Lens-specific regulation of the thioredoxin-1 gene, but not thioredoxin-2, upon in vivo photochemical oxidative stress in the Emory mouse

The angiotensin II (Ang II) type 1 receptor mediates various actions of Ang II, whereas the function of the type 2 (AT2) receptor is not well understood. In the mice lacking the gene encoding the AT2 receptor, the pressor response to Ang II was increased although the underlying mechanism is unknown. We tested the hypothesis that vasoconstrictor response is exaggerated in the AT2 receptor null mice. We measured hemodynamic parameters and evaluated systemic vascular resistance (SVR) in the anesthetized open-chest wild-type and AT2 receptor null mice. Ang II infusion caused dose-dependent increases in SVR in both strains, while the response was significantly higher at 0.5 microgram/kg Ang II in the AT2 receptor null mice (305 +/- 53% of baseline) than in the wild-type mice (179 +/- 27% of baseline). To investigate further the vascular contractility, we examined contraction of aortic rings in vitro. The contraction induced by 1 microM Ang II was increased in the AT2 receptor null mice compared with that in the wild-type mice (0.82 +/- 0.11 vs. 0.54 +/- 0.12 g). Ang II-induced contraction was still greater in the AT2 receptor null mice when calibrated by the maximum tension induced by 90 mM KCl. These data suggest that the AT2 receptor modulates vascular contractility, which may influence blood pressure.     

Genetic deletion of angiotensin AT2 receptor leads to increased cell numbers in different brain structures of mice

Angiotensin II (Ang II) is a potent vasoactive peptide and displays growth factor-like properties. Different high-affinity Ang II receptor subtypes (AT1A, AT1B and AT2) have been cloned. They are expressed in various brain structures. Additionally, it has been assumed that Mas could interact directly or indirectly with the renin-angiotensin system. The AT1 receptor mediates pressor and mitogenic effects of Ang II, whereas physiological function and signaling mechanisms of the AT2 receptor remain poorly understood. Recent reports have shown that Ang II could mediate apoptosis through AT2 receptors. Since the AT1A, AT2 and Mas knockout mice provide new tools for uncovering potential actions of Ang II, the cell number in different brain structures of male adult wild-type mice and mice deficient for AT1A, AT2 or Mas was evaluated to get more insight into the role of Ang II in central nervous system development. In nearly all investigated brain structures (cortex, hippocampus, amygdala, thalamus), the cell number was significantly higher in AT2-deficient mice in comparison to wild-type mice. To the contrary, in AT1A-deficient mice the cell number was significantly less than in controls in the lateral geniculate and the medial amygdaloid nucleus. However, cell numbers were not changed in Mas-knockout mice compared to their wild-types. These results show the contrary effects of both angiotensin receptors on cell growth and represent the first demonstration of their action on neuronal cell development evidenced in the adult mouse brain.     

Renal nuclear angiotensin II receptors in normal and hypertensive rats

Accumulation of Angiotensin II (Ang II) in the kidneys of hypertensive rats infused chronically with Ang II occurs by AT1 receptor mediated internalization of Ang II, which may interact with intracellular targets, including nuclear binding sites. The aims of this study were to determine if kidney cell nuclei have specific Ang II binding sites and if chronic infusion of Ang II (70 ng/min; n=9) influences the nuclear Ang II binding capacity. Kidneys were harvested from control and Ang II infused rats and the renal cortexes were homogenized to obtain crude membrane preparations and nuclear fractions. Ang II binding sites were measured with a single point assay by incubating each fraction with 10 nM 125I-Sar-Ile-Ang II in the absence (total binding sites) or presence of either 2.5 M Sar-Leu-Ang II or 25 microM losartan to detect specific AT or AT1 binding sites. Both fractions exhibited specific Ang II binding sites that were displaced by both saralasin and losartan. In control rats, crude membrane preparations had 792 +/- 218 and the nuclear fraction had 543 +/- 222 fmol/mg protein AT1 receptors. AT1 receptor levels in membrane (885 +/- 170 fmol/mg protein) and nuclear fractions (610 +/- 198 fmol/mg protein) were not significantly different in Ang II infused rats. These data support the presence of nuclear Ang II receptors predominantly of the AT1 subtype in renal cells. Chronic Ang II infusion did not alter overall Ang II receptor densities.     

