Abstract
Does cGMP, via protein kinase G, inhibit cAMP-stimulated Ca2+ current (ICa(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) ([ATPγS]pip). ATPγS is a kinase substrate but thiophosphorylated proteins are not phosphatase substrates. With 5 mM [ATPγS]pip, ICa(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 ICa(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). ICa(L) increased after longer dialysis (≥40 min) with ATPγS; 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 ICa(L). Block of CCh effect by ATPγS, although consistent with cGMP action in muscarinic inhibition, could be explained by guanosine 5′-O-(3-thiotriphosphate) (GTPγS) formation from ATPγS via nucleoside diphosphate kinase. GTPγS uncouples muscarinic and β-adrenoceptors from intracellular effectors. Failure of 8-Br-cGMP to reduce ICa(L) irreversibly excludes calcium channel phosphorylation as an inhibitory mechanism. We propose that cGMP inhibits ICa(L) by activating phosphatase(s) in guinea pig ventricular myocytes.
Footnotes
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This work was supported by U.S. Public Health Service Grant HL-13339.
- Abbreviations:
- PKA
- protein kinase A
- ICa(L)
- L-type calcium current
- ATPγS
- adenosine 5′-O-(3-thiotriphosphate)
- GTPγS
- guanosine 5′-O-(3-thiotriphosphate)
- CCh
- carbachol
- IBMX
- 3-isobutyl-1-methylxanthine
- ISO
- isoproterenol
- PKG
- protein kinase G
- PP
- protein phosphatase
- ACh
- acetylcholine
- PDE
- phosphodiesterase
- 8-Br-cGMP
- 8-bromo-cGMP
- KT5823
- (9S,10R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,-9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-k]pyrrolo[3,4-i][1,6] benzodiazocine-10-carboxylic acid methyl ester
- Received October 31, 2001.
- Accepted January 14, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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