Activation of the cAMP-protein kinase A pathway facilitates Na+ translocation by the Na+-K+ pump in guinea-pig ventricular myocytes

1. The effects of the adenylyl cyclase activator forskolin on steady-state and transient currents generated by the Na+-K+ pump were studied in guinea- pig ventricular myocytes by means of whole-cell voltage clamp at 30 degrees C. 2. In external solution containing 144 mM Na+ (Na-o(+)) and 10 mM K+ (K-o()), steady-state Na+-K+ pump current (I-p) activated by 5 mM pipette Na+ (N a-pip(+)) at -20 mV was reversibly augmented by forskolin (4 mu M) to 133 /- 4% of the control current (n = 15). The forskolin analogue 1,9-dideoxyfo rskolin (10 mu M), which does not activate adenylyl cyclases, did not incre ase I-p (n = 2). Application of the protein kinase a (PKA) inhibitor H-89 ( 10 mu M) in the continued presence of forskolin reversed the forskolin-indu ced elevation of I-p (n = 3). 3. The forskolin effect on I-p persisted in the presence of 50 mM Na-pip(+) which ensured that the internal Na+-binding sites of the Na+-K+ pump were nearly saturated. Under these conditions, the drug increased I-p to 142 +/- 3% of the control I-p when the pipette free Ca2+ concentration ([Ca2+](pip )) was 0.013 nM (n = 5) and to 138 +/- 4% of the control I-p when free [Ca2 +](pip) was 15 nM (n = 9). 4. In Na+-free external solution, I-p activated by 50 mM Na-pip(+) and 1.5 mM K-o(+) was likewise increased by forskolin but to a lesser extent than i n Na+-containing medium (116 +/- 3% of control, (n = 10). 5. In order to investigate exclusively partial reactions in the Na+ limb of the pump cycle, transient pump currents under conditions of electroneutral Na+-Na+ exchange were studied. Transient pump currents elicited by voltage jumps displayed an initial peak and then decayed monoexponentially. Moved charge (Q) and the rate constant of current decay varied with membrane pote ntial (V). The Q- V relationship followed a Boltzmann distribution characte rized by the midpoint voltage (V-0.5) and the maximum amount of movable cha rge (Delta Q(max)). Forskolin (2-10 mu M) shifted V-0.5 to more negative va lues while Delta Q(max) was not affected (n = 11). The effects of forskolin on transient pump currents were mimicked by 8-bromo-cAMP (500 mu M; n = 2) and abolished by a peptide inhibitor of PRA (PKI, 10 mu M; n = 5). 6. We conclude that activation of the cAMP-PKA pathway in guinea-pig ventri cular myocytes increases Na+-K+ pump current at least in part by modulating partial reactions in the Na+ limb of the pump cycle. Under physiological c onditions, the observed stimulation of the cardiac Na+-K+ pump may serve to shorten the action potential duration and to counteract the increased pass ive sarcolemmal Na+ and K+ fluxes during sympathetic stimulation of the hea rt.