Regulation of the beta-stimulation of the Na(+)-K+ pump current in guinea-pig ventricular myocytes by a cAMP-dependent PKA pathway.
1. The whole-cell patch-clamp technique was employed with the free intracellular [Ca2+] fixed at 1.4 microM in order to study the isoprenaline (Iso)-induced increase in the Na(+)-K+ pump current (Ip) in acutely isolated guinea-pig ventricular myocytes. 2. The non-specific protein kinase inhibitor, H-7, eliminated the stimulatory effect of Iso, suggesting a phosphorylation step is involved in the beta-agonist stimulation of Ip. 3. H-7 or the phosphatase inhibitor calyculin A individually had no effect on basal Ip; however, when Ip was first increased by Iso, H-7 inhibited and calyculin A further increased Ip. This suggests phosphorylation is not important to the basal regulation of Ip, but does have an effect during beta-stimulation. 4. The Iso-induced increase in Ip could be mimicked by adding the membrane-permanent cAMP analogue chlorophenylthio-cAMP, blocking cAMP degradation with IBMX or stimulating cAMP production with forskolin. Alternatively the protein kinase A inhibitor PKI blocked the stimulatory effect of Iso. This suggests the Iso-induced phosphorylation responsible for increasing Ip is mediated by cAMP, which then activates protein kinase A (PKA). 5. We conclude that the beta-agonist-induced increase in Ip in the presence of high intracellular [Ca2+] is mediated by a phosphorylation step via the cAMP-dependent PKA pathway. During beta-stimulation, this increase in active Na(+)-K+ transport can serve to offset the effects of increases in passive membrane conductances.
Gao
J
J
Department of Physiology and Biophysics, SUNY, Stony Brook 11794-8661.
Cohen
I S
IS
Mathias
R T
RT
Baldo
G J
GJ
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000818:Animals; D000242:Cyclic AMP / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004553:Electric Conductivity; D006168:Guinea Pigs; D006352:Heart Ventricles; D007545:Isoproterenol / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D008387:Marine Toxins; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects* / Q000502:physiology
D000318:Adrenergic beta-Agonists; D007546:Isoquinolines; D008387:Marine Toxins; D010080:Oxazoles; D010879:Piperazines; D047428:Protein Kinase Inhibitors; C059041:calyculin A; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D000254:Sodium-Potassium-Exchanging ATPase; D007545:Isoproterenol