Deletion of angiotensin II type 1 receptor gene or scavenge of superoxide prevents chronic alcohol-induced aortic damage and remodelling

To investigate whether chronic alcohol consumption induces vascular injury via angiotensin II (Ang II) type 1 (AT1) receptor‐dependent superoxide generation, male transgenic mice with knockout of AT1 gene (AT1‐KO) and age‐matched wild‐type (WT) C57BL/6 mice were pair‐fed a modified Lieber‐DeCarli alcohol or isocaloric maltose dextrin control liquid diet for 2 months. Ethanol content (%, W/V) in the diet was 4.8 (34% of total calories) at initiation, and gradually increased up to 5.4 (38% of total calories). For some WT mice with and without alcohol treatment, superoxide dismutase mimetic (MnTMPyP) was given simultaneously by intraperitoneal injection at 5 mg/kg body weight daily for 2 months. At the end of studies, aortas were harvested for histopathological and immunohistochemical examination. Significant increases in the wall thickness and structural disarrangement of aorta were found in alcohol group, along with significant increases in aortic oxidative and/or nitrosative damage, expressions of NADPH oxidases (NOXs), inflammatory response, cell death and proliferation, and remodelling (fibrosis). However, these pathological changes were completely attenuated in alcohol‐treated AT1‐KO mice or in alcohol‐treated WT mice that were also simultaneously treated with MnTMPyP for 2 months. These results suggest that chronic alcohol consumption may activate NOX via Ang II/AT1 receptor, to generate superoxide and associated peroxynitrite that in turn causes aortic nitrosative damage, inflammation, cell death and proliferation, and remodelling. Therefore, blocking Ang II/AT1 system or scavenging superoxide may become a potential preventive and/therapeutic approach to alcoholic vascular damage.     

Role of AT2 receptor in the brain in regulation of blood pressure and water intake

The effects of intracerebroventricular (ICV) injection of angiotensin II (ANG II) on blood pressure and water intake were examined with the use of ANG II receptor-deficient mice. ICV injection of ANG II increased systolic blood pressure in a dose-dependent manner in wild-type (WT) mice and ANG type 2 AT(2) receptor null (knockout) (AT(2)KO) mice; however, this increase was significantly greater in AT(2)KO mice than in WT mice. The pressor response to a central injection of ANG II in WT mice was inhibited by ICV preinjection of the selective AT(1) receptor blocker valsartan but exaggerated by the AT(2) receptor blocker PD-123319. ICV injection of ANG II also increased water intake. It was partly but significantly suppressed both in AT(2)KO and AT(1)aKO mice. Water intake in AT(2)/AT(1)aKO mice did not respond to ICV injection of ANG II. Both valsartan and PD-123319 partly inhibited water intake in WT mice. These results indicate an antagonistic action between central AT(1)a and AT(2) receptors in the regulation of blood pressure, but they act synergistically in the regulation of water intake induced by ANG II.     

ACE2 deficiency enhances angiotensin II-mediated aortic profilin-1 expression, inflammation and peroxynitrite production

