Role of CAMP-dependent Protein Kinase A in Activation of a Voltage-sensitive Release Mechanism for Cardiac Contraction in Guinea-pig Myocytes
Overview
Authors
Affiliations
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.
Ronkainen J, Hanninen S, Korhonen T, Koivumaki J, Skoumal R, Rautio S J Physiol. 2011; 589(Pt 11):2669-86.
PMID: 21486818 PMC: 3112547. DOI: 10.1113/jphysiol.2010.201400.
Zhang L, Xu C, Hong Y, Zhang J, Liu Y, Zhao M Naunyn Schmiedebergs Arch Pharmacol. 2010; 382(1):63-71.
PMID: 20499050 DOI: 10.1007/s00210-010-0520-y.
Regulation of cardiac excitation and contraction by p21 activated kinase-1.
Ke Y, Lei M, Solaro R Prog Biophys Mol Biol. 2009; 98(2-3):238-50.
PMID: 19351515 PMC: 2709768. DOI: 10.1016/j.pbiomolbio.2009.01.007.
Griffiths H, MacLeod K J Gen Physiol. 2003; 121(5):353-73.
PMID: 12719483 PMC: 2217377. DOI: 10.1085/jgp.200208764.
Excitation contraction coupling in cardiac muscle: is there a purely voltage-dependent component?.
Balke C, Goldman L J Gen Physiol. 2003; 121(5):349-52.
PMID: 12719482 PMC: 2217382. DOI: 10.1085/jgp.200308841.