Cytochrome P450: a novel system modulating Ca2+ channels and contraction in mammalian heart cells.

1. Cytochrome P450 (P450) is a ubiquitous enzyme system that catalyses oxidative reactions of numerous endogenous and exogenous compounds. The modulatory effects of P450 on the L-type Ca2+ current (ICa), intracellular free Ca2+ signals and cell shortening were assessed in adult rat single ventricular myocytes. 2. Bath administration of the imidazole antimycotics, clotrimazole, econazole and miconazole, which are potent P450 inhibitors, significantly suppressed cardiac ICa. While the Ca2+ channel antagonist nifedipine blocked ICa within 30 s, clotrimazole-induced suppression of ICa required 5.1 +/- 0.4 min (n = 14) to reach a steady low level. The suppression of ICa was dose dependent and recovered after washout of clotrimazole. Intracellular dialysis with the P450 antibody anti-rat CYP1A2 also significantly reduced cardiac ICa. 3. Additional administration of the beta-adrenergic agonist isoprenaline (1 microM) or the membrane-permeable 8-bromo-cAMP (2 mM) completely reversed the suppressant effects of clotrimazole and NaCN on ICa. In addition, intracellular dialysis with 2 mM cAMP abolished the P450 inhibitor-induced suppression of ICa. Phosphorylation of the channel with hydrolysis-resistant ATPgammaS prevented the suppressant effect of clotrimazole on ICa. Furthermore, dephosphorylation of the Ca2+ channel with intracellular dialysis with phosphatase types I and II reduced ICa by 85 +/- 3 % and abolished clotrimazole-induced suppression of ICa. 4. Extracellular administration of the phospholipase A2 inhibitors mepacrine and 4-bromophenacyl bromide significantly suppressed ICa. 5. Clotrimazole, econazole, miconazole and CN- also significantly inhibited intracellular free Ca2+ signals and cell shortening in rat single ventricular myocytes. 6. Intracellular cAMP content was significantly reduced in isolated ventricular myocytes incubated with clotrimazole or CN-. Extracellular administration of 11, 12-epoxyeicosatrienoic acid, one of the P450-mediated metabolites of arachidonic acid, enhanced ICa and intracellular cAMP content. The epoxyeicosatrienoic acid also restored the amplitude of the reduced ICa in P450 antibody-dialysed myocytes. 7. The present data suggest that cytochrome P450 modulates cardiac ICa and cell contraction, and the modulation may result from changes in intracellular levels of cAMP by P450- mediated metabolites of arachidonic acid.