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Although the amygdala is known as a negative emotion center for coordinating defensive behaviors, its functions in autonomic control remain unclear. To resolve this issue, we examined effects on cardiovascular responses induced by stimulation and lesions of the amygdala in anesthetized and free-moving rats. Electrical microstimulation of the central nucleus of the amygdala (CeA) induced a gradual increase in arterial pressure (AP) and heart rate (HR), whereas stimulation of adjacent nuclei evoked a phasic AP decrease. The gain of the baroreceptor reflex was not altered by CeA stimulation, suggesting that CeA activity increases both AP and HR by resetting baroreceptor reflex function. Disinhibition of GABAergic input by amygdalar microinjection of the GABAA receptor antagonist induced robust increases in AP and HR. Furthermore, bilateral electrolytic lesions of CeA evoked consistent AP increases over the light/dark cycle. These results suggest that the amygdala exerts ‘bidirectional’ autonomic control over the cardiovascular system.

Background and aims We developed a bio-artificial liver (BAL) using a radial-flow bioreactor and rescued mini-pig models with lethal acute liver failure (ALF). The point of the rescue is the recovery from hepatic encephalopathy (HE). HE on ALF has sometimes resulted in brain death following brain edema with astrocyte swelling. Several factors, including ammonia and glutamine, have been reported to induce astrocyte swelling and injury. However, many clinicians believe that there are any other factors involved in the development of HE. Therefore, the aim of this study was to identify novel HE-inducible factors, particularly those inducing astrocyte dysfunction. Methods Mini-pig plasma samples were collected at three time points: before the administration of toxins (α-amanitin and LPS), when HE occurred after the administration of toxins, and after treatment with extracorporeal circulation (EC) by the BAL. To identify the causative factors of HE, each plasma sample was subjected to a comparative proteome analysis with two-dimensional gel electrophoresis and mass spectrometry. To assess the direct effects of candidate factors on the astrocyte function and injury, in vitro experiments with human astrocytes were performed. Results Using a proteome analysis, we identified alpha-1 antichymotrypsin (ACT), which was increased in plasma samples from mini-pigs with HE and decreased in those after treatment with EC by BAL. In in vitro experiments with human astrocytes, ACT showed growth-inhibitory and cytotoxic effects on astrocytes. In addition, the expression of water channel protein aquaporin-4, which is induced in injured astrocytes, was increased following ACT treatment. Interestingly, these effects of ACT were additively enhanced by adding arginine-vasopressin (AVP) and were canceled by adding an AVP receptor antagonist. Conclusions These results suggest that ACT is involved in astrocyte injury and dysfunction in concert with AVP during the development of acute HE.

Aim The nucleus tractus solitarii (NTS) is a central brainstem structure that plays an important role in regulating cardiovascular homeostasis. In this study, we examined whether H1 receptors in the NTS can control the baroreceptor reflex function by modulating synaptic transmission. Methods Cardiac baroreceptor reflex function was assessed before and after the microinjection of 2-pyridylethylamine (10–25 nmol), a histamine H1 receptor-specific agonist, into the NTS of urethane-anaesthetized Wistar rats. The cardiovascular responses induced by l-glutamate microinjection into the NTS were also examined before and after the NTS administration of 2-pyridylethylamine. Results Nucleus tractus solitarii microinjections of 2-pyridylethylamine significantly inhibited the gain of the cardiac baroreceptor reflex and bradycardiac/depressor responses induced by l-glutamate microinjection into the NTS. These findings suggest that histamine H1 receptors regulate the cardiac baroreceptor reflex in a post-synaptic manner to inhibit barosensitive NTS neurons. Conclusion Taken together with our previous findings, the present results provide further evidence that histamine may play a role within the NTS in regulating cardiovascular homeostasis.

