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

Abstract

Activation of renin-angiotensin- system, nitric oxide (NO) bioavailability and subsequent sympathoexcitation plays a pivotal role in the pathogenesis of many cardiovascular diseases including hypertension. Previously we have shown Ang II enhances PIN (a protein inhibitor of nNOS: neuronal nitric oxide synthase, known to dissociate nNOS dimers into monomers) mediated ubiquitination of nNOS in the PVN of rats with heart failure. To further elucidate the mechanism by which Ang II increases sympathetic outflow by regulating PIN mediated nNOS ubiquitination, we used Sprague-Dawley rats (250-300 g) subjected to intracerebroventricular infusion of Ang II (20 ng/min, 14days, 0.5μl/h) through osmotic mini-pumps and NG108-15 hybrid neuronal cell line treated with Ang II as an in vitro model. Ang II infusion increased baseline mean arterial pressure (126 ± 9* vs. 84 ± 4 mmHg) and renal sympathetic nerve activity (20.5 ± 2.3* vs. 6.4 ± 1.9 % of Max. Activity). Ang II infusion increased the expression of PIN (1.36 ± 0.04* Ang II vs. 0.81 ± 0.02 Veh) with a concomitant 52% decrease in dimeric nNOS and PIN-Ub conjugates (0.73 ± 0.04* Ang II vs. 1 ± 0.03 Veh) in the PVN. Further, Ang II-mediated increase in PIN expression(0.67±0.06* CHX AngII vs. 0.40±0.08 CHX 0h) was independent of CHX (protein synthesis inhibitor) 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. Substrate-dependent ligase 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). Taken together, our studies suggest that increased central levels of Ang II contribute to the enhanced expression of PIN leading to reduced expression of the dimeric form of nNOS, thus diminishing the inhibitory action of NO on pre-autonomic neurons in the PVN resulting in an increase in sympathetic outflow.