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 PKC alpha and PKC zeta (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 Ir-myristate 13-acetate (PMA) pretreatment to downregulate PKC and the PKC inhibitor chelerythrine, arguing that PKC epsilon 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 inactivate 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 PKC epsilon and PTX-sensitive G protein(s). Thus, these studies identify a functional role for PKC epsilon as a mediator of endothelin receptor-dependent increases in cytosolic calcium and MAPK activity ill AT-I cells. Accordingly, the AT-1 cell system should provide a uniquely useful model to identify the intracellular targets for PKC epsilon and investigate their function in the regulation of intracellular calcium homeostasis and the induction of the growth response in cardiac myocytes.