P2 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 their 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 artery endothelial cells by using [Ca2+](i) microfluorimetry. The rank order of potency to increase [Ca2+](i) was 2-methylthio-ATP approximate to ATP approximate to uridine triphosphate (UTP) > adenosine diphosphate (ADP) >> adenosine monophosphate (AMP) > alpha,beta-methylene-ATP > adenosine, the effect was mediated by both P-2y and P-2u receptors. ATP, 2-methylthio-ATP, and UTP mobilized Ca2+ from intracellular stores and triggered Ca2+ entry. The effects of ATP, 2-methylthio-ATP, and UTP were reduced by phospholipase C inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC), but only the effects of ATP and UTP were attenuated by pertussis toxin, indicating that P-2y,, and P-2u receptors 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 inhibitor econazole and by inorganic Ca2+ entry blocker lanthanum. P-2-receptor antagonists suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and reactive blue 2 reduced the effects of ATP and 2-methylthio-ATP, but not studies suggest a coexistence of P-2y and P-2u receptors in cultured those of UTP, in a concentration-dependent manner. These bovine middle cerebral artery endothelial cells.