The Role of P2Y1 Purinergic Receptors and Cytosolic Ca2+ in Hypotonically Activated Osmolyte Efflux from a Rat Hepatoma Cell Line

Abstract

Exposure of HTC rat hepatoma cells to a 33% decrease in extracellular osmolality caused the cytosolic Ca2+ concentration ([Ca2+]i) to increase transiently by ∼90 nm. This rise in [Ca2+]i was inhibited strongly by apyrase, grade VII (which has a low ATP/ADPase ratio) but not by apyrase grade VI (which has a high ATP/ADPase ratio) or hexokinase, indicating that extracellular ADP and/or ATP play a role in the [Ca2+]i increase. The hypotonically induced rise in [Ca2+]i was prevented by the prior discharge of the intracellular Ca2+ store of the cells by thapsigargin. Removal of extracellular Ca2+ or inhibition of Ca2+ influx by 1–10 μm Gd3+depleted the thapsigargin-sensitive Ca2+ stores and thereby diminished the rise in [Ca2+]i. The hypotonically induced rise in [Ca2+]i was prevented by adenosine 2′-phosphate-5′-phosphate (A2P5P) and pyridoxyl-5′-phosphate-6-azophenyl-2′,4′-disulfonate, inhibitors of purinergic P2Y1 receptors for which ADP is a major agonist. Both inhibitors also blocked the rise in [Ca2+]ielicited by addition of ADP to cells in isotonic medium, whereas A2P5P had no effect on the rise in [Ca2+]i elicited by the addition of the P2Y2 and P2Y4 receptor agonist, UTP. HTC cells were shown to express mRNA encoding for rat P2Y1, P2Y2, and P2Y6 receptors. Inhibition of the hypotonically induced rise in [Ca2+]i blocked hypotonically induced K+ (86Rb+) efflux, modulated the hypotonically induced efflux of taurine, but had no significant effect on Cl− (125I−) efflux. The interaction of extracellular ATP and/or ADP with P2Y1purinergic receptors therefore plays a role in the response of HTC cells to osmotic swelling but does not account for activation of all the efflux pathways involved in the volume-regulatory response. Exposure of HTC rat hepatoma cells to a 33% decrease in extracellular osmolality caused the cytosolic Ca2+ concentration ([Ca2+]i) to increase transiently by ∼90 nm. This rise in [Ca2+]i was inhibited strongly by apyrase, grade VII (which has a low ATP/ADPase ratio) but not by apyrase grade VI (which has a high ATP/ADPase ratio) or hexokinase, indicating that extracellular ADP and/or ATP play a role in the [Ca2+]i increase. The hypotonically induced rise in [Ca2+]i was prevented by the prior discharge of the intracellular Ca2+ store of the cells by thapsigargin. Removal of extracellular Ca2+ or inhibition of Ca2+ influx by 1–10 μm Gd3+depleted the thapsigargin-sensitive Ca2+ stores and thereby diminished the rise in [Ca2+]i. The hypotonically induced rise in [Ca2+]i was prevented by adenosine 2′-phosphate-5′-phosphate (A2P5P) and pyridoxyl-5′-phosphate-6-azophenyl-2′,4′-disulfonate, inhibitors of purinergic P2Y1 receptors for which ADP is a major agonist. Both inhibitors also blocked the rise in [Ca2+]ielicited by addition of ADP to cells in isotonic medium, whereas A2P5P had no effect on the rise in [Ca2+]i elicited by the addition of the P2Y2 and P2Y4 receptor agonist, UTP. HTC cells were shown to express mRNA encoding for rat P2Y1, P2Y2, and P2Y6 receptors. Inhibition of the hypotonically induced rise in [Ca2+]i blocked hypotonically induced K+ (86Rb+) efflux, modulated the hypotonically induced efflux of taurine, but had no significant effect on Cl− (125I−) efflux. The interaction of extracellular ATP and/or ADP with P2Y1purinergic receptors therefore plays a role in the response of HTC cells to osmotic swelling but does not account for activation of all the efflux pathways involved in the volume-regulatory response. Cells swollen by a decrease in the osmolality of the external medium return to their original volume by a process known as regulatory volume decrease (RVD). 