In this study, we analyzed in rat myometrial cells the signaling pathways involved in the endothelin (ET)-1-induced extracellular signal-regulated kinase (ERK) activation required for the induction of DNA synthesis. We found that inhibition of protein kinase C (PKC) by Ro-31-8220 abolished ERK activation. Inhibition of phospholipase C (PLC) by U-73122 or of phosphoinositide (PI) 3-kinase by wortmannin partially reduced ERK activation. A similar partial inhibition was observed after treatment with pertussis toxin or PKC downregulation by phorbol ester treatment. The effect of wortmannin was additive with that produced by PKC downregulation but not with that due to pertussis toxin. These results suggest that both diacylglycerol-sensitive PKC, activated by PLC products, and diacylglycerol-insensitive PKC, possibly activated by a G(i)-PI 3-kinase-dependent process, are involved in ET-1-induced ERK activation. These two pathways were found to be activated mainly through the ET(A) receptor subtype. ET-1 and phorbol ester stimulated Src activity in a PKC-dependent manner, both responses being abolished in the presence of Ro-31-8220. Inhibition of Src kinases by PP1 abrogated phorbol ester- and ET-1-induced ERK activation. Finally, ET-1 activated Ras in a PP1- and Ro-31-8220-sensitive manner. Altogether, our results indicate that ET-1 induces ERK activation in rat myometrial cells through the sequential stimulation of PKC, Src, and Ras.

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