CHARACTERIZATION OF THE P-2Y-PURINOCEPTOR INVOLVED IN THE ATP-INDUCEDRISE IN CYTOSOLIC CA2+ CONCENTRATION IN RAT ILEAL MYOCYTES

1 The P-2-purinoceptor subtype and the intracellular signalling mechan ism(s) involved in the rise in the free cytosolic Ca2+ concentration ( [Ca2+](i)) induced by ATP and analogues were analyzed in myocytes isol ated from the longitudinal muscle layer of rat ileum by means of molec ular and physiological techniques. 2 The P-2-purinoceptor expressed by ileal smooth muscle cells shared 100% amino acid identity with the ra t P-2Y1-receptor. 3 Short applications of the purinoceptor agonists in duced a transient rise in [Ca2+](i) in an all-or-nothing manner. The r ank order of potency of the analogues of ATP and ADP, determined by me asuring the percentage of responding cells was 2-methylthioATP=2-chlor o-ATP > ADP > ATP, with concentrations giving [Ca2+](i) response in 50 % of cells ranging between 3 nM and 0.6 mu M. The concentration-respon se curves to ADP and ATP were shifted to the right by 10 mu M pyridoxa l phosphate-6-azopheny1-2',4'-disulphonic acid (PPADS). 4 Although the rise in [Ca2+](i) induced by stimulation of the ileal P-2Y-purinocept or was inhibited by heparin (5 mg ml(-1)), we were not able to detect stimulation of phospholipase C under conditions (37 degrees C) where m uscarinic cholinoceptor activation markedly increased inositol phospha te (InsP) accumulation. However, the carbachol (CCh)-induced increase in InsP accumulation was suppressed when the agonist was applied at 20 degrees C while a CCh-induced [Ca2+](i) rise similar to that obtained in response to the P-2-purinoceptor agonist was still observed. 5 Our results indicate that the rat ileal myocytes express a PPADS-sensitiv e P-2-purinoceptor similar to the P-2Yl-receptor subtype. Although the re is no detectable increase in InsP production, stimulation of these receptors leads to a rise in [Ca2+](i) by activation of the inositol 1 ,4,5-trisphosphate receptor-channel of the intracellular Ca2+ store, i ndicating that they couple to phospholipase C.