The B subunit of coagulation factor XIII is linked to renin and the Duffy blood group to α-spectrin on human chromosome 1

Family linkage studies were used to detect two linkage relationships on human chromosome 1. The B subunit of coagulation factor XIII showed significant linkage to renin with a maximum lod score of 5.071 at a distance of 10 cM. Significant linkage was also shown between the Duffy blood group and α-spectrin with linkage results giving a combined lod score of 3.194 at 5 cM.

The role of catechol-O-methyltransferase (COMT) and the effects of COMT inhibition in brain and in cardiovascular and renal pathophysiology

Catechol-O-methyltransferase (COMT) metabolizes catecholamines such as dopamine (DA), noradrenaline (NA) and adrenaline, which are vital neurotransmitters and hormones that play important roles in the regulation of physiological processes. COMT enzyme has a functional Val158Met polymorphism in humans, which affects the subjects COMT activity. Increasing evidence suggests that this functional polymorphism may play a role in the etiology of various diseases from schizophrenia to cancers. The aim of this project was to provide novel biochemical information on the physiological and especially pathophysiological roles of COMT enzyme as well as the effects of COMT inhibition in the brain and in the cardiovascular and renal system. To assess the roles of COMT and COMT inhibition in pathophysiology, we used four different study designs. The possible beneficial effects of COMT inhibition were studied in double-transgenic rats (dTGRs) harbouring human angiotensinogen and renin genes. Due to angiotensin II (Ang II) overexpression, these animals exhibit severe hypetension, cardiovascular and renal end-organ damage and mortality of approximately 25-40% at the age of 7-weeks. The dTGRs and their Sprague-Dawley controls tissue samples were assessed with light microscopy, immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and high-pressure liquid chromatography (HPLC) to evaluate the tissue damages and the possible protective effects pharmacological intervention with COMT inhibitors. In a second study, the consequence of genetic and pharmacological COMT blockade in blood pressure regulation during normal and high-sodium was elucidated using COMT-deficient mice. The blood pressure and the heart rate were measured using direct radiotelemetric blood pressure surveillance. In a third study, the effects of acute and subchronic COMT inhibition during combined levodopa (L-DOPA) + dopa decarboxylase inhibitor treatment in homocysteine formation was evaluated. Finally, we assessed the COMT enzyme expression, activity and cellular localization in the CNS during inflammation-induced neurodegeneration using Western blotting, HPLC and various enzymatic assays. The effects of pharmacological COMT inhibition on neurodegeneration were also studied. The COMT inhibitor entacapone protected against the Ang II-induced perivascular inflammation, renal damage and cardiovascular mortality in dTGRs. COMT inhibitors reduced the albuminuria by 85% and prevented the cardiovascular mortality completely. Entacapone treatment was shown to ameliorate oxidative stress and inflammation. Furthermore, we established that the genetic and pharmacological COMT enzyme blockade protects against the blood pressure-elevating effects of high sodium intake in mice. These effects were mediated via enhanced renal dopaminergic tone and suggest an important role of COMT enzyme, especially in salt-sensitive hypertension. Entacapone also ameliorated the L-DOPA-induced hyperhomocysteinemia in rats. This is important, since decreased homocysteine levels may decrease the risk of cardiovascular diseases in Parkinson´s disease (PD) patients using L-DOPA. The Lipopolysaccharide (LPS)-induced inflammation and subsequent delayed dopaminergic neurodegeneration were accompanied by up-regulation of COMT expression and activity in microglial cells as well as in perivascular cells. Interestingly, similar perivascular up-regulation of COMT expression in inflamed renal tissue was previously noted in dTGRs. These results suggest that inflammation reactions may up-regulate COMT expression. Furthermore, this increased glial and perivascular COMT activity in the central nervous system (CNS) may decrease the bioavailability of L-DOPA and be related to the motor fluctuation noted during L-DOPA therapy in PD patients.

Direct angiotensin type 2 receptor (AT2R) stimulation attenuates T-cell and microglia activation and prevents demyelination in experimental autoimmune encephalomyelitis in mice.

