Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, 68198-5850, USA.
Department of Physiology and Biophysics, University of Minas Gerais, Belo Horizonte, MG, 31270-910, Brazil.
Nitric Oxide. 2020 Jan 1;94:54-62. doi: 10.1016/j.niox.2019.10.007. Epub 2019 Oct 22.
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 ATR 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.
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许多心血管疾病的发病机制中起着关键作用，包括高血压。之前我们已经发现，心力衰竭大鼠的 PVN 中，PIN（一种神经元型一氧化氮合酶（nNOS）的蛋白抑制剂，已知可将 nNOS 二聚体解离为单体）的蛋白表达增加，同时催化活性 nNOS 二聚体的水平降低。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（250-300g）通过渗透微型泵进行脑室内输注 Ang II（20ng/min，0.5μl/h）或盐水作为载体（Veh），持续 14 天，以及用 Ang II 处理的 NG108-15 杂交神经元细胞系作为体外模型。Ang II 输注显著增加了基础肾交感神经活性和平均动脉压。Ang II 输注增加了 PIN 的表达（1.24±0.04Ang II 与 0.65±0.07Veh），同时 PVN 中二聚体 nNOS 和 PIN-Ub 缀合物减少了 50%（0.73±0.04Ang II 与 1.00±0.03Veh）。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 Lactacystin（LC）处理后，HA-Ub-PIN 缀合物减少（4.5±0.7LC Ang II 与 9.2±2.5LC）。TUBE（串联泛素结合结构域）测定显示，Ang II 处理的细胞中 PIN-Ub 缀合物减少（0.82±0.12LC Ang II 与 1.21±0.06LC），而 ATR 阻断剂 Losartan（Los）处理减少了 Ang II 介导的 PIN 稳定化（1.21±0.07LC Los 与 1.16±0.04*LC Ang II Los）。总之，我们的研究表明，中枢水平升高的 Ang II 导致 PIN 表达增加，导致 nNOS 二聚体形式的表达减少，从而降低了 NO•对 PVN 中自主前神经元的抑制作用，导致交感神经输出增加。
