© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
cGMP-dependent protein kinase mediates stimulation of L-type calcium current by cGMP in rabbit atrial cells
The Todd Franklin Cardiac Research Laboratory, The Children's Heart Center, Department of Pediatrics, Emory University School of Medicine, 2040 Ridgewood Dr. NE, Atlanta, GA 30322, USA
* Corresponding author. Tel.: +1-404-727-5747; fax: +1-404-727-6024 rajiv{at}cellbio.emory.edu
Objectives: cGMP has been shown to exert both stimulatory and inhibitory effects on cardiac L-type calcium current (ICa). The physiological role of cGMP in regulation of cardiac activity is still controversial. cGMP may be of importance in regulation of ICa in atrial cells. The present study was focused on the role of cGMP in the modulation of ICa in rabbit atrial cells. Methods: Enzymatically isolated adult rabbit atrial cells were used to measure ICa using whole cell voltage clamp. Expressed levels of cGMP-dependent protein kinase (PKG) were determined by Western blotting using PKG specific antibody in homogenates from atrial and ventricular cells. Results: Nitrosoglutathione (GSNO), a nitric oxide donor that stimulates soluble guanylyl-cyclase to elevate cGMP levels increased ICa while soluble G-cyclase inhibitors, ODQ or methylene blue inhibited ICa. Intracellular application of 8BrcGMP increased ICa and blocked the inhibitory effect of methylene blue. KT-5823, an inhibitor of PKG inhibited ICa and the stimulatory effect of GSNO was completely blocked ODQ or KT-5823. Inhibition of cAMP dependent protein kinase (PKA) by the 6–22 peptide completely blocked the stimulation of ICa by the β-agonist isoproterenol but not by GSNO. The potency of isoproterenol to stimulate ICa was very high for atrial cells (EC50 2.4±0.6 nM) and only 100 nM isoproterenol was required to stimulate ICa maximally (21.4±0.7 pA/pF) to a level (23.8±1.6 pA/pF) achieved with the inclusion of 100 µM cAMP in the pipette solution. GSNO produced an additive effect on ICa already stimulated by either 10 µM isobutylmethylxanthine (phosphodiesterase inhibitor) or a low concentration (1 nM) isoproterenol but failed to produce any effect on ICa maximally stimulated by 100 nM isoproterenol. Inhibition of PKG by KT-5823 significantly decreased the efficacy of isoproterenol and the maximal ICa achieved with 100 nM isoproterenol was decreased to 8.2±0.6 pA/pF in the presence of KT-5823. Western blot analysis showed much higher expression of PKG in atrial cells compared to ventricular cells. Conclusions: These findings suggest that stimulatory effects of cGMP on ICa in rabbit atrial cells are likely to be mediated via PKG dependent phosphorylation of calcium channels or associated proteins and that the effects of cGMP are not antagonistic to cAMP. PKG is highly expressed in atrial cells and PKG dependent phosphorylation may be necessary for maintaining basal ICa and fully stimulating ICa by β-adrenergic activation in atrial cells.
KEYWORDS Second messengers; Protein kinases; Nitric oxide; Ca-channel; Myocytes; Atrial function
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