Inflammation and oxidative stress play a crucial role in angiotensin (Ang) II-mediated vascular injury. Angiotensin-converting enzyme 2 (ACE2) has recently been identified as a specific Ang II-degrading enzyme but its role in vascular biology remains elusive. We hypothesized that loss of ACE2 would facilitate Ang II-mediated vascular inflammation and peroxynitrite production. 10-week wildtype (WT, Ace2(+/y)) and ACE2 knockout (ACE2KO, Ace2(-/y)) mice received with mini-osmotic pumps with Ang II (1.5 mg.kg?1.d?1) or saline for 2 weeks. Aortic ACE2 protein was obviously reduced in WT mice in response to Ang II related to increases in profilin-1 protein and plasma levels of Ang II and Ang-(1-7). Loss of ACE2 resulted in greater increases in Ang II-induced mRNA expressions of inflammatory cytokines monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-1β, and IL-6 without affecting tumor necrosis factor-α in aortas of ACE2KO mice. Furthermore, ACE2 deficiency led to greater increases in Ang II-mediated profilin-1 expression, NADPH oxidase activity, and superoxide and peroxynitrite production in the aortas of ACE2KO mice associated with enhanced phosphorylated levels of Akt, p70S6 kinase, extracellular signal-regulated kinases (ERK1/2) and endothelial nitric oxide synthase (eNOS). Interestingly, daily treatment with AT1 receptor blocker irbesartan (50 mg/kg) significantly prevented Ang II-mediated aortic profilin-1 expression, inflammation, and peroxynitrite production in WT mice with enhanced ACE2 levels and the suppression of the Akt-ERK-eNOS signaling pathways. Our findings reveal that ACE2 deficiency worsens Ang II-mediated aortic inflammation and peroxynitrite production associated with the augmentation of profilin-1 expression and the activation of the Akt-ERK-eNOS signaling, suggesting potential therapeutic approaches by enhancing ACE2 action for patients with vascular diseases.     

Selective down-regulation of AT2 receptors in uterine arteries from pregnant ewes given 24-h intravenous infusions of angiotensin II

Previously, we showed that uterine arteries from late gestation pregnant ewes infused intravenously with angiotensin II (Ang II) for 24 h, displayed heightened responsiveness to Ang II in vitro. Furthermore, we found that a small population of ewes with a "preeclampsia-like" disorder also displayed this. Therefore, we have investigated the density and affinity of Ang II receptor subtypes in the uterine arteries from these groups. Ang II receptor binding was measured using 125I [Sar1Ile8] Ang II. Proportions of AT1 and AT2 receptors were determined by inhibiting 125I [Sar1Ile8] Ang II with losartan (AT1 antagonist) or PD 123319 (AT2 antagonist). Uterine arteries from 24-h Ang II-infused ewes had a lower proportion of AT2 receptors (56.2+/-2.3%) than control (saline-infused) ewes (84.1+/-1.0%; P<0.05). The density of AT2 receptors was reduced (P<0.05) while the density of AT1 receptors was not different. Thus, 24-h infusions of Ang II selectively down-regulated AT2 receptors in the uterine artery, resulting in heightened Ang II reactivity. By contrast, the binding properties of Ang II receptor subtypes in uterine arteries from ewes with the "preeclampsia-like" disorder were not different from control ewes.     

Angiotensin II and III suppress food intake via angiotensin AT(2) receptor and prostaglandin EP(4) receptor in mice

Intracerebroventricularly administered angiotensin (Ang) II and III dose-dependently suppressed food intake in mice and their anorexigenic activities were inhibited by AT(2) receptor-selective antagonist. Ang II did not suppress food intake in AT(2) receptor-knockout mice, while it did significantly in wild-type and AT(1) receptor-knockout mice. The suppression of food intake in AT(1) receptor-knockout mice was smaller than that in wild-type. The anorexigenic activities of Ang II and III were also blocked by a selective antagonist for prostaglandin EP(4) receptor. Taken together, centrally administered Ang II and III may decrease food intake through AT(2) receptor with partial involvement of AT(1) receptor, followed by EP(4) receptor activation, which is a novel pathway regulating food intake.     

ANG II type 2 receptor regulates smooth muscle growth and force generation in late fetal mouse development

Although evidence from culture studies implicates the angiotensin II (ANG II) type 2 receptor (AT(2)R) in the regulation of growth and differentiation of arterial smooth muscle (SM) cells (SMC), the lack of its expression in adult arteries has precluded direct investigation of its role in vivo. The goal of the present study was to determine the role of AT(2)R in the control of fetal SMC growth, contractility, and differentiation during vascular development. Determination of isometric tension in fetal aortas showed potentiated ANG II-induced contraction by treatment with the selective AT(2)R antagonist PD-123319, demonstrating the presence of functional AT(2)Rs that mediate reduced force development in vascular SMC. In direct contrast to numerous cell culture studies, proliferation indexes were decreased rather than increased in aortic SMC of fetal homozygous AT(2)R knockout compared with wild-type or heterozygous knockout mice. Experiments using SMC tissues from heterozygous female AT(2)R knockout mice, which are naturally occurring chimeras for AT(2)R expression, showed that AT(2)R mRNA expression was exactly 50% of that of wild type. This indicated that loss of AT(2)R expression did not confer a selective advantage or disadvantage for SMC lineage determination and expansion. Real time RT-PCR analyses showed no significant difference in expression of SM-alpha-actin, SM myosin heavy chain, and myocardin in various SM tissues from all three genotypes, suggesting that knockout of AT(2)R had no effect on subsequent SMC differentiation. Taken together, results indicate that functional AT(2)R are expressed in fetal aorta and mediate reduced force development but do not significantly contribute to regulation of SMC differentiation.     

