The Role of P2Y sub(1) Purinergic Receptors and Cytosolic Ca super(2+) in Hypotonically Activated Osmolyte Efflux from a Rat Hepatoma Cell Line

Exposure of HTC rat hepatoma cells to a 33% decrease in extracellular osmolality caused the cytosolic Ca super(2+) concentration ((Ca super(2+)) sub(i)) to increase transiently by 90 nM. This rise in (Ca super(2+)) sub(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 super(2+)) sub(i) increase. The hypotonically induced rise in (Ca super(2+)) sub(i) was prevented by the prior discharge of the intracellular Ca super(2+) store of the cells by thapsigargin. Removal of extracellular Ca super(2+) or inhibition of Ca super(2+) influx by 1-10 mu M Gd super(3+) depleted the thapsigargin-sensitive Ca super(2+) stores and thereby diminished the rise in (Ca super(2+)) sub(i). The hypotonically induced rise in (Ca super(2+)) sub(i) was prevented by adenosine 2'-phosphate-5'-phosphate (A2P5P) and pyridoxyl-5'-phosphate-6-azophenyl-2',4'-disulfonate, inhibitors of purinergic P2Y sub(1) receptors for which ADP is a major agonist. Both inhibitors also blocked the rise in (Ca super(2+)) sub(i) elicited by addition of ADP to cells in isotonic medium, whereas A2P5P had no effect on the rise in (Ca super(2+)) sub(i) elicited by the addition of the P2Y sub(2) and P2Y sub(4) receptor agonist, UTP. HTC cells were shown to express mRNA encoding for rat P2Y sub(1), P2Y sub(2), and P2Y sub(6) receptors. Inhibition of the hypotonically induced rise in (Ca super(2+)) sub(i) blocked hypotonically induced K super(+) ( super(86)Rb super(+)) efflux, modulated the hypotonically induced efflux of taurine, but had no significant effect on Cl super(-) ( super(125)I super(-)) efflux. The interaction of extracellular ATP and/or ADP with P2Y sub(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.