Planejamento e desenvolvimento de ligantes não peptídicos com atividade agonista enviesada no receptor de angiotensina II tipo 1 empregando-se técnicas de modelagem molecular e ensaios in vitro / Drug design of non-peptidic biased agonists for angiotensin II type 1 receptor using molecular modeling techniques and in vitro tests

A insuficiência cardíaca (IC) é uma síndrome de elevada morbimortalidade, correspondendo a um grave problema de saúde pública. Uma das abordagens terapêuticas para IC consiste no uso de antagonistas do receptor de angiotensina II do tipo 1 (AT1R), conhecidos como sartanas. Estudos apontam que uma nova classe de compostos, os agonistas enviesados, é capaz de induzir a sinalização da via da ß-arrestina sem ativação da via da proteína G. Essa seletividade funcional é particularmente interessante, pois a via dependente da proteína G é responsável pelo aumento da pressão arterial, morte celular e fibrose tecidual, levando a hipertrofia cardíaca e progressão da IC. No entanto, a via da ß-arrestina está associada com renovação celular e aumento do inotropismo. Além disso, estudos in vivo sugerem que agonistas enviesados poderiam corresponder a uma terapia superior à dos antagonistas convencionais, que bloqueiam ambas as vias. Apesar do potencial terapêutico, esses compostos possuem estrutura peptídica e, por isso, tem sua administração restrita à via intravenosa. A resolução da estrutura cristalográfica do AT1R permitiu estudos de modelagem molecular mais acurados. Tendo isso em mente, nesse trabalho foram propostos agonistas enviesados de natureza não peptídica para o AT1R por meio de técnicas de modelagem molecular e validação das hipóteses levantadas por ensaios in vitro. Foram realizados estudos de dinâmica molecular com o AT1R (PDB ID: 4YAY) em uma bicamada lipídica e ensaios de ancoramento molecular da angiotensina II (AngII) e do ligante enviesado TRV027. As poses de ancoramento molecular selecionadas foram utilizadas em dinâmicas de complexo, que revelaram diferenças entre os sistemas apo (sem nenhum ligante) e holo (com o ligante no sitio de ligação). Nossos resultados sugerem que o TRV027 induz um padrão exclusivo de ligações de hidrogênio e de estrutura secundária, enquanto que a AngII afeta os resíduos do bolso hidrofóbico do sitio de ligação, principalmente a conformação do Trp2536.48. Com base nas simulações, três farmacóforos foram criados e utilizados de maneira complementar em triagens virtuais na base de dados ZINC15, resultando na seleção de cinco compostos. Um desses compostos apresentou afinidade pelo receptor AT1R e, ainda que estudos complementares de ativação de vias especificas sejam necessários para que o composto possa ser classificado como agonista enviesado, já se constitui em molécula potencialmente promissora. Além disso, esses estudos permitiram a proposição de estruturas inéditas que podem vir a ser hits no processo de desenvolvimento de agonistas enviesados para AT1R. Portanto, como continuidade desse trabalho, essas moléculas serão sintetizadas e investigadas quanto à possível interação com o receptor.

Heart Failure (HF) is a common syndrome with high morbimortality, being considered a serious public health problem. One of the therapeutic approaches for HF consists in the use of the sartan class, which are angiotensin II type 1 receptor (AT1R) antagonists. Recent studies have shown that a new class of compounds, known as biased agonists, is able to induce signaling via ß-arrestin without G-protein activation. This functional selectivity is particularly interesting since G-protein dependent signaling is responsible for cell death and cardiac tissue fibrosis, which leads to cardiac muscle hypertophy and HF progression. On the other hand, ß-arrestin signaling is associated with cellular renewal and increased inotropism. In vivo studies suggests that biased agonists could correspond to a superior therapy over conventional angiotensin II type 1 receptor antagonists, which blocks cell signaling as a whole, however their peptidic structure restricts their use to intravenous administration. Moreover, the AT1R crystal structure determination holds great promise for more accurate molecular modeling studies. With that being said, the aim of this work was to plan and develop new non-peptidic biased agonists for ATR1 employing molecular modeling techniques and in vitro tests for hypothesis validation. Molecular dynamics (MD) simulations of the refined AT1R crystal (PDB ID: 4YAY) embedded in a lipid bilayer and molecular docking studies with angiotensin II (AngII) and TRV027 (biased agonist) were conducted. Selected docking poses from both ligands underwent complex MD simulations revealing differences between apo (ligand free) and holo (ligand in the binding site) systems. Our results suggest that TRV027 induces an exclusive hydrogen bond and secondary structure pattern, while AngII affects the hydrophobic pocket conformation, mainly Trp253. Based on the simulations, three pharmacophore models were created and used in virtual screenings in the ZINC15 database, resulting in the selection of five compounds that were tested in vitro. One of the compounds displayed affinity for AT1R and is a promising molecule. Nonetheless, it needs further pathway activation characterization in order to be a classified as a biased agonist. Furthermore, these results have contributed significantly for the proposition of new structures that could be hits with biased agonist activity for AT1R. Thus, for future works, we point out the necessity for synthesis and characterization of this new compounds

