Abstract 106: Central Angiotensin II Mediates Neurogenic Hypertension Through Post-Translational Regulation of Neuronal Nitric Oxide Synthase in the Paraventricular Nucleus of the Hypothalamus

Hypertension ◽  
2018 ◽  
Vol 72 (Suppl_1) ◽  
Author(s):  
Neeru M Sharma ◽  
Andrea S Haibara ◽  
Kenichi Katsurada ◽  
Xuefei Liu ◽  
Kaushik P Patel
Keyword(s):  
Nitric Oxide ◽  
Angiotensin Ii ◽  
Nitric Oxide Synthase ◽  
Paraventricular Nucleus ◽  
Translational Regulation ◽  
Neuronal Nitric Oxide Synthase ◽  
Neurogenic Hypertension ◽  
Oxide Synthase

scholarly journals Central angiotensin II-Protein inhibitor of neuronal nitric oxide synthase (PIN) axis contribute to neurogenic hypertension

Nitric Oxide ◽  
2020 ◽  
Vol 94 ◽  
pp. 54-62 ◽  
Author(s):  
Neeru M. Sharma ◽  
Andrea S. Haibara ◽  
Kenichi Katsurada ◽  
Xuefei Liu ◽  
Kaushik P. Patel
Keyword(s):  
Nitric Oxide ◽  
Angiotensin Ii ◽  
Nitric Oxide Synthase ◽  
Neuronal Nitric Oxide Synthase ◽  
Protein Inhibitor ◽  
Neurogenic Hypertension ◽  
Oxide Synthase

Abstract 085: CHIP: E3 Ubiquitin Ligase Mediates Proteasomal Degradation of Neuronal Nitric Oxide Synthase in the Paraventricular Nucleus of Rats with Heart Failure

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Neeru M Sharma ◽  
Kenichi Katsurada ◽  
Xuefei Liu ◽  
Kaushik P Patel
Keyword(s):  
Nitric Oxide ◽  
Heart Failure ◽  
Nitric Oxide Synthase ◽  
Paraventricular Nucleus ◽  
Ubiquitin Ligase ◽  
Protein A ◽  
Neuronal Nitric Oxide Synthase ◽  
Proteasomal Degradation ◽  
Oxide Synthase

The exaggerated sympathetic drive is a characteristic of heart failure (HF) due to reduced neuronal nitric oxide synthase (nNOS) within the paraventricular nucleus (PVN). Previously we have shown that there were increased accumulation of nNOS-ubiquitin (nNOS-Ub) conjugates in the PVN of rats with HF (1.0±0.05 Sham vs. 1.29±0.06 HF) due to the increased levels of PIN (a protein inhibitor of nNOS, known to dissociate nNOS dimers into monomers) (0.76±0.10 Sham vs. 1.12±0.09 HF) and decreased levels of tetrahydrobiopterin (BH4): a cofactor required for stabilization of nNOS dimers (0.62±0.02 Sham vs. 0.44±0.03 HF). We also showed that there is blunted nitric oxide-mediated inhibition of sympathetic tone via the PVN in HF. Here we examined whether CHIP(C-terminus of Hsp70 -interacting protein), a chaperone-dependent E3 ubiquitin-protein isopeptide ligase known to ubiquitylate Hsp90-chaperoned proteins could act as an ubiquitin ligase for nNOS in the PVN. Immunofluorescence studies revealed colocalization of nNOS and CHIP in the PVN indicating their possible interaction. CHIP expression was increased by 50% in the PVN of rats with HF(0.96±0.08 Sham vs.1.44±0.10* HF). It is shown that Hsp90 protects nNOS from ubiquitination while Hsp70 promotes the ubiquitination and degradation. We observed significant upregulation of Hsp70 (0.49±0.03 Sham vs. 0.65±0.02* HF) with a trend toward the decrease in Hsp90 expression (0.90±0.07 Sham vs. 0.71±0.06 HF). The opposing effects of the two chaperones could account for the increased CHIP-mediated ubiquitination and degradation of dysfunctional nNOS monomers in the PVN of rats with HF. Furthermore, neuronal NG108-15 cell line transfected with the pCMV3-CHIP-GFP spark (CHIP overexpression plasmid) showed approximately 74% increase in CHIP with concomitant 49% decrease in nNOS expression. In vitro ubiquitination assay in NG108 cells transfected with pCMV-(HA-Ub) 8 and pCMV3-CHIP-GFP spark plasmid reveal increased HA-Ub-nNOS conjugates (1.13 ± 0.09 Scramble vs. 1.65 ± 0.12* CHIP plasmid). Taken together, our results identify CHIP as an E3 ligase for ubiquitination of dysfunctional nNOS and CHIP expression is augmented during HF leading to increased proteasomal degradation of nNOS in the PVN.