Neurogenic hypertension, the primary form of essential hypertension, is one of the most common diseases worldwide. Hypertension is a risk factor for many cardiovascular diseases such as heart attacks and stroke; therefore, it is crucial to maintain arterial blood pressure (BP) within the normal range. Regular aerobic exercise at moderate intensities can lower basal BP, and is a recommended therapy to prevent or improve primary hypertension. However, the mechanisms underlying the anti-hypertensive effects of exercise remain unknown. In this review, we discuss the mechanisms for the anti-hypertensive effects of exercise training/therapy that are hypothesised from recent findings, including our own. In particular, we discuss the nucleus of the solitary tract (NTS) of the brainstem, which is involved in mechanisms underlying the manifestation of neurogenic hypertension. Moreover, the NTS may also be involved in the anti-hypertensive effects of exercise training. However, exercise training does not seem to improve causative genetic factors for neurogenic hypertension in the NTS. Nevertheless, exercise training may affect other mechanisms responsible for neuroactive ligand-receptor interactions within the NTS, which also regulate BP homeostasis. We hope this review will further enhance research in this and promote exercise habits that help delay or even prevent the progression of essential hypertension.

Inflammation within the brain stem microvasculature has been associated with chronic cardiovascular diseases. We found that the expression of several enzymes involved in arachidonic acid-leukotriene B4 (LTB4) production was altered in nucleus tractus solitarii (NTS) of spontaneously hypertensive rat (SHR). LTB4 produced from arachidonic acid by 5-lipoxygenase is a potent chemoattractant of leukocytes. Leukotriene B4-12-hydroxydehydrogenase (LTB4-12-HD), which degrades LTB4, was downregulated in SHR rats compared with that in Wistar-Kyoto rats. Quantitative real-time PCR revealed that LTB4-12-HD was reduced by 63% and 58% in the NTS of adult SHR and prehypertensive SHR, respectively, compared with that in age-matched Wistar-Kyoto rats (n=6). 5-lipoxygenase gene expression was upregulated in the NTS of SHR (≈50%; n=6). LTB4 levels were increased in the NTS of the SHR, (17%; n=10, P P P

Essential hypertension is idiopathic although it is accepted as a complex polygenic trait with underlying genetic components, which remain unknown. Our supposition is that primary hypertension involves activation of the sympathetic nervous system. One pivotal region controlling arterial pressure set point is nucleus tractus solitarii (NTS). We recently identified that pro-inflammatory molecules, such as junctional adhesion molecule-1, were over expressed in endothelial cells of the microvasculature supplying the NTS in an animal model of human hypertension (the spontaneously hypertensive rat: SHR) compared to normotensive Wistar Kyoto (WKY) rats. We have also shown endogenous leukocyte accumulation inside capillaries within the NTS of SHR but not WKY rats. Despite the inflammatory state in the NTS of SHR, transcripts of some inflammatory molecules such as chemokine (C-C motif) ligand 5 (Ccl5), and its receptors, chemokine (C-C motif) receptor 1 and 3 were down-regulated in the NTS of SHR compared to WKY rats. This may be compensatory to avoid further strong inflammatory activity. More importantly, we found that down-regulation of Ccl5 in the NTS of SHR may be pro-hypertensive since microinjection of Ccl5 into the NTS of SHR decreased arterial pressure but was less effective in WKY rats. Leukocyte accumulation of the NTS microvasculature may also induce an increase in vascular resistance and hypoperfusion within the NTS; the latter may trigger release of pro-inflammatory molecules which via paracrine signaling may affect central neural cardiovascular activity conducive to neurogenic hypertension. All told, we suggest that vascular inflammation within the brainstem contributes to neurogenic hypertension by multiple pathways.

OBJECTIVES The brainstem nucleus of the solitary tract (nucleus tractus solitarii, NTS) is a pivotal region for regulating the set-point of arterial pressure, the mechanisms of which are not fully understood. Based on evidence that the NTS exhibits O2-sensing mechanisms, we examined whether a localized disturbance of blood supply, resulting in hypoxia in the NTS, would lead to an acute increase in arterial pressure. METHODS Male Wistar rats were used. Cardiovascular parameters were measured before and after specific branches of superficial dorsal medullary veins were occluded; we assumed these were drainage vessels from the NTS and would produce stagnant hypoxia. Hypoxyprobe-1, a marker for detecting cellular hypoxia in the post-mortem tissue, was used to reveal whether vessel occlusion induced hypoxia within the NTS. RESULTS Following vessel occlusion, blood flow in the dorsal surface of the medulla oblongata including the NTS region showed an approximately 60% decrease and was associated with hypoxia in neurons located predominantly in the caudal part of the NTS as revealed using hypoxyprobe-1. Arterial pressure increased and this response was pronounced significantly in both magnitude and duration when baroreceptor reflex afferents were sectioned. CONCLUSION These results suggest that localized hypoxia in the NTS increases arterial pressure. We suggest this represents a protective mechanism whereby the elevated systemic pressure is a compensatory mechanism to enhance cerebral perfusion. Whether this physiological mechanism has any relevance to neurogenic hypertension is discussed.

