Role of cAMP-dependent protein kinase A in activation of a voltage-sensitive release mechanism for cardiac contraction in guinea-pig myocytes

• Published in: The Journal of physiology 1998-11, Vol.513 (1), p.185-201
• Main Authors: Ferrier, Gregory R., Zhu, Jiequan, Redondo, Isabel M., Howlett, Susan E.
• Format: Article
• Language: English
• Subjects: 8-Bromo Cyclic Adenosine Monophosphate - pharmacology; Adenylyl Cyclase Inhibitors; Algorithms; Animals; Calcium - physiology; Cyclic AMP-Dependent Protein Kinases - antagonists & inhibitors; Cyclic AMP-Dependent Protein Kinases - physiology; Electric Stimulation; Electrophysiology; Enzyme Inhibitors - pharmacology; Guinea Pigs; In Vitro Techniques; Isoquinolines - pharmacology; Male; Membrane Potentials - physiology; Myocardial Contraction - physiology; Myocardium - cytology; Myocardium - enzymology; Original; Patch-Clamp Techniques; Sodium Channel Agonists; Sodium Channel Blockers; Sodium Channels - physiology; Sulfonamides
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1111/j.1469-7793.1998.185by.x

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 MΩ) were used to provide intracellular dialysis with pipette solutions. Na + currents were blocked with 200 μ m lidocaine. Contractions initiated by the voltage-sensitive release mechanism (VSRM) and Ca 2+ -induced Ca 2+ release (CICR) in response to L-type Ca 2+ current ( I Ca,L ) were separated with voltage clamp protocols. 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 I Ca,L -induced contractions were demonstrable in 100 % of cells. Addition of 10 or 50 μ m 8-Br-cAMP to the pipette increased the percentage of cells exhibiting VSRM contractions to 68 and 93 %, respectively. With 50 μ m 8-Br-cAMP, contractions initiated by the VSRM and I Ca,L were not significantly different in amplitude. 8-Br-cAMP-supported VSRM contractions had characteristics of the VSRM shown previously in undialysed myocytes. Cd 2+ (100 μ m ) blocked I Ca,L and I Ca,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. Without 8-Br-cAMP in the pipette, contraction-voltage relations determined with steps from a post-conditioning potential ( V pc ) of either −40 or −65 mV were bell shaped, with a threshold near −35 mV. With 50 μ m 8-Br-cAMP in the pipette, contraction-voltage relations from a V pc 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 V pc was −40 mV. H-89, which inhibits cAMP-dependent protein kinase A (PKA), significantly reduced the amplitudes of VSRM contractions by approximately 84 % with 50 μ m 8-Br-cAMP in the pipette. H-89 also significantly reduced the amplitudes of peak I Ca,L and I Ca,L contractions, although to a lesser extent. 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 excitatio