1The abbreviations used are: RVD, regulatory volume decrease; AM, acetoxymethyl ester; A2P5P, adenosine 2′-phosphate-5′-phosphate; A3P5P, adenosine 3′-phosphate-5′-phosphate; Δ[Ca2+]i, maximum increase in cytosolic Ca2+ concentration; Me2SO, dimethyl sulfoxide; FBS, fetal bovine serum; HBS, HEPES-buffered saline; MAPK, mitogen-activated protein kinase; 2-MeSADP, 2-methylthioadenosine-5′-diphosphate; PPADS, pyridoxyl-5′-phosphate-6-azophenyl-2′,4′-disulfonate; RT, reverse transcriptase; ER, endoplasmic reticulum. The signaling pathways activated during RVD have not yet been elucidated fully but result in the efflux of the inorganic ions, K+and Cl−, and organic molecules, with a concomitant efflux of water and a decrease in cell volume. Transient increases in cytoplasmic Ca2+ concentration ([Ca2+]i) have been observed in response to osmotic swelling in many, but not all, cell types (1Foskett J.K. Strange K. Cellular and Molecular Physiology of Cell Volume Regulation. CRC Press, Inc., Boca Raton, FL1994: 259-277Google Scholar, 2Lang F. Busch G.L. Volkl H. Cell. Physiol. Biochem. 1998; 8: 1-45Google Scholar, 3Pasantes-Morales H. Morales Mulia S. Nephron. 2000; 86: 414-427Google Scholar) and may contribute to RVD by direct activation or modulation of K+, Cl−, or organic osmolyte channels and/or by activating signaling pathways involved in RVD. Recent reports (4–16) have demonstrated that cells respond to mechanical stimuli, including osmotic swelling, by releasing ATP. ATP is known to bind to two main classes of purinergic receptors present on the cell plasma membrane; P2Y receptors are G-protein-linked receptors, and P2X receptors are intrinsic ion channels (17Ralevic V. Burnstock G. Pharmacol. Rev. 1998; of of receptor result in a increase in [Ca2+]i. a of cell types apyrase and receptor as and have been shown to swelling and cytoplasmic Ca2+ S. Scholar, Physiol. 2000; K. Physiol. 2000; Scholar, K. K. H. K. Pharmacol. Physiol. for a extracellular ATP and increases in [Ca2+]i. a of HTC rat hepatoma and S. Scholar, Scholar) that extracellular in response to a decrease in the extracellular in by to purinergic receptors and thereby signaling pathways for activating the Cl− efflux for RVD. Scholar) demonstrated a rise in [Ca2+]i in HTC cells in response to osmotic swelling and that of thapsigargin-sensitive intracellular stores of Ca2+ inhibited K+ and Cl− to the in cell types Physiol. 2000; Scholar, K. Physiol. 2000; Scholar, K. K. H. K. Pharmacol. Physiol. the rise in [Ca2+]i was not inhibited by apyrase or inhibitors of purinergic receptors, which was that the of ATP was not for the rise in [Ca2+]i have the a decrease in extracellular extracellular and ion and organic osmolyte efflux in HTC the of that that the hypotonically induced rise in [Ca2+]i was inhibited in which extracellular ADP as as ATP was The hypotonically induced rise in [Ca2+]i in HTC cells was with the increase in [Ca2+]i elicited by the addition of or ADP to cells in isotonic medium, and the effect of of ATP on the efflux of and the organic osmolyte cells in isotonic medium was with the effect of a decrease in extracellular the by and Scholar) that the hypotonically induced rise in [Ca2+]i in HTC cells is a of the of Ca2+ intracellular also in to the by and extracellular play a role in rise in [Ca2+]i and in the volume-regulatory efflux of and taurine, but not the and were Molecular was or were and were HTC hepatoma cells were as a in medium with fetal bovine and in a HEPES-buffered was of the Physiol. S. S. to with The osmolality was by in and to of with The used in efflux were as that the was to the of [Ca2+]i cells were to a decrease in the extracellular osmolality by of the isotonic with a of the as but which the was The osmolality of the medium in all the in which was the the osmolality was to the with in which extracellular Ca2+ was with the of the used to the osmolality to and the concentration of the was by that the osmolality of the in the The concentration of in the extracellular was and that of Ca2+ a on the in Biochem. Scholar) was nm. VII and and were in water a