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

Abstract

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 °C. In external solution containing 144 mM Na+ (Na+o) and 10 mM K+ (K+o), steady‐state Na+‐K+ pump current (Ip) activated by 5 mM pipette Na+ (Na+pip) at ‐20 mV was reversibly augmented by forskolin (4 μM) to 133 ± 4 % of the control current (n= 15). The forskolin analogue 1,9‐dideoxyforskolin (10 μM), which does not activate adenylyl cyclases, did not increase Ip (n= 2). Application of the protein kinase A (PKA) inhibitor H‐89 (10 μM) in the continued presence of forskolin reversed the forskolin‐induced elevation of Ip (n= 3). The forskolin effect on Ip 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 Ip to 142 ± 3 % of the control Ip when the pipette free Ca2+ concentration ([Ca2+]pip) was 0·013 nM (n= 5) and to 138 ± 4 % of the control Ip when free [Ca2+]pip was 15 nM (n= 9). In Na+‐free external solution, Ip activated by 50 mM Na+pip and 1·5 mM K+o was likewise increased by forskolin but to a lesser extent than in Na+‐containing medium (116 ± 3 % of control, n= 10). 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 potential (V). The Q‐V relationship followed a Boltzmann distribution characterized by the midpoint voltage (V0·5) and the maximum amount of movable charge (ΔQmax). Forskolin (2‐10 μM) shifted V0·5 to more negative values while ΔQmax was not affected (n= 11). The effects of forskolin on transient pump currents were mimicked by 8‐bromo‐cAMP (500 μM; n= 2) and abolished by a peptide inhibitor of PKA (PKI, 10 μM; n= 5). We conclude that activation of the cAMP‐PKA pathway in guinea‐pig ventricular myocytes increases Na+‐K+ pump current at least in part by modulating partial reactions in the Na+ limb of the pump cycle. Under physiological conditions, the observed stimulation of the cardiac Na+‐K+ pump may serve to shorten the action potential duration and to counteract the increased passive sarcolemmal Na+ and K+ fluxes during sympathetic stimulation of the heart.

Activation of cAMP-dependent Signaling Induces Oxidative Modification of the Cardiac Na+-K+ Pump and Inhibits Its Activity

“…ϩ -K ϩ pump inhibition mediated by cAMP-dependent pathways in cardiac myocytes was reported in early studies (8,9), but many more recent studies have reported that these pathways mediate pump stimulation either by increasing maximal pump rate or by increasing pump affinity for intracellular Na ϩ (7,(11)(12)(13)(14)(15)(16)30). In our study, forskolin induced glutathionylation of the Na ϩ -K ϩ pump ␤ 1 subunit.…”

“…We have found the ␤ 3 adrenergic receptor mediates stimulation of the Na ϩ -K ϩ pump in cardiac myocytes (5). 5 We, therefore, used forskolin, as have most of the recent studies (7,11,12,15,16).…”

Differences in the protein-kinase-A-dependent regulation of CFTR Cl - channels and Na + -K + pumps in guinea-pig ventricular myocytes

“…As noted in an earlier study of guinea-pig ventricular myocytes [25], the time courses of I CFTR activation and I p stimulation by forskolin are strikingly different. This finding suggests differences in the regulation of the two currents by the same intracellular signalling pathway.…”

“…Furthermore, I p contributes to the resting membrane potential and the AP form. Recent studies of guinea-pig and rat ventricular myocytes have shown that activation of the cAMP-PKA cascade by either β-adrenergic agonists (noradrenaline, isoprenaline) or the adenylyl cyclase activator forskolin causes an increase in I p [7,18,25]. Stimulation of pump activity by PKA is thought to be important for maintaining the Na + and K + gradients across the sarcolemma since passive Na + and K + fluxes are increased in the presence of β-mimetics.…”

Mechanisms of Isoform-Specific Na/K Pump Regulation by Short- and Long-Term Adrenergic Activation in Rat Ventricular Myocytes

“…After a short-term (10 min) treatment of guinea pig myocytes, α-adrenoreceptor (α-AR) agonists increased the I PH without affecting the I PL , and β-adrenoreceptor (β-AR) agonists inhibited the I PL at low intracellular Ca 2+ concentrations ([Ca 2+ ] i ) but did not affect the I PH [5,6,7,8]. However, although some evidence has indicated no effect [9,10] or even inhibition during β-AR activation [5,11,12], most results demonstrate α 1 - or α 2 -isoform stimulation after α-AR or β-AR activation [13,14,15,16,17]. These data are discrepant with the results from Mathias's laboratory, in which the short-term α-AR or β-AR agonist exposure had opposite effects on the I P by affecting specific NKP α-isoforms.…”