AT1B Receptor Research Articles

AT1b receptors contribute to regional disparities in angiotensin II mediated aortic remodelling in mice.

The renin-angiotensin system plays a key role in regulating blood pressure, which has motivated many investigations of associated mouse models of hypertensive arterial remodelling. Such studies typically focus on histological and cell biological changes, not wall mechanics. This study explores tissue-level ramifications of chronic angiotensin II infusion in wild-type (WT) and type 1b angiotensin II (AngII) receptor null (Agtr1b -/-) mice. Biaxial biomechanical and immunohistological changes were quantified and compared in the thoracic and abdominal aorta in these mice following 14 and 28 days of angiotensin II infusion. Preliminary results showed that changes were largely independent of sex. Associated thickening and stiffening of the aortic wall in male mice differed significantly between thoracic and abdominal regions and between genotypes. Notwithstanding multiple biomechanical changes in both WT and Agtr1b -/- mice, AngII infusion caused distinctive wall thickening and inflammation in the descending thoracic aorta of WT, but not Agtr1b -/-, mice. Our study underscores the importance of exploring differential roles of receptor-dependent angiotensin II signalling along the aorta and its influence on distinct cell types involved in regional histomechanical remodelling. Disrupting the AT1b receptor primarily affected inflammatory cell responses and smooth muscle contractility, suggesting potential therapeutic targets.

Enalapril Diminishes the Diabetes-Induced Changes in Intestinal Morphology, Intestinal RAS and Blood SCFA Concentration in Rats.

Evidence suggests that microbiota-derived metabolites, including short-chain fatty acids (SCFAs) and trimethylamine-oxide (TMAO), affect the course of diabetic multiorgan pathology. We hypothesized that diabetes activates the intestinal renin–angiotensin system (RAS), contributing to gut pathology. Twelve-week-old male rats were divided into three groups: controls, diabetic (streptozotocin-induced) and diabetic treated with enalapril. Histological examination and RT-qPCR were performed to evaluate morphology and RAS expression in the jejunum and the colon. SCFA and TMAO concentrations in stools, portal and systemic blood were evaluated. In comparison to the controls, the diabetic rats showed hyperplastic changes in jejunal and colonic mucosa, increased plasma SCFA, and slightly increased plasma TMAO. The size of the changes was smaller in enalapril-treated rats. Diabetic rats had a lower expression of Mas receptor (MasR) and angiotensinogen in the jejunum whereas, in the colon, the expression of MasR and renin was greater in diabetic rats. Enalapril-treated rats had a lower expression of MasR in the colon. The expression of AT1a, AT1b, and AT2 receptors was similar between groups. In conclusion, diabetes produces morphological changes in the intestines, increases plasma SCFA, and alters the expression of renin and MasR. These alterations were reduced in enalapril-treated rats. Future studies need to evaluate the clinical significance of intestinal pathology in diabetes.

Recharacterizing the Murine Chronic Angiotensin II Infusion Model of Cardiovascular and Renal Injury

