title: Role of phosphodiesterase in regulation of calcium current in isolated cardiac myocytes.
abstract: It has previously been shown that intracellular perfusion of isolated cardiac myocytes with cGMP reduces the amplitude of the trans-sarcolemmal calcium current (ICa) elevated by cAMP-dependent mechanisms. To test the hypothesis that cGMP acts by stimulating a cyclic nucleotide phosphodiesterase (PDE) activity, PDE activity and the effects of methylisobutylxanthine (MIX), a PDE inhibitor, on ICa were examined in cardiomyocytes dissociated from frog ventricle. PDE activity was determined by measuring hydrolysis of [33P]cAMP in subcellular fractions. Using 100 microM cAMP as substrate, approximately 50% of the PDE activity was found in the 20,000 x g particulate fraction. Basal activity in this fraction had a Vmax of 15.4 nmol [corrected] of cAMP hydrolyzed/min/mg of protein and a Km of 113 microM cAMP. The PDE activity of the particulate fraction was stimulated significantly by cGMP. Half-maximal stimulation was observed with 1.1 microM cGMP. This value is virtually identical to the value for the concentration of intracellular cGMP that produced a half-maximal reduction of cAMP-elevated ICa in electrophysiological experiments. The cGMP-stimulated PDE activity had a Vmax of 9.5 nmol/min/mg [corrected] and a Km of 12.3 microM cAMP. MIX (100 microM) selectively inhibited the cGMP-stimulated PDE activity (IC50 = 20 microM). To determine whether PDEs modulate the amplitude of ICa, the effects of MIX were examined on basal ICa and cAMP-elevated ICa. MIX produced small increases in the basal ICa and increased ICa in the presence of 1 microM intracellular cAMP. MIX at 100 microM potentiated the effects of submaximal doses of isoproterenol and shifted the dose-response curve for cAMP to the left but did not affect the dose-response curve for 8-bromo-cAMP. MIX reversed the effect of cGMP on the cAMP-elevated ICa. We conclude that cyclic nucleotide PDEs play an important role in modulating the cardiac calcium current. The hypothesis that cGMP inhibits the cAMP-elevated ICa by activating a PDE is supported by the finding that MIX inhibited both the cGMP-stimulated PDE activity and the effect of cGMP on ICa at similar concentrations.
authors: Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322.
authors: M A
authors: MA
authors: Simmons
authors: H C
authors: HC
authors: Hartzell
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D066298:In Vitro Techniques; D007473:Ion Channels / Q000187:drug effects*; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000032:analysis / Q000502:physiology*; D011898:Ranidae
chemical_list: D007473:Ion Channels; D010726:Phosphodiesterase Inhibitors; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases; D006152:Cyclic GMP; D007545:Isoproterenol; D000109:Acetylcholine; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine