Premium
Activation of the cAMP–protein kinase A pathway facilitates Na + translocation by the Na + –K + pump in guinea‐pig ventricular myocytes
Author(s) -
Kockskämper Jens,
Erlenkamp Simone,
Glitsch Helfried G.
Publication year - 2000
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.2000.t01-2-00561.x
Subject(s) - forskolin , adenylyl cyclase , chemistry , pipette , protein kinase a , patch clamp , guinea pig , biophysics , endocrinology , kinase , biology , biochemistry , enzyme , receptor
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 °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 + (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 I p ( 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 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 Ca 2+ concentration ([Ca 2+ ] pip ) was 0·013 nM ( n = 5 ) and to 138 ± 4 % of the control I p when free [Ca 2+ ] 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 in 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 potential ( V ). The Q ‐ V relationship followed a Boltzmann distribution characterized by the midpoint voltage ( V 0·5 ) and the maximum amount of movable charge (Δ Q max ). Forskolin (2‐10 μM) shifted V 0·5 to more negative values while Δ Q max 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). 6 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.