Nitric Oxide Modulates Cardiac Na ⁺ Channel via Protein Kinase A and Protein Kinase G

Abstract

Abstract-We directly tested the effects of nitric oxide (NO) on Na ϩ channels in guinea pig and mouse ventricular myocytes using patch-clamp recordings. We have previously shown that NO donors have no observed effects on expressed Na ϩ channels. In contrast, NO (half-blocking concentration of 523 nmol/L) significantly reduces peak whole-cell Na ϩ current (I Na ) in isolated ventricular myocytes. The inhibitory effect of NO on I Na was not associated with changes in activation, inactivation, or reactivation kinetics. At the single-channel level, the reduction in macroscopic current was mediated by a decrease in open probability and/or a decrease in the number of functional channels with no change in single-channel conductance. Application of cell permeable analogs of cGMP or cAMP mimics the inhibitory effects of NO. Furthermore, the effects of NO on I Na can only be blocked by inhibition of both cGMP and cAMP pathways. Sulfhydryl-reducing agent does not reverse the effect of NO. In summary, although NO exerts its action via the known guanylyl cyclase (GC)/cGMP pathway, our findings provide evidence that NO can mediate its function via a GC/cGMP-independent mechanism involving the activation of adynylyl cyclase (AC) and cAMP-dependent protein kinase. Key Words: nitric oxide Ⅲ cardiac Na ϩ current Ⅲ protein kinase A Ⅲ protein kinase G N itric oxide (NO) is a uniquely diffusible and reactive molecular messenger, which is found in abundance and plays important regulatory roles in different systems throughout the body. 1 In the cardiovascular system, NO is the major endothelium-derived relaxing factor (EDRF) and causes vasodilation and reduces blood pressure. 2 In addition, NO functions as an important endogenous inhibitor of vascular lesion formation. 3 The coronary endothelium is responsible for the bulk of the endogenous, physiological production of NO. 4 However, NO can also be produced within the cardiac myocytes themselves by the constitutive NO synthase. 5 NO modulates cardiac contractility both in vitro and in vivo. 6 More recently, it was shown that NO can regulate both adenylyl cyclase (AC) and guanylyl cyclase (GC) in cardiac myocytes. High levels of NO induce large increases in cGMP and a negative inotropic effect, whereas low levels of NO increase cAMP and induce a positive contractile response. 7 We have previously shown using heterologous expression systems that although Ca 2ϩ channel can be directly modulated by NO, Na ϩ channels are unaffected by direct NO modulation. 8 Here, we show that NO modulates Na ϩ channels in native cardiac myocytes. In addition, we provide evidence to demonstrate that NO modulates Na ϩ channels via second messenger pathways through activation of protein kinase G (PKG) and protein kinase A (PKA). Materials and MethodsSingle left ventricular myocytes were isolated from guinea pigs and mice (Charles River Laboratories, Wilmington, Mass) as previously described. 9 See further details in the expanded Materials and Methods section that can be found in the online data supplement av...

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