COEXPRESSION OF P-2Y AND P-2U RECEPTORS ON AORTIC ENDOTHELIAL-CELLS -COMPARISON OF CELL LOCALIZATION AND SIGNALING PATHWAYS

Depending on the vascular bed considered, the actions of ATP on the en dothelium are mediated by either P-2Y or P-2U receptors. The two types of receptors seem to coexist on bovine aortic endothelial cells, wher e they are both coupled to phospholipase C. In this study, we have inv estigated whether they are truly coexpressed on the same cells and whe ther their signaling pathways diverge beyond phospholipase C activatio n. Measurements of [Ca2+](i) in single cells showed that almost all bo vine aortic endothelial cells are responsive to both 2-methylthio-ATP (2MeSATP), an agonist of P-2Y receptors, and UTP, an agonist of P-2U r eceptors. UTP stimulated the release of prostacyclin from freshly isol ated bovine aortic endothelial cells, even when they were exposed to c ycloheximide at the time of their collection: this indicates that P-2U receptors must already be expressed on endothelial cells in situ and do not appear during cell culture. The time course of inositol phospha te (InsP) accumulation and the relative proportion of Ins(1,4,5)P-3, I ns(1,3,4,5)P-4, and Ins(1,3,4)P-3 were similar in cells stimulated by 2MeSATP or UTP. UTP and 2MeSATP both stimulated the hydrolysis of phos phatidylcholine by phospholipase D, as reflected by the release of [H- 3]choline from prelabeled cells. The responses to both agents were blo cked after downregulation of protein kinase C, resulting from a prolon ged exposure to phorbol 12-myristate 13-acetate: this blockade occurre d at a step distal to phospholipase C activation. A single difference between the two pathways has been identified: the effect of 2MeSATP on InsP(3) was significantly more inhibited after a short exposure to ph orbol 12-myristate 13-acetate than that of UTP. This discrepancy is co nsistent with the involvement of distinct G proteins in the activation of phospholipase C by P-2Y and P-2U receptors.