Functional adenylyl cyclase inhibition in murine cardiomyocytes by 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio]triphosphate.
Beta1-adrenergic receptor activation stimulates cardiac L-type Ca2+ channels via adenylyl cyclases (ACs), with AC5 and AC6 being the most important cardiac isoforms. Recently, we have identified 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio-]triphosphate (MANT-GTPgammaS) as a potent competitive AC inhibitor. Intriguingly, MANT-GTPgammaS inhibits AC5 and -6 more potently than other cyclases. These data prompted us to study the effects of MANT-GTPgammaS on L-type Ca2+ currents (ICa,L) in ventricular myocytes of wild-type (WT) and AC5-deficient (AC5-/-) mice by whole-cell recordings. In wild-type myocytes, MANT-GTPgammaS attenuated ICa,L stimulation following isoproterenol application in a concentration-dependent manner (control, +77+/-13%; 100 nM MANT-GTPgammaS, +43+/-6%; 1 microM MANT-GTPgammaS, +21+/-9%; p<0.05). The leftward shift of current-voltage curves was abolished by 1 microM but not by 100 nM MANT-GTPgammaS. In myocytes from AC5-/- mice, the residual stimulation of ICa,L was not further attenuated by the nucleotide, indicating AC5 to be the major AC isoform mediating acute beta-adrenergic stimulation in WT mice. Interestingly, basal ICa,L was lowered by 1 microM but not by 100 nM MANT-GTPgammaS. The decrease was less pronounced in myocytes from AC5-/- mice compared with wild types (-23+/-1 versus -40+/-7%), indicating basal ICa,L to be partly driven by AC5. Collectively, we found a concentration-dependent inhibition of ICa,L by MANT-GTPgammaS, both under basal conditions and following beta-adrenergic stimulation. Comparison of data from wild-type and AC5-deficient mice indicates that AC5 plays a major role in ICa,L activation and that MANT-GTPgammaS predominantly acts via AC5 inhibition.
Rottlaender
Dennis
D
Department of Pharmacology, Center for Molecular Medicine, University of Cologne, Gleueler Str. 24, D-50931 Köln, Germany.
Matthes
Jan
J
Vatner
Stephen F
SF
Seifert
Roland
R
Herzig
Stefan
S
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000502:physiology; D000818:Animals; D020746:Calcium Channels, L-Type / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D032383:Myocytes, Cardiac / Q000201:enzymology*; D062367:ortho-Aminobenzoates / Q000494:pharmacology*
C520023:2'-O-(N-methylanthraniloyl)guanosine 5'-(gamma-thio)triphosphate; C520024:3'-O-(N-methylanthraniloyl)guanosine 5'-(gamma-thio)triphosphate; D000067956:Adenylyl Cyclase Inhibitors; D020746:Calcium Channels, L-Type; D004791:Enzyme Inhibitors; D062367:ortho-Aminobenzoates; D006160:Guanosine Triphosphate; D000262:Adenylyl Cyclases; D007545:Isoproterenol