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 increased protein expression of 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 heart failure. To elucidate the molecular mechanism by which Angiotensin II (Ang II) increases PIN expression, we used Sprague-Dawley rats (250-300 g) subjected to intracerebroventricular infusion of Ang II (20 ng/min, 0.5 μl/h) or saline as vehicle (Veh) for 14 days 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 renal sympathetic nerve activity and mean arterial pressure. Ang II infusion increased the expression of PIN (1.24 ± 0.04* Ang II vs. 0.65 ± 0.07 Veh) with a concomitant 50% decrease in dimeric nNOS and PIN-Ub conjugates (0.73 ± 0.04* Ang II vs. 1.00 ± 0.03 Veh) in the PVN. 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 a proteasome inhibitor, Lactacystin (LC), treatment (4.5 ± 0.7* LC Ang II vs. 9.2 ± 2.5 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. 1.21 ± 0.06 LC) while AT1R blocker, Losartan (Los) treatment diminished the Ang II-mediated stabilization of PIN (1.21 ± 0.07 LC Los vs. 1.16 ± 0.04* LC Ang II 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 increased sympathetic outflow.

中文翻译：

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神经元一氧化氮合酶（PIN）轴的中央血管紧张素II-蛋白质抑制剂可导致神经源性高血压。

肾素-血管紧张素系统的激活，一氧化氮（NO•）的生物利用度以及随后的交感神经兴奋在包括高血压在内的许多心血管疾病的发病机理中起着关键作用。以前，我们已经显示出PIN（nNOS的蛋白抑制剂：神经型一氧化氮合酶，已知将nNOS二聚体解离为单体）的蛋白表达增加，同时伴有心力衰竭的PVN中nNOS的催化活性二聚体水平降低。为了阐明血管紧张素II（Ang II）增加PIN表达的分子机制，我们使用Sprague-Dawley大鼠（250-300 g）进行脑室内脑室灌注Ang II（20 ng / min，0.5μl/ h）或生理盐水通过渗透微型泵和以Ang II处理的NG108-15混合神经元细胞系体外模型（Veh）持续14天。Ang II输注显着增加基线肾交感神经活动和平均动脉压。Ang II输注增加了PIN的表达（1.24±0.04 * Ang II vs. 0.65±0.07 Veh），同时二聚nNOS和PIN-Ub共轭物降低了50％（0.73±0.04 * Ang II vs. 1.00±0.03 Veh）在PVN中。用pCMV-（HA-Ub）8载体转染的细胞中的底物依赖性连接酶测定显示，Ang II和蛋白酶体抑制剂乳杆菌素（LC）处理（4.5±0.7 * LC Ang II）后，HA-Ub-PIN共轭物减少相对于9.2±2.5 LC）。TUBE（串联泛素结合实体）分析显示，Ang II处理的细胞中PIN-Ub缀合物减少（0.82±0.12 * LC Ang II vs. 1.21±0.06 LC），而AT1R阻滞剂，氯沙坦（Losartan）处理减少了Ang II-介导的PIN稳定化（1.21±0.07 LC损失vs.1.16±0.04 * LC Ang II损失）。