Extracellular nucleotide-induced [Ca2+](i) elevation in rat basilar smooth muscle cells

Background and Purpose Extracellular nucleotides play an important role in the regulation of vascular tone and may be involved in cerebral vasospasm after subarachnoid hemorrhage. The objective of this study was to investigate the receptor subtypes for nucleotides and their mechanisms of [Ca2+](i) mobilization in cerebral vasculature. Methods Rat basilar smooth muscle cells were isolated by an enzymatic method. [Ca2+](i) mobilization in freshly isolated cells was monitored using fura 2 microfluorimetry. Result Extracellular nucleotides produced a concentration-dependent biphasic [Ca2+](i) response, a large transient peak followed by a slowly decaying plateau. The potency of nucleotides to raise [Ca2+](i) was ATP gamma S greater than or equal to UDP greater than or equal to ATP approximate to UDP approximate to TTP, indicating that P-2u receptors were expressed in the rat basilar smooth muscle cells. The effect of UTP to release Ca2+ from internal stores was reduced by pertussis toxin, by the phospholipase C inhibitor 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate, and by the Ca2+-pump inhibitor thapsigargin. The Ca2+ entry induced by UTP was partially attenuated by the receptor-operated Ca2+ channel blocker SK&F96365 and by the voltage-dependent Ca2+ channel blocker verapamil. P-2 receptor antagonists suramin and, at higher concentrations, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid reduced the effect of UTP. Conclusions The results are the first demonstration that nucleotides activate G protein-coupled P-2u receptors to mobilize [Ca2+](i) in rat basilar smooth muscle cells.