EFFECTS OF ENDOTHELIN ON MITOGEN-ACTIVATED PROTEIN-KINASE ACTIVITY AND PROTEIN-SYNTHESIS IN ISOLATED ADULT FELINE CARDIAC MYOCYTES

The growth-promoting effects of endothelin-1 (ET-1) were examined in a dult heart cells. The activity of mitogen-activated protein kinases (M APKs) was measured in cytosolic extracts of isolated adult feline card iac myocytes incubated with and without ET-1. Kinase activity was asse ssed by phosphorylation of the exogenous substrate, myelin basic prote in. ET-1 stimulated the activity of MAPK up to 4-fold, with peak activ ation occurring between five and ten minutes after addition of ET-1. P olyclonal antisera raised against a 14-amino acid sequence of the erk- 2 gene product, a MAPK isoform, identified two major bands in cytosoli c extracts of the cardiac myocytes. Partial purification of kinase act ivities using Mono Q anion-exchange chromatography demonstrated two ma jor peaks of myelin basic protein kinase activity. Subsequent immunobl ots of the eluted fractions demonstrated that the immunoreactive bands observed in the cytosolic extracts eluted in those fractions possessi ng kinase activity. Overnight pretreatment of the cardiac myocytes wit h 100 ng/ml pertussis toxin inhibited the ET-1-stimulated increase in MAPK activity by 50 - 70%, but did not alter stimulation by 100 nM pho rbol 12-myristate 13-acetate (PMA). These data suggest that stimulatio n of MAPK by ET-1 may be mediated by more than one pathway. MAPK has b een shown to be activated in the intracellular transmission of growth factor signals. Indicative of a growth effect in this adult heart cell model, myocytes exposed to increasing concentrations of ET-1 demonstr ated a dose dependent increase in [H-3]-phenylalanine incorporation in to cellular protein. This response was blocked by staurosporine and pa rtially inhibited by pretreatment with pertussis toxin, again suggesti ng the possible involvement of multiple early signals. These data from isolated adult cardiac myocytes further support the hypothesis that E T-1 has a role in the regulation of cardiac growth.