P2 receptor regulation of [Ca²⁺]_i in cultured mouse mesangial cells

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 [Ca ²⁺]_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 [Ca ²⁺]_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 [Ca ²⁺]_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.