The renin-angiotensin system in Barrett's esophagus.

OBJECTIVE: Barrett's esophagus (BE) is a risk factor for esophageal adenocarcinoma. In addition to its classical endocrine character known for hemodynamic regulation, the renin-angiotensin system (RAS) can be associated with inflammation, wound healing, and cancer. The aim of this study was to explore a potential expression of the RAS in BE, with or without the presence of dysplasia. MATERIAL AND METHODS: Biopsy material was prepared for western blotting and immunohistochemistry. Non-BE patients (controls) were compared with BE patients regarding RAS in the squamous epithelium. In the columnar BE mucosa, RAS expression was studied in patients with and without dysplasia. Key components of the 'classical' RAS were assessed: the angiotensin-converting enzyme (ACE) and the angiotensin II subtype 1 and 2 receptors (AT1R and AT2R). RESULTS: The presence of RAS factors was confirmed in the esophageal mucosa of both control and BE patients. ACE protein expression was 48% lower (p = 0.001) whereas AT1R was 45% higher (p = 0.039) in the squamous epithelium of BE patients compared to epithelia from non-BE controls. In the metaplastic intestinal-like epithelium, AT1R expression was 37% higher in BE patients with confirmed dysplasia than in patients without dysplasia (p = 0.009). Immunohistochemistry showed an altered distribution of RAS proteins in BE patients with dysplasia. CONCLUSIONS: The differential RAS expression observed may prove to be useful as a biomarker or a pharmaceutical target.

The potential repertoire of the innate immune system in the bladder: expression of pattern recognition receptors in the rat bladder and a rat urothelial cell line (MYP3 cells).

PURPOSE: The urothelium is a frontline sensor of the lower urinary tract, sampling the bladder lumen and stimulating an immune response to infectious and noxious agents. Pattern recognition receptors (PRRs) recognize such agents and coordinate the innate response, often by forming inflammasomes that activate caspase-1 and the release of interleukin-1. We have shown the presence of one PRR (NLRP3) in the urothelia and its central role in the inflammatory response to cyclophosphamide. The purpose of this study was to (1) assess the likely range of the PPR response by assessing the repertoire present in the rat bladder and (2) determine the utility of the MYP3 rat urothelia cell line for in vitro studies by assessing its PPR repertoire and functional responsiveness. METHODS: Immunohistochemistry was performed for seven PPRs (NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, NLRC4 and AIM2) on bladder sections and MYP3 cells. For functionality, MYP3 cells were challenged with the quintessential NLRP3 activator ATP and assessed for caspase-1 activation. RESULTS: All PPRs examined were expressed in the bladder and localized to the urothelial layer with several also in the detrusor (none in the interstitia). MYP3 cells also expressed all PRRs with a variable intracellular location. ATP-stimulated caspase-1 activity in MYP3 cells in a dose-dependent manner was reduced by knockdown of NLRP3 expression. CONCLUSION: The results suggest that the bladder possesses the capacity to initiate an innate immune response to a wide array of uropathological agents and the MYP3 cells will provide an excellent investigational tool for this field.

Urinary angiotensinogen as a potential biomarker of intrarenal renin-angiotensin system activity in Chinese chronic kidney disease patients.

