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 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.
School of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra ACT 0200, Australia. pauline.junankar@anu.edu.au
Pauline R
PR
Junankar
Ari
A
Karjalainen
Kiaran
K
Kirk
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism*; D017931:DNA Primers; D008114:Liver Neoplasms, Experimental / Q000378:metabolism* / Q000473:pathology; D009994:Osmolar Concentration; D058919:Purinergic P2 Receptor Antagonists; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058472:Receptors, Purinergic P2Y1; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D017931:DNA Primers; C546384:P2ry1 protein, rat; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D002118:Calcium