Detection of angiotensin II mediated nitric oxide release within the nucleus of the solitary tract using electron-paramagnetic resonance (EPR) spectroscopy.

We previously identified an action of nitric oxide (NO) within the nucleus tractus solitarii (NTS) that attenuates the cardiac component of the baroreceptor reflex. In the present study we have tested the hypothesis that angiotensin II (AngII), acting on angiotensin type 1 receptors (AT1R), can release NO within the NTS and that its actions are mediated by soluble guanylate cyclase (sGC). Utilising cryogenic electron paramagnetic resonance (EPR), we have detected NO release in brainstem samples following AngII, but not saline, microinjections into the NTS. In these experiments, we confirmed that both AngII and a NO donor (diethylamine NONOate) in the NTS both depressed the baroreflex bradycardia. In additional studies, we showed that the latter effects were both sensitive to blockade of sGC using 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ). To initiate studies to resolve the cellular source of NO released by angiotensin II in the NTS, we performed immunohistochemical/electron microscopy studies on the distribution of AT1R. We found AT1R located on NTS neurones and blood vessels. Since a rise in intracellular calcium [Ca]i levels is prerequisite for nNOS activation, we imaged responses in [Ca]i in NTS neurones during exposure to AngII in vitro using confocal microscopy. Our data indicate a paucity of neurones showing changes in [Ca]i when exposed to AngII (200 nM). We suggest that AngII-induced release of NO is from non-neuronal sites. With the presence of AT1R on blood vessel endothelial cells we propose that AngII released NO in the NTS is due to activation of endothelial nitric oxide synthase located within the endothelium. The present study supports the novel concept that AngII can trigger NO release in the NTS by a mechanism of vascular-neuronal signalling that affects central neuronal networks regulating cardiovascular function.