BACKGROUND: Urinary angiotensinogen (AGT) mainly derives from the AGT produced in proximal tubular cells. Evidence exists that supports the correlation between urinary AGT and circulating AGT. AIM: To investigate the role of urinary AGT as a potential biomarker of intrarenal renin-angiotensin system activity in Chinese chronic kidney disease (CKD) patients. METHODS: ELISA-based method used to quantify urinary AGT. Analyzed the relationship between urinary AGT and intrarenal angiotensin II (Ang II) activity in 128 CKD patients. ELISA was applied to measure the urinary and plasma renin activity, AGT, Ang II and aldosterone. Furthermore expression levels of intrarenal renin, AGT, Ang II and Ang II receptor were examined by immunohistochemistry staining (IHCS) in 72 CKD patients undergoing renal biopsy. RESULTS: The logarithmic transformation Log(urinary AGT/UCre) levels showed a normal distribution. Therefore, Log(urinary AGT/UCre) levels were used for the analyses. Average urinary AGT was 2.02 ± 0.55 ng/(mg Cr). Hypertension, urinary protein, urinary Ang II and urinary type IV collagen (Col IV) positively correlated with urinary AGT. Estimated glomerular filtration rate (eGFR), urinary sodium and serum AGT negatively correlated with urinary AGT. Multiple regression analysis indicated that low serum AGT, high urinary protein, urinary Ang II and urinary Col IV correlated significantly with high urinary AGT. CONCLUSIONS: We observed positive correlation between urinary AGT and positive IHCS area of AGT, Ang II and Ang II type 1 receptor in renal tissue. These data suggest that urinary AGT might be a potential biomarker of intrarenal Ang II activity in CKD patients.

Variety of angiotensin receptors in 3T3-L1 preadipose cells and differentiated adipocytes.

A local paracrine acting angiotensin (ANG) system of preadipocytes and mature adipocytes is involved in metabolic effects and tissue differentiation. The present study reports on the investigation of binding affinities for various angiotensin receptors including their relevance in 3T3-L1 adipocytes and preadipocytes and 3T3-442A preadipocytes. Competitive binding studies using both 125I-ANG II and its more stable analogue 125I-SARILE for investigating AT1/AT2 binding sites in 3T3-L1 preadipocytes reveal a biphasic competition curve with KDs at a low and high nanomolar range. By using the AT2 receptor selective ligand 125I-CGP4112A the presence of high affinity AT2 binding sites in preadipocytes was observed. High nonspecific binding and a low receptor number is characteristic for all these experiments. An AT4 binding site (binding site for ANG IV) exists in 3T3-L1 and F442A preadipocytes and adipocytes with a high nanomolar KD. This low binding affinity was confirmed by a biological assay, the IRAP assay (=insulin regulated aminopeptidase assay). IRAP is associated with the AT4 receptor, which is a binding site at the luminal part of membrane bound IRAP. The curves for competition binding and for inhibition of IRAP activity are superimposable with respect to angiotensin IV. In conclusion, AT1 and AT2 binding sites are present in preadipocytes. AT2 receptor binding affinities are shown in preadipocytes for the first time. The description of a non-AT1/AT2 binding site with low affinity remains speculative albeit of high interest because antidiabetic and obesity related effects of angiotensin peptides and sartanes as antagonists are observed at these high concentrations. Local concentrations of ANG II and their degradation products may be extremely high. The low amounts of AT1 and AT2 binding sites emphasize the relevance of other binding sites in adipose tissue development and metabolic effects. The AT4 binding site seems to be one of the predominant receptors in adipose cells. Other degraded, but still bioactive peptides like ANG III, IV and ANG(1-7), activating receptors not influenced by ANG II, could be of importance.

ACE2 and AT4R are present in diseased human blood vessels.

A growing body of evidence suggests that the angiotensin II fragments, Ang(1-7) and Ang(3-8), have a vasoactive role, however ACE2, the enzyme that produces Ang(1-7), or AT4R, the receptor that binds Ang (3-8), have yet been simultaneously localised in both normal and diseased human conduit blood vessels. We sought to determine the immunohistochemical distribution of ACE2 and the AT4R in human internal mammary and radial arteries from patients undergoing coronary artery bypass surgery. We found that ACE2 positive cells were abundant in both normal and diseased vessels, being present in neo-intima and in media. ACE2 positive immunoreactivity was not present in the endothelial layer of the conduit vessels, but was clearly evident in small newly formed angiogenic vessels as well as the vaso vasorum. Endothelial AT4R immunoreactivity were rarely observed in either normal and diseased arteries, but AT4R positive cells were observed adjacent to the internal elastic lamine in the internal mammary artery, in the neo-intima of radial arteries, as well as in the media of both internal mammary artery and radial artery. AT4R was abundant in vaso vasorum and within small angiogenic vessels. Both AT4R and ACE2 co-localised with smooth muscle cell alpha actin. This study identifies smooth muscle cell alpha actin positive ACE2 and AT4R in human blood vessels as well as in angiogenic vessels, indicating a possible role for these enzymes in pathological disease.

