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Am J Physiol Renal Physiol 280: F1054-F1061, 2001;
0363-6127/01 $5.00
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Vol. 280, Issue 6, F1054-F1061, June 2001

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

Steven M. White1, John D. Imig2, Thu-Thuy Kim2, Benjamin C. Hauschild2, and Edward W. Inscho2

1 Louisiana State University School of Medicine; and 2 Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana 70112

This study tested the hypothesis that P2X receptor activation increases intracellular Ca2+ concentration ([Ca2+]i) in preglomerular microvascular smooth muscle cells (MVSMC) by evoking voltage-dependent calcium influx. MVSMC were obtained and loaded with the calcium-sensitive dye fura 2 and studied by using single-cell fluorescence microscopy. The effect of P2X receptor activation on [Ca2+]i was assessed by using the P2X receptor-selective agonist alpha ,beta -methylene-ATP and was compared with responses elicited by the endogenous P2 receptor agonist ATP. alpha ,beta -Methylene-ATP increased [Ca2+]i dose dependently. Peak increases in [Ca2+]i averaged 37 ± 11, 73 ± 15, and 103 ± 21 nM at agonist concentrations of 0.1, 1, and 10 µM, respectively. The average peak response elicited by 10 µM alpha ,beta -methylene-ATP was ~34% of the response obtained with 10 µM ATP. alpha ,beta -Methylene-ATP induced a transient increase in [Ca2+]i before [Ca2+]i returned to baseline, whereas ATP induced a biphasic response including a peak response followed by a sustained plateau. In Ca2+-free medium, ATP induced a sharp transient increase in [Ca2+]i, whereas the response to alpha ,beta -methylene-ATP was abolished. Ca2+ channel blockade with 10 µM diltiazem or nifedipine attenuated the response to alpha ,beta -methylene-ATP, whereas nonspecific blockade of Ca2+ influx pathways with 5 mM Ni2+ abolished the response. Blockade of P2X receptors with the novel P2X receptor antagonist NF-279 completely but reversibly abolished the response to alpha ,beta -methylene-ATP. These results indicate that P2X receptor activation by alpha ,beta -methylene-ATP increases [Ca2+]i in preglomerular MVSMC, in part, by stimulating voltage-dependent Ca2+ influx through L-type Ca2+ channels.

microvascular smooth muscle cells; adeonsine 5'-triphosphate; afferent arteriole; renal microvasculature; 8,8'-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-napthalenetrisulfonic acid hexasodium salt; alpha ,beta -methylene-adeonsine 5'-triphosphate; nifedipine; diltiazem; nickel; calcium channels; P2X receptors


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