Endothelin-dependent actions in cultured AT-1 cardiac myocytes. The role of the epsilon isoform of protein kinase C.

The consequences of endothelin receptor activation were examined in atrial tumor myocytes derived from transgenic mice (AT-1 cells). Endothelin-1 (endothelin) stimulates phosphoinositide hydrolysis in a dose-dependent manner. Endothelin also induces the rapid and transient translocation of protein kinase C (PKC)-epsilon immunoreactivity from the soluble to the particulate cell fraction. The subcellular distributions of PKCalpha and PKCzeta (also expressed by AT-1 cells) are not influenced by endothelin. Using quantitative fluorescence microscopy with fura 2, we examined the effects of endothelin on intracellular calcium. In electrically driven myocytes, endothelin induces a rapid and transient increase in the amplitude of the calcium transient. This is blocked by both phorbol 12-myristate 13-acetate (PMA) pretreatment to downregulate PKC and the PKC inhibitor chelerythrine, arguing that PKCepsilon plays a critical role in endothelin receptor-dependent increases in intracellular calcium. Endothelin also stimulates mitogen-activated protein kinase (MAPK). MAPK activation is markedly attenuated by pretreatment with PMA or pertussis toxin (PTX, to activate susceptible G protein alpha subunits); it is completely prevented by combined pretreatment with PMA and PTX. In contrast, it is not attenuated by chelation of intracellular calcium with BAPTA. These findings indicate that the pathway for endothelin receptor stimulation of MAPK involves PKCepsilon and PTX-sensitive G protein(s). Thus, these studies identify a functional role for PKCepsilon as a mediator of endothelin receptor-dependent increases in cytosolic calcium and MAPK activity in AT-1 cells. Accordingly, the AT-1 cell system should provide a uniquely useful model to identify the intracellular targets for PKCepsilon and investigate their function in the regulation of intracellular calcium homeostasis and the induction of the growth response in cardiac myocytes.