The chronic angiotensin II (AngII) infusion model is one of the most commonly used to study cardiovascular and kidney injury. However, the dosages of AngII used to induce cardiovascular or renal injury vary significantly in the literature. Therefore, we set out to determine the dosages of angiotensin II needed to produce renal injury and hypertension in mice. All experiments were performed in accordance with protocols approved by the Liberty University IACUC and conform to the FASEB standards for the use of animals in research and education. Male C57Bl/6J mice (~20 grams body weight) were purchased from The Jackson Laboratory and acclimated to our environment for one week. Mice were then assigned to receive 0, 400, 800, or 1200 ng AngII/kg BW*min for four weeks via osmotic minipumps (subcutaneous). Blood pressure (tail‐cuff) was recorded each week of the study. At baseline, 2 and 4 weeks, renal blood flow and renal hemodynamics were estimated using contrast‐enhanced ultrasonography. After 4 weeks, mice were placed in metabolic cages for the quantification of water intake and urinary output. Urine was stored for determination of albumin concentration using a commercially available ELISA. Mice were then euthanized and kidney tissues collected to measure the mRNA expression of several genes involved in the intrarenal renin‐angiotensin system (RAS) (renin, angiotensinogen, angiotensin converting enzyme (ACE), ACE2, angiotensin II‐receptor 1a (AT1a), and AT1b receptor) and tissue inflammation and remodeling [CCL5, α‐smooth muscle actin (αSMA), collagen 1, collagen 3, and tumor growth factor β (TGFβ)]. All data were analyzed using general linear models in SPSS. Mice administered the 800 dose had the greatest and most consistent increase in systolic blood pressure (147 ± 8mmHg) as compared to mice administered the saline (114 ± 9, P=0.004) and the 400 dose (121 ± 11mmHg, P=0.01). Mice administered the 1200 dose tended (P=0.08) to have a greater total renal blood flow throughout the study, with a significant increase (P=0.047) in cortical blood flow as compared to mice administered saline. Despite the modest changes in estimated renal blood flow, mice administered the 800 and 1200 doses of AngII had similar, yet significantly greater (P≤0.001) water intake and urinary output as compared to mice treated with saline or the 400 dose. Similarly, mice with the 800 and 1200 doses had significantly greater evidence of tissue damage evident by increased albuminuria (P≤0.05) and increased renal gene expression of CCL5, TGFβ, collagen 1, and collagen 3. There was also an effect on the intrarenal RAS, with mice administered the 800 and 1200 doses having significant (P<0.05) increase of renin, angiotensinogen, ACE1, ACE2, AT1a, and AT1b expression. In our hands, it appears that a dosage of greater than 400 ng/kg*min is needed to elicit the pressor effect of AngII and its associated effects on kidney structure and function. Our data also suggests that doses greater than 800 do not result in a further increase in blood pressure or several markers of kidney health. Taken together, more studies are needed to better refine this popular animal model and identify potential causes of variability throughout the literature.Support or Funding InformationProject was supported by LUCOM Intramural Grant #2018‐01.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interaction between angiotensin II (ANGII) receptor 1A (AT1AR) and adenosine receptors in the control of vascular function in mouse aorta and mesenteric arteries

ANG II, a systemic vasoconstrictor and adenosine through its receptors (ARs) regulate vascular function. Recently, we have shown that acute ANG II stimulation enhanced A1AR dependent CYP4A mediated contraction in mesenteric arteries (MA). In addition, A2BAR dependent and partly KATP channel mediated relaxation was also reduced in MAs after ANG II stimulation (Yadav et al., 2015). Thus, suggesting a possible interaction between ARs and AT1R in the control of vascular function. The present study is designed to explore interaction between AR and AT1R. There are two types of AT1 receptors; AT1A is ubiquitously expressed where as AT1B expressed in aorta, resistance arteries, adrenal glands, pituitary, and hypothalamus. Considering ubiquitous tissue expression of AT1A receptors, we performed muscle tension studies in isolated MA and aorta from WT and AT1A KO mice. We used DMT wire myograph for MAs and organ bath for aortas to perform concentration response curves (CRCs) to various agonists. Our data showed a significant increase in contraction to A1AR agonist CCPA in MAs isolated from AT1A KO. The contraction observed at 10 and 100 nM of CCPA was 24.8 ± 13.7 (% PE) and 29.7 ± 14.8 in AT1A KO mice which was significantly higher compared to −4.8 ± 3.7 and 0.5 ± 3.2 observed in WT mice. NECA (non‐selective AR agonist) induced CRC was similar in WT and AT1A KO MAs. Pinacidil (KATP channel opener) and C21 (AT2 agonist) produced comparable CRCs in WT and AT1A KO MAs. In addition, comparable ACh, PE and KCl‐induced responses were present in MAs. In aorta, CRCs to CCPA, NECA, C21 and Pinacidil were comparable between WT and AT1A KO. However, PE‐induced contraction was significantly reduced in aorta of AT1A KO mice. ACh induced relaxation in aorta was 54.1 ± 4.4 (% PE) in AT1A KO compared to WT, 40.7±4.6 (p<0.05). Comparable contraction to ANG II in MAs suggests the involvement of AT1B receptors which needs further investigation. In conclusion, our data suggest an interaction between ARs and AT1A receptor in the control of vascular function.Support or Funding InformationSupported by HL 027339

Mechanosensitive Gq/11 Protein-Coupled Receptors Mediate Myogenic Vasoconstriction.