Since the nucleus tractus solitarii (NTS) is a pivotal region for regulating the set-point of arterial pressure, we proposed a role for it in the development of neurogenic hypertension. Recent studies have suggested that proinflammatory molecules, such as junctional adhesion molecule 1 (JAM-1) are highly expressed in the NTS of an animal model of human essential hypertension, the spontaneously hypertensive rat (SHR), compared with normotensive rats (Wistar-Kyoto, WKY). Moreover, we have also shown endogenous leukocyte accumulation inside capillaries within the NTS of SHR but not WKY rats. Based on this evidence, we hypothesized that gene expression of cytokines/chemokines is altered in the NTS of SHR. We have screened for abnormally expressed inflammatory mediators in the NTS of SHR using the RT2 Profiler PCR arrays, which were designed specifically to target major cytokines/chemokines. The specific PCR array revealed that seven genes were less expressed in the NTS of SHR compared with WKY rats (more than twofold differences), while only two genes were more expressed in the SHR. Moreover, we identified that some of these validated molecules exhibit important functional roles for cardiovascular homeostasis at the level of the NTS. We suggest that abnormal gene expression of proinflammatory molecules, such as JAM-1, causes leukocyte accumulation within the vasculature in the NTS of SHR. Consequently, gene expression of specific cytokines/chemokines may be downregulated to avoid further strong inflammatory activity in the NTS of SHR at the expense of an alteration in neuronal activity that leads to cardiovascular autonomic pathology. Importantly, to allow translation of our work, these novel insights need to be assessed in hypertensive human brainstem tissue; their confirmation could lead to novel therapeutic approaches for one of the world's most prevalent diseases.

Recent gene array and molecular studies have suggested that an abnormal gene expression profile of interleukin-6 (IL-6) in the nucleus tractus solitarii (NTS), a pivotal region for regulating arterial pressure, may be related to the development of neurogenic hypertension. However, the precise functional role of IL-6 in the NTS remains unknown. In the present study, we have tested whether IL-6 affects cardiovascular control at the level of the NTS. IL-6 (1, 10, and 100 fmol) was microinjected in the NTS of Wistar rats (280–350 g) under urethane anesthesia. Although the baseline levels of arterial pressure and heart rate did not change following IL-6 injections, the cardiac baroreflex in response to increased arterial pressure was dose-dependently attenuated. In addition, IL-6 (100 fmol) microinjections also attenuated l-glutamate-induced bradycardia at the level of the NTS. Immunohistochemical detection of IL-6 in naïve rats demonstrated that it was predominantly observed in neurons within the brain stem, including the NTS. These findings suggest that IL-6 within the NTS may play an important role for regulating cardiovascular control via modulation of input signals from baroreceptor afferents. Whether the abnormal gene expression of IL-6 in the NTS is associated in a causal way with hypertension remains to be resolved.

