On the role of phosphatase in regulation of cardiac L-type calcium current by cyclic GMP.
Does cGMP, via protein kinase G, inhibit cAMP-stimulated Ca(2+) current (I(Ca(L))) in mammalian ventricular myocytes by phosphorylating the calcium channel at a site different from that acted on by cAMP or by dephosphorylating the calcium channel through phosphatase(s)? We tested these possibilities in guinea pig ventricular myocytes superfused with Tyrode's solution (35 degrees C) and dialyzed with adenosine 5'-O-(3-thiotriphosphate) ([ATPgammaS](pip)). ATPgammaS is a kinase substrate but thiophosphorylated proteins are not phosphatase substrates. With 5 mM [ATPgammaS](pip), I(Ca(L)) increased gradually over 20 to 25 min and then rapidly in the presence of 3-isobutyl-1-methylxanthine. 8-Bromo-cGMP (8-Br-cGMP; 1 mM) did not inhibit I(Ca(L)) significantly (-3 +/- 11.8%, n = 21) in contrast to results with ATP dialysis (). Similar results were obtained with 0.1 mM carbachol (CCh). I(Ca(L)) increased after longer dialysis (>/=40 min) with ATPgammaS; again, 8-Br-cGMP had no effect. Also, isoproterenol (ISO) did not stimulate and CCh, alone or in the presence of ISO, did not inhibit I(Ca(L)). Block of CCh effect by ATPgammaS, although consistent with cGMP action in muscarinic inhibition, could be explained by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) formation from ATPgammaS via nucleoside diphosphate kinase. GTPgammaS uncouples muscarinic and beta-adrenoceptors from intracellular effectors. Failure of 8-Br-cGMP to reduce I(Ca(L)) irreversibly excludes calcium channel phosphorylation as an inhibitory mechanism. We propose that cGMP inhibits I(Ca(L)) by activating phosphatase(s) in guinea pig ventricular myocytes.
Shen
Jian-Bing
JB
Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
Pappano
Achilles J
AJ
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D020746:Calcium Channels, L-Type / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology; D002316:Cardiotonic Agents / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006152:Cyclic GMP / Q000494:pharmacology*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D004594:Electrophysiology; D006168:Guinea Pigs; D006321:Heart / Q000187:drug effects* / Q000502:physiology; D066298:In Vitro Techniques; D009206:Myocardium / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000502:physiology*
D020746:Calcium Channels, L-Type; D002316:Cardiotonic Agents; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D002217:Carbachol; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D010744:Phosphoric Monoester Hydrolases; D006152:Cyclic GMP