Coexpression of P 2Y and P 2U Receptors on Aortic Endothelial Cells

Abstract

Abstract Depending on the vascular bed considered, the actions of ATP on the endothelium are mediated by either P 2Y or P 2U receptors. The two types of receptors seem to coexist on bovine aortic endothelial cells, where they are both coupled to phospholipase C. In this study, we have investigated whether they are truly coexpressed on the same cells and whether their signaling pathways diverge beyond phospholipase C activation. Measurements of [Ca 2+ ] i in single cells showed that almost all bovine aortic endothelial cells are responsive to both 2-methylthio-ATP (2MeSATP), an agonist of P 2Y receptors, and UTP, an agonist of P 2U receptors. UTP stimulated the release of prostacyclin from freshly isolated bovine aortic endothelial cells, even when they were exposed to cycloheximide 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 phosphate (InsP) accumulation and the relative proportion of Ins(1,4,5)P 3 , Ins(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 phosphatidylcholine by phospholipase D, as reflected by the release of [ 3 H]choline from prelabeled cells. The responses to both agents were blocked after downregulation of protein kinase C, resulting from a prolonged exposure to phorbol 12-myristate 13-acetate: this blockade occurred 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 phorbol 12-myristate 13-acetate than that of UTP. This discrepancy is consistent with the involvement of distinct G proteins in the activation of phospholipase C by P 2Y and P 2U receptors.