The inhibitory effect of β-stimulation on the Na/K pump current in guinea pig ventricular myocytes is mediated by a cAMP-dependent PKA pathway

Abstract

 The β-agonist isoproterenol (ISO) reduces the Na/K pump current (I _p) via β-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 μM), or the adenylyl cyclase activator forskolin (50 μM). Intracellular application of the synthetic peptide inhibitor of protein kinase A (PKA), PKI (5 μM), prevented the effect of ISO. These results suggest that the inhibitory effect of ISO on I _p is mediated via a phosphorylation step induced by a cAMP-dependent PKA pathway. Neither the non-specific protein kinase inhibitor H₇ (100 μM) nor the protein phosphatase inhibitor calyculin A (0.5 μM) had any effect on I _p in the absence of ISO. However, H₇ could increase I _p and calyculin A could reduce it in the presence of ISO (1 μM 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.