Peptide-conjugated quantum dots: imaging the angiotensin type 1 receptor in living cells.

Peptide-quantum dot conjugates have been prepared by attaching angiotensin II (Ang II) to cadmium selenide/zinc sulfide core-shell nanocrystals using an 1-[3-(Dimethyamino)propyl]-3-ethylcarbo diimide hydrochloride (EDC) coupling. These conjugates have been used to image angiotensin I-expressing Chinese hamster ovary (CHO) cells in vitro. When CHO cells were incubated with Ang II before incubating with Ang II-conjugated quantum dots, we were able to block the binding of the dots. The Ang II-quantum dot conjugates did not bind to parental cells and showed similar staining patterns when compared with a commercially available Ang II Alexa 488 conjugate.

Differential regulation of angiotensin II receptors during renal injury and compensatory hypertrophy in the rat.

1. The renin-angiotensin system may be involved in the compensatory adaptations occurring after the reduction of renal mass and during the consecutive changes leading to chronic renal failure. We therefore investigated the regulation of angiotensin II receptors in two models of renal hypertrophy in the rat: hypertrophy following uninephrectomy (UNx) or subtotal nephrectomy (STNx). The level of angiotensin type 1 (AT1A-R and AT1B-R) and type 2 (AT2-R) receptor mRNA was quantified by competitive reverse transcription-polymerase chain reaction (RT-PCR) in specific renal zones and the intrarenal distribution of angiotensin II receptors was analysed by immunohistochemistry. 2. In the UNx rats, AT1-R mRNA expression was not modified in the cortex or in the inner stripe of the outer medulla of the residual kidney at any time after the surgery (1, 4 and 12 weeks). In contrast, AT1-R mRNA expression was significantly reduced in these zones in STNx rats (-33% and -40%, respectively). This downregulation was organ-specific, as AT1-R mRNA levels were not modified in the liver. The proportions of AT1-R subtype (AT1A and AT1B) mRNA were unchanged by UNx or STNx. Very low levels of AT2-R mRNA were found in the cortex of all groups. Immunostaining revealed a similar localization of AT1-R in mesangial cells, proximal tubule, basolateral membrane of thick ascending limb, in both models of hypertrophy. AT1-R labelling was also detected in the apical membrane of intercalated cells of cortical collecting ducts. 3. This differential mRNA expression of angiotensin II receptors during compensatory hypertrophy and renal injury suggests that the development of renal hypertrophy is independent of AT1-R and AT2-R gene expression levels.

Angiotensin II induces fibronectin expression in human peritoneal mesothelial cells via ERK1/2 and p38 MAPK.

BACKGROUND: The renin-angiotensin system has been implicated in the pathogenesis of fibrosis in various organs. However, its involvement in peritoneal fibrosis, a crucial complication of peritoneal dialysis, is unclear. Human peritoneal mesothelial cells (HPMC) play a major role in peritoneal fibrosis by producing extracellular matrix (ECM). However, there is scant data regarding the effect of angiotensin II (Ang II) on ECM expression and signal transduction pathways in HPMC. METHODS: The concentration of Ang II in the peritoneal dialysis effluent was measured by radioimmunoassay. We investigated the expression of Ang II type 1 (AT1) and type 2 (AT2) receptors by HPMC. We also examined the effect of Ang II upon fibronectin production by HPMC, and dissected the receptor and intracellular signaling pathways involved. RESULTS: Ang II levels in the peritoneal dialysis effluent at the onset of peritonitis were 30 times higher than baseline levels. HPMC expression of AT1 and AT2 receptors was confirmed at the mRNA and protein level by reverse transcriptase-polymerase chain reaction (PCR), Western blotting, and immunocytochemistry. Quantitative reverse transcriptase-PCR and Western blotting showed that 10 nmol/L Ang II increased fibronectin mRNA expression followed by secretion of fibronectin protein. This response was completely inhibited by the AT1 receptor antagonist RNH6270, while the AT2 receptor antagonist PD123319 had no effect. Ang II-induced fibronectin expression was mediated by the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK), but not c-Jun N-terminal kinase. CONCLUSION: These results indicate the potential importance of ERK1/2 and p38 MAPK signaling pathways in Ang II-induced fibronectin expression in HPMC, and suggest the therapeutic potential of AT1 receptor blockers in the prevention or treatment of peritoneal fibrosis in patients on peritoneal dialysis.

Localization of angiotensin II in pig ovary and its effects on oocyte maturation in vitro.