Upregulation of ERK1/2-eNOS via AT2 Receptors Decreases the Contractile Response to Angiotensin II in Resistance Mesenteric Arteries from Obese Rats

It has been clearly established that mitogen-activated protein kinases (MAPKS) are important mediators of angiotensin II (Ang II) signaling via AT1 receptors in the vasculature. However, evidence for a role of these kinases in changes of Ang II-induced vasoconstriction in obesity is still lacking. Here we sought to determine whether vascular MAPKs are differentially activated by Ang II in obese animals. The role of AT2 receptors was also evaluated. Male monosodium glutamate-induced obese (obese) and non-obese Wistar rats (control) were used. The circulating concentrations of Ang I and Ang II, determined by HPLC, were increased in obese rats. Ang II-induced isometric contraction was decreased in endothelium-intact resistance mesenteric arteries from obese compared with control rats and exhibited a retarded AT1 receptor antagonist response. Blocking of AT2 receptors and inhibition of either endothelial nitric oxide synthase (eNOS) or extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) restored Ang II-induced contraction in obese rats. Western blot analysis revealed increased protein expression of AT2 receptors in arteries from obese rats. Basal and Ang II-induced ERK1/2 phosphorylation was also increased in obese rats. Blockade of either AT1 or AT2 receptors corrected the increased ERK1/2 phosphorylation in arteries from obese rats to levels observed in control preparations. Phosphorylation of eNOS was increased in obese rats. Incubation with the ERK1/2 inhibitor before Ang II stimulation did not affect eNOS phosphorylation in control rats; however, it corrected the increased phosphorylation of eNOS in obese rats. These results clearly demonstrate that enhanced AT2 receptor and ERK1/2-induced, NO-mediated vasodilation reduces Ang II-induced contraction in an endothelium-dependent manner in obese rats.     

A proteomic analysis of aorta from spontaneously hypertensive rat: RhoGDI alpha upregulation by angiotensin II via AT(1) receptor

Arteries undergo remodeling as a consequence of increased wall stress during hypertension. However, the molecular mechanisms of the vascular remodeling are largely unknown. Proteomics is a powerful tool to screen for differentially expressed proteins, but little effort was made on vascular disease research, especially on hypertension. In the present study, the differentially expressed proteins in aortas from 18-week-old spontaneously hypertensive rats (SHR) and their normotensive counterpart, Wistar Kyoto rats (WKY), were examined by two-dimensional electrophoresis (2-DE). We found 50 proteins to be differentially expressed, among which 27 were highly or only expressed in SHR and 23 in WKY. Using matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF-MS) and online data search, nine proteins, including Rho GDP dissociation inhibitor alpha (RhoGDIalpha), were identified with high confidence. Further, the upregulation of RhoGDIalpha was verified at both mRNA and protein level in SHR. In addition, when cultured vascular smooth muscle cells (VSMCs) from aortas of SHR and WKY were treated with angiotensin II (Ang II) and antagonist of angiotensin II type I (AT(1)) receptor, L158809, respectively, RhoGDIalpha was upregulated by Ang II and downregulated by L158809 in VSMCs of SHR. These results demonstrate that vascular remodeling results in significant alterations in the protein expression profile of the aorta during hypertension and suggest that the upregulation of RhoGDIalpha in hypertension is induced by Ang II via AT(1) receptor.     

