Ca2+ channels mediate protein tyrosine kinase activation by endothelin-1.

To investigate the novel interaction between endothelin-1 (ET-1) and cellular protein tyrosine kinases (PTK), we asked whether Ca2+ influx links ET-1 receptors to PTK activation. In glomerular mesangial cells, ET-1 stimulated a biphasic increase in PTK activity in anti-phosphotyrosine immunoprecipitates that temporally correlated with increased tyrosine phosphorylation of cellular proteins. ET-1 increased tyrosine phosphorylation of proteins in the cytosol and in a puncture distribution consistent with focal adhesions. Addition of ionomycin to increase Ca2+ influx stimulated PTK activity, and inhibition of extracellular Ca2+ influx blocked PTK activation by ET-1. ET-1 increased autophosphorylation of pp60c-src, which was mimicked by addition of ionomycin and inhibited by chelation of extracellular Ca2+. In addition, a selective PTK inhibitor blocked induction of c-fos mRNA by ionomycin, suggesting that Ca(2+)-stimulated PTKs contribute to a signaling pathway regulating immediate early gene expression. Taken together, these results demonstrate that ET-1 stimulates nonreceptor PTK activity, including pp60c-src, by activating Ca2+ channels and subsequent influx of extracellular Ca2+.