concentration of and as of the concentration were in and in the on the of the 2-MeSADP, PPADS, and A2P5P were as in water were on the of and on ADP was for with and in to ATP. and were as and in dimethyl and as was on the of the as a in was in and HTC cells were on in for as for cell The cells were with in FBS, and for The cells were for to in for of the The were with a and in a in the were the a of were by and and the in a with a The were for the that the with the of [Ca2+]i was as by G. Scholar) the [Ca2+]i and a of G. The were all in cells with μm the by Cell are the on of Ca2+ with and in the of are the of the on to that on of Ca2+ with and in the of The maximum increase in in response to was by of prior to the of during the the rise in [Ca2+]i was was HTC cells the to the of was with for in a volume of and on and were and for to was and the was for the by to for to of the by were in in which the reverse was were for rat P2Y receptor and as as for the the The P2Y to a receptor are shown in The of of the and of and μm The were and were on a by used to of rat P2Y receptor in a HTC cells were in and for with for and efflux were cells with for and for efflux were on the and with the of the extracellular was by addition of of The was by the addition to the cells of of the extracellular was to a for and the extracellular was by with or grade VII apyrase or a the was that also the The in the cells the of the in medium was by the cells with were efflux were the is the of in the cells is the of the is the the two are as the of to the of efflux to the of on and efflux were and efflux was of was used to for the of [Ca2+]i and of for efflux were significant of [Ca2+]i in HTC cells with were in the nm. the cells were to a decrease in to [Ca2+]i a of to a of the in The maximum increase in was nm. the [Ca2+]i to the [Ca2+]i is shown in in the osmolality rise to in with significant increases observed in response to the osmolality to VII apyrase is with a low ATP/ADPase of of in the extracellular prior to and during a decrease in the osmolality caused a in the hypotonically induced to of the in which the [Ca2+]i was to isotonic the addition of apyrase also caused a significant decrease in the [Ca2+]i not extracellular in the [Ca2+]i. VI apyrase is with a high ATP/ADPase to the with grade VII apyrase, of grade VI apyrase not decrease the hypotonically induced The in the of grade VI apyrase was not the of for the which with ATP to and was also significant effect on the hypotonically induced observed in response to a in the extracellular osmolality The in the of was not the increase for the The increase in [Ca2+]i observed on of the osmolality of the extracellular was not to mechanical or to the decrease in of volume of isotonic not the [Ca2+]i the addition of isotonic which osmolality but of the extracellular not The role of extracellular Ca2+ in the hypotonically induced rise in [Ca2+]i was by the Ca2+ the extracellular extracellular with the in and cells with in to the endoplasmic Ca2+ stores S. Scholar) prior to the in HTC cells were for in which Ca2+ had been the [Ca2+]i to the observed in response to a decrease in the extracellular osmolality to was to with for in for in is that extracellular Ca2+ is the major of Ca2+ for the observed on osmotic cells that had been to a decrease in osmolality were with in to Ca2+ in the Ca2+ stores S. the in cells in the was that in cells in extracellular Ca2+ Exposure of HTC cells to a therefore caused a decrease in the of the intracellular Ca2+ are shown in the observed in response to a decrease in the osmolality cells in and is to the observed in response to the addition of thapsigargin. cells in the is the are with the that the Ca2+ rise to the hypotonically induced increase in [Ca2+]i was the Ca2+ The of Ca2+ in store was the cells were a to a the of the of store Ca2+ store that was in response to a was the in to that of extracellular Ca2+ is not for the hypotonically