Myogenic vasoconstriction (Bayliss effect) is mediated by vascular smooth muscle cells (VSMCs) of small resistance arteries sensing mechanical forces. During the last three decades, several proteins have been proposed as VSMC mechanosensors. Our previous studies highlighted agonist-independent mechanical activation of Gq/11 protein-coupled receptors (Gq/11 PCRs) in VSMCs of resistance arteries. In particular, angiotensin II AT1 receptors (AT1 Rs) emerged as mechanosensors mediating myogenic tone. Moreover, we found that the AT1B receptor isoform was more mechanosensitive than the AT1A receptor. Interestingly, cysteinyl leukotriene 1 receptors (CysLT1 Rs) were up-regulated in AT1 R-deficient arteries as an essential backup strategy to compensate for the loss of vasoconstrictor receptors. Up-regulation of CysLT1 Rs resulted in increased myogenic tone at low intraluminal pressures resulting in hyperactivity of AT1 R-deficient arteries. Only at high intraluminal pressures myogenic tone was reduced, thus reflecting the loss of AT1 Rs. Further, CysLT1 Rs were involved in myogenic vasoconstriction of wild-type arteries. Simultaneous blockade of AT1 Rs and CysLT1 Rs in wild-type arteries caused reduction in myogenic tone of more than 60% comparable to the application of the selective Gq/11 -protein inhibitor YM-254890. Our findings suggest that AT1 Rs and CysLT1 Rs are crucial mechanosensors in resistance arteries mediating 60% of myogenic vasoconstriction via the Gq/11 -protein pathway without involvement of endogenous agonists.

Erythropoiesis and Blood Pressure Are Regulated via AT1 Receptor by Distinctive Pathways.

The renin–angiotensin system (RAS) plays a central role in blood pressure regulation. Although clinical and experimental studies have suggested that inhibition of RAS is associated with progression of anemia, little evidence is available to support this claim. Here we report that knockout mice that lack angiotensin II, including angiotensinogen and renin knockout mice, exhibit anemia. The anemia of angiotensinogen knockout mice was rescued by angiotensin II infusion, and rescue was completely blocked by simultaneous administration of AT1 receptor blocker. To genetically determine the responsible receptor subtype, we examined AT1a, AT1b, and AT2 knockout mice, but did not observe anemia in any of them. To investigate whether pharmacological AT1 receptor inhibition recapitulates the anemic phenotype, we administered AT1 receptor antagonist in hypotensive AT1a receptor knockout mice to inhibit the remaining AT1b receptor. In these animals, hematocrit levels barely decreased, but blood pressure further decreased to the level observed in angiotensinogen knockout mice. We then generated AT1a and AT1b double-knockout mice to completely ablate the AT1 receptors; the mice finally exhibited the anemic phenotype. These results provide clear evidence that although erythropoiesis and blood pressure are negatively controlled through the AT1 receptor inhibition in vivo, the pathways involved are complex and distinct, because erythropoiesis is more resistant to AT1 receptor inhibition than blood pressure control.

Vascular Type 1A Angiotensin II Receptors Control BP by Regulating Renal Blood Flow and Urinary Sodium Excretion

