Characterization of protein kinase C isotype expression in adult rat heart. Protein kinase C-epsilon is a major isotype present, and it is activated by phorbol esters, epinephrine, and endothelin.

Abstract

The pattern of protein kinase C (PKC) isotype expression in whole extracts of dispersed, freshly isolated adult rat ventricular myocytes and adult rat heart ventricle was examined by immunoblot analysis using antisera specific for PKC-alpha, -beta 1, -gamma, -delta, -epsilon, -zeta, or -eta isotypes. This analysis revealed significant levels of expression of the Ca(2+)-independent isotype PKC-epsilon, which was detected as band of 97-kd molecular mass. PKC-zeta was detected principally as a 66-kd band that probably represented a proteolytic product of the holoenzyme. PKC-eta was detected only in whole ventricle as a doublet at 75 and 81 kd and was therefore probably present in nonmyocytic cells. PKC-alpha, -beta 1, -gamma, and -delta could not be detected. Because of our inability to detect PKC-alpha, -beta 1, -gamma, and -delta in whole extracts, PKC isotypes were partially purified from whole heart by DEAE Sepharose chromatography. PKC-alpha, -beta 1, -gamma, and -delta could still not be detected in the appropriate fractions. All PKC isotypes were detectable in appropriate positive control extracts (brain or certain cultured cell lines). In unstimulated isolated cardiomyocytes, the majority (80-95%) of the PKC-epsilon immunoreactivity was present in the soluble fraction of the extract. On exposure of the cardiomyocytes to 1 microM phorbol 12-myristate 13-acetate (PMA), PKC-epsilon undergoes a rapid (< 30 seconds), sustained (at least 60 minutes), and virtually complete association with the Triton X-100-soluble membrane fraction. There was an associated loss of PKC-epsilon from the soluble fraction. The EC50 for PMA of the translocation event was 15-37 nM. Exposure of cardiomyocytes to 1 microM 4 beta-phorbol 12,13-didecanoate or 1 microM phorbol 12,13-dibutyrate also resulted in translocation of PKC-epsilon to the membrane fraction, whereas exposure to 1 microM 4 alpha-phorbol 12,13-didecanoate was without effect. PKC-epsilon also translocated on exposure of cardiomyocytes to 50 microM epinephrine or 100 nM endothelin-1. However, in both cases, the extent of translocation was significantly less than that after exposure to PMA. We conclude that interventions that lead to hypertrophy of cardiomyocytes (phorbol esters, epinephrine, and endothelin-1) activate PKC-epsilon.