My research goals are to understand cellular and molecular mechanisms underlying baroreflex- and hypoxia-linked regulation of circulation, which include the following three areas: (1) the roles of reactive oxygen species in angiotensin II-elicited excitation of brainstem neurons, and (2) hypoxia sensitive ion channels in brainstem neurons.

1. The roles of reactive oxygen species in angiotensin II-elicited neural excitation.
A selected group of brainstem nuclei, belonging to the central autonomic nervous system, plays a critical role in the maintenance of cardiovascular homeostasis. These centers, including the nucleus of the solitary tract (NTS) and rostral ventrolateral medulla (RVLM), among others, process incoming signals from arterial baroreceptors and cardiopulmonary receptors, and coordinate complex vascular adjustments by regulating cardiac function, peripheral vascular resistance, and fluid balance. Dysregulation within these centers disrupts cardiovascular homeostasis and leads to fatal conditions, such as hypertension and heart failure.
The octapeptide angiotensin II (AngII) has emerged as a critical mediator in central autonomic regulation. AngII is produced from the sequential enzymatic cleavage of angiotensinogen by renin and angiotensin converting enzyme, and exerts its effects by activating specific G-protein coupled receptors, mainly angiotensin type 1 (AT1) and type 2 (AT2) receptors. In addition to the "classical" renin-angiotensin system, which generates circulating AngII, tissue-specific AngII synthesis occurs in many organs including the brain. In brain, AngII and its receptors are present in autonomic nuclei, and have been implicated in central mechanisms leading to hypertension, cardiac dysfunction and volume dysregulation, effects mediated by brain AT1 receptors. Recent evidence suggests that reactive oxygen species (ROS) may be involved in signaling by AngII in central autonomic networks. However, the sources of AngII-derived ROS within functionally characterized autonomic neurons and their intracellular targets have not been defined. In vascular cells, the main source of AngII-derived radicals is NADPH oxidase, an enzyme first described in phagocytes. NADPH oxidase is comprised of two membrane-bound subunits, gp91phox and p22phox, and several cytoplasmic subunits, p47phox, p40phox, p67phox, and the small G-proteins Rac and Rap1a. Upon stimulation of AT1 receptors by AngII, the cytoplasmic subunits bind to the membrane subunits and activate the enzyme resulting in production of the radical superoxide. While some NADPH oxidase subunits (gp91phox and p47phox) have been described in neurons in culture, their presence in central autonomic neurons and their relationships to AT1 receptors have not been established.
The specific aims of my recent research are to use ultrastructural and functional approaches to determine whether ROS are involved in AngII signaling in well-characterized central autonomic neurons and to define their enzymatic source to determine whether NADPH oxidase is present in central autonomic neurons and, if so, whether NADPH oxidase-derived ROS are involved in the effects of AngII on these neurons. These studies focused on the dorsomedial nucleus of the solitary tract (dmNTS) because this region receives afferents from arterial baroreceptors and cardiopulmonary receptors via the vagus nerve, and is critical for maintaining cardiovascular homeostasis. Using double-label immunoelectronmicroscopy we have found that the essential NADPH oxidase subunit gp91phox is present in NTS neurons receiving vagal-like afferents that also contain AT1 receptors. In parallel experiments using patch-clamp of dissociated dmNTS neurons anterogradely labeled via the vagus, I have found that AngII potentiates the L-type Ca2+ currents, an effect mediated by AT1 receptors and abolished by the ROS scavenger MnTBAP. The NADPH oxidase assembly inhibitor apocynin and the peptide inhibitor gp91ds, but not its scrambled version, also blocked the potentiation. The results provide evidence that NADPH oxidase-derived ROS are involved in the effects of AngII on Ca2+ signaling in dmNTS neurons receiving vagal afferents, and support the notion that ROS are important signaling molecules in central autonomic networks.

2. Oxygen-sensitive potassium channel in RVLM neurons
The brainstem RVLM neuron plays a key role in detection of local O2 levels as well as in regulation of arterial blood pressure. The acute adaptive responses of the RVLM neurons to hypoxia allow to reduce hypoxia-induced injury by modulation of patterned autonomic responses, resulting in redistribution of peripheral blood to the brain. The long-term goal of the present study is to understand the molecular mechanisms underlying rapid and reversible hypoxia-induced excitation of adrenergic vasomotor RVLM neurons. Although recent studies have provided several models for oxygen-sensing in chemoreceptor cells, it is largely unknown how low level O2 is transduced into the cellular excitation of the RVLM neuron. My central hypothesis is that the acute hypoxia affects voltage-gated, O2-sensitive K+ channels via modulation of nitric oxide (NO), triggering excitation and an increased catecholamine or glutamate release from C1 bulbospinal RVLM neurons. Using anatomical, electrophysiological and molecular biological approaches, I have systematically investigated acute hypoxia-induced signal transduction pathways in the identified C1-RVLM neurons. First, I have identified the rat or mice C1 bulbospinal RVLM neuron using a combination of retrograde fluorescence labeling and single-cell RT-PCR or single cell TH-immunostaining. Second, I have investigated acute responses of electrical activities and voltage-gated ion currents of the identified C1-RVLM neuron to hypoxic or chemical hypoxia, finding that hypoxia, cyanide and TEA exerted similar effects on membrane potentials, spontaneous discharges and sustained outward K+ current. These data strongly indicate that the hypoxia-induced bursting of spontaneous discharges is attributed to acute suppression of sustained, maxi Ca2+-activated K+ current in the RVLM neurons. Third, I am also focusing on regulatory mechanisms of the hypoxia-sensitive K+ current in RVLM neurons, finding that O2-sensitive K+ current was also regulated by NO. Finally, we will perform the single-cell RT-PCR to determine the source of NO in TH-positive RVLM neuron. Collectively, my preliminary data support my central hypothesis that NO plays an critical role in the regulation of the acute hypoxia-elicited excitatory responses in the C1 RVLM neurons. My long-term goal will be focusing on whether the AT1 receptor is also involved in the hypoxia-induced pre-sympathetic excitation in the RVLM area.

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