THE INHIBITORY EFFECT OF BETA-STIMULATION ON THE NA K PUMP CURRENT INGUINEA-PIG VENTRICULAR MYOCYTES IS MEDIATED BY A CAMP-DEPENDENT PKA PATHWAY/

The beta-agonist isoproterenol (ISO) reduces the Na/K pump current (I- p) via beta-adrenergic receptors when the intracellular calcium concen tration ([Ca2+](i)) is below 150 nM [8]. In the present study, the int racellular signaling pathway was investigated with whole-cell patch-cl amp of isolated guinea pig ventricular myocytes. The inhibitory effect of ISO could be mimicked by external application of the membrane-perm eant cAMP analog chlorophenylthio-cAMP (0.5 mM), the phosphodiesterase inhibitor isobutyl-1-methylxanthine (IBMX, 100 mu M), or the adenylyl cyclase activator forskolin (50 mu M) Intracellular application of th e synthetic peptide inhibitor of protein kinase A (PKA), PKI (5 mu M), Prevented the effect of ISO. These results suggest that the inhibitor y effect of ISO on I-p is mediated via a phosphorylation step induced by a cAMP-dependent PKA pathway. Neither the non-specific protein kina se inhibitor H-7 (100 mu M) nor the protein phosphatase inhibitor caly culin A (0.5 mu M) had any effect on I-p in the absence of ISO. Howeve r, H-7 could increase I-p and calyculin A could reduce it in the prese nce of ISO (1 mu M and 12 nM respectively). These results indicate tha t there is a low basal level of phosphorylation which makes the effect s of H-7 and calyculin A difficult to detect in the absence of an ISO- induced increase in phosphorylation level.