The renin-angiotensin system (RAS) has been found in mammalian ovarian tissue; however, its physiological role is unclear. This study examined the content of angiotensin II (Ang II) in porcine follicular fluid (pFF), Ang II localization and its receptors in ovary, and the effects of Ang II on porcine oocyte maturation. The concentrations of Ang II were 6951.82 +/- 1295.83, 3502.99 +/- 679.10, 3147.89 +/- 690.60, and 2545.92 +/- 407.01 pg/ml in pFF from small, medium, large, and extra-large follicles, respectively. In addition, Ang II was found on zona pellucidae (ZP) and granulosa cells by immunoreactive staining. The distribution of AT1, an Ang II receptor subtype, was in accordance with that of Ang II. However, AT2, another Ang II receptor, was mainly distributed in the stroma and thecal layers of follicles. When oocytes were cultured in media containing various concentrations of Ang II, a higher (P<0.05) proportion of oocytes reached metaphase II (MII) in the medium with 100 ng/ml (87.0%) than without Ang II (61%). When oocytes from different sizes of follicles were separately cultured in media containing 100 ng/ml Ang II, maturation rates were significantly higher in oocytes from small (61.5%) and medium (85.1%) follicles than that of their controls (45.1 and 72.6%, respectively). However, addition of Ang II inhibited nuclear maturation in oocytes from large follicles (77.8% versus 87.3%). Fertilization and male pronuclear (MPN) formation rates of oocytes matured in medium containing 100 or 1000 ng/ml of Ang II were higher (P<0.05) than that of oocytes matured in medium containing 0 or 10 ng/ml Ang II. Glutathione content in oocytes cultured for 44 h in medium containing 100 or 1000 ng/ml of Ang II was also higher (P<0.01) than that of oocytes cultured in medium containing 0 or 10 ng/ml Ang II. In conclusion, Ang II was present in porcine ovaries and may regulate follicle growth and oocyte maturation.

AT1 blockade prevents glucose-induced cardiac dysfunction in ventricular myocytes: role of the AT1 receptor and NADPH oxidase.

Enhanced tissue angiotensin (Ang) II levels have been reported in diabetes and might lead to cardiac dysfunction through oxidative stress. This study examined the effect of blocking the Ang II type 1 (AT1) receptor on high glucose-induced cardiac contractile dysfunction. Rat ventricular myocytes were maintained in normal- (NG, 5.5 mmol/L) or high- (HG, 25.5 mmol/L) glucose medium for 24 hours. Mechanical and intracellular Ca2+ properties were assessed as peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening/relengthening (+/-dL/dt), and intracellular Ca2+ decay (tau). HG myocytes exhibited normal PS; decreased +/-dL/dt; and prolonged TPS, TR90, and tau. Interestingly, the HG-induced abnormalities were prevented with the AT1 blocker L-158,809 (10 to 1000 nmol/L) but not the Janus kinase-2 (JAK2) inhibitor AG-490 (10 to 100 micromol/L). The only effect of AT1 blockade on NG myocytes was enhanced PS at 1000 nmol/L. AT1 antagonist-elicited cardiac protection against HG was nullified by the NADPH oxidase activator sodium dodecyl sulfate (80 micromol/L) and mimicked by the NADPH oxidase inhibitors diphenyleneiodonium (10 micromol/L) or apocynin (100 micromol/L). Western blot analysis confirmed that the protein abundance of NADPH oxidase subunit p47phox and the AT1 but not the AT2 receptor was enhanced in HG myocytes. In addition, the HG-induced increase of p47phox was prevented by L-158,809. Enhanced reactive oxygen species production observed in HG myocytes was prevented by AT1 blockade or NADPH oxidase inhibition. Collectively, our data suggest that local Ang II, acting via AT1 receptor-mediated NADPH oxidase activation, is involved in hyperglycemia-induced cardiomyocyte dysfunction, which might play a role in diabetic cardiomyopathy.

Role of host angiotensin II type 1 receptor in tumor angiogenesis and growth.

