Dual regulation of cardiac Na+-K+ pumps and CFTR Cl- channels by protein kinases A and C.
Regulation of Na+-K+ pump current (I(p)) and cystic fibrosis transmembrane conductance regulator (CFTR) Cl- current (I(CFTR)) by protein kinases A and C (PKA and PKC) was compared under identical experimental conditions by simultaneous measurement of the two currents in guinea-pig ventricular myocytes whole-cell voltage-clamped at 30-32 degrees C. Membrane current (I) was monitored at a holding potential (V) of -20 mV. I/ V relationships were obtained by hyperpolarizing voltage ramps. Phorbol 12,13-dibutyrate (PDBu, 0.1-1 microM) and chelerythrine (10 microM) were used to stimulate and inhibit, respectively, PKC activity. PKA was stimulated by forskolin (4 microM) and inhibited by H-89 (50 microM). At -20 mV, stimulation of PKC by PDBu increased I(p) to 121-123% of control. Addition of chelerythrine completely reversed this effect. The PDBu-induced augmentation of I(p) was voltage dependent. The ratio I(p)(PDBu)/ I(p)(control) increased from 1.10 at -100 mV to ~1.35 at positive membrane potentials. Stimulation of PKA by forskolin also increased I(p) voltage dependently (128% of control at -20 mV). The effects of PKC and PKA stimulation on I(p) were additive. The maximum I(p) observed in the presence of PDBu and forskolin was 141% of control. Application of either H-89 or chelerythrine reversibly decreased I(p) by 40% and 24%, respectively, suggesting that basal PKA and PKC activities were involved in the regulation of I(p). In the presence of H-89, PDBu was unable to increase I(p). Likewise, pre-application of chelerythrine abolished the forskolin-induced augmentation of I(p). In contrast to I(p), I(CFTR) (measured simultaneously) was absent under basal conditions. Stimulation of PKA by forskolin activated a pronounced I(CFTR). Stimulation of PKC by PDBu, on the other hand, neither activated the Cl(-) current significantly nor increased I(CFTR) pre-activated by forskolin. Inhibition of PKC by chelerythrine, however, attenuated the PKA-mediated activation of I(CFTR). The results reveal a complex interplay between PKA and PKC in regulating cardiac I(p) and I(CFTR) with some similarities but also important differences. I(p) is increased voltage dependently and additively by stimulation of both kinases. The steady-state activity of each of the kinases is involved in the modulation of basal I(p) and obligatory for the augmentation of I(p) induced by stimulation of the other kinase. In contrast, there appears to be no basal I(CFTR). I(CFTR) is activated significantly only after stimulation of PKA. PKC activity, however, appears to facilitate this activation.
Erlenkamp
Simone
S
Arbeitsgruppe Muskelphysiologie, Fakultät für Biologie, Ruhr-Universität, 44780 Bochum, Germany.
Glitsch
Helfried G
HG
Kockskämper
Jens
J
D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D002301:Cardiac Glycosides / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006168:Guinea Pigs; D066298:In Vitro Techniques; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D010617:Phenanthridines / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020239:Receptor Cross-Talk / Q000502:physiology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000470:Alkaloids; D053119:Benzophenanthridines; D002301:Cardiac Glycosides; D018118:Chloride Channels; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D010617:Phenanthridines; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D015240:Phorbol 12,13-Dibutyrate; C016299:chelerythrine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase