Abstract
Exposure of HTC rat hepatoma cells to a 33% decrease in extracellular osmolality caused the cytosolic Ca(2+) concentration ([Ca(2+)](i)) to increase transiently by approximately 90 nm. This rise in [Ca(2+)](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 [Ca(2+)](i) increase. The hypotonically induced rise in [Ca(2+)](i) was prevented by the prior discharge of the intracellular Ca(2+) store of the cells by thapsigargin. Removal of extracellular Ca(2+) or inhibition of Ca(2+) influx by 1-10 microm Gd(3+) depleted the thapsigargin-sensitive Ca(2+) stores and thereby diminished the rise in [Ca(2+)](i). The hypotonically induced rise in [Ca(2+)](i) was prevented by adenosine 2'-phosphate-5'-phosphate (A2P5P) and pyridoxyl-5'-phosphate-6-azophenyl-2',4'-disulfonate, inhibitors of purinergic P2Y(1) receptors for which ADP is a major agonist. Both inhibitors also blocked the rise in [Ca(2+)](i) elicited by addition of ADP to cells in isotonic medium, whereas A2P5P had no effect on the rise in [Ca(2+)](i) elicited by the addition of the P2Y(2) and P2Y(4) receptor agonist, UTP. HTC cells were shown to express mRNA encoding for rat P2Y(1), P2Y(2), and P2Y(6) receptors. Inhibition of the hypotonically induced rise in [Ca(2+)](i) blocked hypotonically induced K(+) ((86)Rb(+)) efflux, modulated the hypotonically induced efflux of taurine, but had no significant effect on Cl(-) ((125)I-) efflux. The interaction of extracellular ATP and/or ADP with P2Y(1) purinergic 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.
Highlights
Recent reports (4 –16) have demonstrated that cells respond to mechanical stimuli, including osmotic swelling, by releasing ATP
The hypotonically induced rise in [Ca2ϩ]i in HTC cells was compared with the increase in [Ca2ϩ]i elicited by the addition of ATP, UTP, or ADP to cells in isotonic medium, and the effect of submicromolar concentrations of ATP on phate; PPADS, pyridoxyl-5Ј-phosphate-6-azophenyl-2Ј,4Ј-disulfonate; RT, reverse transcriptase; ER, endoplasmic reticulum
HTC Cells Undergo a Hypotonically Induced Rise in [Ca2ϩ]i Due to the Release of Ca2ϩ from Intracellular Stores—Exposure of HTC cells to a reduction of the extracellular osmolality resulted in a transient increase in [Ca2ϩ]i
Summary
In a study of HTC rat hepatoma cells, Fitz and co-workers (4, 6) proposed that extracellular ATP, released in response to a decrease in the extracellular osmolality, could act in an autocrine manner by binding to purinergic receptors and thereby stimulating signaling pathways responsible for activating the ClϪ efflux required for RVD. The hypotonically induced rise in [Ca2ϩ]i in HTC cells was compared with the increase in [Ca2ϩ]i elicited by the addition of ATP, UTP, or ADP to cells in isotonic medium, and the effect of submicromolar concentrations of ATP on phate; PPADS, pyridoxyl-5Ј-phosphate-6-azophenyl-2Ј,4Ј-disulfonate; RT, reverse transcriptase; ER, endoplasmic reticulum. Our results show that, in contrast to the conclusion drawn by Roe and colleagues (18), extracellular adenine nucleotides do play a role in this rise in [Ca2ϩ]i and in activating/modulating the consequent volume-regulatory efflux of Kϩ (86Rbϩ) and taurine, but not ClϪ (125IϪ), from the cells
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