title: The role of constitutive PKA-mediated phosphorylation in the regulation of basal I(Ca) in isolated rat cardiac myocytes.
abstract: 1. Pharmacological inhibitors of protein kinase A (PKA) and protein phosphatases 1/2A were used to determine whether basal L-type Ca(2+) current (I(Ca)) observed in the absence of exogenous beta-adrenergic receptor stimulation is sustained by PKA-mediated phosphorylation. Amphotericin B was used to record whole-cell I(Ca) in the perforated patch-clamp configuration. 2. Calyculin A and isoprenaline (both 1 micromol l(-1)) increased basal I(Ca) (P<0.05), whereas H-89 inhibited I(Ca) in a concentration-dependent manner with an IC(50) approximately 5 micromol l(-1). H-89 also inhibited the response to 1.0 micromol l(-1) isoprenaline, although relatively high concentrations (30 micromol l(-1)) were required to achieve complete suppression of the response. 3. Double-pulse protocols were used to study the effects of 10 micromol l(-1) H-89 on time-dependent recovery of I(Ca) from voltage-dependent inactivation as well as the steady-state gating of I(Ca). T(0.5) (time for I(Ca) to recover to 50% of the preinactivation amplitude) increased in the presence of H-89 (P<0.05) but was unaffected by calyculin A or isoprenaline. 4. Steady-state activation/inactivation properties of I(Ca) were unaffected by 10 micromol l(-1) H-89 or 1 micromol l(-1) calyculin A, whereas isoprenaline caused a leftward shift in both curves so that V(0.5) for activation and inactivation became more negative. 5. Data show that basal I(Ca) is regulated by cAMP-PKA-mediated phosphorylation in the absence of externally applied beta-receptor agonists and that relatively high concentrations of H-89 are required to fully suppress the response to beta-adrenergic receptor stimulation, thereby limiting the value of H-89 as a useful tool in dissecting signalling pathways in intact myocytes.
authors.affiliation: Department of Medicine, School of Clinical Sciences, University Clinical Departments, University of Liverpool, Daulby Street, Liverpool L69 3GA.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Bracken
authors.forename: Moutaz
authors.initials: M
authors.lastname: Elkadri
authors.forename: George
authors.initials: G
authors.lastname: Hart
authors.forename: Munir
authors.initials: M
authors.lastname: Hussain
mesh_terms: D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006352:Heart Ventricles / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide