Angiotensin II-mediated vascular brain inflammation emerged as a novel pathophysiological mechanism in neurogenic hypertension. However, the precise underlying mechanisms and functional consequences in relation to blood-brain barrier (BBB) integrity and central angiotensin II actions mediating neurohumoral activation in hypertension are poorly understood. Here, we aimed to determine whether BBB permeability within critical hypothalamic and brain stem regions involved in neurohumoral regulation was altered during hypertension. Using digital imaging quantification after intravascularly injected fluorescent dyes and immunohistochemistry, we found increased BBB permeability, along with altered key BBB protein constituents, in spontaneously hypertensive rats within the hypothalamic paraventricular nucleus, the nucleus of the solitary tract, and the rostral ventrolateral medulla, all critical brain regions known to contribute to neurohumoral activation during hypertension. BBB disruption, including increased permeability and downregulation of constituent proteins, was prevented in spontaneously hypertensive rats treated with the AT1 receptor antagonist losartan, but not with hydralazine, a direct vasodilator. Importantly, we found circulating angiotensin II to extravasate into these brain regions, colocalizing with neurons and microglial cells. Taken together, our studies reveal a novel angiotensin II-mediated feed-forward mechanism during hypertension, by which circulating angiotensin II evokes increased BBB permeability, facilitating in turn its access to critical brain regions known to participate in blood pressure regulation.

Although communication between neurons is considered a function of the synapse, neurons also release neurotransmitter from their dendrites. We found that dendritic transmitter release coordinates activity across distinct neuronal populations to generate integrative homeostatic responses. We show that activity-dependent vasopressin release from hypothalamic neuroendocrine neurons in the paraventricular nucleus stimulates neighboring (~100 μm soma-to-soma) presympathetic neurons, resulting in a sympathoexcitatory population response. This interpopulation crosstalk was engaged by an NMDA-mediated increase in dendritic Ca(2+), influenced by vasopressin's ability to diffuse in the extracellular space, and involved activation of CAN channels at the target neurons. Furthermore, we demonstrate that this interpopulation crosstalk plays a pivotal role in the generation of a systemic, polymodal neurohumoral response to a hyperosmotic challenge. Because dendritic release is emerging as a widespread process, our results suggest that a similar mechanism could mediate interpopulation crosstalk in other brain systems, particularly those involved in generating complex behaviors.

Dendritic release of vasopressin (VP) in the SON and PVN nuclei plays an important role in the autoregulation of VP neuronal activity. Here, we tested the hypothesis that dendritically‐released VP acts in a diffusible manner to influence the activity of neighboring presympathetic neurons. Patch‐clamp recordings in brain slices showed that focally applied VP depolarized and increased presympathetic PVN‐RVLM neurons (~450%, P< 0.01), an effect that persisted in the presence of GABA/glutamate receptor blockers. Activation of a single, identified eGFP‐VP neuron using photolysis of caged NMDA induced a Ca2+‐dependent, V1a‐mediated increased firing discharge in neighboring PVN‐RVLM neurons. Blockade of V1a receptors per se reduced PVN‐RVLM firing discharge (~75%, P< 0.02). Increasing (high K+, aminopeptidase blockade) and decreasing (κ opiod agonist) endogenous VP ambient levels, respectively, further increased/decreased PVN‐RVLM firing activity. Finally, a central osmotic stimulation (NaCl 0.3–2.1 omosl/l ICA) induced a prompt increase in RSNA, and effect that was blunted (~50%) when V1a receptors were locally blocked within the PVN (P<0.001). Collectively, our results demonstrate that dendritic release of VP mediates a functional crosstalk between neurosecretory and presympathetic PVN neurons, playing an important role in the generation of osmotically‐driven homeostatic responses. Supported by NIH HL‐090948 to JES and NIH HL‐62222 to KKP.

Elevated sympathetic outflow and altered autonomic reflexes, including impaired baroreflex function, are common findings observed in hypertensive disorders. Although a growing body of evidence supports a contribution of preautonomic neurons in the hypothalamic paraventricular nucleus (PVN) to altered autonomic control during hypertension, the precise underlying mechanisms remain unknown. Here, we aimed to determine whether the intrinsic excitability and repetitive firing properties of preautonomic PVN neurons that innervate the nucleus tractus solitarii (PVN-NTS neurons) were altered in spontaneously hypertensive rats (SHR). Moreover, given that exercise training is known to improve and/or correct autonomic deficits in hypertensive conditions, we evaluated whether exercise is an efficient behavioral approach to correct altered neuronal excitability in hypertensive rats. Patch-clamp recordings were obtained from retrogradely labeled PVN-NTS neurons in hypothalamic slices obtained from sedentary (S) and trained (T) Wistar-Kyoto (WKY) and SHR rats. Our results indicate an increased excitability of PVN-NTS neurons in SHR-S rats, reflected by an enhanced input-output function in response to depolarizing stimuli, a hyperpolarizing shift in Na(+) spike threshold, and smaller hyperpolarizing afterpotentials. Importantly, we found exercise training in SHR rats to restore all these parameters back to those levels observed in WKY-S rats. In several cases, exercise evoked opposing effects in WKY-S rats compared with SHR-S rats, suggesting that exercise effects on PVN-NTS neurons are state dependent. Taken together, our results suggest that elevated preautonomic PVN-NTS neuronal excitability may contribute to altered autonomic control in SHR rats and that exercise training efficiently corrects these abnormalities.

