Role of cAMP-dependent protein kinase A in activation of a voltage-sensitive release mechanism for cardiac contraction in guinea-pig myocytes.
1. Ionic currents and unloaded cell shortening were recorded from guinea-pig ventricular myocytes with single electrode voltage clamp techniques and video edge detection at 37 C. Patch pipettes (1-3 MOmega) were used to provide intracellular dialysis with pipette solutions. 2. Na+ currents were blocked with 200 microM lidocaine. Contractions initiated by the voltage-sensitive release mechanism (VSRM) and Ca2+-induced Ca2+ release (CICR) in response to L-type Ca2+ current (ICa,L) were separated with voltage clamp protocols. 3. Without 8-bromo cyclic adenosine 3',5'-monophosphate (8-Br-cAMP) in the pipette, small VSRM-induced contractions occurred transiently in only 13% of myocytes. In contrast, large ICa,L-induced contractions were demonstrable in 100% of cells. 4. Addition of 10 or 50 microM 8-Br-cAMP to the pipette increased the percentage of cells exhibiting VSRM contractions to 68 and 93%, respectively. With 50 microM 8-Br-cAMP, contractions initiated by the VSRM and ICa,L were not significantly different in amplitude. 5. 8-Br-cAMP-supported VSRM contractions had characteristics of the VSRM shown previously in undialysed myocytes. Cd2+ (100 microM) blocked ICa,L and ICa,L contractions but not VSRM contractions. 8-Br-cAMP-supported contractions exhibited steady-state inactivation with parameters characteristic of the VSRM, as well as sigmoidal contraction-voltage relations. 6. Without 8-Br-cAMP in the pipette, contraction-voltage relations determined with steps from a post-conditioning potential (Vpc) of either -40 or -65 mV were bell shaped, with a threshold near -35 mV. With 50 microM 8-Br-cAMP in the pipette, contraction-voltage relations from a Vpc of -65 mV were sigmoidal and the threshold shifted to near -55 mV. Contraction-voltage relations remained bell shaped in the presence of 8-Br-cAMP when the Vpc was -40 mV. 7. H-89, which inhibits cAMP-dependent protein kinase A (PKA), significantly reduced the amplitudes of VSRM contractions by approximately 84% with 50 microM 8-Br-cAMP in the pipette. H-89 also significantly reduced the amplitudes of peak ICa, L and ICa,L contractions, although to a lesser extent. 8. We conclude that intracellular dialysis with patch pipettes disrupts the adenylyl cyclase-PKA phosphorylation cascade, and that the VSRM requires intracellular phosphorylation to be available for activation. Intracellular dialysis with solutions that do not maintain phosphorylation levels inhibits a major mechanism in cardiac excitation- contraction coupling.
Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. gregory.ferrier@dal.ca
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Ferrier
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Zhu
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Redondo
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Howlett
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000067956:Adenylyl Cyclase Inhibitors; D000465:Algorithms; D000818:Animals; D002118:Calcium / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D004558:Electric Stimulation; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D009200:Myocardial Contraction / Q000502:physiology*; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D018408:Patch-Clamp Techniques; D062687:Sodium Channel Agonists; D026941:Sodium Channel Blockers; D015222:Sodium Channels / Q000502:physiology; D013449:Sulfonamides
D000067956:Adenylyl Cyclase Inhibitors; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D062687:Sodium Channel Agonists; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D013449:Sulfonamides; D015124:8-Bromo Cyclic Adenosine Monophosphate; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D002118:Calcium