Although both α1- and α2-adrenergic receptors (ARs) are known to be expressed in the nucleus of the solitary tract (NTS), the functional significance of these receptors is still not fully established. In this study, we microinjected α1- and α2-AR agonists into the NTS of urethane-anaesthetized Wister rats to study the cardiovascular effects in response to their activation. When the α1-AR agonist phenylephrine was microinjected into the area where barosensitive neurons are dominantly located (baro-NTS), mean arterial pressure (MAP) and heart rate (HR) were significantly elevated. When tested in the area where chemosensitive neurons are dominantly located (chemo-NTS), however, MAP and HR were significantly decreased. Pretreatment with the non-specific α-AR antagonist phentolamine into the NTS inhibited the phenylephrine-induced cardiovascular responses. In contrast, microinjection of the α2-AR agonist clonidine into either the baro-NTS or the chemo-NTS decreased MAP and HR; they were also inhibited by the α2-adrenergic antagonist yohimbine. Moreover, we immunohistochemically identified that cardiovascular responses induced by α1-ARs may be mediated by NTS neurons while those induced by α2-ARs may be mediated by astrocytes located in the barosensitive and chemosensitive areas of the NTS. These results suggest that both types of α-AR expressed in the NTS may be involved in regulating cardiovascular homeostasis via modulation of input signals from baroreceptor and chemoreceptor afferents; however, cardiovascular responses produced by stimulation of α1-ARs are strictly location specific within the NTS.

Recent studies have demonstrated that pro-inflammatory molecules such as junctional adhesion molecules-1 are highly expressed in the nucleus tractus solitarii (NTS) of the spontaneously hypertensive rat (SHR), compared to normotensive rats (Wistar-Kyoto rats: WKY), suggesting that the NTS of SHR may exhibit an abnormal inflammatory state. In the present study, we tested whether gene expression of inflammatory markers such as cytokines and chemokines is altered in the NTS of SHR and whether this contributes to the hypertensive phenotype in the SHR.We have performed RT Profiler PCR arrays in the NTS of SHR and WKY, which were designed to specifically target major cytokines/chemokines and their receptors. To validate PCR array results quantitative RT-PCR was performed. Microinjection studies using anesthetized rats were also carried out to examine whether validated inflammatory molecules exhibit functional roles on cardiovascular regulation at the level of the NTS.Five inter-related transcripts were identified to be differentially expressed between the NTS of SHR and WKY. They include chemokine (C-C motif) ligand 5 (Ccl5), and its receptors, chemokine (C-C motif) receptor 1 and 3. All of them were down-regulated in the NTS of SHR compared to WKY. Moreover, we found that the protein Ccl5 microinjected into the NTS significantly decreased baseline arterial pressure and that the response was greater in the SHR compared to the WKY (-33.2±3.2 vs. -8.8±1.6 mmHg, P0.001), demonstrating that its down-regulation in the NTS may contribute to hypertension in the SHR.We suggest that gene expression of specific chemokines may be down-regulated to protect further inflammatory reactions in the NTS of SHR at the expense of arterial hypertension.

Adrenomedullin 2 (AM2), a novel member of the calcitonin gene-related peptide family, has emerged as a multifunctional peptide controlling endocrine and cardiovascular functions and physiological behaviors. Both central and peripheral administration of AM2 can have profound effects on systemic and/or pulmonary circulation in mammals. However, the target nuclei of AM2 and the role of central AM2 in cardiovascular regulation remain unknown. In the present study, we microinjected AM2 into the rat nucleus tractus solitarius (NTS), the central termination site of baroreceptor afferents. Consistent with previous reports showing the hypertensive effect of intracerebroventricular administration of AM2, the direct microinjection of the peptide into the NTS increased arterial pressure as well as heart rate in rats. Importantly, this effect of AM2 on cardiovascular regulation was significantly attenuated by an antagonist of receptor components for AM2 that were abundant in the rat NTS. Our results indicate that AM2 may play an important role in the regulation of the cardiovascular system at the NTS level.

Human essential hypertension is a complex polygenic trait with underlying genetic components that remain unknown. Since the brainstem structure--the nucleus of the solitary tract (NTS)--is a pivotal region for regulating the set-point of arterial pressure, we proposed a role for it in the development of primary hypertension. Using microarray and real-time RT-PCR, we have recently identified that some pro-inflammatory molecules, such as junctional adhesion molecule-1 (JAM-1; a leukocyte/platelet adhesion molecule), were over expressed in endothelial cells in the NTS of an animal model of human essential hypertension--the spontaneously hypertensive rat (SHR) compared to normotensive Wistar Kyoto rats (WKY). Adenoviral mediated over expression of JAM-1 in NTS of WKY rats produced both hypertension and localized leukocyte adherence to the microvasculature. With a known effect of leukocyte adhesion causing cytokine release, we predicted differences in the level of gene expression of cytokines in the NTS of SHR relative to WKY. Gene expression of monocyte chemoattractant protein-1 (MCP-1) was higher in the NTS of SHR while inter-leukin-6 (IL-6) was lower in the NTS of SHR compared to the WKY. Because both inflammatory molecules are known to affect neural functions, our data suggest that the microvasculature of NTS of the SHR exhibits a specific inflammatory state. We propose a new hypothesis that as a consequence of enhanced expression of leukocyte adhesion molecules within the microvasculature of NTS there is a specific inflammatory response that leads to cardiovascular autonomic dysfunction contributing to neurogenic hypertension.

