Angiotensin II regulates growth of the developing papillas ex vivo

We tested the hypothesis that lack of angiotensin (ANG) II production in angiotensinogen (AGT)-deficient mice or pharmacologic antagonism of ANG II AT(1) receptor (AT(1)R) impairs growth of the developing papillas ex vivo, thus contributing to the hypoplastic renal medulla phenotype observed in AGT- or AT(1)R-null mice. Papillas were dissected from Hoxb7(GFP+) or AGT(+/+), (+/-), (-/-) mouse metanephroi on postnatal day P3 and grown in three-dimentional collagen matrix gels in the presence of media (control), ANG II (10(-5) M), or the specific AT(1)R antagonist candesartan (10(-6) M) for 24 h. Percent reduction in papillary length was attenuated in AGT(+/+) and in AGT(+/-) compared with AGT(-/-) (-18.4 ± 1.3 vs. -32.2 ± 1.6%, P < 0.05, -22.8 ± 1.3 vs. -32.2 ± 1.6%, P < 0.05, respectively). ANG II blunted the decrease in papilla length observed in respective media-treated controls in Hoxb7(GFP+) (-1.5 ± 0.3 vs. -10.0 ± 1.4%, P < 0.05) or AGT(+/+), (+/-), and (-/-) papillas (-12.8 ± 0.7 vs. -18.4 ± 1.3%, P < 0.05, -16.8 ± 1.1 vs. -23 ± 1.2%, P < 0.05; -26.2 ± 1.6 vs. -32.2 ± 1.6%, P < 0.05, respectively). In contrast, percent decrease in the length of Hoxb7(GFP+) papillas in the presence of the AT(1)R antagonist candesartan was higher compared with control (-24.3 ± 2.1 vs. -10.5 ± 1.8%, P < 0.05). The number of proliferating phospho-histone H3 (pH3)-positive collecting duct cells was lower, whereas the number of caspase 3-positive cells undergoing apoptosis was higher in candesartan- vs. media-treated papillas (pH3: 12 ± 1.4 vs. 21 ± 2.1, P < 0.01; caspase 3: 3.8 ± 0.5 vs. 1.7 ± 0.2, P < 0.01). Using quantitative RT-PCR, we demonstrate that AT(1)R signaling regulates the expression of genes implicated in morphogenesis of the renal medulla. We conclude that AT(1)R prevents shrinkage of the developing papillas observed ex vivo via control of Wnt7b, FGF7, β-catenin, calcineurin B1, and α3 integrin gene expression, collecting duct cell proliferation, and survival.     

Aortic stiffness affects the coronary blood flow response to percutaneous coronary intervention

Chronic subcutaneous infusion of ouabain causes hypertension via central pathways involving angiotensin type 1 (AT(1)) receptor stimulation. The present study assessed plasma and tissue ANG I and II levels as well as AT1 receptor and angiotensin-converting enzyme (ACE) mRNA levels and binding densities by real-time PCR and in vitro autoradiography in relevant brain nuclei and peripheral tissues (heart and kidney) in rats at 1 and/or 2 wk after start of ouabain infusion at 50 microg/day. After 2 wk (but not after 1 wk), blood pressures significantly increased (+15 mmHg). At 2 wk, plasma ANG I and II levels were markedly suppressed by ouabain. In contrast, in the heart and kidneys, ANG I levels were not affected, and ANG II levels tended to decrease, whereas in the hypothalamus ANG II content clearly increased. At 1 wk, no changes in ACE and AT1 receptor densities were seen. After 2 wk, there were significant decreases in AT(1) receptor mRNA and densities in the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), and paraventricular nucleus (PVN). ACE densities decreased only in the OVLT and SFO, but ACE mRNA showed more variable responses (decrease in OVLT vs. increase in PVN). In the kidneys, at 2 wk both AT1 receptor and ACE densities were decreased, but mRNA abundance did not change. The heart showed no significant changes. The increase in hypothalamic ANG II content and associated decreases in central AT1 receptor and ACE densities support the involvement of the brain renin-angiotensin system in the central hypertensive mechanism of action of ouabain.     

