Abstract-It is well established that the central cardiovascular effects of angiotensin II (Ang II) involve superoxide production. However, the intracellular mechanism by which reactive oxygen species (ROS) signaling regulates neuronal Ang II actions remains to be elucidated. In the present study, we have used neuronal cells in primary cultures from the hypothalamus and brain stem areas to study the role of ROS on the cellular actions of Ang II. Ang II increases neuronal firing rate, an effect mediated by the AT 1 receptor subtype and involving inhibition of the delayed rectifier potassium current (I Kv ). This increase in neuronal activity was associated with increases in NADPH oxidase activity and ROS levels within neurons, the latter evidenced by an increase in ethidium fluorescence. The increases in NADPH oxidase activity and ethidium fluorescence were blocked by either the AT 1 receptor antagonist losartan or by the selective NAD(P)H oxidase inhibitor gp91ds-tat. Extracellular application of the ROS scavenger, Tempol, attenuated the Ang II-induced increase in neuronal firing rate by 70%. In addition, gp91ds-tat treatment resulted in a 50% inhibition of Ang II-induced increase in firing rate. In contrast, the ROS generator Xanthine-Xanthine oxidase significantly increased neuronal firing rate. Finally, Ang II inhibited neuronal I Kv, and this inhibition was abolished by gp91ds-tat treatment.

The role of soluble epoxide hydrolase (sEH) in the central control of blood pressure (BP) has not been elucidated in spite of peripheral sEH overexpression being linked to hypertension. Thus, our objective was to investigate the involvement of brain sEH in BP control. sEH expression in the hypothalamus and brain stem, two cardioregulatory brain areas, was increased in the spontaneously hypertensive rat (SHR) compared to the Wistar Kyoto (WKY) rat. Inhibition of the enzyme by intracerebroventricular (icv) delivery of AUDA further increased both BP and heart rate (HR) by 32 +/- 6 mmHg and 54 +/- 10 bpm, respectively, (P<0.05) in the SHR. Analysis of waveform telemetry data revealed a decrease in spontaneous baroreceptor reflex gain following sEH inhibition, indicating the sustained increase in BP may be due to a decrease in baroreceptor reflex function. The hypertensive effect of sEH inhibition is likely a result of an increase in epoxyeicosatrienoic acid (EET)-mediated generation of ROS. This view is supported by the following: 1) Inhibition of EET formation attenuates AUDA-induced increase in BP; 2) delivery of an EET agonist increases BP and HR in the WKY rat, and 3) inhibition of NAD(P)H oxidase by gp91ds-tat prevents AUDA-induced increases in BP and HR. Finally, electrophysiological studies demonstrate that AUDA increased neuronal firing rate exclusively in the SHR, an effect completely abolished by gp91ds-tat. These observations suggest that EETs and sEH inhibition are involved in increasing BP in the SHR. We suggest that an increased expression of sEH is a futile central nervous system response in protection against hypertension.

Abstract-Gene profiling data coupled with adducin polymorphism studies led us to hypothesize that decreased expression of this cytosolic protein in the brain could be a key event in the central control of hypertension. Thus, our objectives in the present study were to (1) determine which adducin subunit gene demonstrates altered expression in the hypothalamus and brainstem (two cardioregulatory-relevant brain areas) in two genetic strains of hypertensive rats and (2) analyze the role of adducins in neurotransmission at the cellular level. All three adducin subunits (␣, ␤, and ␥) were present in the hypothalamus and brainstem of Wistar Kyoto (WKY) and spontaneously hypertensive (SH) rats. However, only the ␥-adducin subunit expression was 40% to 60% lower in the SH rat compared with WKY rat. A similar decrease in ␥-adducin expression was observed in the hypothalamus and brainstem of the renin transgenic rat compared with its normotensive control. Losartan treatment of the SH rat failed to normalize ␥-adducin gene expression.A hypertension-linked decrease of ␥-adducin was confirmed by demonstrating a decrease in ␥-adducin expression in hypothalamic/brainstem neuronal cultures from prehypertensive SH rats. Neuronal firing rate was evaluated to analyze the role of this protein in neurotransmission. Perfusion of a ␥-adducin-specific antibody caused a 2-fold increase in the neuronal firing rate, an effect similar to that observed with angiotensin II. Finally, we observed that preincubation of neuronal cultures for 8 hours with 100 nmol/L angiotensin II caused a 60% decrease in endogenous ␥-adducin and was associated with a 2-fold increase in basal firing rate. These observations support our hypothesis that a decrease in ␥-adducin expression in cardioregulatory-relevant brain areas is linked to hypertension possibly by regulating the release of neurotransmitters. Key Words: hypothalamus/brainstem Ⅲ gene profiling Ⅲ neurons Ⅲ ␥-adducin Ⅲ hypertension T he central nervous system (CNS) plays a key role in the control of cardiovascular functions. Anatomical and physiological evidence have established that specific hypothalamic and brainstem nuclei participate in the control of sympathetic nerve activity (SNA), vasopressin release, and baroreceptor reflexes, which are all aspects involved in normal cardiovascular functions. 1,2 The significance of these pathways in central control of cardiovascular functions is further underscored by the evidence that an increase in SNA and secretion of neurotransmitters/neurohormones is associated with the development and establishment of hypertension. 3 In spite of this evidence, little is known about the cellular and molecular basis of hypertension-linked dysregulation in the CNS. In an attempt to elucidate the molecular basis of this dysregulation, we hypothesized that an inherent change in the expression profiling of gene(s) in cardiovascular-relevant brain nuclei is responsible for the development of hypertension. In this regard, our present studies have indicated that the expression ...

Centrally mediated increases in sympathetic nerve activity and attenuated arterial baroreflexes contribute to the pathogenesis of hypertension. Despite the characterization of cellular and physiological mechanisms that regulate blood pressure and alterations that contribute to hypertension, the genetic and molecular basis of this pathophysiology remains poorly understood. Strategies to identify genes that contribute to central pathophysiologic mechanisms in hypertension include integrative biochemistry and physiology as well as functional genomics. This article summarizes recent progress in applying functional genomics to elucidate the genetic basis of altered central blood pressure regulatory mechanisms in hypertension. We describe approaches others and we have undertaken to investigate gene expression profiles in hypertensive models in order to identify genes that contribute to the pathogenesis of hypertension. Finally, we provide the readers a roadmap for negotiating the route from experimental findings of gene expression profiling to translating their therapeutic potential. The combination of gene expression profiling and the phenotypic characterization of in vitro and in vivo loss or gain of function experiments for candidate genes have the potential to identify genes involved in the pathogenesis of hypertension and may present novel targets for therapy.