P-2 PURINOCEPTORS IN CULTURED BOVINE MIDDLE CEREBRAL-ARTERY ENDOTHELIAL-CELLS

Extracellular adenosine triphosphate (ATP) plays an important role in the regulation of endothelial function. However, its receptors and the ir signal-transduction pathways in major cerebral arterial endothelial cells are largely unknown. This study was undertaken functionally to classify the P-2 purinoceptors in cultured bovine middle cerebral arte ry endothelial cells by using [Ca2+](i) microfluorimetry. The rank ord er of potency to increase [Ca2+](i) was 2-methylthio-ATP approximate t o ATP approximate to uridine triphosphate (UTP) > adenosine diphosphat e (ADP) >> adenosine monophosphate (AMP) > alpha,beta-methylene-ATP > adenosine, the effect was mediated by both P-2y and P-2u receptors. AT P, 2-methylthio-ATP, and UTP mobilized Ca2+ from intracellular stores and triggered Ca2+ entry. The effects of ATP, 2-methylthio-ATP, and UT P were reduced by phospholipase C inhibitor 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate (NCDC), but only the effects of ATP and UTP were a ttenuated by pertussis toxin, indicating that P-2y,, and P-2u receptor s may activate the same effector mechanisms by coupling to different G proteins. The [Ca2+](i) entry caused by UTP was significantly reduced by the receptor-regulated Ca2+ channel blocker SK&F 96365, by P-450 i nhibitor econazole and by inorganic Ca2+ entry blocker lanthanum. P-2- receptor antagonists suramin, pyridoxal-phosphate-6-azophenyl-2',4'-di sulfonic acid (PPADS), and reactive blue 2 reduced the effects of ATP and 2-methylthio-ATP, but not studies suggest a coexistence of P-2y an d P-2u receptors in cultured those of UTP, in a concentration-dependen t manner. These bovine middle cerebral artery endothelial cells.