Vol. 301, Issue 2, 501-506, May 2002

On the Role of Phosphatase in Regulation of Cardiac L-Type Calcium Current by Cyclic GMP

Jian-Bing Shen and Achilles J. Pappano

Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut

Does cGMP, via protein kinase G, inhibit cAMP-stimulated Ca²⁺ 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°C) and dialyzed with adenosine 5'-O-(3-thiotriphosphate) ([ATPS]_pip). ATPS is a kinase substrate but thiophosphorylated proteins are not phosphatase substrates. With 5 mM [ATPS]_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 (Imai et al., 2001). Similar results were obtained with 0.1 mM carbachol (CCh). I_Ca(L) increased after longer dialysis (40 min) with ATPS; 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 ATPS, although consistent with cGMP action in muscarinic inhibition, could be explained by guanosine 5'-O-(3-thiotriphosphate) (GTPS) formation from ATPS via nucleoside diphosphate kinase. GTPS uncouples muscarinic and -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.