suggested that an impairment of the insulin-dependent PI3K-PIP3 pathway is responsible for the attenuation of VRAC currents in STZ-diabetic cells. Taken together, we propose that VRAC in mouse ventricular cells is regulated by PIP3 and/or its down stream signaling pathways.

Mechanosensor TRPM7 channel and its physiological role in cell volume regulation

Mechanosensor TRPM7 channel and its physiological role in cell volume regulation

Regulation of volume-regulated outwardly rectifying anion channels by phosphatidylinositol 3,4,5-trisphosphate in mouse ventricular cells

Volume-regulated outwardly rectifying anion channel (VRAC) plays an important role in cell-volume regulation in many types of cells. Little is known about the regulation of VRAC by phosphatidylinositides (PIs), which include phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PIP2). We examined the effect of PIs on the VRAC current activated in hypotonic solution in mouse ventricular cells. VRAC current was inhibited strongly by intracellular application of LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor) or anti-PIP3 antibody (PIP3-Ab), and less strongly by anti-PIP2 antibody (PIP2-Ab). LY294002 inhibited regulatory volume decrease in hypotonically swollen cells, which was in parallel with the VRAC inhibition. Intracellular PIP3 or PIP2 influenced neither the basal background current in isotonic solution nor the VRAC current in hypotonic solution. However, PIP3, but not PIP2, restored the VRAC current suppressed by LY294002 or PIP2-Ab. These results suggest that the activation of VRAC current requires the presence of intracellular PIP3, that PI3K-mediated increase in PIP3 level is sufficient to fully activate VRAC current, and that PIP3 alone without osmotic stimulation cannot induce VRAC current. We propose that VRAC in mouse ventricular cells is regulated by PIP3 and/or its down stream signaling pathways.