MODIFICATION OF CARDIAC SODIUM CURRENT BY INTRACELLULAR APPLICATION OF CAMP

We examined the effects of intracellular perfusion of cyclic adenosine monophosphate (cAMP) on the sodium current (I-Na) of guinea-pig ventricular myocytes, using the whole-cell clamp technique. I-Na was elicited by depolarizing voltage steps (-20 mV) from a variety of holding potentials (-120 to -50 mV), under conditions of 60 mM extracellular Na+ concentration ([Na+](0)) and at the temperature of 24-26 degrees C. Intracellular perfusion of cAMP decreased the I-Na elicited from the holding potentials less negative than -90 mV. In the presence of 1 mM cAMP, for example, the peak I-Na elicited from -80 mV decreased from 6.0 +/- 2.0 nA to 4.0 +/- 2.2 nA (mean +/- SD, P < 0.02, n = 7) within 3-6 min. In the presence of extracellular 3-isobutyl-1-methylxanthine (IBMX, 20 mu M), much lower concentrations of cAMP (0.2 mM) yielded a comparable effect. On the other hand, intracellular perfusion of cAMP increased the I-Na elicited from very negative holding potentials (< -100 mV). For instance, the application of cAMP (1 mM) increased the I-Na elicited by step depolarizations from -120 mV (to -20 mV), from 9.9 +/- 2.1 nA to 11.0 +/- 3.1 nA (P < 0.05, n = 5). The former effect was attributed to a marked shift of the steady-state inactivation curve of I-Na to the negative direction; the voltage of half-inactivation shifted from -77.9 +/- 1.0 to -83.5 +/- 1.4 mV, or by -5.6 mV. The latter effect may be explained by increases in maximum available conductance of I-Na. Extracellular application of isoproterenol (1 mu M) also decreased the I-Na evoked from a holding potential of -80 mV, whereas it increased the I-Na elicited from more negative potentials of -120 mV. These effects of isoproterenol were reversible and markedly attenuated in the presence of a specific inhibitor of cAMP-dependent protein kinase, H-89 {N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide}, , an isoquinolinesulphonamide derivative, in the extracellular medium (2-10 mu M) and a protein kinase inhibitor (Walsh inhibitor) in the pipette solution (40 mu M). H-89 (10 and 30 mu M) affected neither the adenylate cyclase activity prepared from rabbit ventricular muscles, nor the isoproterenol-mediated increases in the cAMP content in guinea-pig ventricular muscles. Our observations suggest that the increase in intracellular cAMP modulates the function of cardiac Na channels, preferentially by stimulating cAMP-dependent protein kinase, with subsequent phosphorylation of the channel protein.