University of Nebraska Medical Center, Omaha, 68198-5850, USA.
Am J Physiol Cell Physiol. 2011 Jan;300(1):C58-64. doi: 10.1152/ajpcell.00369.2010. Epub 2010 Oct 27.
Actions of angiotensin-(1-7) [Ang-(1-7)], a heptapeptide of the renin-angiotensin system, in the periphery are mediated, at least in part, by activation of nitric oxide (NO) synthase (NOS) and generation NO(·). Studies of the central nervous system have shown that NO(·) acts as a sympathoinhibitory molecule and thus may play a protective role in neurocardiovascular diseases associated with sympathoexcitation, such as hypertension and heart failure. However, the contribution of NO in the intraneuronal signaling pathway of Ang-(1-7) and the subsequent modulation of neuronal activity remains unclear. Here, we tested the hypothesis that neuronal NOS (nNOS)-derived NO(·) mediates changes in neuronal activity following Ang-(1-7) stimulation. For these studies, we used differentiated catecholaminergic (CATH.a) neurons, which we show express the Ang-(1-7) receptor (Mas R) and nNOS. Stimulation of CATH.a neurons with Ang-(1-7) (100 nM) increased intracellular NO levels, as measured by 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) fluorescence and confocal microscopy. This response was significantly attenuated in neurons pretreated with the Mas R antagonist (A-779), a nonspecific NOS inhibitor (nitro-L-arginine methyl ester), or an nNOS inhibitor (S-methyl-L-thiocitrulline, SMTC), but not by endothelial NOS (eNOS) or inhibitory NOS (iNOS) inhibition {L-N-5-(1-iminoethyl)ornithine (L-NIO) and 1400W, respectively}. To examine the effect of Ang-(1-7)-NO(·) signaling on neuronal activity, we recorded voltage-gated outward K(+) current (I(Kv)) in CATH.a neurons using the whole cell configuration of the patch-clamp technique. Ang-(1-7) significantly increased I(Kv), and this response was inhibited by A-779 or S-methyl-L-thiocitrulline, but not L-NIO or 1400W. These findings indicate that Ang-(1-7) is capable of increasing nNOS-derived NO(·) levels, which in turn, activates hyperpolarizing I(Kv) in catecholaminergic neurons.
血管紧张素-(1-7)[Ang-(1-7)]是肾素-血管紧张素系统的七肽，其在外周的作用至少部分是通过一氧化氮（NO）合酶（NOS）的激活和 NO·的产生介导的。对中枢神经系统的研究表明，NO·作为一种交感抑制分子，因此可能在与交感兴奋相关的神经心血管疾病中发挥保护作用，例如高血压和心力衰竭。然而，NO 在 Ang-(1-7)的神经元内信号通路中的贡献以及随后对神经元活性的调节仍不清楚。在这里，我们检验了这样一个假设，即神经元 NOS（nNOS）衍生的 NO·介导 Ang-(1-7)刺激后神经元活性的变化。为此，我们使用分化的儿茶酚胺能（CATH.a）神经元进行了研究，我们证明这些神经元表达 Ang-(1-7)受体（Mas R）和 nNOS。用 Ang-(1-7)（100 nM）刺激 CATH.a 神经元会增加细胞内 NO 水平，这可以通过 4-氨基-5-甲基氨基-2'，7'-二氟荧光素二乙酸酯（DAF-FM）荧光和共聚焦显微镜测量。用 Mas R 拮抗剂（A-779）、非特异性 NOS 抑制剂（硝基-L-精氨酸甲酯）或 nNOS 抑制剂（S-甲基-L-硫代瓜氨酸，SMTC）预处理神经元会显著减弱这种反应，但内皮 NOS（eNOS）或抑制性 NOS（iNOS）抑制（分别为 L-N-5-（1-亚氨基乙基）鸟氨酸（L-NIO）和 1400W）不会减弱这种反应。为了研究 Ang-(1-7)-NO·信号对神经元活性的影响，我们使用膜片钳技术的全细胞构象记录 CATH.a 神经元的电压门控外向 K+电流（I(Kv))。Ang-(1-7) 显著增加了 I(Kv)，并且该反应被 A-779 或 S-甲基-L-硫代瓜氨酸抑制，但 L-NIO 或 1400W 则没有。这些发现表明，Ang-(1-7)能够增加 nNOS 衍生的 NO·水平，而后者转而激活儿茶酚胺能神经元中的超极化 I(Kv)。