Effects of ANG II type 1 and 2 receptors on oxidative stress,renal NADPH oxidase,and SOD expression

Oxidative stress accompanies angiotensin (ANG) II infusion, but the role of ANG type 1 vs. type 2 receptors (AT1-R and AT2-R, respectively) is unknown. We infused ANG II subcutaneously in rats for 1 wk. Excretion of 8-isoprostaglandin F2alpha (8-Iso) and malonyldialdehyde (MDA) were related to renal cortical mRNA abundance for subunits of NADPH oxidase and superoxide dismutases (SODs) using real-time PCR. Subsets of ANG II-infused rats were given the AT1-R antagonist candesartan cilexetil (Cand) or the AT2-R antagonist PD-123,319 (PD). Compared to vehicle (Veh), ANG II increased 8-Iso excretion by 41% (Veh, 5.4 +/- 0.8 vs. ANG II, 7.6 +/- 0.5 pg/24 h; P < 0.05). This was prevented by Cand (5.6 +/- 0.5 pg/24 h; P < 0.05) and increased by PD (15.8 +/- 2.0 pg/24 h; P < 0.005). There were similar changes in MDA excretion. Compared to Veh, ANG II significantly (P < 0.005) increased the renal cortical mRNA expression of p22phox (twofold), Nox-1 (2.6-fold), and Mn-SOD (1.5-fold) and decreased expression of Nox-4 (2.1-fold) and extracellular (EC)-SOD (2.1-fold). Cand prevented all of these changes except for the increase in Mn-SOD. PD accentuated changes in p22phox and Nox-1 and increased p67phox. We conclude that ANG II infusion stimulates oxidative stress via AT1-R, which increases the renal cortical mRNA expression of p22phox and Nox-1 and reduces abundance of Nox-4 and EC-SOD. This is offset by strong protective effects of AT2-R, which are accompanied by decreased expression of p22phox, Nox-1, and p67phox.     

Angiotensin II induces monocyte chemoattractant protein-1 expression via a nuclear factor-kappaB-dependent pathway in rat preadipocytes

Both monocyte chemoattractant protein-1 (MCP-1), a member of chemokine family, and angiotensinogen, a precursor of angiotensin (ANG) II, are produced by adipose tissue and increased in obese state. MCP-1 has been shown to decrease insulin-stimulated glucose uptake and several adipogenic genes expression in adipocytes in vitro, suggesting its pathophysiological significance in obesity. However, the pathophysiological interaction between MCP-1 and ANG II in adipose tissue remains unknown. The present study was undertaken to investigate the potential mechanisms by which ANG II affects MCP-1 gene expression in rat primary cultured preadipocytes and adipose tissue in vivo. ANG II significantly increased steady-state MCP-1 mRNA levels in a time- and dose-dependent manner. The ANG II-induced MCP-1 mRNA and protein expression was completely abolished by ANG II type 1 (AT1)-receptor antagonist (valsartan). An antioxidant/NF-kappaB inhibitor (pyrrolidine dithiocarbamate) and an inhibitor of 1kappaB-alpha phosphorylation (Bay 11-7085) also blocked ANG II-induced MCP-1 mRNA expression. ANG II induced translocation of NF-kappaB p65 subunit from cytoplasm to nucleus by immunocytochemical study. Luciferase assay using reporter constructs containing MCP-1 promoter region revealed that two NF-kappaB binding sites in its enhancer region were essential for the ANG II-induced promoter activities. Furthermore, basal mRNA and protein of MCP-1 during preadipocyte differentiation were significantly greater in preadipocytes than in differentiated adipocytes, whose effect was more pronounced in the presence of ANG II. Exogenous administration of ANG II to rats led to increased MCP-1 expression in epididymal, subcutaneous, and mesenteric adipose tissue. In conclusion, our present study demonstrates that ANG II increases MCP-1 gene expression via ANG II type 1 receptor-mediated and NF-kappaB-dependent pathway in rat preadipocytes as well as adipose MCP-1 expression in vivo. Thus the augmented MCP-1 expression by ANG II in preadipocytes may provide a new link between obesity and cardiovascular disease.     