induced Δ[Ca2+]i, the isotonic was with a low osmolality that the extracellular concentration of Ca2+ was to with the in osmolality to is shown in the decrease in osmolality caused [Ca2+]i to rise of not in cells to Ca2+ The rise was by a decrease in to nm. isotonic was that the was was no increase in [Ca2+]i to to the in cells to no Ca2+ cells in the the of the thapsigargin-sensitive Ca2+ store was to that in cells in Ca2+ and with as was the Ca2+ was the was to HTC cells in isotonic medium [Ca2+]i to a maximum of and to a of Ca2+ is shown in the cells were to a decrease in the extracellular osmolality or in the of [Ca2+]i no of with the thapsigargin-sensitive Ca2+ store the of Ca2+ for the hypotonically induced increase in [Ca2+]i. the decrease in osmolality caused [Ca2+]i to to as a increase in cell volume and of the The was by a a of the cells RVD a of as as of Ca2+ channels Pharmacol. Rev. Scholar) has been to RVD in a of hypotonically swollen cells including HTC cells Physiol. S. Scholar, Physiol. has also been that ATP with of and μm Physiol. 1998; Scholar, Physiol. Scholar, S. S. Physiol. Scholar, S. Physiol. 2000; the to which is has been has been to with the for ATP F. Physiol. and for and Scholar) that μm of ATP by as low as the [Ca2+]i in HTC cells and inhibited the hypotonically induced increase in [Ca2+]i. are shown in and in μm the hypotonically induced to of the whereas μm prevented the hypotonically induced rise in [Ca2+]i. The that μm the [Ca2+]i in HTC cells is with Ca2+ influx pathways a role in the [Ca2+]i of the cells the used not was to the of extracellular to in HTC cells isotonic with for was also to the of Ca2+ that thapsigargin-sensitive Ca2+ The observed on addition of was with for the cells not are with the effect of on the hypotonically induced increase in [Ca2+]i a of the of the intracellular Ca2+ The of grade VI apyrase (which ATP to ADP the of ADP to or of (which ATP to to the hypotonically induced increase in [Ca2+]i is with the that ADP is to purinergic involved in the hypotonically induced increase in [Ca2+]i. The that the rise to the hypotonically induced increase in is intracellular the extracellular P2Y receptors in the ADP is a for P2Y1, but not for P2Y2, or P2Y6 purinergic receptors (17Ralevic V. Burnstock G. Pharmacol. Rev. 1998; Scholar, Pharmacol. 2000; two purinergic receptor A2P5P and PPADS, were for their to the rise in [Ca2+]i caused by a decrease in osmolality to A2P5P has been shown to P2Y1 but not P2Y receptors Pharmacol. Scholar, Pharmacol. is a of purinergic receptors a significant inhibition of P2Y1 but or no inhibition of the P2Y receptors (17Ralevic V. Burnstock G. Pharmacol. Rev. 1998; Scholar, Pharmacol. 2000; The hypotonically induced increase in [Ca2+]i was inhibited by by μm A2P5P and by μm are shown in with shown in the of to a with the of in the of for of the was therefore in the of The purinergic receptor inhibitors and not used in as strongly with the the effect of A2P5P and on the hypotonically activated in HTC cells for by inhibition of purinergic receptors that are for ADP as P2Y2, or P2Y6 for which ATP and/or or are major Pharmacol. 2000; Scholar, Pharmacol. the of ADP and on [Ca2+]i in cells in isotonic medium were The concentration of were to a maximum of the of and no that observed in response to a in the extracellular osmolality to of ADP were with to by and the ADP were in the of with in the extracellular HTC cells in isotonic medium to μm ADP with a rise in to The maximum response was is shown in response was observed 2-MeSADP, P2Y1 Pharmacol. 