Inappropriate activation of the type 1A angiotensin (AT1A) receptor contributes to the pathogenesis of hypertension and its associated complications. To define the role for actions of vascular AT1A receptors in BP regulation and hypertension pathogenesis, we generated mice with cell-specific deletion of AT1A receptors in smooth muscle cells (SMKO mice) using Loxp technology and Cre transgenes with robust expression in both conductance and resistance arteries. We found that elimination of AT1A receptors from vascular smooth muscle cells (VSMCs) caused a modest (approximately 7 mmHg) yet significant reduction in baseline BP and exaggerated sodium sensitivity in mice. Additionally, the severity of angiotensin II (Ang II)-dependent hypertension was dramatically attenuated in SMKO mice, and this protection against hypertension was associated with enhanced urinary excretion of sodium. Despite the lower BP, acute vasoconstrictor responses to Ang II in the systemic vasculature were largely preserved (approximately 80% of control levels) in SMKO mice because of exaggerated activity of the sympathetic nervous system rather than residual actions of AT1B receptors. In contrast, Ang II-dependent responses in the renal circulation were almost completely eliminated in SMKO mice (approximately 5%-10% of control levels). These findings suggest that direct actions of AT1A receptors in VSMCs are essential for regulation of renal blood flow by Ang II and highlight the capacity of Ang II-dependent vascular responses in the kidney to effect natriuresis and BP control.

AT1a Receptor Upregulation in the MnPO Following CIH Leads to Sustained Hypertension

Angiotensin type 1 (AT1) receptors play a central role in many forms of neurogenic hypertension. Two distinct subtypes, At1a and AT1b, exist for AT1 receptors. Activation of the AT1a subtype is characterized by increased sympathetic nerve activity and hypertension. The Median Preoptic Nucleus (MnPO) expresses both subtypes of the angiotensin receptor and our lab has previously demonstrated that AT1a message is increased in MnPO brain punches following CIH. This study sought to determine if changes in expression of the angiotensin receptor subtypes play a role in the sustained hypertension that develops from chronic intermittent hypoxia (CIH). We hypothesized that AT1a receptor expression increased and AT1b receptor expression remained unchanged resulting in the sustained elevation in blood pressure from CIH. Rats were microinjected in MnPO with an adeno‐associated virus loaded with a shRNA construct to prevent AT1a expression (shAT1a) or a scramble construct (shSCR). After recovery, rats were exposed to a 7 day CIH or normoxic protocol. Laser Capture Microdissection and qRTPCR was performed to determine the receptor subtype expression in MnPO with ribosomal subunit S18 as the control. shSCR rats exposed to CIH had significantly more AT1a RNA than their normoxic counterparts (P<.05), but their expression of AT1b remained constant. shAT1a CIH rats did not exhibit the sustained component of hypertension found in shSCR CIH animals and their MnPO AT1a message was reduced compared to control. Overall this indicates that the pathophysiological blood pressure increase during CIH is in part mediated by increases in AT1a receptor subtype expression in the MnPO.

Cysteinyl leukotriene 1 receptors as novel mechanosensors mediating myogenic tone together with angiotensin II type 1 receptors-brief report.

Myogenic vasoconstriction is mediated by vascular smooth muscle cells of resistance arteries sensing mechanical stretch. Angiotensin II AT1 receptors and in particular AT1BRs in murine vascular smooth muscle cells have been characterized as mechanosensors that cannot fully account for myogenic vasoconstriction observed. Therefore, we aimed at uncovering novel vascular mechanosensors by expression profiling and functional characterization of candidate proteins. Analyzing myogenic tone of isolated murine mesenteric arteries of AT1A and AT1B receptor double gene-deficient (AT1A/1B (-/-)) mice ex vivo, we observed a decreased myogenic tone at high intraluminal pressures and an unexpected hyper-reactivity at low intraluminal pressures because of upregulation of cysteinyl leukotriene 1 receptors (CysLT1Rs). Pharmacological blockade of CysLT1Rs with pranlukast significantly reduced myogenic tone not only in AT1A/1B (-/-) but also in wild-type arteries. In wild-type arteries, additional blockade of angiotensin II AT1 receptors with candesartan resulted in an additive reduction of myogenic tone. Furthermore, calcium imaging experiments were performed with fura-2-loaded human embryonic kidney 293 cells overexpressing CysLT1Rs and with isolated mesenteric vascular smooth muscle cells. Hypo-osmotically induced membrane stretch provoked calcium transients that were significantly reduced by pranlukast. Incubations of isolated mesenteric vascular smooth muscle cells with the 5-lipoxygenase inhibitor zileuton had no effect. Furthermore, the Gq/11-protein inhibitor YM 254890 profoundly reduced myogenic tone to the same extent as induced by the application of pranlukast plus candesartan. Here, we identify a novel, hitherto unappreciated role of CysLT1Rs in vascular regulation. We identified CysLT1Rs as novel mechanosensors in the vasculature involved in myogenic vasoconstriction. Moreover, our findings suggest that myogenic tone is determined by AT1 and CysLT1 receptors acting together as mechanosensors via Gq/11-protein activation.

