Post-translational regulation of neuronal nitric oxide synthase: implications for sympathoexcitatory states

Abstract

Nitric oxide (NO) synthesized via neuronal nitric oxide synthase (nNOS) plays a significant role in regulation/modulation of autonomic control of circulation. Various pathological states are associated with diminished nNOS expression and blunted autonomic effects of NO in the central nervous system (CNS) including heart failure, hypertension, diabetes mellitus, chronic renal failure etc. Therefore, elucidation of the molecular mechanism/s involved in dysregulation of nNOS is essential to understand the pathogenesis of increased sympathoexcitation in these diseased states. Areas covered: nNOS is a highly regulated enzyme, being regulated at transcriptional and posttranslational levels via protein-protein interactions and modifications viz. phosphorylation, ubiquitination, and sumoylation. The enzyme activity of nNOS also depends on the optimal concentration of substrate, cofactors and association with regulatory proteins. This review focuses on the posttranslational regulation of nNOS in the context of normal and diseased states within the CNS. Expert opinion: Gaining insight into the mechanism/s involved in the regulation of nNOS would provide novel strategies for manipulating nNOS directed therapeutic modalities in the future, including catalytically active dimer stabilization and protein-protein interactions with intracellular protein effectors. Ultimately, this is expected to provide tools to improve autonomic dysregulation in various diseases such as heart failure, hypertension, and diabetes.

Keywords: Cardiovascular diseases; PVN; nNOS; sympathoexcitation.

Figure 1

(A) Illustration of the molecular structure of NOS dimer. All NOS monomers include an oxygenase and a reductase domain and calmodulin (CaM) binding site. The oxygenase moiety comprises the L-arginine, heme, and tetrahydrobiopterin (BH4)-binding domains; whereas the reductase moiety includes the flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide phosphate (NADPH)- binding domains. NOS monomers are unable to bind the cofactor BH4 or the substrate arginine to catalyze the NO production. In the presence of heme, NOS can form a functional dimer that can bind to BH4 and L-Arg that allows interdomain electron transfer. (B) Schematic illustration of the protein structure of NOS isoforms.

Figure 2

(A) Composite data were showing the expression of nNOS, CAPON, and PIN in the PVN of CHF rats. Values are mean ± SEM (n = 4–5 rats per group). *P < 0.05 vs. Sham. Modified from Sharma et al (2011)[36] and Sharma et al (2013)[48](B) Immunofluorescent photomicrographs from the sections of the PVN stained for nNOS, CAPON, and PIN. The intensity of nNOS staining (green) is decreased while the intensity of CAPON and PIN as shown in red is increased in the PVN of CHF rats. Yellow staining showed colocalization of nNOS-PIN or nNOS-CAPON in the same neuron. Blue spots showed the nucleus stained by Dap1. Modified from Sharma et al (2011)[36] and Sharma et al (2013)[48] (C) Effect of silencing of CAPON on nNOS expression in NG108 neuronal cell line. siRNA CAPON or negative control (20 pmol) was transfected in NG108 cells using Lipofectamine 2000 as per the manufacturer’s instructions. Values were mean±SE from four Independent experiments. From Sharma et al (2011)[36]. (D) Effect of upregulation of nNOS on the CAPON expression. Adenovirus vectors encoding nNOS (AdnNOS, 10⁵~10⁸pfu/ml, 48hrs) or adenovirus vectors encoding EGFP (AdEGFP, 10⁵ pfu/ml, 48hrs) were transfected into NG108 cells

Figure 3

(A) Assessment of monomers and dimers of nNOS represented as a ratio of dimer to monomer and (B) high molecular weight ubiquitinated forms of immuno-detectable nNOS in the PVN of Sham and CHF rats. Dimeric and monomeric nNOS were separated by low temperature-PAGE in the cold room and visualized by immunoblotting using an anti-nNOS antibody. (C) Composite data of Dimer/Monomer ratio and nNOS-Ub in the PVN of Sham and CHF rats. Values are mean ± SEM (n = 4–5 rats per group). *P < 0.05 vs. Sham. (D) High magnification images of the coronal sections of PVN (63X) showing the colocalization of Ub(red) and nNOS (green). From Sharma et al (2013)[48]

Figure 4

Schematic depicting presumptive pathway of nNOS regulation in the PVN of CHF rats. CAPON and PIN are over-expressed due to augmented levels of Ang II and AT₁ receptors in the PVN. Increased CAPON competes with PSD95 for binding to nNOS and sequesters nNOS therefore decreasing NMDAR/PSD95/nNOS complexes at the synaptic membrane. Binding of PIN to nNOS in cytosol destabilizes nNOS dimers, which renders nNOS catalytically inactive, either by interfering with the assembly or dimer stability. Inactive nNOS monomers are susceptible to ubiquitination and subsequent proteasomal degradation. This results in decreased levels of nNOS as well as NO production in the PVN during CHF causing an increase in sympathetic nerve activity (SNA). Modified from Sharma et al (2013)[48]