Extracellular calcium antagonizes forskolin-induced aquaporin 2 trafficking in collecting duct cells. Background Urinary concentrating defects and polyuria are the most important renal manifestations of hypercalcemia and the resulting hypercalciuria. In this study, we tested the hypothesis that hypercalciuria-associated polyuria in kidney collecting duct occurs through an impairment of the vasopressin-dependent aquaporin 2 (AQP2) water channel targeting to the apical membrane possibly involving calcium-sensing receptor (CaR) signaling. Methods AQP2-transfected collecting duct CD8 cells were used as experimental model. Quantitation of cell surface AQP2 immunoreactivity was performed using an antibody recognizing the extracellular AQP2 C loop. Intracellular cyclic adenosine monophosphate (cAMP) accumulation was measured in CD8 cells using a cAMP enzyme immunoassay kit. To study the translocation of protein kinase C (PKC), membranes or cytosol fractions from CD8 cells were subjected to Western blotting using anti-PKC isozymes antibodies. The amount of F-actin was determined by spectrofluorometric techniques. Intracellular calcium measurements were performed by spectrofluorometric analysis with Fura-2/AM. Results We demonstrated that extracellular calcium (Ca 2+ o ) (5 mmol/L) strongly inhibited forskolin-stimulated increase in AQP2 expression in the apical plasma membrane. At least three intracellular pathways activated by extracellular calcium were found to contribute to this effect. Firstly, the increase in cAMP levels in response to forskolin stimulation was drastically reduced in cells pretreated with Ca 2+ o compared to untreated cells. Second, Ca 2+ o activated PKC, known to counteract vasopressin response. Third, quantification of F-actin demonstrated that Ca 2+ o caused a nearly twofold increase in F-actin content compared with basal conditions. All these effects were mimicked by a nonmembrane permeable agonist of the extracellular CaR, Gd 3+ . Conclusion Together, these data demonstrate that extracellular calcium, possibly acting through the endogenous CaR, antagonizes forskolin-induced AQP2 translocation to the apical plasma membrane in CD8 cells. In hypercalciuria, this mechanism might blunt water reabsorption and prevent further calcium concentration, thus protecting against a potential risk of urinary calcium-containing stone formation.

In this study, we analyzed the effect of a therapeutic intervention in 46 enuretic children, 26 (57%) of whom were hypercalciuric. All the patients ( n = 46) were treated with DDAVP for 3–6 mo. The hypercalciuric patients ( n = 26) received a low-calcium diet (∼500 mg/day) for the same period. After the therapy, the bed-wetting episodes stopped in 80% of the 46 patients tested. In those patients having low-AVP levels before the therapy, circulating AVP concentration returned to normal (>4 pg/ml), and the hypercalciuria was resolved in the hypercalciuric patients (calcium/creatinine ratio

One likely mechanism of essential hypertension(EH) is increased sympathoexcitation due to abnormal functions in the cardiovascular center of the brain. Recent findings obtained using experimental animal models of EH have shown that abnormal inflammation in the cardiovascular center may contribute to the onset of hypertension. Inflammatory molecules such as cytokines and reactive oxygen species released from the inflamed vasculature and glial cells in the medulla oblongata and hypothalamus might directly or indirectly affect neuronal functions. This in turn could increase sympathetic nerve activity and consequently arterial pressure. Abnormal inflammatory responses in the brain could also be central mechanisms underlying angiotensin Ⅱ-related EH. In this review, we present the current understanding of EH mechanisms with regard to inflammatory responses in the cardiovascular center.