Angiotensin II mediates postischemic leukocyte-endothelial interactions: role of calcitonin gene-related peptide

Vascular inflammation and enhanced production of angiotensin II (ANG II) are involved in the pathogenesis of hypertension and diabetes, disease states that predispose the afflicted individuals to ischemic disorders. In light of these observations, we postulated that ANG II may play a role in promoting leukocyte rolling (LR) and adhesion (LA) in postcapillary venules after exposure of the small intestine to ischemia-reperfusion (I/R). Using an intravital microscopic approach in C57BL/6J mice, we showed that ANG II type I (AT(1)) or type II (AT(2)) receptor antagonism (with valsartan or PD-123319, respectively), inhibition of angiotensin-converting enzyme (ACE) with captopril, or calcitonin gene-related peptide (CGRP) receptor blockade (CGRP8-37) prevented postischemic LR but did not influence I/R-induced LA. However, both postischemic LR and LA were largely abolished by concomitant AT(1) and AT(2) receptor blockade or chymase inhibition (with Y-40079). Additionally, exogenously administered ANG II increased LR and LA, effects that were attenuated by pretreatment with a CGRP receptor antagonist or an NADPH oxidase inhibitor (apocynin). Our work suggests that ANG II, formed by the enzymatic activity of ACE and chymase, plays an important role in inducing postischemic LR and LA, effects that involve the engagement of both AT(1) and AT(2) receptors and may be mediated by CGRP and NADPH oxidase.     

ACE2 is expressed in mouse adipocytes and regulated by a high-fat diet

Adipose tissue expresses components of the renin-angiotensin system (RAS). Angiotensin converting enzyme (ACE2), a new component of the RAS, catabolizes the vasoconstrictor peptide ANG II to form the vasodilator angiotensin 1-7 [ANG-(1-7)]. We examined whether adipocytes express ACE2 and its regulation by manipulation of the RAS and by high-fat (HF) feeding. ACE2 mRNA expression increased (threefold) during differentiation of 3T3-L1 adipocytes and was not regulated by manipulation of the RAS. Male C57BL/6 mice were fed low- (LF) or high-fat (HF) diets for 1 wk or 4 mo. At 1 wk of HF feeding, adipose expression of angiotensinogen (twofold) and ACE2 (threefold) increased, but systemic angiotensin peptide concentrations and blood pressure were not altered. At 4 mo of HF feeding, adipose mRNA expression of angiotensinogen (twofold) and ACE2 (threefold) continued to be elevated, and liver angiotensinogen expression increased (twofold). However, adipose tissue from HF mice did not exhibit elevated ACE2 protein or activity. Increased expression of ADAM17, a protease responsible for ACE2 shedding, coincided with reductions in ACE2 activity in 3T3-L1 adipocytes, and an ADAM17 inhibitor decreased media ACE2 activity. Moreover, ADAM17 mRNA expression was increased in adipose tissue from 4-mo HF-fed mice, and plasma ACE2 activity increased. However, HF mice exhibited marked increases in plasma angiotensin peptide concentrations (LF: 2,141 +/- 253; HF: 6,829 +/- 1,075 pg/ml) and elevated blood pressure. These results demonstrate that adipocytes express ACE2 that is dysregulated in HF-fed mice with elevated blood pressure compared with LF controls.     