Endothelin-1 but not angiotensin II contributes to functional aging in murine carotid arteries

AimsAging is a major risk factor for carotid artery disease and stroke. Endothelin-1 (ET-1) and angiotensin II (Ang II) are important modifiers of vascular disease, partly through increased activity of NADPH oxidase and vasoconstrictor prostanoids. Since the renin–angiotensin and endothelin systems become activated with age, we hypothesized that aging affects NADPH oxidase- and prostanoid-dependent contractions to ET-1 and Ang II. Main methodsCarotid artery rings of young (4month-old) and old (24month-old) C57BL6 mice were pretreated with the NO synthase inhibitor L-NAME to exclude differential effects of NO. Contractions to ET-1 and Ang II were determined in the presence and absence of the NADPH oxidase-selective inhibitor gp91ds-tat or the thromboxane-prostanoid receptor antagonist SQ 29,548. Gene expression of endothelin and angiotensin receptors was measured by qPCR. Key findingsAging reduced ET-1-induced contractions and diminished ETA but increased ETB receptor gene expression levels. Gp91ds-tat inhibited contractions to ET-1 in young and to a greater extent in old animals, whereas SQ 29,548 had no effect. Ang II-induced contractions were weak compared to ET-1 and unaffected by aging, gp91ds-tat, and SQ 29,548. Aging had also no effect on AT1A and AT1B receptor gene expression levels. SignificanceAging in carotid arteries decreases ETA receptor gene expression and responsiveness to ET-1, which nevertheless becomes increasingly dependent upon NAPDH oxidase activity with age; responses to Ang II and gene expression of its receptors are however unaffected. These findings suggest that physiological aging differentially regulates functional responses to G protein-coupled receptor agonists and the signaling pathways associated with their activation.

Heterogeneous Downregulation of Angiotensin II AT1-A and AT1-B Receptors in Arterioles in STZ-Induced Diabetic Rat Kidneys

Introduction. The renin granulation of kidney arterioles is enhanced in diabetes despite the fact that the level of angiotensin II in the diabetic kidney is elevated. Therefore, the number of angiotensin II AT1-A and AT1-B receptors in afferent and efferent arteriole's renin-positive and renin-negative smooth muscle cells (SMC) was estimated. Method. Immunohistochemistry at the electron microscopic level was combined with 3D stereological sampling techniques. Results. In diabetes the enhanced downregulation of AT1-B receptors in the renin-positive than in the renin-negative SMCs in both arterioles was resulted: the significant difference in the number of AT1 (AT1-A + AT1-B) receptors between the two types of SMCs in the normal rats was further increased in diabetes and in contrast with the significant difference observed between the afferent and efferent arterioles in the normal animals, there was no such difference in diabetes. Conclusions. The enhanced downregulation of the AT1-B receptors in the renin-negative SMCs in the efferent arterioles demonstrates that the regulation of the glomerular filtration rate by the pre- and postglomerular arterioles is changed in diabetes. The enhanced downregulation of the AT1-B receptors in the renin-positive SMCs in the arterioles may result in an enhanced level of renin granulation in the arterioles.