Neurohumoral activation, a hallmark in heart failure (HF), is linked to the progression and mortality of HF patients. Thus, elucidating its precise underlying mechanisms is of critical importance. Other than its classic peripheral vasodilatory actions, the gas NO is a pivotal neurotransmitter in the central nervous system control of the circulation. While accumulating evidence supports a contribution of blunted NO function to neurohumoral activation in HF, the precise cellular sources, and NO synthase (NOS) isoforms involved, remain unknown. Here, we used a multidisciplinary approach to study the expression, cellular distribution, and functional relevance of the endothelial NOS isoform within the hypothalamic paraventricular nucleus in sham and HF rats. Our results show high expression of endothelial NOS in the paraventricular nucleus (mostly confined to astroglial cells), which contributes to constitutive NO bioavailability, as well as tonic inhibition of presympathetic neuronal activity and sympathoexcitatory outflow from the paraventricular nucleus. A diminished endothelial NOS expression and endothelial NOS-derived NO availability were found in the paraventricular nucleus of HF rats, resulting, in turn, in blunted NO inhibitory actions on neuronal activity and sympathoexcitatory outflow. Taken together, our study supports blunted central nervous system endothelial NOS-derived NO as a pathophysiological mechanism underlying neurohumoral activation in HF.

We previously showed an altered integrity of the blood‐brain barrier (BBB) during chronic hypertension in cardiovascular associated brain regions. However, the differential contribution of AT1 receptors and high blood pressure (BP) was not assessed. To address this issue, SHR rats received either losartan or hydralazin orally (4 weeks), followed by a bolus carotid infusion of fluorescently labeled dextran dyes or FITC‐AngII. Brains were removed and processed for confocal imaging. BBB integrity was assessed by the degree of dye leakage into the extravascular CNS space. Results showed that while losartan prevented the increased BBB permeability in the PVN, NTS and RVLM in SHRs (P<0.05), hydralazin only had a partial effect in the RVLM. In addition to dye extravasation, we found differences in the expression of proteins that represent constituents of the BBB. Immunoreactivities for endothelial barrier antigen (EBA) and transferrin receptor (TfR), both markers endothelial cells at intact BBB, were decreased in the RVLM and NTS of SHRs, compared to WKYs (30–35%, P< 0.05). Conversely, dysferlin, a marker observed in disease conditions with a leaky BBB, was found in SHRs, but was almost absent in WKYs. Our results support an altered BBB integrity in SHRs, mediated by activation of AT1 receptors, and to a lesser degree, by the high blood pressure itself, which occurs in a region‐specific manner. Supported by AHA 0640092N.

Hypothalamic glucose sensing neurons in the arcuate and ventromedial nuclei adjust their firing activity according to extracellular glucose levels, playing a major role in glucose/energy homeostasis. The paraventricular nucleus (PVN), via descending preautonomic projection to the brainstem and spinal cord, is also critically involved in energy/glucose metabolism. However, whether preautonomic PVN neurons are also glucose sensitive is unknown. Here, we performed patch clamp recordings from preautonomic neurons (PVN‐RVLM) in brain slices obtained from control and diabetic (STZ 65mg/ml/kg, 28days) rats. Lowering extracellular glucose from 10 or 5 mM to 0.1mM diminished PVN‐RVLM firing activity (~65%, P<0.001, n=6) in control rats. The effect was reversed by the ATP‐sensitive K+ channel (K(ATP)) blocker tolbutamide (20μM). Moreover, membrane hyperpolarization and inhibition of firing (~90%, P<0.01, n=7) was observed when the K(ATP) channel opener diazoxide (0.5mM) was used. In STZ‐treated rats, PVN‐RVLM glucose sensing was impaired (n=9, P<0.0001 vs. controls), and a diminished effect of diazoxide was observed (~65% inhibition, P<0.04 vs. controls). Our results support (a) K(ATP)‐dependent glucose sensing in preautonomic PVN neurons, and (b) impared glucose sensitivity in diabetic rats, likely involving a diminished expression/function of K(ATP) channels. Supported by RO1 HL 090948.