In the present study, we evaluated stimulation of the angiotensin type 2 receptor (AT2R) by the selective non-peptide agonist Compound 21 (C21) as a novel therapeutic concept for the treatment of multiple sclerosis using the model of experimental autoimmune encephalomyelitis (EAE) in mice. C57BL-6 mice were immunized with myelin-oligodendrocyte peptide and treated for 4 weeks with C21 (0.3 mg/kg/day i.p.). Potential effects on myelination, microglia and T-cell composition were estimated by immunostaining and FACS analyses of lumbar spinal cords. The in vivo study was complemented by experiments in aggregating brain cell cultures and microglia in vitro. In the EAE model, treatment with C21 ameliorated microglia activation and decreased the number of total T-cells and CD4+ T-cells in the spinal cord. Fluorescent myelin staining of spinal cords further revealed a significant reduction in EAE-induced demyelinated areas in lumbar spinal cord tissue after AT2R stimulation. C21-treated mice had a significantly better neurological score than vehicle-treated controls. In aggregating brain cell cultures challenged with lipopolysaccharide (LPS) plus interferon-γ (IFNγ), AT2R stimulation prevented demyelination, accelerated re-myelination and reduced the number of microglia. Cytokine synthesis and nitric oxide production by microglia in vitro were significantly reduced after C21 treatment. These results suggest that AT2R stimulation protects the myelin sheaths in autoimmune central nervous system inflammation by inhibiting the T-cell response and microglia activation. Our findings identify the AT2R as a potential new pharmacological target for demyelinating diseases such as multiple sclerosis.

In vitro functionality of isolated embryonic hypothalamic vasopressinergic and oxytocinergic neurons: modulatory effects of brain-derived neurotrophic factor and angiotensin II.

There are only a few studies on the ontogeny and differentiation process of the hypothalamic supraoptic-paraventriculo-neurohypophysial neurosecretory system. In vitro neuron survival improves if cells are of embryonic origin; however, surviving hypothalamic neurons in culture were found to express small and minimal amounts of arginine-vasopressin (AVP) and oxytocin (OT), respectively. The aim of this study was to develop a primary neuronal culture design applicable to the study of magnocellular hypothalamic system functionality. For this purpose, a primary neuronal culture was set up after mechanical dissociation of sterile hypothalamic blocks from 17-day-old Sprague-Dawley rat embryos (E17) of both sexes. Isolated hypothalamic cells were cultured with supplemented (B27)-NeuroBasal medium containing an agent inhibiting non-neuron cell proliferation. The neurosecretory process was characterized by detecting AVP and OT secreted into the medium on different days of culture. Data indicate that spontaneous AVP and OT release occurred in a culture day-dependent fashion, being maximal on day 13 for AVP, and on day 10 for OT. Interestingly, brain-derived neurotrophic factor (BDNF) and Angiotensin II (A II) were able to positively modulate neuropeptide output. Furthermore, on day 17 of culture, non-specific (high-KCl) and specific (Angiotensin II) stimuli were able to significantly (P < 0.05) enhance the secretion of both neuropeptides over respective baselines. This study suggests that our experimental design is useful for the study of AVP- and OT-ergic neuron functionality and that BDNF and A II are positive modulators of embryonic hypothalamic cell development.

The roles of ERK1/2 and PI3K in abnormal vascular functions in angiotensin II-infused hypertensive rats