2000; was to the cells not Both A2P5P and inhibited the response to ADP the to of the response. the of inhibitors on the increase in [Ca2+]i their on the hypotonically induced increase in [Ca2+]i addition of or ATP to cells in isotonic medium, [Ca2+]i to a maximum and to that of the cell [Ca2+]i for the of the The observed in response to the addition of and ATP were and for for the P2Y1 A2P5P not the response of HTC cells to a significant in the with a on P2Y2 and/or of the purinergic receptors in HTC Inhibition of the by was that observed for the The and in the response of HTC cells to a decrease in the extracellular which of the P2Y receptors are in HTC for of the rat P2Y receptors and were by The the significant of and mRNA were also to was with the were the reverse was the indicating the of by of the extracellular osmolality to in increase in the efflux for for and of a organic HTC cells the of grade VII apyrase which was shown in to the hypotonically induced the hypotonically induced increase in efflux was inhibited had no significant effect on hypotonically induced efflux but caused a inhibition of the hypotonically induced efflux also inhibited the hypotonically induced the effect of ion on the efflux of HTC cells in isotonic and μm caused a significant inhibition of in not medium, whereas μm the hypotonically efflux the of taurine, was a decrease in hypotonically induced efflux with hypotonically induced efflux was not inhibited by or μm strongly inhibited efflux in a as as significant inhibition of hypotonically and efflux a and K. in a of K+ were for their on the efflux of HTC cells a decrease in the osmolality to not The and blocked the hypotonically induced efflux by and which the and channels Pharmacol. Scholar, 1998; 8: Scholar) and the S. Pharmacol. inhibited the hypotonically induced efflux by whereas which channels 1998; 8: had no significant effect The effect of ATP on efflux was with that of a 33% decrease in osmolality in of [Ca2+]i on the as the efflux a for of on the addition of ATP in isotonic not in response to a decrease in Exposure of HTC cells in isotonic to ATP caused the efflux to increase by a of to This increase was that observed to in response to a 33% in the to The was inhibited strongly by μm and not as was the hypotonically efflux efflux HTC were for and the addition of ATP to cells in isotonic medium, in the and of μm The are but increase in efflux to was observed on the addition of ATP This rise was by the increase in efflux that in response to a 33% in osmolality to ATP had no significant effect on efflux ATP μm was to increases in the of efflux of whereas efflux not Exposure of HTC cells to a of the extracellular osmolality in a increase in [Ca2+]i. The has been by Scholar) also that is to the of Ca2+ intracellular for of shown to decrease the of the intracellular Ca2+ or the hypotonically induced increase in [Ca2+]i. of cells in a medium of the cells with or of Ca2+ influx by of extracellular Ca2+ with of the extracellular osmolality the extracellular Ca2+ as a of had no significant effect on the of the observed The that of extracellular Ca2+ and the of μm the [Ca2+]i as the of Ca2+ that was the thapsigargin-sensitive are of a and extracellular Ca2+ in HTC with the [Ca2+]i and the of Ca2+ in the stores a the influx and efflux of of Ca2+ the external medium, or addition of decrease the influx of thereby cytoplasmic [Ca2+]i and the of Ca2+ in the is as a result of the of Ca2+ stores that a decrease in the in response to a decrease in the extracellular is that cells to a of including ATP. The by which has yet to elucidated but a process that inhibited by and is of cell S. Scholar, S. 1998; Scholar, Physiol. 2000; Scholar, H. Physiol. 2000; Scholar, Physiol. their and Scholar) that grade VI apyrase not the hypotonically induced increase in [Ca2+]i in HTC and that the increase was not by extracellular receptors. have the by Scholar) with grade VI apyrase, but have also for the of P2Y1 purinergic receptors in the hypotonically induced increase in [Ca2+]i and the volume-regulatory response. of apyrase have been has of and the has of Biochem. Scholar, V. S. VI apyrase is the the high and grade VII apyrase is the the low grade VI ADP to to increase in whereas the grade VII to the of ADP a that with VII apyrase not or apyrase was shown to the hypotonically induced increase in [Ca2+]i are with the that as as or of is in the hypotonically induced increase in [Ca2+]i in HTC cells the of purinergic receptors. The that the Ca2+ is intracellular in the G-protein-linked P2Y receptors the P2X in rise in [Ca2+]i. was shown by that mRNA for P2Y1, P2Y2, and but not receptors are present in HTC cells that the rat of the and receptors Pharmacol. 