Angiotensin-(1–7) increases neuronal potassium current via a nitric oxide-dependent mechanism

Abstract

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_Kvin catecholaminergic neurons.