Novel role of mechanosensitive AT1B receptors in myogenic vasoconstriction

Myogenic vasoconstriction is an inherent property of vascular smooth muscle cells (VSMCs) of resistance arteries harboring ill-defined mechanosensing and mechanotransducing elements. G protein-coupled receptors (GPCRs) are discussed as mechanosensors in VSMCs. In this study, we sought to identify and characterize the role and impact of GPCRs on myogenic vasoconstriction. Thus, we analyzed mRNA expression levels of GPCRs in resistance versus preceding conduit arteries revealing a significant enrichment of several GPCRs in resistance vessels. Selective pharmacological blockade of the highly expressed GPCRs in isolated murine mesenteric arteries ex vivo decreased myogenic vasoconstriction. In particular, candesartan and losartan most prominently suppressed myogenic tone, suggesting that AT1 receptors play a central role in myogenic vasoconstriction. Analyzing angiotensinogen(-/-) mice, a relevant contribution of locally produced angiotensin II to myogenic tone could be excluded. Investigation of AT1A (-/-) and AT1B (-/-) murine mesenteric arteries revealed that AT1B, but not AT1A, receptors are key components of myogenic regulation. This notion was supported by examining fura-2-loaded isolated AT1A (-/-) and AT1B (-/-) VSMCs. Our results indicate that in VSMCs, AT1B receptors are more mechanosensitive than AT1A receptors even at comparable receptor expression levels. Furthermore, we demonstrate that the mechanosensitivity of GPCRs is agonist-independent and positively correlates with receptor expression levels.

Regional Variation in Aortic AT1b Receptor mRNA Abundance Is Associated with Contractility but Unrelated to Atherosclerosis and Aortic Aneurysms

BackgroundAngiotensin II (AngII), the main bioactive peptide of the renin angiotensin system, exerts most of its biological actions through stimulation of AngII type 1 (AT1) receptors. This receptor is expressed as 2 structurally similar subtypes in rodents, termed AT1a and AT1b. Although AT1a receptors have been studied comprehensively, roles of AT1b receptors in the aorta have not been defined.Methodology/ResultsWe initially compared the regional distribution of AT1b receptor mRNA with AT1a receptor mRNA in the aorta. mRNA abundance of both subtypes increased from the proximal to the distal aorta, with the greatest abundance in the infra-renal region. Corresponding to the high mRNA abundance for both receptors, only aortic rings from the infra-renal aorta contracted in response to AngII stimulation. Despite the presence of both receptor transcripts, deletion of AT1b receptors, but not AT1a receptors, diminished AngII-induced contractility. To determine whether absence of AT1b receptors influenced aortic pathologies, we bred AT1b receptor deficient mice into an LDL receptor deficient background. Mice were fed a diet enriched in saturated fat and infused with AngII (1,000 ng/kg/min). Parameters that could influence development of aortic pathologies, including systolic blood pressure and plasma cholesterol concentrations, were not impacted by AT1b receptor deficiency. Absence of AT1b receptors also had no effect on size of aortic atherosclerotic lesions and aortic aneurysms in both the ascending and abdominal regions.Conclusions/SignificanceRegional abundance of AT1b receptor mRNA coincided with AngII-induced regional contractility, but it was not associated with AngII-induced aortic pathologies.

Blood pressure response to angiotensin II is enhanced in obese Zucker rats and is attributed to an aldosterone‐dependent mechanism

Plasma aldosterone levels correlate positively with obesity, suggesting a link between the hypertension associated with obesity and increased mineralocorticoid levels. We tested the hypothesis that aldosterone is involved in the BP response to angiotensin II (AngII) in obese rats. Lean (LZR) and obese (OZR) Zucker rats were treated with AngII (9 µg·h(-1) ; 4 weeks), and BP and plasma AngII and aldosterone were determined. Chronic AngII increased the BP in OZR markedly more so than in LZR. Plasma AngII levels in LZR and OZR were similar after AngII treatment. The AngII stimulated a rise in plasma aldosterone that was sixfold more in OZR than in LZR. The thickness of the zona glomerulosa of the adrenal glands was selectively increased by AngII in OZR. Adrenal mRNA levels of CYP11B2 aldosterone synthase and the AT(1B) receptor were selectively increased in AngII-treated OZR. The BP response to chronic AngII stimulation was diminished in OZR after adrenalectomy when plasma aldosterone was absent. Acute bolus injections of AngII did not increase the BP response or aldosterone release in OZR. The AngII-induced BP response is enhanced in obesity and this is associated with a specific increase in circulating aldosterone. Due to the AngII-induced growth of the zona glomerulosa in OZR, the AT(1B) receptors and aldosterone synthase may be selectively enhanced in obesity under concomitant AngII stimulation, increasing the adrenal synthesis of aldosterone. Our results confirm functionally that aldosterone plays a major role in obesity-related hypertension.