Molecular mechanisms of angiotensin II stimulation on aquaporin-2 expression and trafficking

ANG II plays a major role in renal water and sodium regulation. In the immortalized mouse renal collecting duct principal cells (mpkCCD(cl4)) cell line, we treated cells with ANG II and examined aquaporin-2 (AQP2) protein expression, trafficking, and mRNA levels, by immunoblotting, immunofluorescence, and RT-PCR. After 24-h incubation, ANG II-induced AQP2 protein expression was observed at the concentration of 10(-10) M and increased in a dose-dependent manner. ANG II (10(-7) M) increased AQP2 protein expression and mRNA levels at 0.5, 1, 2, 6, and 24 h. Immunofluorescence studies showed that ANG II increased the apical membrane targeting of AQP2 from 30 min to 6 h. Next, the signaling pathways underlying the ANG II-induced AQP2 expression were investigated. The PKC inhibitor Ro 31-8220 (5 × 10(-6) M) and the PKA inhibitor H89 (10(-5) M) blocked ANG II-induced AQP2 expression, respectively. Calmodulin inhibitor W-7 markedly reduced ANG II- and/or dDAVP-stimulated AQP2 expression. ANG II (10(-9) M) and/or dDAVP (10(-10) M) stimulated AQP2 protein levels and cAMP accumulation, which was completely blocked by pretreatment with the vasopressin V2 receptor (V2R) antagonist SR121463B (10(-8) M). Pretreatment with the angiotensin AT(1) receptor (AT1R) antagonist losartan (3 × 10(-6) M) blocked ANG II (10(-9) M)-stimulated AQP2 protein expression and cAMP accumulation, and partially blocked dDAVP (10(-10) M)- and dDAVP+ANG II-induced AQP2 protein expression and cAMP accumulation. In conclusion, ANG II regulates AQP2 protein, trafficking, and gene expression in renal collecting duct principal cells. ANG II-induced AQP2 expression involves cAMP, PKC, PKA, and calmodulin signaling pathways via V2 and AT(1) receptors.     

AT(1) and AT(2) receptor expression and blockade after acute ischemia-reperfusion in isolated working rat hearts

We assessed ANG II type 1 (AT(1)) and type 2 (AT(2)) receptor (R) expression and functional recovery after ischemia-reperfusion with or without AT(1)R/AT(2)R blockade in isolated working rat hearts. Groups of six hearts were subjected to global ischemia (30 min) followed by reperfusion (30 min) and exposed to no drug and no ischemia-reperfusion (control), ischemia-reperfusion and no drug, and ischemia-reperfusion with losartan (an AT(1)R antagonist; 1 micromol/l), PD-123319 (an AT(2)R antagonist; 0.3 micromol/l), N(6)-cyclohexyladenosine (CHA, a cardioprotective adenosine A(1) receptor agonist; 0.5 micromol/l as positive control), enalaprilat (an ANG-converting enzyme inhibitor; 1 micromol/l), PD-123319 + losartan, ANG II (1 nmol/l), or ANG II + losartan. Compared with controls, ischemia-reperfusion decreased AT(2)R protein (Western immunoblots) and mRNA (Northern immunoblots, RT-PCR) and impaired functional recovery. PD-123319 increased AT(2)R protein and mRNA and improved functional recovery. Losartan increased AT(1)R mRNA (but not AT(1)R/AT(2)R protein) and impaired recovery. Other groups (except CHA) did not improve recovery. The results suggest that, in isolated working hearts, AT(2)R plays a significant role in ischemia-reperfusion and AT(2)R blockade induces increased AT(2)R protein and cardioprotection.     

Age-related changes of Renin-Angiotensin system genes in white adipose tissue of rats.

The adipose tissue renin-angiotensin system has been implicated in the regulation of adipocyte growth and differentiation. We studied the influence of age, body weight, total body fat content, anatomical localization, and diet on the expression of angiotensinogen (AGT) and angiotensin II type 1 (AT 1 )-receptor genes in white adipose tissue of normal and postnatal overfed rats. Relative gene expression was measured in epididymal adipose tissue and liver of control and postnatal overfed (PNO) rats at the age of 4, 8, and 12 weeks using real time RT-PCR. Body fat content was determined by carcass analysis. Body weight and body fat content were only significantly greater in PNO rats when compared to control rats at the age of 4 weeks. At the age of 12 weeks, AGT expression was significantly decreased in both tissues. Furthermore, expression of the AT 1 -receptor gene was significantly decreased in liver but not in adipose tissue at 12 weeks of age. Postnatal overfeeding did not influence the expression levels of either gene at any time-point in either liver or adipose tissue. At the age of 24 weeks, AGT expression was significantly greater in epididymal than in subcutaneous adipose tissue, whereas no site-specific differences could be found for the AT 1 -receptor. We conclude that age and depot-specific mechanisms are of more importance for the expression of AGT and AT 1 -receptor genes during the first 12 weeks of age than a short period of overfeeding.     

