Experiments were performed to establish the pharmacological profile of purinoceptors and to identify the signal transduction pathways responsible for increases in intracellular calcium concentration ([Ca2+]i) for cultured mouse mesangial cells. Mouse mesangial cells were loaded with fura 2 and examined using fluorescent spectrophotometry. Basal [Ca2+]i averaged 102 ± 2 nM ( n = 346). One hundred micromolar concentrations of ATP, ADP, 2′,3′-(benzoyl-4-benzoyl)-ATP (BzATP), ATP-γ-S, and UTP in normal Ca2+ medium evoked peak increases in [Ca2+]i of 866 ± 111, 236 ± 18, 316 ± 26, 427 ± 37, and 808 ± 73 nM, respectively. UDP or 2-methylthio-ATP (2MeSATP) failed to elicit significant increases in [Ca2+]i, whereas identical concentrations of adenosine, AMP, and α,β-methylene ATP (α,β-MeATP) had no detectable effect on [Ca2+]i. Removal of Ca2+ from the extracellular medium had no significant effect on the peak increase in [Ca2+]i induced by ATP, ADP, BzATP, ATP-γ-S, or UTP compared with normal Ca2+; however, Ca2+-free conditions did accelerate the rate of decline in [Ca2+]i in cells treated with ATP and UTP. [Ca2+]i was unaffected by membrane depolarization with 143 mM KCl. Western blot analysis for P2 receptors revealed expression of P2X2, P2X4, P2X7, P2Y2, and P2Y4 receptors. No evidence of P2X1 and P2X3 receptor expression was detected, whereas RT-PCR analysis reveals mRNA expression for P2X1, P2X2, P2X3, P2X4, P2X7, P2Y2, and P2Y4 receptors. These data indicate that receptor-specific P2 receptor activation increases [Ca2+]i by stimulating calcium influx from the extracellular medium and through mobilization of Ca2+ from intracellular stores in cultured mouse mesangial cells.
P2Y Receptor Modulation of ATP Release in the Urothelium