Enhanced downregulation of AT1b receptors on afferent and efferent arterioles in STZ-induced diabetic rat kidney

Enhanced downregulation of AT1b receptors on afferent and efferent arterioles in STZ-induced diabetic rat kidney

Lack of specificity for AT1 angiotensin receptor antibodies in Western blot analysis

The angiotensin II type 1 receptor (AT1R) mediates the classical actions of angiotensin II (Ang II). In rodents, there are two AT1R, AT1a and AT1b, which are highly homologous (>90% at the amino acid level) and encoded by different genes. Detection of AT1R protein is important for a broad range of experimental approaches to understand molecular mechanisms of disease. While several anti AT1R antibodies are available commercially and used by investigators for these purposes, we have had concerns about their specificity. To address this, we examined the specificity of a panel of commercial anti AT1R antibodies (Santa Cruz 1173, AbCam 18801, Alomone AAR 011) for Western blot analysis. For these studies, we used kidney tissue from wild type mice and mice with genetic deficiencies of AT1a receptors (AT1a KO) or combined deficiency of AT1a and AT1b receptors (AT1ab KO), which completely lack AT1. For all three antibodies tested, a ~42 kDa band consistent with the predicted size of the AT1 was detected in tissues from WT mice. However, identical bands were also detected in tissues from the AT1a KO and AT1ab KO mice (n=3). Along with genotyping, we verified the absence of mRNA for the AT1a and AT1b receptors in KO mice and confirmed the absence of pressor responses to Ang II. We conclude that many of the available anti‐AT1R antibodies exhibit non‐specific binding that may lead to anomalous results.

Angiotensin II does not acutely regulate conduction velocity in rat atrial tissue

Aim. Atrial angiotensin II (Ang II) levels are increased in atrial fibrillation and are believed to be important in the pathogenesis of atrial arrhythmias. Ang II reduces intercellular coupling by inhibiting gap junctions (connexins) and may thereby increase the risk of reentry arrhythmia. The aim of the current study was to investigate the acute effect of Ang II on conduction velocity (CV) in atrial tissue from normal and chronically infarcted rats. Methods. Contractile force was measured and CV was determined from the conduction time between electrodes placed on the tissue preparation. Expression of AT1a and AT1b receptors was examined by real-time PCR. Results. Acute stimulation with Ang II did not affect CV in tissue from auricle or atrial free wall. A transient 6.5 ± 3.6% increase in resting tension was observed in atrial free wall preparations, indicating that receptors are present and functional in the free wall preparation. The difference between free wall and auricle was probably not caused by differences in receptor expression since equal amounts of AT1 mRNA were present. To test if myocardial infarction (MI) sensitizes the atrium to Ang II, free atrial wall from rats subjected to 4–5 weeks ventricular MI was examined. Although CV was significantly reduced by MI, no effect on CV of Ang II was seen. Conclusion. Ang II does not acutely regulate CV in tissue preparations from the free wall of the left atria or the left auricle. Although ventricular MI reduces CV, this does not sensitize the atria to Ang II.