Angiotensin II type 1 receptor interaction is an important regulator for the development of pancreatic fibrosis in mice

The renin-angiotensin system (RAS) plays important roles in various pathophysiological processes. However, the role of the RAS in pancreatic fibrosis has not been established. We investigated the role of angiotensin II (ANG II)-ANG II type 1 (AT(1)) receptor pathway in the development of pancreatic fibrosis with AT(1a) receptor-deficient [AT(1a)(-/-)] mice. To induce pancreatic fibrosis, AT(1a)(-/-) and wild-type (WT) mice were submitted to three episodes of acute pancreatitis induced by six intraperitoneal injections of 50 microg/kg body wt cerulein at hourly intervals, per week, for four consecutive weeks. Pancreatic fibrosis was assessed by histology and hydroxyproline content. Pancreatic stellate cell (PSC) activation and the localization of AT(1) receptors were assessed by Western blot analysis for alpha-smooth muscle actin and immunostaining. Transforming growth factor-beta(1) (TGF-beta(1)) mRNA expression in the pancreas was assessed by RT-PCR. Six intraperitoneal injections of cerulein induced acute pancreatitis in both AT(1a)(-/-) and WT mice. There were no significant differences between two groups with regard to serum amylase and histological changes. Pancreatic fibrosis induced by repeated episodes of acute pancreatitis was significantly attenuated in AT(1a)(-/-) mice compared with that in WT mice. This finding was accompanied by a reduction of activated PSCs. Dual-immunofluorescence staining in WT mice revealed that activated PSCs express AT(1) receptors. The level of TGF-beta(1) mRNA was lower in AT(1a)(-/-) mice than in WT mice. Our results demonstrate that the ANG II-AT(1) receptor pathway is not essential for the local pancreatic injury in acute pancreatitis but plays an important role in the development of pancreatic fibrosis through PSC activation and proliferation.     

Adipocyte-specific deficiency of angiotensinogen decreases plasma angiotensinogen concentration and systolic blood pressure in mice

Previous studies demonstrated that overexpression of angiotensinogen (AGT) in adipose tissue increased blood pressure. However, the contribution of endogenous AGT in adipocytes to the systemic renin-angiotensin system (RAS) and blood pressure control is undefined. To define a role of adipocyte-derived AGT, mice with loxP sites flanking exon 2 of the AGT gene (Agt(fl/fl)) were bred to transgenic mice expressing Cre recombinase under the control of an adipocyte fatty acid-binding protein 4 promoter (aP2) promoter to generate mice with adipocyte AGT deficiency (Agt(aP2)). AGT mRNA abundance in adipose tissue and AGT secretion from adipocytes were reduced markedly in adipose tissues of Agt(aP2) mice. To determine the contribution of adipocyte-derived AGT to the systemic RAS and blood pressure control, mice were fed normal laboratory diet for 2 or 12 mo. In males and females of each genotype, body weight and fat mass increased with age. However, there was no effect of adipocyte AGT deficiency on body weight, fat mass, or adipocyte size. At 2 and 12 mo of age, mice with deficiency of AGT in adipocytes had reduced plasma concentrations of AGT (by 24-28%) compared with controls. Moreover, mice lacking AGT in adipocytes exhibited reduced systolic blood pressures compared with controls (Agt(fl/fl), 117 ± 2; Agt(aP2), 110 ± 2 mmHg; P < 0.05). These results demonstrate that adipocyte-derived AGT contributes to the systemic RAS and blood pressure control.     

