Calcium signaling pathways utilized by P2X receptors in freshly isolated preglomerular MVSMC

This study tested the hypothesis that P2X receptor activation increases int racellular Ca2+ concentration ([Ca2+](i)) in preglomerular microvascular sm ooth muscle cells (MVSMC) by evoking voltage-dependent calcium influx. MVSM C were obtained and loaded with the calcium-sensitive dye fura 2 and studie d by using single-cell fluorescence microscopy. The effect of P2X receptor activation on [Ca2+](i) was assessed by using the P2X receptor-selective ag onist alpha,beta -methylene-ATP and was compared with responses elicited by the endogenous P2 receptor agonist ATP. alpha,beta -Methylene-ATP increase d [Ca2+](i) dose dependently. Peak increases in [Ca2+](i) averaged 37 +/- 1 1, 73 +/- 15, and 103 +/- 21 nM at agonist concentrations of 0.1, 1, and 10 muM, respectively. The average peak response elicited by 10 mM alpha,beta -methylene-ATP was similar to 34% of the response obtained with 10 muM ATP. alpha,beta -Methylene-ATP induced a transient increase in [Ca2+](i) before [Ca2+](i) returned to baseline, whereas ATP induced a biphasic response in cluding a peak response followed by a sustained plateau. In Ca2+-free mediu m, ATP induced a sharp transient increase in [Ca2+](i), whereas the respons e to alpha,beta -methylene-ATP was abolished. Ca2+ channel blockade with 10 mM diltiazem or nifedipine attenuated the response to alpha,beta -methylen e-ATP, whereas nonspecific blockade of Ca2+ influx pathways with 5 mM Ni2abolished the response. Blockade of P2X receptors with the novel P2X recept or antagonist NF-279 completely but reversibly abolished the response to al pha,beta -methylene-ATP. These results indicate that P2X receptor activatio n by alpha,beta -methylene-ATP increases [Ca2+](i) in preglomerular MVSMC, in part, by stimulating voltage-dependent Ca2+ influx through L-type Ca2+ c hannels.