Extracellular Nucleotide–Induced [Ca 2+ ] i Elevation in Rat Basilar Smooth Muscle Cells

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Abstract
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 [Ca 2+ ] i mobilization in cerebral vasculature. Methods Rat basilar smooth muscle cells were isolated by an enzymatic method. [Ca 2+ ] i mobilization in freshly isolated cells was monitored using fura 2 microfluorimetry. Result Extracellular nucleotides produced a concentration-dependent biphasic [Ca 2+ ] i response, a large transient peak followed by a slowly decaying plateau. The potency of nucleotides to raise [Ca 2+ ] i was ATPγS≥UDP≥ATP≈UDP≈TTP, indicating that P 2u receptors were expressed in the rat basilar smooth muscle cells. The effect of UTP to release Ca 2+ from internal stores was reduced by pertussis toxin, by the phospholipase C inhibitor 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate, and by the Ca 2+ -pump inhibitor thapsigargin. The Ca 2+ entry induced by UTP was partially attenuated by the receptor-operated Ca 2+ channel blocker SK&F96365 and by the voltage-dependent Ca 2+ 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 [Ca 2+ ] i in rat basilar smooth muscle cells.