A brain leptin-renin angiotensin system interaction in the regulation of sympathetic nerve activity

The sympathetic nervous system, leptin, and renin-angiotensin system (RAS) have been implicated in obesity-associated hypertension. There is increasing evidence for the presence of both leptin and angiotensin II receptors in several key brain cardiovascular and metabolic control regions. We tested the hypothesis that the brain RAS plays a facilitatory role in the sympathetic nerve responses to leptin. In rats, intracerebroventricular (ICV) administration of losartan (5 μg) selectively inhibited increases in renal and brown adipose tissue (BAT) sympathetic nerve activity (SNA) produced by leptin (10 μg ICV) but did not reduce the SNA responses to corticotrophin-releasing factor (CRF) or the melanocortin receptor agonist MTII. In mice with deletion of angiotensin II type-1a receptors (AT(1a)R(-/-)), increases in renal and BAT SNA induced by leptin (2 μg ICV) were impaired whereas SNA responses to MTII were preserved. Decreases in food intake and body weight with ICV leptin did not differ in AT(1a)R(-/-) vs. AT(1a)R(+/+) mice. ICV leptin in rats increased AT(1a)R and angiotensin-converting enzyme (ACE) mRNA in the subfornical organ and AT(1a)R mRNA in the arcuate nucleus, suggesting leptin-induced upregulation of the brain RAS in specific brain regions. To evaluate the role of de novo production of brain angiotensin II in SNA responses to leptin, we treated rats with captopril (12.5 μg ICV). Captopril attenuated leptin effects on renal and BAT SNA. In conclusion, these studies provide evidence that the brain RAS selectively facilitates renal and BAT sympathetic nerve responses to leptin while sparing effects on food intake.     

Influences of AT1 receptor blockade on tissue metabolism in obese men

ANG II applied to the interstitial space influences carbohydrate and lipid metabolism in a tissue-specific fashion. Thus endogenous ANG II may have a tonic effect on tissue metabolism that could be reversed with ANG II type 1 (AT1) receptor blockade, particularly during adrenergic stimulation. We studied 14 obese men. They were treated for 10 days with the AT1 receptor blocker irbesartan or with placebo in a double-blind and crossover fashion. At the end of each treatment period, we assessed skeletal muscle and adipose tissue metabolism using the microdialysis technique. The ethanol dilution technique was applied to follow changes in tissue blood flow. Measurements were obtained at baseline and during application of incremental isoproterenol concentrations through the microdialysis catheter. Blood pressure decreased from 133 +/- 3/84 +/- 3 to 128 +/- 3/79 +/- 2 mmHg for systolic and diastolic blood, respectively (P = 0.02 and 0.006, respectively) with AT1 receptor blockade. Isoproterenol perfusion caused a dose-dependent increase in dialysate glycerol in adipose tissue and in skeletal muscle. Irbesartan slightly reduced the isoproterenol-induced glycerol response in adipose tissue (P < 0.05 by ANOVA). Ethanol ratio, interstitial glucose supply, and lactate production in adipose tissue and skeletal muscle were similar with placebo and irbesartan. We conclude that AT1 receptor blockade in obese men does not reveal a major tonic ANG II effect on interstitial glucose supply, lipolysis, or glycolysis in skeletal muscle, either at rest or during beta-adrenergic stimulation. Endogeneous ANG II may slightly increase adipose tissue lipolysis. The mechanism may promote the redistribution of triglycerides from adipose tissue toward other organs.     

Angiotensin II infusion to the midgestation ovine fetus: effects on the fetal kidney

Renal and cardiovascular responses to an intravenous infusion of ANG II (1 microg/h) or saline for 3 days were examined in ovine fetuses at midgestation (75-85 days of gestation, term 150 days). ANG II caused an increase in fetal blood pressure (36 +/- 2 to 44 +/- 3 mmHg) and urine flow rate (8 +/- 2 to a maximum of 18 +/- 6 ml/h). Plasma renin concentrations decreased in ANG II-infused fetuses. Fetal fluids (amniotic and allantoic) did not differ in volume or composition between the groups when measured at postmortem. There was no difference in the expression levels of the mRNA for the angiotensin (AT(1) or AT(2)) receptors between the two groups when measured by an RNase protection assay. However, there was a significant decline in renin and AT(1) receptor gene expression when measured by a real-time polymerase chain reaction method. These results indicate that ANG II is diuretic and pressor when infused at midgestation. ANG II can feedback to decrease renin secretion by the fetal kidney, and this may occur by decreased renin gene expression.     

