The inhibitory effect of beta-stimulation on the Na/K pump current in guinea pig ventricular myocytes is mediated by a cAMP-dependent PKA pathway.
The beta-agonist isoproterenol (ISO) reduces the Na/K pump current (Ip) via beta-adrenergic receptors when the intracellular calcium concentration ([Ca2+]i) is below 150 nM [8]. In the present study, the intracellular signaling pathway was investigated with whole-cell patch-clamp of isolated guinea pig ventricular myocytes. The inhibitory effect of ISO could be mimicked by external application of the membrane-permeant cAMP analog chlorophenylthio-cAMP (0.5 mM), the phosphodiesterase inhibitor isobutyl-1-methylxanthine (IBMX, 100 microM), or the adenylyl cyclase activator forskolin (50 microM). Intracellular application of the synthetic peptide inhibitor of protein kinase A (PKA), PKI (5 microM), prevented the effect of ISO. These results suggest that the inhibitory effect of ISO on Ip is mediated via a phosphorylation step induced by a cAMP-dependent PKA pathway. Neither the non-specific protein kinase inhibitor H7 (100 microM) nor the protein phosphatase inhibitor calyculin A (0.5 microM) had any effect on Ip in the absence of ISO. However, H7 could increase Ip and calyculin A could reduce it in the presence of ISO (1 microM and 12 nM respectively). These results indicate that there is a low basal level of phosphorylation which makes the effects of H7 and calyculin A difficult to detect in the absence of an ISO-induced increase in phosphorylation level.
Gao
J
J
Department of Physiology and Biophysics, Health Sciences Center, 8661 SUNY, Stony Brook, NY 11794-8661, USA.
Cohen
I S
IS
Mathias
R T
RT
Baldo
G J
GJ
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000818:Animals; D000242:Cyclic AMP / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006168:Guinea Pigs; D006352:Heart Ventricles / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D008387:Marine Toxins; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009206:Myocardium / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D010080:Oxazoles / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects / Q000502:physiology*
D000318:Adrenergic beta-Agonists; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; C059041:calyculin A; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000254:Sodium-Potassium-Exchanging ATPase