Background: Ang II plays a major role in cardiovascular regulation. Recently, it has become apparent that vascular superoxide anion may play an important role in hypertension development. Treatment with antisense NAD(P)H oxidase or SOD decreased BP in Ang II-infused rats. Wang et al recently reported mice which lack one of the subunits of NAD(P)H oxidase developed hypertension at a much lower extent when compared to the wild type animals infused with Ang II, indicating that superoxide anion contributes to elevation in BP in the Ang II-infused hypertensive model. In the Ang II-infused hypertensive model, altered reactivity of blood vessels is often associated with the elevation of systolic blood pressure. We have observed abnormal tension development and impaired endothelium-dependent relaxation in the isolated aorta of Ang II-infused and DOCA-salt hypertensive rats. Recently, several other cellular signal molecules, including ERK1I2 and PI3K, have been determined to play important roles in the regulation of smooth muscle contraction and relaxation. ERKl/2 and PI3K pathways are also reported to contribute to Ang II induced cell growth, hypertrophy, remodeling and contraction. Moreover, these signaling pathways have shown ROS-sensitive properties. Therefore, the aim of the present study is to investigate the roles of ERKl12 and PI3K in vascular oxidative stress, spontaneous tone and impaired endothelium relaxation in Ang II-infused hypertensive model. Hypothesis: We hypothesize that the activation of ERKl12 and PI3K are elevated in response to an Ang II infusion for 6 days. The elevated activation of phospho-ERKl/2 and PI3K mediated the increased level of vascular superoxide anion, the abnormal vascular contraction and impaired endothelium-dependent vascular relaxation in Ang II-infused hypertensive rats. Methods: Vascular superoxide anion level is measured by lucigenin chemiluminescence. Spontaneous tone and ACh-induced endothelium-dependent relaxation was measured by isometric tension recording in organ chamber. The activity of ERK pathway will be measured by its Western blot of phosphorylation of ERK. PI3K activity was evaluated indirectly by Western blot of the phosphorylation of PDKl, a downstream protein of PI3K signaling pathway. The role of each pathway was also addressed via comparing the responses to the specific inhibitors. Results: Superoxide anion was markedly increased in the isolated thoracic aorta from Ang II-infused rats. There was spontaneous tone developed in rings from Ang II-induced hypertensive but not sham-operated normotensive rats. ACh-induced endothelium-dependent relaxation function is impaired in Ang II-infused hypertensive rats. Superoxide dismutase and NAD(P)H oxidase inhibitor, apocynin, inhibited the abnormal spontaneous tone and ameliorated impaired endothelium-dependent relaxation. The expression of phopho-ERKII2 was enhanced in Ang II-infused rats, indicating the activity of ERK1I2 could be increased. MEK1I2 inhibitors, PD98059 and U126, but not their inactive analogues, SB203580 and U124, significantly reduced the vascular superoxide anion in aortas from Ang II-infused rats. The MEK1I2 inhibitors reduced the spontaneous tone and improved the impaired endothelium-dependent relaxation in aorta of hypertension. These findings supported the role of ERKII2 signaling pathway in vascular oxidative stress, spontaneous tone and impaired endothelium-dependent relaxation in Ang II-infused hypertensive rats. The amount of phospho-PDK, a downstream protein of PI3K was increased in Ang II rats indicating the activity of PI3K activity was elevated. Strikingly, PI3K significantly inhibited the increase of superoxide anion level, abnormal spontaneous tone and restored endothelium-dependent relaxation in Ang II-infused hypertensive rats. These findings indicated the important role of PI3K in Ang II-infused hypertensive rats. Conclusion: ERKII2 and PI3K signaling pathways are sustained activated in Ang II-infused hypertensive rats. The activated ERKII2 and PI3K mediate the increase of vascular superoxide anion level, vascular abnormal spontaneous tone and impaired endothelium-dependent relaxation.

Interaction of severe acute respiratory syndrome-coronavirus and NL63 coronavirus spike proteins with angiotensin converting enzyme-2

Although in different groups, the coronaviruses severe acute respiratory syndrome-coronavirus (SARS-CoV) and NL63 use the same receptor, angiotensin converting enzyme (ACE)-2, for entry into the host cell. Despite this common receptor, the consequence of entry is very different; severe respiratory distress in the case of SARS-CoV but frequently only a mild respiratory infection for NL63. Using a wholly recombinant system, we have investigated the ability of each virus receptor-binding protein, spike or S protein, to bind to ACE-2 in solution and on the cell surface. In both assays, we find that the NL63 S protein has a weaker interaction with ACE-2 than the SARS-CoV S protein, particularly in solution binding, but the residues required for contact are similar. We also confirm that the ACE-2-binding site of NL63 S lies between residues 190 and 739. A lower-affinity interaction with ACE-2 might partly explain the different pathological consequences of infection by SARS-CoV and NL63.

