High affinity regulation of cardiac Cl– and Ca2+ conductances by (13R)-spiroforskolin

Sendhoff

Pfl�gers Archiv European Journal of Physiology

et al. 2001

Protein-kinase-A- (PKA-) dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- current (I(CFTR)) and Na+-K+ pump current (Ip) was studied in single guinea-pig ventricular myocytes. Both currents were measured simultaneously by means of whole-cell recording at 30 degrees C. The adenylyl cyclase activator forskolin was used to stimulate PKA activity. At -20 mV, forskolin (4 microM) induced a fast activation of I(CFTR) and a delayed stimulation of Ip. Despite the strikingly different time courses, however, the potency of the drug to regulate both currents was identical. Half-maximal activation of I(CFTR) and stimulation of Ip, respectively, were observed at 9.6 x 10(-8) M and 9.9 x 10(-8) M forskolin. Inclusion of a specific peptide inhibitor of PKA in the pipette solution (PKI, 20 microM) blocked forskolin's effect on Ip. However, regardless of the time allowed for cell dialysis, there still was a marked, transient activation of I(CFTR), which could be prevented by: (1) a short pre-activation of I(CFTR) with forskolin or (2) the additional inclusion in the pipette solution of a synthetic peptide (Ht31 peptide, 60 microM) that interferes with PKA binding to its anchoring proteins. Thus, there is a tight functional coupling between PKA and CFTR Cl- channels in guinea-pig ventricular myocytes. The coupling is probably due to the close physical proximity of channels and kinases mediated by PKA anchoring proteins. Na+-K+ pumps, on the other hand, though also regulated by PKA, appear to be loosely coupled to the kinases.

Section: Discussioncontrasting

confidence: 65%

Section: Discussionmentioning

confidence: 99%

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

Sendhoff

Pfl�gers Archiv European Journal of Physiology

et al. 2001

“…1 and ). By contrast, application of 4 μM forskolin, a concentration previously shown to maximally stimulate I Cl(cAMP) in these cells (Traebert et al 1998), activated, after a lag of approximately 20 s, an outward current which was identified as I Cl(cAMP) (see Fig. 8).…”

Section: Resultsmentioning

confidence: 93%

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

The Journal of Physiology

Glitsch

2000

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.

“…Figure 4A shows an original recording of the membrane current of a ventricular myocyte (C m : 209 pF) first challenged with 4 µM forskolin (to stimulate PKA maximally [33,43]) and then, in addition, with 1 µM PDBu (to stimulate PKC maximally). Forskolin caused an outward current shift that was due to the activation of I CFTR (as evidenced by the increase in holding current at zero K + o of +55 pA or +0.26 pA/pF; see below) and a stimulation of I p .…”

Section: Resultsmentioning

confidence: 99%

Dual regulation of cardiac Na+-K+ pumps and CFTR Cl– channels by protein kinases A and C

Glitsch

Pflügers Arch - Eur J Physiol

2002

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.