The role of P2Y₁ purinergic receptors and cytosolic Ca²⁺ 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 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-azo-phenyl-2′,4′-disulfonate, inhibitors of purinergic P2Y₁, receptors for which ADP is a major agonist. Both inhibitors also blocked the rise in [Ca²⁺]_i elicited 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 P2Y₁ 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.