The Central Nervous System as Target for Antihypertensive Actions of a Proline-Rich Peptide from Bothrops jararaca Venom

Pyroglutamyl proline-rich oligopeptides, present in the venom of the pit viper Bothrops jararaca (Bj-PROs), are the first described naturally occurring inhibitors of the angiotensin I-converting enzyme (ACE). The inhibition of ACE by the decapeptide Bj-PRO-10c (system. In agreement with its supposed neuronal action, this peptide dose-dependently evoked elevations of intracellular calcium concentration ([Ca(2+)](i)) in primary culture from postnatal rat brain. The N-terminal sequence of the peptide was not essential for induction of calcium fluxes, while any changes of C-terminal Pro or Ile residues affected Bj-PRO-10c`s activity. Using calcium imaging by confocal microscopy and fluorescence imaging plate reader analysis, we have characterized Bj-PRO-10c-induced [Ca(2+)](i) transients in rat brain cells as being independent from bradykinin-mediated effects and ACE inhibition. Bj-PRO-10c induced pertussis toxin-sensitive G(i/o)-protein activity mediated through a yet unknown receptor, influx and liberation of calcium from intracellular stores, as well as reduction of intracellular cAMP levels. Bj-PRO-10c promoted glutamate and GABA release that may be responsible for its antihypertensive activity and its effect on HR. (C) 2010 International Society for Advancement of Cytometry

Elevated intracardiac angiotensin II leads to cardiac hypertrophy and mechanical dysfunction in normotensive mice

Introduction
Angiotensin II (Ang II) is known to induce cardiac growth and modulate myocardial contractility. It has been reported that elevated levels of endogenous Ang II contribute to the development of cardiac hypertrophy in hypertensives. However, the long-term functional effects of cardiac exposure to Ang II in normotensives is unclear.

A recently developed transgenic mouse (TG1306/1R), in which cardiac-specific overproduction of Ang II produces primary hypertrophy, provides a new experimental model for investigation of this phenotype. The aim of the present study was to use this model to investigate whether there is a functional deficit in primary hypertrophy that may predispose to cardiac failure and sudden death. We hypothesised that primary cardiac hypertrophy is associated with mechanical dysfunction in the basal state.

Methods
Normotensive heterozygous TG1306/1R mice harbouring multiple copies of a cardiac-specific rat angiotensinogen gene were studied at age 30—40 weeks and compared with age-matched wild-type littermates. Left ventricular function was measured ex vivo in bicarbonate buffer-perfused, Langendorffmounted hearts ( at a perfusion pressure of 80 mmHg, 37°C) using a fluid-filled PVC balloon interfaced to a pressure transducer and digital data acquisition system.

Results
There was no difference in the mean (±SEM) intrinsic heart rate of TG1306/1R and wild-type control mice (357.4±11.8 vs. 367.5±20.9 bpm, n=9 & 7). Under standardised end-diastolic pressure conditions, TG1306/1R hearts exhibited a significant reduction in peak developed pressure (132.2±9.4 vs. 161.5±3.1 mmHg, n=9 & 7, p<0.05) and maximum rate of pressure development (3566.7±323.7 vs. 4486.3±109.4 mmHg, n=9 & 7, p<0.05). TG1306/1R mice show a significant correlation between incidence of arrhythmia and increasing heart size (Spearman's correlation coefficient 0.61).

Conclusion
These data demonstrate that chronic in vivo exposure to elevated levels of intra-cardiac Ang II is associated with significant contractile abnormalities evident in the ex vivo intact heart. Our findings suggest that endogenous overproduction of cardiac Ang II, independent of changes in blood pressure, is sufficient to induce ventricular remodelling that culminates in impaired cardiac function which may precede failure.