Intradermal angiotensin II administration attenuates the local cutaneous vasodilator heating response

The vasodilation response to local cutaneous heating is nitric oxide (NO) dependent and blunted in postural tachycardia but reversed by angiotensin II (ANG II) type 1 receptor (AT(1)R) blockade. We tested the hypothesis that a localized infusion of ANG II attenuates vasodilation to local heating in healthy volunteers. We heated the skin of a calf to 42 degrees C and measured local blood flow to assess the percentage of maximum cutaneous vascular conductance (%CVC(max)) in eight healthy volunteers aged 19.5-25.5 years. Initially, two experiments were performed; in one, Ringer solution was perfused in three catheters, the response to heating was measured, 2 microg/l losartan, 10 mM nitro-l-arginine (NLA), or NLA + losartan was added to perfusate, and the heat response was remeasured; in another, 10 microM ANG II was given, the heat response was measured, losartan, NLA, or NLA + losartan was added to ANG II, and the heat response was reassessed. The heat response decreased with ANG II, particularly the plateau phase (47 +/- 5 vs. 84 +/- 3 %CVC(max)). Losartan increased baseline conductance in both experiments (from 8 +/- 1 to 20 +/- 2 and 12 +/- 1 to 24 +/- 3). Losartan increased the ANG II response (83 +/- 4 vs. 91 +/- 6 in Ringer). NLA decreased both angiotensin and Ringer responses (31 +/- 4 vs. 43 +/- 3). NLA + losartan blunted the Ringer response (48 +/- 2), but the ANG II response (74 +/- 5) increased. In a second set of experiments, we used dose responses to ANG II (0.1 nM to 10 microM) with and without NLA + losartan to confirm graded responses. Sodium ascorbate (10 mM) restored the ANG II-blunted heating plateau. NO synthase and AT(1)R inhibition cause an NO-independent angiotensin-mediated vasodilation with local heating. ANG II mediates the AT(1)R blunting of local heating, which is not exclusively NO dependent, and is improved by antioxidant supplementation.     

Angiotensin peptides modulatory system: how is it implicated in the control of seizure susceptibility?

Accumulated studies support the concept that angiotensin peptides, ANG II, ANG III, and ANG IV act as neurotransmitters or neuromodulators in specific neuronal pathways in the brain stem, the hypothalamus, and the forebrain. They have been implicated in the regulation of several physiological processes, particularly in excitable brain structures that express high concentration of their receptors. With the help of pharmacological approaches it was shown that angiotensin peptides appear to be anticonvulsant in a variety of experimental seizure models. Thus, ANG II increases the threshold for pentylenetetrazol (PTZ)-, bicuculline-and picrotoxin-induced seizures in mice. It also attenuates the intensity of clonic seizures evoked by PTZ and 3-mercaptopropionic acid and is effective in the maximal electroshock test. Furthermore, ANG II, ANG III, and ANG IV protect against the clonic convulsions in the PTZ kindling model of epilepsy in mice. From the accumulated results it could be assumed that the angiotensin peptides appear to realize their effects acting directly on their receptors (AT(1), AT(2) and AT(4)) and through close interaction with different neurotransmitter/neuromodulator systems as dopamine (DA)-, gamma-aminobutyric acid (GABA)-and adenosine. This may contribute to a new potential use of angiotensin drugs either alone or in combination with other neuroprotective agents acting through the above mentioned systems, thus providing a more rational strategy for the treatment of neurodegenerative disorders such as epilepsy.     