2000; Scholar, S. V. Scholar) have not been as mRNA for P2Y2 receptors have been Physiol. Scholar) in HTC cells and for P2Y1, P2Y2, and P2Y6 receptors have been in rat Pharmacol. 2000; and the hepatoma cell and H. H. S. Physiol. by and Physiol. Scholar) have for the of P2Y2 receptors in HTC The of the present and for the of P2Y1 receptors. which with with that does ATP G. Biochem. Pharmacol. 2000; caused increase in [Ca2+]i to cells in isotonic medium The increase was blocked by A2P5P, to for Pharmacol. as as by PPADS, a purinergic receptor (17Ralevic V. Burnstock G. Pharmacol. Rev. 1998; Scholar, Pharmacol. 2000; The addition of for P2Y2 and P2Y4 receptors but not P2Y1 also caused increase in [Ca2+]i to cells in isotonic The by A2P5P with A2P5P as a of P2Y1 receptors in but in the of μm ATP for rat P2Y1, P2Y2, and P2Y4 caused increase in [Ca2+]i to cells in isotonic medium, and was by in the of The inhibition of the by was the inhibition of the This for the response of HTC cells to extracellular ATP was to the of a P2Y1 and a P2Y2 whereas the response of HTC cells to extracellular was to P2Y2 receptors. The of P2Y6 receptors to increases in [Ca2+]i in HTC cells was not The used in the rise to may have been with and therefore the observed increase in [Ca2+]i in to a P2Y6 response. which express P2Y1 and P2Y2 receptors, have been shown to in a to that for HTC rat the A3P5P, a of A2P5P, the [Ca2+]i rise in response to of P2Y1 but does not the rise in response to Pharmacol. 2000; A2P5P and inhibited the rise in [Ca2+]i in cells to a decrease in the extracellular The of inhibition of the hypotonically activated increase in [Ca2+]i that for the inhibition of the increase in [Ca2+]i induced by the addition of the P2Y1 ADP to cells in isotonic medium and are with the that in HTC cells P2Y1, and not P2Y2, receptors are activated by in response to a The by and Scholar) that a of receptors which P2Y1 and P2Y2 receptors, does not the rise in [Ca2+]i in response to a is to Both and have been shown to result in inhibition of the increase in [Ca2+]i in cell types Physiol. 2000; Scholar, K. Physiol. 2000; Scholar, K. K. H. K. Pharmacol. Physiol. were to in to in the of the The that in response to with P2Y1 receptors and thereby the of Ca2+ intracellular stores the of the ATP that is and has been Physiol. Scholar) the to significant activation of P2Y2 receptors. for the activation of P2Y1 P2Y2 receptors may in the of the two receptors for ATP. The activation of P2Y1 receptors by ATP has been to G. Biochem. Pharmacol. 2000; whereas that for activation of P2Y2 receptors by ATP is Physiol. Scholar, S. the concentration of ATP in the of the of hypotonically swollen cells is not may to significant activation of the low P2Y2 receptors yet high to activation of the P2Y1 receptors. for the activation of P2Y1 receptors by hypotonically ATP in a of P2Y1 receptors with the ATP and/or with that are known to in cell plasma Burnstock G. 2000; Scholar, 2000; Scholar, H. Pharmacol. 2000; The for activation by ADP of P2Y1 receptors has been as the for ATP G. Biochem. Pharmacol. 