Abstract 15532: Altered Ubiquitination and Stability of Protein Inhibitor of Neuronal Nitric Oxide Synthase in the Paraventricular Nucleus of Chronic Heart Failure Rats: Role of Angiotensin II

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Neeru Sharma ◽  
Xuefei Liu ◽  
Hong Zheng ◽  
Kaushik Patel
Keyword(s):  
Nitric Oxide ◽  
Heart Failure ◽  
Angiotensin Ii ◽  
Chronic Heart Failure ◽  
Paraventricular Nucleus ◽  
Neuronal Nitric Oxide Synthase ◽  
Ang Ii ◽  
Protein Inhibitor ◽  
Ubiquitin Proteasome ◽  
Oxide Synthase

Introduction and Hypothesis: Expression of neuronal nitric oxide synthase (nNOS) is decreased in the paraventricular nucleus (PVN) of rats with chronic heart failure (CHF), however the underlying molecular mechanism/s remain unclear. Recently, we demonstrated, Angiotensin II (Ang II) mediated increase in PIN: protein inhibitor of nNOS (0.76±0.10 Sham vs 1.12±0.09* CHF) which is known to down-regulate nNOS through disruption of active dimers (~60% decrease in dimer/monomer ratio) in the PVN of rats with CHF. Functionally impeded monomeric enzyme is degraded by ubiquitin proteasome system. Interestingly, PIN transcript levels remain unchanged in the PVN in CHF (1.00±0.23 Sham vs. 1.1±0.28 CHF). This observation prompted us to elucidate the molecular mechanism for the accumulation of PIN post-transcriptionally in the PVN in CHF Methods and Results: We used coronary artery ligation model of CHF in rats (6-8 weeks past ligation) and neuronal NG108-15 hybrid cell line. PIN translation was inhibited using cyclohexamide (CHX) for 0-4h after 20h of pretreatment with Ang II in NG108 cells. CHX mediated decrease in PIN expression was ameliorated with Ang II (0.19±0.04 vs 0.41±0.06* 4h). Proteasome inhibitor lactacystin (LC) treatment dramatically elevates PIN level suggesting the involvement of proteasome system in PIN regulation. Immunoprecipitation with ubiquitin antibody showed decrease PIN-Ub conjugates in Ang II-treated cells (1.04 ± 0.05 LC vs. 0.62 ± 0.07* LC AngII). In vitro ubiquitination assay in cells transfected with pCMV-(HA-Ub)8 vector revealed reduction of HA-Ub-PIN conjugates after Ang II treatment (9.2 ± 2.2 LC vs. 4.5 ± 0.6* LC Ang II). Furthermore, there was decreased accumulation of PIN-Ub conjugates in the PVN of CHF rats compared to Sham as revealed by immunohistochemistry. Conclusions: Taken together, our studies revealed that PIN is targeted for rapid degradation by the ubiquitin-proteasome pathway and Ang II delays the rate of degradation resulting in accumulation of PIN. We conclude that post-translational accumulation of PIN, mediated by Ang II, leads to a decrease in the dimeric active form of nNOS as well as protein levels of nNOS, which may lead to reduced nitric oxide resulting in over-activation of sympathetic drive during CHF.

scholarly journals Translational Regulation of Human Neuronal Nitric-oxide Synthase by an Alternatively Spliced 5′-Untranslated Region Leader Exon

2002 ◽  
Vol 278 (1) ◽  
pp. 636-644 ◽  
Author(s):  
Derek C. Newton ◽  
Siân C. Bevan ◽  
Stephen Choi ◽  
G. Brett Robb ◽  
Adam Millar ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Untranslated Region ◽  
Translational Regulation ◽  
Neuronal Nitric Oxide Synthase ◽  
Alternatively Spliced ◽  
Oxide Synthase
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