Role of angiotensin II in the hypertension induced by renal artery stenosis

Identical degrees of renal artery stenosis were induced in 5 dogs on two separate occasions; once during continuous inhibition of angiotensin I converting enzyme with enalapril, and once with the dogs untreated. Arterial pressure rose about 25 mm Hg during 3 days of stenosis in untreated dogs, due to increased total peripheral resistance. When the dogs were treated with enalapril, blood pressure had risen 14.5 ± 3.4 mm Hg 24 hours after stenosis due to a 35% increase in cardiac output while total peripheral resistance fell by 16%. By the third day, blood pressure had returned to pre-stenosis levels, cardiac output was close to normal and total peripheral resistance had increased. The stenosis on the renal artery increased the resistance to blood flow of the kidneys in both untreated and enalapril treated dogs. This increase in kidney resistance in the untreated dogs accounted for about 30% of the change in total peripheral resistance. In the enalapril treated dogs, the increased kidney resistance helped offset the vasodilatation in the rest of the vasculature. These results suggest that angiotensin II mediated vasoconstriction of nonrenal vascular beds was responsible for about ⅔ of the hypertension following renal artery stenosis, and the resistance of the stenosis responsible for about ⅓.

Angiotensin II-derived reactive oxygen species underpinning the processing of the cardiovascular reflexes in the medulla oblongata

The brainstem is a major site in the central nervous system involved in the processing of the cardiovascular reflexes such as the baroreflex and the peripheral chemoreflex. The nucleus tractus solitarius and the rostral ventrolateral medulla are 2 important brainstem nuclei, and they play pivotal roles in autonomic cardiovascular regulation. Angiotensin II is one of the neurotransmitters involved in the processing of the cardiovascular reflexes within the brainstem. It is well-known that one of the mechanisms by which angiotensin II exerts its effect is via the activation of pathways that generate reactive oxygen species (ROS). In the central nervous system, ROS are reported to be involved in several pathological diseases such as hypertension, heart failure and sleep apnea. However, little is known about the role of ROS in the processing of the cardiovascular reflexes within the brainstem. The present review mainly discussed some recent findings documenting a role for ROS in the processing of the baroreflex and the peripheral chemoreflex in the brainstem.

Subfornical organ mediates pressor effect of angiotensin: Influence of nitric oxide synthase inhibitors, AT(1) and AT(2) angiotensin antagonist's receptors

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Hypothalamic disconnection caudal to paraventricular nucleus affects cardiovascular and drinking responses to central angiotensin II and carbachol

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Regulation of angiotensin type 2 receptor in bovine granulosa cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Angiotensin II, progesterone, and prostaglandins are sequential steps in the pathway to bovine oocyte nuclear maturation

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Brain versus peripheral angiotensin II receptors in hypovolaemia: Behavioural and cardiovascular implications

1. Angiotensin (Ang)II is involved in responses to hypovolaemia, such as sodium appetite and increase in blood pressure, Target areas subserving these responses for AngII include the cardiovascular system in the periphery and the circumventricular organs in the brain.2. Conflicting data have been reported for the role of systemic versus brain AngII in the mediation of sodium appetite.3. The role for systemic AngII and systemic AngII receptors in the control of blood pressure in hypovolaemia is well established. In contrast with systemic injections, i.c.v injections of AngII non-peptide AT(1) and AT(2) receptor antagonists, such as losartan and PD123319, do not reduce arterial pressure in sodium-depleted (furosemide injection plus removal of ambient sodium for 24 h) rats. Thus, brain AngII receptors are likely not important for cardiovascular responses to hypovolaemia induced by sodium depletion.4. Intracerebroventricular injections of losartan or PD 123319 increase arterial pressure when injected at relatively high doses. This hypertensive effect is unlikely to be an agonist effect on brain AngII receptors, Increases in arterial pressure produced by i.c.v, losartan are attenuated by lesions of the tissue surrounding the anterior third ventricle (AV3V). The hypertensive effect of i.c.v, AngII is abolished by lesions of the AV3V.5. Hypertension induced by AngII receptor antagonists is consistent with hypotension induced by AngII acting in the brain, However, the full physiological significance of this hypotensive effect mediated by brain AngII receptors remains to be determined.