Although the renin angiotensin system (RAS) is a major regulator of vascular homeostasis, the role of the RAS in tumor angiogenesis is little understood. Here we show that host angiotensin II (ATII) type 1 (AT1) receptor plays an important role in angiogenesis and growth of tumor cells engrafted in mice. Subcutaneous B16-F1 melanoma-induced angiogenesis as assessed by tissue capillary density and microangiography was prominent in WT mice but was reduced in AT1a receptor-deficient (AT1a-/-) mice. Consequently, tumor growth rate was significantly slower, and the mouse survival rate was greater, in AT1a-/- mice than in WT mice. Tumor growth was also reduced in WT mice treated with TCV-116, a selective blocker of AT1 receptor. Because the beta-galactosidase gene was inserted into the AT1a gene locus in AT1a-/- mice, the site of beta-galactosidase expression represents the AT1a receptor expression in these mutant mice. In tumor-implanted AT1a-/- mice, the major site of the beta-galactosidase expression was macrophages in tissues surrounding tumors. Moreover, the number of infiltrated macrophages was significantly lower in AT1a-/- mice than in WT mice, and double-immunofluorescence staining revealed that these macrophages expressed VEGF protein intensively. Therefore, the host ATII-AT1 receptor pathway supports tumor-associated macrophage infiltration, which results in enhanced tissue VEGF protein levels. The host ATII-AT1 receptor pathway thereby plays important roles in tumor-related angiogenesis and growth in vivo.

Attenuated responses to angiotensin II in follitropin receptor knockout mice, a model of menopause-associated hypertension.

Activation of the renin-angiotensin system has been implicated in the development of hypertension in menopausal women. We investigated whether blood pressure is elevated and whether angiotensin II (Ang II)-induced vascular reactivity is increased in follitropin receptor knockout (FORKO) female mice. These mice are estrogen-deficient and have characteristics similar to postmenopausal women. Serum estradiol levels were significantly reduced in FORKO versus wild-type mice (1.4+/-0.2 versus 15+/-3 pg/mL, P<0.01). Blood pressure, measured by telemetry, was significantly increased in FORKO (120+/-2/92+/-2 mm Hg) compared with wild-type counterparts (110+/-1/85+/-2 mm Hg, P<0.05). Vascular dose responses to acetylcholine (endothelium-dependent dilation) and sodium nitroprusside (endothelium-independent dilation) were not different. Ang II-induced vasoconstriction was blunted in FORKO compared with wild-type mice (P<0.05). Media-to-lumen ratio was significantly increased in FORKO (6.2+/-0.5%) versus control mice (5.2+/-0.3%), indicating vascular remodeling. Aortic*O2- levels, NADH-inducible.O2- generation, and plasma levels of thiobarbituric acid reactive substances (TBARS), indexes of oxidative stress, were not significantly different between wild-type and FORKO mice. Vascular AT1 receptor content, assessed by immunoblotting, was reduced by 40% in FORKO compared with wild-type mice (P<0.01). This was associated with decreased circulating Ang II levels in FORKO versus control mice. These data indicate that FORKO mice have increased blood pressure, vascular remodeling, and attenuated vascular responses to Ang II. Our findings suggest that vascular Ang II signaling is downregulated in female FORKO mice and that Ang II may not play an important role in blood pressure elevation in this model of menopause-associated hypertension.

Anti-inflammatory effects of angiotensin II AT1 receptor antagonism prevent stress-induced gastric injury.

Stress reduces gastric blood flow and produces acute gastric mucosal lesions. We studied the role of angiotensin II in gastric blood flow and gastric ulceration during stress. Spontaneously hypertensive rats were pretreated for 14 days with the AT1 receptor antagonist candesartan before cold-restraint stress. AT1 receptors were localized in the endothelium of arteries in the gastric mucosa and in all gastric layers. AT1 blockade increased gastric blood flow by 40-50%, prevented gastric ulcer formation by 70-80% after cold-restraint stress, reduced the increase in adrenomedullary epinephrine and tyrosine hydroxylase mRNA without preventing the stress-induced increase in adrenal corticosterone, decreased the stress-induced expression of TNF-alpha and that of the adhesion protein ICAM-1 in arterial endothelium, decreased the neutrophil infiltration in the gastric mucosa, and decreased the gastric content of PGE2. AT1 receptor blockers prevent stress-induced ulcerations by a combination of gastric blood flow protection, decreased sympathoadrenal activation, and anti-inflammatory effects (with reduction in TNF-alpha and ICAM-1 expression leading to reduced neutrophil infiltration) while maintaining the protective glucocorticoid effects and PGE2 release. Angiotensin II has a crucial role, through stimulation of AT1 receptors, in the production and progression of stress-induced gastric injury, and AT1 receptor antagonists could be of therapeutic benefit.

A role for angiotensin II AT1 receptors in ureteric bud cell branching.