Endogenous angiotensin II suppresses stretch-induced ANP secretion via AT1 receptor pathway

Angiotensin II (Ang II) is released by stretch of cardiac myocytes and has paracrine and autocrine effects on cardiac myocytes and fibroblasts. However, the direct effect of Ang II on the secretion of atrial natriuretic peptide (ANP) is unclear. The aim of the present study is to test whether Ang II affects stretch-induced ANP secretion. The isolated perfused beating atria were used from control and two-kidney one-clip hypertensive (2K1C) rats. The volume load was achieved by elevating the height of outflow catheter connected with isolated atria from 5 cmH₂O to 7.5 cmH₂O. Atrial stretch by volume load caused increases in atrial contractility by 60% and in ANP secretion by 100%. Ang II suppressed stretch-induced ANP secretion and tended to increase atrial contractility whereas losartan stimulated stretch-induced ANP secretion. Neither PD123319 nor A779 had direct effect on stretch-induced ANP secretion. The suppressive effect of Ang II on stretch-induced ANP secretion was blocked by the pretreatment of losartan but not by the pretreatment of PD123319 or A779. In hypertrophied atria from 2K1C rats, stretch-induced ANP concentration attenuated and atrial contractility augmented. The response of stretch-induced ANP secretion to Ang II and losartan augmented. The expression of AT1 receptor protein and mRNA increased but AT2 and Mas receptor mRNA did not change in 2K1C rat atria. Therefore, we suggest that Ang II generated endogenously by atrial stretch suppresses stretch-induced ANP secretion through the AT1 receptor and alteration of Ang II effect in 2K1C rat may be due to upregulation of AT1 receptor.     

Pharmacological characterization of angiotensin II binding sites in the canine pancreas.

High affinity 125I-angiotensin II (Ang II) binding sites were characterized in the canine pancreas. Total binding increased with protein concentration and equilibrium was reached within 60-90 min at 22 degrees C. Specific binding was saturable and averaged 70% of total. Scatchard analysis of binding yielded a KD of 0.48 +/- 0.18 nM with a Bmax of 32.8 +/- 6.5 fmol/mg protein (mean +/- SEM, n = 6). The addition of the reducing agent dithiothreitol increased specific binding two-fold. The rank order of displacement of 125I-Ang II binding by native angiotensin peptides was Ang II greater than or equal to Ang III greater than AngI greater than Ang(1-7) much greater than Ang(1-6). The use of the specific Ang II antagonists CGP 42112A, PD 123177, and DuP 753 revealed that the pancreas expresses two receptor subtypes. The majority of Ang II binding sites in the pancreas could be classified as type 2 (AT2), although type 1 (AT1) sites were also detected. In vitro autoradiography revealed binding sites localized over islet cells, acinar and duct cells, as well as the pancreatic vasculature. In addition, the autoradiographic studies confirmed the predominance of the AT2 receptor subtype throughout the pancreas.     

Enhanced central response to dehydration in mice lacking angiotensin AT(1a) receptors

The objective was to determine the central nervous system (CNS) responses to dehydration (c-Fos and vasopressin mRNA) in mice lacking the ANG AT(1a) receptor [ANG AT(1a) knockout (KO)]. Control and AT(1a) KO mice were dehydrated for 24 or 48 h. Baseline plasma vasopressin (VP) was not different between the groups; however, the response to dehydration was attenuated in AT(1a) KO (24 +/- 11 vs. 10.6 +/- 2.7 pg/ml). Dehydration produced similar increases in plasma osmolality and depletion of posterior pituitary VP content. Neuronal activation was observed as increases in c-Fos protein and VP mRNA. The supraoptic responses were not different between groups. In the paraventricular nucleus (PVN), c-Fos-positive neurons (57.4 +/- 10.7 vs. 98.4 +/- 7.4 c-Fos cells/PVN, control vs. AT(1a) KO) and VP mRNA levels (1.0 +/- 0.1 vs. 1.4 +/- 0.1 microCi, control vs. AT(1a) KO) were increased with greater responses in AT(1a) KO. A comparison of 1- to 2-day water deprivation showed that plasma VP, brain c-Fos, and VP mRNA returned toward control on day 2, although plasma osmolality remained high. Data demonstrate that AT(1a) KO mice show a dichotomous response to dehydration, reduced for plasma VP and enhanced for PVN c-Fos protein and VP mRNA. The results illustrate the importance of ANG AT(1a) receptors in the regulation of osmotic and endocrine balance.     

