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 este r, phorbol-12 myristate 13-acetate (PMA), was used as a probe to exami ne the short term effects of augmented protein kinase C activity on th e adenylyl cyclase system and on beta adrenoceptors. Methods: beta Adr enoceptors were measured by radioligand binding, cAMP by radioimmunoas say, and adenylyl cyclase activity by a single column method. Results: After 10 minutes of incubation with 100 nM PMA beta adrenoceptor dens ity was reduced by 25% (p < 0.002) with no change in antagonist affini ty. Competition curves showed no increase in agonist affinity for (-)- isoprenaline. Surprisingly, cAMP content stimulated by 1 mu M (-)-isop renaline increased by 62% from 47 (SEM 6) to 76(15) pmol.100 mu l(-1) (n = 8, p < 0.05). Both effects could be suppressed by incubation with the protein kinase C inhibitor 1-(5-isoquinoline-sulphonyl)-2-methylp iperazine 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 a ctivity over a wide range of concentrations. PMA did not alter the eff ects of pertussis toxin on (-)-isoprenaline-stimulated cAMP content. C onclusions: These data indicate that protein kinase C modifies both th e catalytic subunit of adenylyl cyclase and the guanine nucleotide sti mulatory protein (Gs), and also suggest that NaF and GppNHp act at dif ferent sites on Gs alpha. PMA enhances adenylyl cyclase responsiveness despite loss of beta adrenoceptors in cultured neonatal rat ventricul ar myocytes. These findings suggest that Ca2+ and phospholipid depende nt protein kinase C acting at multiple sites in the beta adrenoceptor- adenylyl cyclase cascade may be involved in the regulation of cAMP con centrations in myocardial cells.