Ca2+ mobilization in bovine corneal endothelial cells by P2 purinergic receptors

Abstract

PURPOSE. To characterize Ca 2+ mobilization by P2 receptors in the bovine corneal endothelial cells (BCEC). METHODS. Changes in intracellular Ca 2+ ([Ca 2+ ] i) were measured by fluorescence imaging of cultured and fresh BCEC cells loaded with the Ca 2+ -sensitive dye Fura-PE3. Relative rates of Ca 2+ influx were measured employing Mn 2+ as a surrogate for Ca 2+. RESULTS. Exposure of cultured cells to uridine 5'-triphosphate (UTP), 2-methyl-thio ATP (msATP) and ATP caused biphasic changes in [Ca 2+ ] i consisting of a peak followed by a plateau phase. Based on the peak responses to 100 µM agonist, the magnitude of UTP responses were similar to that of ATP but greater than that of msATP or ADP. UTP and msATP stimulated Mn 2+ influx following [Ca 2+ ] i peak similar to that observed in response to cyclopiazonic acid (CPA), an inhibitor of ER Ca 2+ -ATPase. Under Ca 2+ -free conditions, peak responses were similar to those in the presence of external Ca 2+, but reduced when the cells were pre-exposed to CPA. Reactive Blue-2 (RB2), inhibited msATP responses by 60.4 ± 18.8% but UTP responses by only 10.6 ± 9.5%. Repeated exposures to UTP or msATP reduced [Ca 2+ ] i mobilization indicating homologous desensitization. Response to UTP was not affected by a prior exposure to msATP. However, response to msATP was reduced by a prior exposure to UTP indicating mixed heterologous desensitization. Fresh cells responded to UTP (50 mM) with temporal characteristics of [Ca 2+ ] i mobilization similar to that of cultured cells. CONCLUSION. BCEC express P2 receptors belonging to the P2Y subfamily. The emptying of the IP3-sensitive stores, leading to the initial peak in [Ca2+] i response, subsequently caused capacitative Ca 2+ influx leading to the onset of the plateau phase. A significant homologous desensitization to UTP and msATP, selective heterologous desensitization between UTP and msATP, and selective inhibition by RB2 indicate the coexistence of multiple P2Y receptors.