Nox2, Ca ²⁺ , and Protein Kinase C Play a Role in Angiotensin II-Induced Free Radical Production in Nucleus Tractus Solitarius

Abstract

Abstract-The dorsomedial portion of the nucleus tractus solitarius (dmNTS) is the site of termination of baroreceptor and cardiorespiratory vagal afferents and plays a critical role in cardiovascular regulation. Angiotensin II (Ang II) is a powerful signaling molecule in dmNTS neurons and exerts some of its biological effects by modulating Ca 2ϩ currents via reactive oxygen species (ROS) derived from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. We investigated whether a Nox2-containing NADPH oxidase is the source of the Ang II-induced ROS production and whether the signaling mechanisms of its activation require intracellular Ca 2ϩ or protein kinase C (PKC). Second-order dmNTS neurons were anterogradely labeled with 4-(4-[didecylamino]styryl)-N-methylpyridinium iodide transported from the vagus and isolated from the brain stem. ROS production was assessed in 4-(4-[didecylamino]styryl)-N-methylpyridinium iodide-positive dmNTS neurons using the fluorescent dye 6-carboxy-2Ј,7Ј-dichlorodihydro-fluorescein di(acetoxymethyl ester). Ang II (3 to 2000 nmol/L) increased ROS production in dmNTS neurons (EC 50 ϭ38.3 nmol/L). The effect was abolished by the ROS scavenger Mn (III) porphyrin 5,10,20-tetrakis (benzoic acid) porphyrin manganese (III), the Ang II type 1 receptor antagonist losartan, or the NADPH oxidase inhibitors apocynin or gp91ds-tat. Ang II failed to increase ROS production or to potentiate L-type Ca 2ϩ currents in dmNTS neurons of mice lacking Nox2. The PKC inhibitor GF109203X or depletion of intracellular Ca 2ϩ attenuated Ang II-elicited ROS production. We conclude that the powerful effects of Ang II on Ca 2ϩ currents in dmNTS neurons are mediated by PKC activation leading to ROS production via Nox2. Thus, a Nox2-containing NADPH oxidase is the critical link between Ang II and the enhancement of Key Words: arterial hypertension Ⅲ baroreflex Ⅲ calcium channels Ⅲ oxidative stress Ⅲ blood pressure Ⅲ autonomic nervous system A select group of brain stem nuclei regulates the systemic circulation by modulating cardiac output, vascular resistance, and fluid balance. 1,2 In particular, the dorsomedial region of the nucleus of the solitary tract (dmNTS), wherein vagal afferents from aortic baroreceptors and cardiorespiratory chemoreceptors terminate, plays a major role in cardiovascular regulation. [3][4][5] There is increasing evidence that angiotensin II (Ang II) is a critical neuromodulator in central autonomic nuclei, 6 -9 including the dmNTS. 10 -13 Within the dmNTS, activation of Ang II type 1 (AT 1 ) receptors alters cardiorespiratory reflexes including baroreceptor excitability and ion channel permeability. 10,14 These changes may contribute to Ang II-induced sympathoexcitation, 15-18 hypertension, 15,16 and heart failure. 17,18 Superoxide generated by the enzyme NADPH oxidase has emerged as a key intermediary in the central and peripheral effects of Ang II. 10,14 -16,18 -21 NADPH oxidase, initially described in neutrophils, 22,23 is now known to be present in diverse cell types, inc...

Changes in the subcellular distribution of NADPH oxidase subunit p47phox in dendrites of rat dorsomedial nucleus tractus solitarius neurons in response to chronic administration of hypertensive agents

“…Within dendrites, the major site of NADPH oxidase subunit immunolabeling in dmNTS neurons, p47 phox immunoreactivity was associated with intracellular organelles that are involved in protein transport and calcium signaling, particularly ER-like tubulovesicular organelles, which were frequently associated with the outer mitochondrial membrane. Thus, p47 phox immunolabeling in dendritic smooth ER, which are sites for calcium storage and release, is consistent with physiological evidence that calcium currents in dmNTS neurons are modulated by superoxide produced by NADPH oxidase (23,47,48). In addition to intracellular sites, p47 phox immunolabeling was also seen at synaptic and extra-synaptic areas of the plasmalemma, similar to other reports in the literature indicating that NADPH oxidase has a synaptic distribution in other brain regions (41).…”

“…In the NTS, previous ultrastructural evidence indicates that NADPH oxidase subunits p47 phox, gp91 phox , and p22 phox are present in association with endomembranes, as well as the plasmalemma in neurons (16). In addition, previous neurophysiological results also indicate that functional NADPH oxidase potentiates AT-1A mediated calcium currents in dissociated second-order neurons in the dmNTS (47,48). The present results demonstrate that the subcellular distribution of p47 phox , and presumably functional NADPH oxidase-mediated superoxide production, is not spatially static, but subject to repartitioning in dendrites of dmNTS neurons in response to regimens of AngII or phenylephrine that produce a similar degree of hypertension.…”

“…In the dorsomedial NTS (dmNTS), an area that receives a high density of baroreceptors (9), the activation of AT-1A receptors produces a superoxide-dependent potentiation of calcium currents (47,48). In dmNTS, NADPH oxidase subunits are also present in membranous organelles in astroglia and dendritic processes of neurons (16).…”

Selective Loss of Catecholaminergic Wake–Active Neurons in a Murine Sleep Apnea Model

“…This enzyme has been identified in subpopulations of neurons in many of the regions most susceptible to hypoxia/ reoxygenation injury: cortex, striatum, amygdala, hippocampus, and thalamus (Serrano et al, 2003). Apocynin, the NADPH oxidase inhibitor used in the present studies to prevent injury to wake-active groups, has been shown to protect against hippocampal ischemic injury in rodents (Wang et al, 2006). Whether NADPH oxidase inhibition will prevent hippocampal injury and memory impairments should now be examined.…”

Inhibition of Rac1-Derived Reactive Oxygen Species in Nucleus Tractus Solitarius Decreases Blood Pressure and Heart Rate in Stroke-Prone Spontaneously Hypertensive Rats

“…7,23 Nox2-containing NAD(P)H oxidase in the NTS is the source of the Ang II-induced ROS generation in vitro. 23 Consistent with those studies, our results indicate that the Rac1/NAD(P)H pathway is involved in neuronal activation in the NTS and further indicate that activation of this pathway and the subsequent ROS generation in the NTS occur in the NTS of SHRSPs. More importantly, we demonstrated that the inhibition of Rac1 or overexpression of Cu/Zn-SOD in the NTS decreased blood pressure and HR in SHRSPs but not in WKYs.…”