REGULATION OF MITOGEN-ACTIVATED PROTEIN-KINASE CASCADE IN ADULT-RAT HEART PREPARATIONS IN-VITRO

The regulation of mitogen-activated protein kinase (MAPK) and MAPK kin ase (MEK) was studied in freshly isolated adult rat heart preparations . In contrast to the situation in Ventricular myocytes cultured from n eonatal rat hearts, stimulation of MAPK activity by 1 mu mol/L phorbol 12-myristate 13-acetate (PMA) was not consistently detectable in crud e extracts. After fast protein liquid chromatography, MAPK isoforms p4 2(MAPK) and p44(MAPK) and two peaks of MEK were shown to be activated > 10-fold in perfused hearts or ventricular myocytes exposed to 1 mu m ol/L PMA for 5 minutes. The identities of MAPK or MEK were confirmed b y immunoblotting and, for MAPK, by the ''in-gel'' myelin basic protein phosphorylation assay. In retrogradely perfused hearts, high coronary perfusion pressure (120 mm Hg for 5 minutes), norepinephrine (50 mu m ol/L for 5 minutes), or isoproterenol (50 mu mol/L for 5 minutes) stim ulated MAPK and MEK approximate to 2- to 5-fold. In isolated myocytes, endothelin 1 (100 nmol/L for 5 minutes) also stimulated MAPK, but sti mulation by norepinephrine or isoproterenol was difficult to detect. I mmunoblotting showed that the relative abundances of MAPK and MEK prot ein in ventricles declined to < 20% of their postpartal abundances aft er 50 days. This may explain the difficulties encountered in assaying the activity of MAPK in crude extracts from adult hearts. We conclude that potentially hypertrophic agonists and interventions stimulate the MAPK cascade in adult rats and suggest that the MAPK cascade may be a n important intracellular signaling pathway in this response.