2000; ADP is hypotonically ATP to P2Y1 receptors, may to receptors. This the addition of and apyrase VI of which have all ATP to not a significant increase in the hypotonically induced The efflux of K+ (86Rb+) HTC cells a in the extracellular osmolality was inhibited by grade VII apyrase, μm and all of which prevented the hypotonically induced increase in [Ca2+]i. This is in with the of Scholar) that K+ in HTC cells were inhibited by of intracellular with in that the hypotonically activated efflux of was inhibited strongly by but not are with the that a K+ is involved in the response of HTC cells to The induced by a 33% in the extracellular osmolality was not that observed on of ATP to HTC whereas the increase in the efflux elicited in response to the was that activated by ATP the hypotonically activated is a result of the activation of P2Y1 receptors and the addition of ATP to cells in isotonic medium in activation of P2Y1 and P2Y2 receptors, that the [Ca2+]i rise elicited by the is in activating K+ efflux the caused by a of P2Y1 and P2Y2 receptors. The that the efflux activated by ATP was blocked by the was by is with the that the activating K+ is by P2Y1 activation ATP is to the external and is to P2Y1 and P2Y2 receptors. This for of P2Y1 receptors and K+ channels in HTC The increase in efflux in response to of ATP to HTC cells in isotonic medium was that observed in cells to a 33% decrease in the extracellular of ATP a increase efflux not and have been to Cl− in HTC cells Physiol. The hypotonically activated efflux not a rise in [Ca2+]i as extracellular grade VII apyrase of which inhibited the rise in caused a significant inhibition of efflux and The inhibition efflux in the of is to to direct of the efflux organic and purinergic have been to hypotonically activated Biochem. Scholar, F. G. Physiol. with of and have shown that apyrase hypotonically activated in HTC cells S. Scholar, 2000; and in a cell Physiol. Cl− in HTC cells were also inhibited on of intracellular the for are in with reports in cells and cells in which the of ATP was not for activation of a Cl− S. S. Physiol. Scholar, Biochem. S. Physiol. 2000; Scholar) for hypotonically induced efflux Biochem. efflux was by the addition of ATP to HTC cells in isotonic of the hypotonically induced by apyrase and to a inhibition of efflux. This may to the signaling pathways involved in the induced activation of efflux [Ca2+]i a increase in response to efflux was inhibited by of intracellular Ca2+ Physiol. The that hypotonically activated efflux was to [Ca2+]i efflux is to the the efflux pathways K. Cell. Physiol. Biochem. 2000; has been that and efflux a in HTC K. Scholar) and cell types K. Scholar, G. V. G. a may the that hypotonically activated efflux a on whereas hypotonically activated efflux is with the of organic osmolyte that is to [Ca2+]i are efflux on the in present a indicating hypotonically ATP may on extracellular purinergic receptors in to the activation of ion and osmolyte efflux during RVD in HTC cells swelling the of ATP which on P2Y1 receptors and/or is the cell to ADP which is a of P2Y1 receptors. P2Y1 receptor activation to the of Ca2+ intracellular the activation of K+ channels as as the of the organic osmolyte efflux This in S. Scholar, Scholar) in two of purinergic receptors the hypotonically induced rise in and activation rise in is not for the hypotonically activated efflux of are with the by and Scholar) that intracellular plays a role in hypotonically induced K+ the in cell volume increases in response to as and the and the of the cell has been shown to a in the in cell 1998; and 1998; Scholar, Biochem. Scholar) have that activation is a in the signaling the mitogen-activated protein that cell swelling to and have shown that the G-protein-linked is activated by extracellular in response to osmotic swelling in HTC a rat hepatoma cell a increase in [Ca2+]i. the osmotic used for was the a decrease in osmolality to (which to swelling with in the 1998; result in a significant rise in [Ca2+]i not in response to swelling, has been shown the in cells Biochem. and the of to P2Y1 receptors has been shown to the in addition to activating K+ efflux in response to osmotic the of extracellular to purinergic receptors also involved in activating the signaling for the observed in swollen for the of HTC and for with the for in the of and and for on the