Central Angiotensin II regulates Protein Inhibitor of Neuronal Nitric Oxide Synthase through post‐translational mechanisms in the Paraventricular Nucleus resulting in increased Sympathetic outflow

Abstract

Increased activation of Renin‐Angiotensin System in the paraventricular nucleus (PVN) of the hypothalamus has been associated with the increased sympathetic outflow observed in hypertension and chronic heart failure (CHF). Previously we have shown increased protein expression of the PIN (a protein inhibitor of nNOS: neuronal nitric oxide synthase, known to dissociate nNOS dimers into monomers) with concomitantly reduced levels of catalytically active dimers of nNOS in the PVN of rats with CHF. However, PIN transcripts levels remain unchanged. To elucidate the molecular mechanism of increased PIN expression post‐transcriptionally, we used Sprague‐Dawley rats (250–300 g) subjected to intracerebroventricular infusion of Ang II (20 ng/min, 14 days, 0.5 μl/h) through osmotic mini‐pumps and NG108‐15 hybrid neuronal cell line treated with Ang II as an in vitro model. Ang II infusion significantly increased baseline mean arterial pressure (126 ± 9 vs. 84 ± 4 mmHg) and baseline renal sympathetic nerve activity (20.5 ± 2.3 vs. 6.4 ± 1.9 % of Max activity). Ang II infusion increased the expression of the PIN (1.36 ± 0.04* Ang II vs. 0.81 ± 0.02 Veh) a with concomitant decrease in PIN‐Ub conjugates (0.73 ± 0.04* Ang II vs. 1 ± 0.03 Veh) in the PVN. Further, PIN translation was inhibited in vitro using protein synthesis inhibitor cycloheximide (CHX) for 0–4h after 20h of pretreatment with Ang II. Ang II‐mediated increase in PIN expression(0.67±0.06* CHX AngII vs. 0.40±0.08 CHX 0h) was independent of CHX mediated decrease in PIN expression (0.41±0.06* CHX AngII vs. 0.19±0.04 CHX 4h) suggesting Ang II‐mediated post‐translational stabilization of PIN. Ubiquitination assay in cells transfected with pCMV‐(HA‐Ub) 8 vector revealed a reduction of HA‐Ub‐PIN conjugates after Ang II and proteasome inhibitor lactacystin (LC) treatment (4.5 ± 0.6* LC Ang II vs. 9.2 ± 2.2 LC). TUBE (Tandem Ubiquitin‐Binding Entities) assay showed decrease PIN‐Ub conjugates in Ang II‐treated cells (0.82 ± 0.12* LC Ang II vs. 1.23 ± 0.05 LC) while AT1R blocker Losartan(Los) treatment diminishes the Ang II‐mediated stabilization of PIN (1.21 ± 0.07 LC Los vs. 1.14 ± 0.04* LC AngII Los). These results suggest that PIN is targeted for rapid degradation by the ubiquitin‐proteasome and Ang II via AT1R delays the rate of degradation resulting in accumulation of PIN. It of interest to note that previously we observed that there was a decreased accumulation of PIN‐Ub conjugates in the PVN of CHF rats compared to Sham (0.63 ± 0.05* CHF vs. 1.00 ± 0.10 Sham). Taken together, our studies suggest that increased central levels of Ang II contribute to the upregulation of PIN expression in the PVN via decreasing the ubiquitination which may be involved in reducing the expression of dimeric nNOS of pre‐autonomic neurons in the PVN resulting in the increased sympathetic outflow.Support or Funding InformationSupported by AHA ‐ 14SDG19980007 and NIH grants HL124104 & HL62222.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.