PHORBOL ESTER STIMULATES THE SYNTHESIS AND SECRETION OF BRAIN NATRIURETIC PEPTIDE FROM NEONATAL RAT VENTRICULAR CARDIOCYTES - A COMPARISON WITH THE REGULATION OF ATRIAL-NATRIURETIC-FACTOR

During left ventricular hypertrophy, brain natriuretic peptide (BNP) and atrial natriuretic factor (ANF) mRNA levels increase, possibly due to stretch-induced activation of protein kinase C. Phorbol ester treatment of primary cultures of neonatal rat ventricular cardiocytes represents an in vitro model of hypertrophic cell growth and has previously been shown to stimulate ANF synthesis and secretion. Using this model, we studied the synthesis and secretion of BNP to determine whether its regulation in cardiac cells is similar to ANF. Addition of 10(-7) M phorbol 12-myristate 13-acetate (PMA) resulted in a 3- to 4-fold increase in immunoreactive BNP (irBNP) secretion 24-48 h after treatment. Over a concentration range of 10(-8)-10(-6) m, PMA increased irBNP secretion to equivalent levels. Another phorbol ester agonist, phorbol 12,13-didecanoate, stimulated irBNP secretion, while the inactive analog 4alpha-phorbol 12,13-didecanoate had no effect. Inhibition of protein kinase C (PKC) with 10(-8) m staurosporine decreased basal secretion of irBNP 60% and prevented PMA induction of irBNP, whereas both stimulated and basal secretion of ANF were minimally affected. BNP mRNA increased 6-fold by 3 h of PMA treatment and remained elevated above control levels for 48 h. Staurosporine prevented the increase in BNP mRNA. To determine whether PKC or a PKC-dependent pathway was involved in persistent stimulation of BNP and ANF in cells chronically treated with PMA, ventricular cardiocytes were treated with PMA for 24 h, followed by PMA plus 10(-8) m staurosporine for 24 h. BNP mRNA was reduced to control levels, while ANF mRNA was reduced by an average of 20%. To test whether mRNA stability was involved in the differential effect of chronic phorbol ester treatment, cardiocytes were treated with the protein synthesis inhibitor cycloheximide (20 mug/ml). BNP mRNA levels were stimulated as early as 30 min after treatment, but ANF mRNA remained unaffected. Cycloheximide also potentiated PMA's effect on BNP mRNA after 1.5, 9.5, and 24 h of treatment. To test whether a transcriptional mechanism was involved in the stimulation of BNP mRNA by PMA, cells were treated with the inhibitor actinomycin D (5 mug/ml) for 24 h in the presence of PMA. Actinomycin D reduced the stimulatory effect of PMA on BNP mRNA. These findings suggest that: 1) PMA, presumably acting through activation of PKC, mediates the synthesis and secretion of BNP by neonatal ventricular cardiocytes, as previously shown for ANF and BNP synthesized in atrial cardiocytes; 2) staurosporine inhibits basal and PMA-stimulated secretion of BNP, but only minimally affects ANF secretion; 3) activation of the PKC signaling pathway with PMA stimulates BNP and ANF mRNAs; 4) inhibition of PKC or a PKC-dependent pathway with staurosporine after persistent activation with PMA results in more rapid turnover of BNP than ANF mRNA; 5) acute blockade of protein synthesis enhances BNP but not ANF mRNA, suggesting post-transcriptional regulation of BNP mRNA; and 6) a transcriptional mechanism, involving either the ANF and BNP genes or a gene(s) encoding for a regulatory protein, may also be involved.