Angiotensin II Type 1 Receptor Antagonist Attenuates Lacrimal Gland,Lung, and Liver Fibrosis in a Murine Model of Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGVHD), a serious complication following allogeneic HSCT (hematopoietic stem cell transplantation), is characterized by systemic fibrosis. The tissue renin-angiotensin system (RAS) is involved in the fibrotic pathogenesis, and an angiotensin II type 1 receptor (AT1R) antagonist can attenuate fibrosis. Tissue RAS is present in the lacrimal gland, lung, and liver, and is known to be involved in the fibrotic pathogenesis of the lung and liver. This study aimed to determine whether RAS is involved in fibrotic pathogenesis in the lacrimal gland and to assess the effect of an AT1R antagonist on preventing lacrimal gland, lung, and liver fibrosis in cGVHD model mice. We used the B10.D2→BALB/c (H-2^d) MHC-compatible, multiple minor histocompatibility antigen-mismatched model, which reflects clinical and pathological symptoms of human cGVHD. First, we examined the localization and expression of RAS components in the lacrimal glands using immunohistochemistry and quantitative real-time polymerase chain reaction (PCR). Next, we administered an AT1R antagonist (valsartan; 10 mg/kg) or angiotensin II type 2 receptor (AT2R) antagonist (PD123319; 10 mg/kg) intraperitoneally into cGVHD model mice and assessed the fibrotic change in the lacrimal gland, lung, and liver. We demonstrated that fibroblasts expressed angiotensin II, AT1R, and AT2R, and that the mRNA expression of angiotensinogen was greater in the lacrimal glands of cGVHD model mice than in controls generated by syngeneic-HSCT. The inhibition experiment revealed that fibrosis of the lacrimal gland, lung, and liver was suppressed in mice treated with the AT1R antagonist, but not the AT2R antagonist. We conclude that RAS is involved in fibrotic pathogenesis in the lacrimal gland and that AT1R antagonist has a therapeutic effect on lacrimal gland, lung, and liver fibrosis in cGVHD model mice. Our findings point to AT1R antagonist as a possible target for therapeutic intervention in cGVHD.     

Angiotensin II type 2 receptor-coupled nitric oxide production modulates free radical availability and voltage-gated Ca2+ currents in NTS neurons

The medial region of the nucleus tractus solitarius (mNTS) is a key brain stem site controlling cardiovascular function, wherein ANG II modulates neuronal L-type Ca(2+) currents via activation of ANG II type 1 receptors (AT(1)R) and production of reactive oxygen species (ROS). ANG II type 2 receptors (AT(2)R) induce production of nitric oxide (NO), which may interact with ROS and modulate AT(1)R signaling. We sought to determine whether AT(2)R-mediated NO production occurs in mNTS neurons and, if so, to elucidate the NO source and the functional interaction with AT(1)R-induced ROS or Ca(2+) influx. Electron microscopic (EM) immunolabeling showed that AT(2)R and neuronal NO synthase (nNOS) are coexpressed in neuronal somata and dendrites receiving synapses in the mNTS. In the presence of the AT(1)R antagonist losartan, ANG II increased NO production in isolated mNTS neurons, an effect blocked by the AT(2)R antagonist PD123319, but not the angiotensin (1-7) antagonist D-Ala. Studies in mNTS neurons of nNOS-null or endothelial NOS (eNOS)-null mice established nNOS as the source of NO. ANG II-induced ROS production was enhanced by PD123319, the NOS inhibitor N(G)-nitro-l-arginine (LNNA), or in nNOS-null mice. Moreover, in the presence of losartan, ANG II reduced voltage-gated L-type Ca(2+) current, an effect blocked by PD123319 or LNNA. We conclude that AT(2)R are closely associated and functionally coupled with nNOS in mNTS neurons. The resulting NO production antagonizes AT(1)R-mediated ROS and dampens L-type Ca(2+) currents. The ensuing signaling changes in the NTS may counteract the deleterious effects of AT(1)R on cardiovascular function.