The paraventricular nucleus of the hypothalamus (PVN) is known to be involved in increased neurohumoral drive in heart failure (HF). While abundant evidence supports blunted nitric oxide (NO) function as an underlying mechanism, the potential role of carbon monoxide (CO), another gas molecule highly related to NO, has not yet been explored. Thus, we aimed in this study to determine whether the expression of heme ‐ oxygenase 1 (HO1), a CO‐synthesizing enzyme was altered in the PVN during heart failure. HO1 immunohistochemical studies were performed in sham and ischemic congestive heart failure rats. Both neuronal and glial‐like staining patterns were observed in the PVN. Quantification of immunofluorescence intensity within individual PVN neurons demonstrated a significant increase (~20%) of HO1 in the PVN of heart failure rats. No differences however were observed in the supraoptic nucleus. Ongoing studies aim to determine whether changes in HO1 levels affect both neurosecretory and autonomic‐related neurons, as well as their electrophysiological activity. Based on our recent observation supporting CO as an excitatory gas molecule, our results suggest that enhanced CO levels within the PVN of heart failure rats may contribute to enhanced neurohumoral activation in this disease. Supported by NIH HL68725.

The blood brain barrier (BBB) acts as a physical barrier and a transport mechanism in the CNS, restricting access of systemic harmful chemicals while allowing entrance of essential nutrients. Growing evidence supports compromised BBB integrity under pathological conditions. Here, we aimed to assess if BBB integrity is altered in hypertensive rats. WKY and SHR rats at two developmental stages (7–8 & 11–13 week old) received intracarotid infusions of two fluorescently labeled dyes, RHO‐dextran 70kD and FITC‐dextran 10kD (28 mg/kg.bw, 30 sec). Dyes were allowed to circulate for 20min before the brains were removed, fixed, and processed for confocal imaging. BBB integrity was assessed based on the degree of dye leakage into the extravascular CNS space of hypothalamic and brainstem nuclei. RHO‐70 (larger dye) was largely confined within microvessels, and no differences in extravascular leakage were detected between WKY and SHRs (P>0.5). Conversely, extravascular FITC‐10 mean fluorescence intensity significantly increased in the PVN, NTS and RVLM of SHR compared to WKY rats at both stages (20–200% increase). These changes were largely prevented in SHR rats treated with Losartan (0.2mg/ml, 7 weeks). Our results supports the notion of a disrupted BBB within key CNS areas involved in cardiovascular control in hypertensive rats, and further suggests an AngII‐dependent mechanism. Supported by AHA 0640092N.

In addition to neurohypophysial secretion, vasopressin (VP) is locally released within the supraoptic and paraventricular (PVN) nuclei, autoregulating VP neuronal activity. Here, we tested the hypothesis that locally produced VP can also influence presympathetic PVN neuronal activity. Patch‐clamp recordings from presympathetic PVN‐RVLM neurons show that focal application of VP (1μM) depolarized, and increased the firing activity of PVN‐RLVM neurons (∼450%, P< 0.01). The effect persisted in the presence of the GABAA and glutamate receptor blockers BIC (20 μM) and KYN (1 mM), respectively. In the voltage‐clamp mode, VP activated an inward current of ∼6–10 pA. VP effects were blocked by the manning compound (1 μM), a V1a receptor antagonist. The expression of V1a receptors in PVN‐RVLM neurons was further confirmed by immunohistochemical studies. Importantly, the V1a receptor antagonist per se induced membrane hyperpolarization (∼4 mV) and reduced the ongoing firing activity of PVN‐RVLM neurons (∼75%, P< 0.02). In summary, our results indicate that locally produced VP tonically excites presympathetic PVN neurons, and suggest that activity‐dependent VP release from magnocellular neurosecretory neurons may constitute an important intercellular signaling pathway in the generation of homeostatic patterns of neuroendocrine and autonomic activities by the PVN. Supported by NIH HL68725.

Objective: Expression of adhesion molecules on endothelial cells and subsequent monocyte adhesion are initial events in the development of atherosclerosis. The purpose of this study was to investigate the role of apurinic/apyrmidinic endonuclease1/redox factor-1 (APE1/ref-1) in the interaction of monocytes with vascular endothelial cells.Methods: Human umbilical vein endothelial cells (HUVECs) were transfected with an adenovirus encoding human APE1/ref-1. The effect of APE1/ref-1 overexpression on monocyte adhesion, vascular cell adhesion molecule-1 (VCAM-1) protein expression, and intracellular superoxide production in tumor necrosis factor (TNF)-alpha-activated HUVECs was examined.Results: Adhesion of the monocytic cell line U937 to TNF-alpha-stimulated HUVECs in which APE1/ref-1 was overexpressed was suppressed. APE1/ref-1 overexpression also suppressed expression of VCAM-1 induced by TNF-alpha. APE1/ref-1-mediated suppression of VCAM-1 was blocked by pretreatment with the nitric oxide synthase (NOS) inhibitor L-nitroarginine methyl ester. Furthermore, APE1/ref-1 overexpression inhibited the TNF-alpha-induced increase in intracellular superoxide and p38 MAPK phosphorylation.Conclusions: These data provide evidence that APE1/ref-1 in endothelial cells mitigates TNF-alpha-induced monocyte adhesion and expression of vascular cell adhesion molecules, and this anti-adhesive property of APE1/ref-1 is primarily mediated by a NOS-dependent mechanism. Furthermore, APE1/ref-1 may inhibit VCAM-1 expression by inhibiting superoxide production and p38 MAPK activation. (C) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.