Gene-targeting studies in mice demonstrate that the renin-angiotensin system is required for the proper development of the renal medulla. In the absence of angiotensin II (ANG II) or the ANG II type 1 (AT1) receptor, mice exhibit poor papillary development and a severe urinary-concentrating defect. These findings imply that the ureteric bud (UB) and its branches are targets for ANG II actions during renal development. However, direct evidence linking ANG II with UB-branching morphogenesis does not exist. Using immunohistochemistry, we demonstrated that UB-derived epithelia express angiotensinogen (Ao) and the AT1 receptor during murine metanephrogenesis. Ao and AT1 receptors are expressed in the UB branches and to a lesser extent in the stromal mesenchyme. AT1 receptor expression in UB-derived epithelia increased from embryo day 12 to day 16 and was observed on both luminal and basolateral membranes. In accord with these findings, cultured murine UB cells express AT1 receptor protein and mRNA. Treatment of UB cells cultured in three-dimensional type I collagen gels with ANG II (10-7 to 10-5 M) elicits a dose-related increase in the number of cells that have primary and secondary branches. These effects of ANG II on UB branching are abrogated by pretreatment with the AT1 receptor antagonist candesartan. These data demonstrate a direct and independent role for ANG II acting via AT1 receptors on UB cell branching in vitro. The presence of Ao in the stroma and AT1 on UB cells supports the notion that cross talk between stroma and epithelial cells is crucial to epithelial branching morphogenesis in the developing kidney.

Estrogen upregulates renal angiotensin II AT(2) receptors.

AT(2) receptors may act in opposition to and in balance with AT(1) receptors, their stimulation having beneficial effects. We found renal AT(2) receptor expression in female mice higher than in male mice. We asked the question of whether such expression might be estrogen dependent. In male, female, ovariectomized, and estrogen-treated ovariectomized mice, we studied renal AT(1) and AT(2) receptors by immunocytochemistry and autoradiography, AT(2) receptor mRNA by RT-PCR, and cAMP, cGMP, and PGE(2) by RIA. AT(1) receptors predominated. AT(2) receptors were present in glomeruli, medullary rays, and inner medulla, and in female kidney capsule. AT(1) and AT(2) receptors colocalized in glomeruli. Female mice expressed fewer glomerular AT(1) receptors. Ovariectomy decreased AT(1) receptors in medullary rays and capsular AT(2) receptors. Estrogen administration normalized AT(1) receptors in medullary rays and increased AT(2) receptors predominantly in capsule and inner medulla, and also in glomeruli, medullary rays, and inner stripe of outer medulla. In medullas of estrogen-treated ovariectomized mice there was higher AT(2) receptor mRNA, decreased cGMP, and increased PGE(2) content. We propose that the protective effects of estrogen may be partially mediated through enhancement of AT(2) receptor stimulation.

Angiotensin II evokes calcium-mediated signaling events in isolated dog pancreatic epithelial cells.

INTRODUCTION: Calcium-activated chloride conductance has been identified in normal pancreatic duct cells. Recent in vitro evidence suggests that angiotensin II (AngII) stimulates pancreatic secretion in both cystic fibrosis (CFPAC) and transformed pancreatic cells. AIMS: To investigate calcium-mediated stimulatory effects of AngII in both nontransformed dog pancreatic duct epithelial (DPDE) and CFPAC cells. METHODS: Western blots were performed in both cells seeking AngII receptors. In additional studies, DPDE and CFPAC cells were grown on vitrogen-coated glass cover slips and loaded with Indo-1-AM dye. Cells were placed in a confocal microscope's perfusion chamber and perfused with 100 microM AngII or ATP (control). Cells were excited with UV light, and intracellular calcium ([Ca+2]i) was read using fluorescence emission at 405 and 530 nm. Finally, single channels in the DPDE cells were examined using cell-attached patch clamps. Current amplitude histograms provided estimates of the conductance and open probability of channels. RESULTS: Western blots demonstrated presence of both AT and AT AngII receptors in DPDE and CFPAC cells; the density of AT receptors appeared lower than that of AT receptors. Basal intracellular calcium concentrations did not differ between DPDE (109 +/- 11 nM) and CFPAC (103 +/- 8 nM) cells. AngII significantly increased measured intracellular calcium concentrations in both DPDE (909 +/- 98 nM) and CFPAC (879 +/- 207 nM) cells, as did ATP (DPDE = 1722 +/- 228 nM; CFPAC = 1522 +/- 245 nM). In the patch clamp studies, a variety of different channels were observed; they appeared to be an 11pS nonselective cation (NSC) channel, a 4.6pS Na+ channel, a 3pS anion channel, and an 8pS chloride channel. The latter channel had characteristics similar to cystic fibrosis transmembrane conductance regulator (CFTR). Apical or basolateral application of AngII activated both the 11pS NSC and the 3pS channels. CONCLUSION: In nontransformed DPDE and CFPAC cells, specific AngII receptors mediate increases in [Ca ]. The latter effect of AngII may elicit activation of calcium-mediated chloride channels, suggesting a role for AngII as an alternative mediator of pancreatic ductal secretion.

