Involvement of protein tyrosine kinase in the InsP3-induced activation of Ca2+-dependent Cl- currents in cultured cells of the rat retinal pigment epithelium

This combined study of patch-clamp and intracellular Ca2+ ([Ca2+](i)) measu rement was undertaken in order to identify signaling pathways that lead to activation of Ca2+-dependent Cl- channels in cultured rat retinal pigment e pithelial (RPE) cells. Intracellular application of InsP3 (10 mu M) led to an increase in [Ca2+](i) and activation of Cl- currents. In contrast, intra cellular application of Ca2+ (10 mu M) only induced transient activation of Cl- currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca2+ and to the blocker of I-CRAC, La3+ (10 mu M) , despite the fact that both maneuvers led to a decline in [Ca2+](i). The I nsP3-induced rise in Cl- conductance could be prevented either by thapsigar gin-induced (1 mu M) depletion of intracellular Ca2+ stores or by removal o f Ca2+ prior to the experiment. The effect of InsP3 could be mimicked by in tracellular application of the Ca2+-chelator BAPTA (10 mM). Block of PKC (c helerythrine, 1 mu M) had no effect. Inhibition of Ca2+/calmodulin kinase ( KN-63, KN-92; 5 mu M) reduced Cl--conductance in 50% of the cells investiga ted without affecting [Ca2+](i). Inhibition of protein tyrosine kinase (50 mu M tyrphostin 51, 5 mu M genistein, 5 mu M lavendustin) reduced an increa se in [Ca2+](i) and Cl- conductance. In summary, elevation of [Ca](i) by In sP3 leads to activation of Cl- channels involving cytosolic Ca2+ stores and Ca2+ influx from extracellular space. Tyrosine kinases are essential for t he Ca2+ independent maintenance of this conductance.