Contributions of ACE and mast cell chymase to endogenous angiotensin II generation and leucocyte recruitment in vivo

In vitro studies suggest that mast cell chymase (MCP) is more important than angiotensin-converting enzyme (ACE) for generating angiotensin II (Ang II) within the cardiovascular system. We investigated in vivo the relative contributions of ACE and MCP to leucocyte recruitment induced by endogenously generated Ang II. Exposure of the murine cremasteric microcirculation of C57BL/6 mice to Ang I (100 nM for 4 h) induced leucocyte-endothelium interactions. Either losartan (an Ang II receptor-1 antagonist, AT(1)) or enalapril (an ACE inhibitor), but not chymostatin (a chymase inhibitor), inhibited Ang I-induced responses. Mast cell degranulation with compound 48/80 (CMP48/80, 1 μg/mL) also induced leucocyte adhesion but this was only weakly affected by the inhibitors. When Ang I and CMP48/80 were co-administered, AT(1B) receptor expression was increased, MCP-4 was found surrounding the vessel wall, and ACE was detected in the endothelium. Ang I + CMP48/80 induced enhanced leucocyte adhesion that was attenuated by losartan, enalapril, enalapril + chymostatin, and cromolyn (a mast cell stabilizer). The use of male mast cell-deficient WBB6F1/J-Kit(w)/Kit(w-v) mice (C57BL/6 background) confirmed these findings. In vivo, Ang II is primarily generated by ACE under basal conditions, but in inflammatory conditions, the release of MCP amplifies local Ang II concentrations and the associated inflammatory process. Thus, AT(1) receptor antagonists may be more effective than ACE inhibitors for treating ongoing Ang II-mediated vascular inflammation.

Long-term systemic angiotensin II type 1 receptor blockade regulates mRNA expression of dorsomedial medulla renin-angiotensin system components

In Fischer 344 (F344) rats, renin-angiotensin system (RAS) blockade for 1 yr with the angiotensin II type 1 (AT(1)) receptor blocker L-158,809 prevents age-related impairments in metabolic function, similar to transgenic rats with low glial angiotensinogen (Aogen). Brain RAS regulation may contribute to the benefits of long-term systemic AT(1) antagonism. We assessed the mRNA of RAS components in the dorsomedial medulla of F344 rats at 3 (young; n = 8) or 15 mo of age (old; n = 7) and in rats treated from 3 to 15 mo of age with 20 mg/l of the AT(1) receptor antagonist L-158,809 (Old+L; n = 6). Aogen and renin mRNA were lower in the young compared with old group. Angiotensin-converting enzyme (ACE) mRNA was lower in the old and Old+L compared with the young group. ACE2 and neprilysin expression were significantly higher in Old+L compared with young or old rats. AT(1b), AT(2), and Mas receptor mRNA were higher with treatment. Leptin receptor mRNA was lower in the old rats and this was prevented by L-158,809 treatment. Dual-specificity phosphatase 1 (DUSP1) mRNA was highest in the Old+L group. Aggregate correlate summation revealed a positive relationship for Mas receptor mRNA with food intake. The findings provide evidence for regulation of dorsomedial medullary renin and Aogen mRNA during aging. Long-term AT(1) receptor blockade increases the mRNA of the enzymes ACE2 and neprilysin and the MAS receptor, which could potentially shift the balance from ANG II to ANG-(1-7) and prevent age-related declines in the leptin receptor and its signaling pathway.

Angiotensin AT1 receptor subtypes AT1A and AT1B mRNAs are expressed in tyrosine hydroxylase immunoreactive (TH‐ir) neurons in the rat caudal nucleus of the solitary tract (NTS)

Identifying the gene expression profiles in cells in the caudal NTS is a major challenge due to the heterogeneity of cellular populations in this region. We utilized quick immunohistochemical labeling coupled with laser capture microdissection to identify and capture TH‐ir neurons from the caudal NTS. Six groups of 10–13 TH‐ir caudal NTS neurons were captured from adult, male Sprague‐Dawley rats and total RNA was extracted from each group. mRNA levels of TH and the angiotensin II receptor subtypes AT1A and AT1B were assessed by quantitative real time reverse transcription polymerase chain reaction using GAPDH as the housekeeping gene. The 2−ΔCt for TH averaged .171 ± .028. mRNA of both AT1A and AT1B subtypes of the angiotensin AT1 receptor was expressed in the TH neurons. The 2−ΔCt for AT1A and AT1B averaged .023 ± .004 and .025 ± .003 respectively. Our data show the existence of AT1A and AT1B receptor subtype mRNAs in catecholaminergic neurons in the caudal NTS.