Modulation of ATP-sensitive Potassium Channels by cGMP-dependent Protein Kinase in Rabbit Ventricular Myocytes

Abstract

This investigation used a patch clamp technique to test the hypothesis that protein kinase G (PKG) contributes to the phosphorylation and activation of ATP-sensitive K ؉ (K ATP ) channels in rabbit ventricular myocytes. Nitric oxide donors and PKG activators facilitated pinacidilinduced K ATP channel activities in a concentration-dependent manner, and a selective PKG inhibitor abrogated these effects. In contrast, neither a selective protein kinase A (PKA) activator nor inhibitor had any effect on K ATP channels at concentrations up to 100 and 10 M, respectively. Exogenous PKG, in the presence of both cGMP and ATP, increased channel activity, while the catalytic subunit of PKA had no effect. PKG activity was prevented by heat inactivation, replacing ATP with adenosine 5-O-(thiotriphosphate) (a nonhydrolyzable analog of ATP), removing Mg 2؉ from the internal solution, applying a PKG inhibitor, or by adding exogenous protein phosphatase 2A. The effects of cGMP analogs and PKG were observed under conditions in which PKA was repressed by a selective PKA inhibitor. The results suggest that K ATP channels are regulated by a PKG-signaling pathway that acts via PKG-dependent phosphorylation. This mechanism may, at least in part, contribute to a signaling pathway that induces ischemic preconditioning in rabbit ventricular myocytes.Protein phosphorylation is a putative effector mechanism in the infarct size-limiting effect of ischemic preconditioning (1), a phenomenon whereby a brief period of ischemia and reperfusion can protect the heart against subsequent prolonged ischemia and reperfusion injury (2). Indeed, it was shown that phosphorylation levels decrease during ischemia in nonpreconditioned hearts, whereas they increase during ischemia in preconditioned hearts (3). A number of cellular components, including cytoskeletal and stress proteins, have been proposed as potential phosphorylation targets in the ischemic preconditioned heart (4 -6).ATP-sensitive K ϩ channels (K ATP channels) 1 in sarcolemma and mitochondria are also modulated by phosphorylation (7, 8).The majority of the studies on the contribution of phosphorylation to K ATP channel activity to the cardioprotective effects of ischemic preconditioning have centered on the role of protein kinase C. Protein kinase C may act as a link between one or more receptor-mediated pathways and increased K ATP channel activity and thus lead to ischemic preconditioning (9). The release of endogenous substances, such as adenosine, bradykinin, nitric oxide (NO), and prostacyclin (10 -12), has been proposed as a potential mechanism of ischemic preconditioning. These substances increase cGMP via direct stimulation of myocardial cells or via the endothelium. It was reported that the cGMP levels in preconditioned hearts are higher than in nonpreconditioned hearts (13-15). Since cGMP can induce protein phosphorylation via protein kinase G (PKG) activation, the involvement of PKG-dependent phosphorylation in ischemic preconditioning is expected. To date, however, the role of PKG ...

Controlling anoxic tolerance in adult Drosophila via the cGMP–PKG pathway

“…The role of PKG in K ATP channel regulation remains controversial, with some studies suggesting that PKG activates K ATP (Han et al, 2001) while others report K ATP suppression by guanyl cyclase (GC) (Ropero et al, 1999). Recent patch clamp work by Chai and Lin et al (Chai and Lin et al, 2008) confirms that stimulation of the PKG pathway increases K ATP activity, and the authors suggest that this is a means to manipulate neuronal excitability and/or survival (Chai and Lin, 2008).…”

A role for nitric oxide in hypoxia-induced activation of cardiac KATP channels in goldfish (Carassius auratus)

“…Even more NOS would be available in the intact heart, in light of the fact that the endocardial endothelium in fish has a much greater surface area relative to the volume of the ventricle than does the equivalent tissue in mammals (Imbrogno et al, 2001). In mammals, the hypoxia-induced activation of sarcolemmal KATP channels is thought to involve NO and to be a cGMPdependent mechanism (Chen et al, 2000;Han et al, 2001). Prior experiments with teleost fish involving the stable and lipid soluble cGMP analog 8-Br-cGMP and the guanylate cyclase inhibitor ODQ indicate that an NO-cGMP-dependent pathway is also operative in the vasculature and the heart of the eel Anguilla anguilla (Imbrogno et al, 2001;Pellegrino et al, 2002).…”

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