PHORBOL ESTER REGULATION OF OPIOID PEPTIDE GENE-EXPRESSION IN MYOCARDIAL-CELLS - ROLE OF NUCLEAR-PROTEIN KINASE-C

Opioid peptide gene expression was characterized in adult rat ventricular cardiac myocytes that had been cultured in the absence or the presence of phorbol 12-myristate 13-acetate. The phorbol ester induced a concentration- and time-dependent increase of prodynorphin mRNA, the maximal effect being reached after 4 h of treatment, The increase in mRNA expression was suppressed by incubation of cardiomyocytes with staurosporine, a putative protein kinase C inhibitor, and was not observed when the cells were cultured in the presence of the inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate, Incubation of cardiac myocytes with phorbol 12-myristate 13-acetate also elicited a specific and staurosporine-sensitive increase in immunoreactive dynorphin B, a biologically active end product of the precursor, both in the myocardial cells and in the culture medium, In vitro run-off transcription assays indicated that transcription of the prodynorphin gene was increased both in nuclei isolated hom phorbol ester-treated myocytes and in nuclei isolated from control cells and then exposed to phorbol 12-myristate 13-acetate, No transcriptional effect was observed when cardiac myocytes or isolated nuclei where exposed to 4 alpha-phorbol 12,13-didecanoate. The phorbol ester-induced increase in prodynorphin gene transcription was prevented by pretreatment of myocytes or isolated nuclei with staurosporine, suggesting that myocardial opioid gene expression may be regulated by nuclear protein kinase C, In this regard, cardiac myocytes expressed protein kinase C-alpha, -delta, -epsilon, and -zeta, as shown by immuno-blotting, Only protein kinase C-delta and protein kinase C-epsilon were expressed in nuclei that have been isolated from control myocytes, suggesting that these two isotypes of the enzyme may be part of the signal transduction pathway involved in the effect elicited by the phorbol ester on opioid gene transcription in isolated nuclei, The incubation of myocardial nuclei isolated from control cells in the presence of a protein kinase C activator induced the phosphorylation of the myristoylated alanine-rich protein kinase C substrate peptide, a specific fluorescent substrate of the enzyme, The possibility that prodynorphin gene expression may control the heart function through autocrine or paracrine mechanisms is discussed.