A phorbol ester augments cAMP content and adenylyl cyclase activity in neonatal rat cardiac myocytes despite reduced beta adrenoceptor density.

OBJECTIVES: In cultured neonatal rat myocardial cells the phorbol ester, phorbol-12 myristate 13-acetate (PMA), was used as a probe to examine the short term effects of augmented protein kinase C activity on the adenylyl cyclase system and on beta adrenoceptors. METHODS: beta Adrenoceptors were measured by radioligand binding, cAMP by radioimmunoassay, and adenylyl cyclase activity by a single column method. RESULTS: After 10 minutes of incubation with 100 nM PMA beta adrenoceptor density was reduced by 25% (p < 0.002) with no change in antagonist affinity. Competition curves showed no increase in agonist affinity for (-)-isoprenaline. Surprisingly, cAMP content stimulated by 1 microM (-)-isoprenaline increased by 62% from 47 (SEM 6) to 76(15) pmol.100 microliters-1 (n = 8, p < 0.05). Both effects could be suppressed by incubation with the protein kinase C inhibitor 1-(5-isoquinoline-sulphonyl)-2-methylpiperazine dihydrochloride (H7). Preincubation with PMA also augmented NaF, Mn2+, and forskolin stimulated adenylyl cyclase activity but had no effect on guanylyl-5'-imidodiphosphate (GppNHp) stimulated enzyme activity over a wide range of concentrations. PMA did not alter the effects of pertussis toxin on (-)-isoprenaline-stimulated cAMP content. CONCLUSIONS: These data indicate that protein kinase C modifies both the catalytic subunit of adenylyl cyclase and the guanine nucleotide stimulatory protein (Gs), and also suggest that NaF and GppNHp act at different sites on Gs alpha. PMA enhances adenylyl cyclase responsiveness despite loss of beta adrenoceptors in cultured neonatal rat ventricular myocytes. These findings suggest that Ca2+ and phospholipid dependent protein kinase C acting at multiple sites in the beta adrenoceptor-adenylyl cyclase cascade may be involved in the regulation of cAMP concentrations in myocardial cells.

Duke Scholars

Author Alexander Paraschos Medicine, Cardiology