In situ demonstration of angiotensin-dependent and independent pathways for hyperaldosteronism during chronic extracellular fluid volume depletion.

In wild-type mice, 2-wk administration of losartan, an angiotensin (Ang) II type 1 (AT1) receptor antagonist, along with dietary sodium restriction, resulted in an elevation of plasma aldosterone greater than that seen with sodium restriction alone (2.75 +/- 0.35 vs. 1.38 +/- 0.16 ng/ml, P < 0.01). Plasma potassium increased in sodium-restricted, losartan-treated mice (6.0 +/- 0.2 mEq/liter), while potassium remained unchanged in mice with sodium restriction alone. To study the effect of Ang II on glomerulosa cells that may operate independently of plasma potassium in situ, we used chimeric mice made of cells with or without the intact AT1A gene (Agtr1a). When animals were fed a normal diet or chronically infused with Ang II, the aldosterone synthase mRNA was detectable only in Agtr1a+/+ but not Agtr1a-/- zona glomerulosa cells. After 2 wk of sodium restriction, plasma aldosterone increased (1.51 +/- 0.27 ng/ml) and potassium remained on average at 4.5 +/- 0.2 mEq/liter, with aldosterone synthase mRNA expressed intensively in Agtr1a+/+, but not detectable in Agtr1a-/- cells. Simultaneous sodium restriction and losartan treatment caused increases in plasma potassium (5.5 +/- 0.1 mEq/liter) and aldosterone (1.84 +/- 0.38 ng/ml), with both Agtr1a-/- and Agtr1a+/+ cells intensively expressing aldosterone synthase mRNA. Thus, aldosterone production is regulated by Ang II in the adrenal gland during chronic alterations in extracellular fluid volume when plasma potassium is maintained within the normal range. In the light of a previous observation that dietary potassium restriction superimposed on sodium restriction abolished secondary hyperaldosteronism in angiotensinogen null-mutant mice, the present findings demonstrate that when the renin-Ang system is compromised, plasma potassium acts as an effective alternative mechanism for the volume homeostasis through its capacity to induce hyperaldosteronism.     

Expression of angiotensin type 1 and 2 receptors in brain after transient middle cerebral artery occlusion in rats

Angiotensin II (Ang II) type 2 receptors (AT2Rs) have been associated with apoptosis. We hypothesized that AT2Rs are increased in stroke and may contribute effects of stroke to the brain. To test this, we have examined the expression of Ang II type 1 receptor (AT1R), AT2R and Ang II levels in the brain 24 h after transient middle cerebral artery occlusion (MCAO). The densities of AT1R and AT2R were measured by quantitative autoradiography (n=6). The levels of Ang II were measured by radioimmunoassay (RIA) (n=6) and by immunohistochemistry (n=3). AT1R levels on autoradiography showed a significant decrease (0.87±0.06 to 1.39±0.07 fmol/mg, p<0.01) in the ventral cortex of the stroke side compared to the cortices of non-stroke (NS) rats (n=4). There was no significant difference on ATIR in the contralateral verbal cortex of the stroke rats compared to NS control. In contrast, levels of AT2R in the ventral cortex of both the stroke and the contralateral sides were significantly increased (0.77±0.06, p<0.05 and 0.91±0.05, p<0.01 compared to 0.60±0.03 fmol/mg tissue, respectively). RIA showed that Ang II in the ventral cortex of both the stroke and the contralateral sides were significantly increased (241.63±47.72, p<0.01 and 165.51±42.59, p<0.05 compared to 76.80±4.10 pg/g tissue, respectively). Also, Ang II in the hypothalamus was significantly increased (179.50±17.49 to 118.50±6.65 pg/g tissue, p<0.05). Immunohistochemistry confirmed the increase of Ang II. These results demonstrate that brain Ang II and AT2Rs are increased whereas AT1Rs are decreased after transient MCAO in rats. We conclude that in stroke, Ang II and AT2R are activated and may contribute neural effects to brain ischemia.