Augmented upregulation by c-fos of angiotensin subtype 1 receptor in nucleus tractus solitarii of spontaneously hypertensive rats.

Our laboratory demonstrated previously that spontaneously hypertensive rats (SHR) exhibited an elevated basal Fos expression in the nucleus tractus solitarii (NTS), the terminal site for primary baroreceptor afferents, and that Fos protein is required for the re-expression of angiotensin subtype 1 receptor (AT1R) mRNA in the NTS after baroreceptor activation. The present study evaluated the hypothesis that this re-expression of AT1R is augmented in SHR and is promoted by the heightened Fos expression. Reverse transcription-polymerase chain reaction analysis revealed that baroreceptor activation via sustained increase in systemic arterial pressure resulted in a discernible reduction in the expression of AT1R mRNA at the dorsomedial medulla of SHR and normotensive Wistar-Kyoto rats. However, SHR manifested an appreciably larger magnitude of decline, followed by a faster time course of re-expression in AT1R mRNA. Parallel findings were obtained from the pressor response induced by microinjection unilaterally of angiotensin II (40 pmol) into the NTS. Whereas the re-expression of AT1R at both transcriptional and functional expression levels after baroreceptor activation was discernibly blunted by prior bilateral application into the NTS of an antisense c-fos oligonucleotide (50 pmol), the suppression in SHR was again significantly more intense. Control pretreatment with the corresponding sense or scrambled c-fos oligonucleotide was ineffective. We conclude that the heightened Fos expression in SHR is causatively related to the augmented re-expression of AT1R in the NTS at both transcriptional and functional levels.

Immunohistochemical study of angiotensin receptors in human anagen hair follicles and basal cell carcinoma.

BACKGROUND: Angiotensin receptors are the specific receptors of angiotensin II of the renin-angiotensin system. However, expression of the receptors in hair follicles has not been determined. OBJECTS: To clarify the expression and localization of angiotensin receptors in human anagen hair follicles and basal cell carcinomas. METHODS: We studied immunohistochemically the expression of angiotensin type 1(AT1) and type 2 (AT2) receptors in human anagen hair follicles and in 16 cases of basal cell carcinoma (BCC) (nine of solid BCC of the circumscribed type, two of adenoid BCC, five of BCC with follicular differentiation). RESULTS: Our experiments demonstrated the localization of AT1 in the inner root sheath and the inner layers of the outer root sheath. In BCC, positive staining with AT1 was revealed in the tumour cells of basal cell carcinoma with follicular differentiation. CONCLUSIONS: AT1 may have a role in association with follicular keratinization. Studying AT1 distribution may be useful in understanding the pathophysiology of human hair follicles and the hair follicle-associated tumours.

The renin-angiotensin system in experimental radiation nephropathy.

Irradiation of the kidneys is followed by a well-defined sequence of changes leading eventually to kidney failure. In the rat, inhibition of angiotensin-converting enzyme or blockade of angiotensin II receptors can prevent the structural and functional changes that occur after kidney irradiation. These interventions are particularly effective between 3 and 10 weeks after irradiation. However, in a series of studies with the rat model we failed to find any evidence that the renin-angiotensin system (RAS) is activated in the first 10 weeks after kidney irradiation. First, if the RAS was activated during this interval, one would expect hypertension followed by proteinuria and azotemia. However, hypertension is significant only at the end of this period and is preceded by significant proteinuria and azotemia. This evolution is not in favor of an obviously activated RAS during the 3- to 10-week postirradiation interval that is critical for interventions aimed at the RAS. Second, plasma renin activity and active plasma renin protein concentrations are not significantly increased over the first 10 weeks after irradiation. Third, whole-blood and intrarenal angiotensin II levels are not increased and may even be decreased over this interval. This last observation is particularly important because the assay used is sensitive enough to detect the effects of dietary salt manipulation. We hypothesize that even the normal activity of the RAS contributes to injury after kidney irradiation, possibly by supporting the proliferation of cells that carry potentially lethal radiation injuries.