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

18 F-Labeled Derivatives of Irbesartan for Angiotensin†II Receptor PET Imaging.

The renin angiotensin aldosterone system (RAAS) is a hormonal cascade involved in the regulation of blood pressure and electrolyte balance, and represents a common target for the treatment of various diseases including hypertension, heart failure, and diabetes. Herein we present a novel 18 F-labeled derivative of the drug irbesartan, one of the most prescribed angiotensin II type 1 receptor (AT1 R) antagonists, for in vivo positron emission tomography (PET). This allows the in vivo measurement of AT1 R expression, and thus the evaluation of functional changes in its expression under pathophysiological conditions. We followed various synthetic approaches optimized for the introduction of fluorine into different positions of the aliphatic side chain of irbesartan. Radioligand binding studies revealed that fluorine atoms at specified positions (α-position (IC50 =6.6 nm) and δ-position (IC50 =8.5 nm) of the aliphatic side chain) do not alter the binding properties of irbesartan (IC50 =1.6 nm). After successful radiolabeling with fluorine-18 in a radiochemical yield of 11 %, we observed high renal uptake in healthy rats and pigs, which could be decreased by pretreatment with the parent compound irbesartan.

Fluorescence-Based Binding Assay for Screening Ligands of Angiotensin Receptors.

Binding assay is a common technique used to characterize ability of a ligand to interact with a specific biological target. A number of parameters, such as binding affinity, receptor density, and association/dissociation rate constants, can be measured by means of this technique. In most cases, implementation of the binding assay requires specific infrastructure for labeling and detecting the ligand, which impedes realization of this technique in a standard laboratory. Here we describe a simple fluorescence-based binding assay for angiotensin peptides and receptors, which does not require complex equipment and can be used for initial screening of the novel ligands or mutational studies.

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors.

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping. Tissue specimens are harvested and stored at -80 °C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in (125)I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: 'non-specific binding' (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and 'total binding' with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 µM) is also present in the incubation buffer to limit (125)I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 °C, NSP slides are exposed to 10 µM PD123319 and losartan, while 'total binding' slides are exposed to 10 µM PD123319. Slides are then incubated with (125)I-SI-Ang II in the presence of PD123319 for 'total binding', and PD123319 and losartan for NSP in assay buffer, followed by several 'washes' in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons. The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

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.

Functional Local Renin-Angiotensin System in Human and Rat Periodontal Tissue.

The initiation or progression of periodontitis might involve a local renin-angiotensin system (RAS) in periodontal tissue. The aim of this study was to further characterize the local RAS in human and rat periodontal tissues between healthy and periodontally-affected tissue. Components of the RAS were investigated using in vitro, ex vivo and in vivo experiments involving both human and Wistar rat periodontium. Although not upregulated when challenged with P. gingivalis-lipopolysaccharide, human gingival and periodontal ligament fibroblasts expressed RAS components. Likewise, healthy and inflamed human gingiva expressed RAS components, some of which were shown to be functional, yet no differences in expression were found between healthy and diseased gingiva. However, in inflamed tissue the immunoreactivity was greater for the AT1R compared to AT2R in fibroblasts. When compared to healthy tissue, ACE activity was increased in human gingiva from volunteers with gingivitis. Human-gingiva homogenates generated Ang II, Ang 1-9 and Ang 1-7 when incubated with precursors. In gingiva homogenates, Ang II formation from Ang I was nearly abolished only when captopril and chymostatin were combined. Ang 1-7 formation was significantly greater when human gingiva homogenates were incubated with chymostatin alone compared to incubation without any inhibitor, only captopril, or captopril and chymostatin. In rat gingiva, RAS components were also found; their expression was not different between healthy and experimentally induced periodontitis (EP) groups. However, renin inhibition (aliskiren) and an AT1R antagonist (losartan) significantly blocked EP-alveolar-bone loss in rats. Collectively, these data are consistent with the hypothesis that a local RAS system is not only present but is also functional in both human and rat periodontal tissue. Furthermore, blocking AT1R and renin can significantly prevent periodontal bone loss induced by EP in rats.

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.

Ang II receptor expression and effect of Ang II receptor blockade in thyrotoxic rat myocardium.

AIM: To investigate the relationship between expression of the angiotensin II (Ang II) receptors and thyroid hormones in the myocardium of rats with thyrotoxicosis. MATERIALS AND METHODS: Forty-four adult male Sprague-Dawley rats were divided into four groups: control group (saline), losartan group (10 mg/kg), thyrotoxicosis group (0.5 mg/kg L-thyroid hormone sodium) and thyrotoxicosis-plus-losartan group (0.5 mg/kg L-thyroid hormone plus 10 mg/kg losartan) and treated intragastrically daily for four weeks. The heart weight (HW), body weight (BW) and HW/BW ratios were determined. The Ang II protein contents in cardiac homogenates and serum were determined by ELISA. The serum concentrations of levothyroxine (T3), trilodothyronine (T4) and thyroid stimulating hormone (TSH) were measured by radioimmunoassay. The expression of angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) were quantified by real-time PCR and Western blotting. RESULTS: The thyrotoxicosis group had an increased BW/HW and higher cardiac AT1R and AT2R expression compared to controls. AT1R and AT2R expressions significantly reduced in the thyrotoxicosis-plus-losartan group, compared to the thyrotoxicosis group. CONCLUSIONS: Thyroid hormone upregulated cardiac AT1R and AT2R, leading to cardiac remodeling, which was reversed by losartan. Cardiac damage in thyrotoxic rats may be related to upregulation of the Ang II receptors.

Transition from oxidative stress to nitrosative stress in rostral ventrolateral medulla underlies fatal intoxication induced by organophosphate mevinphos.

As the most widely used pesticides in the world, fatal incidence of suicidal poisoning by organophosphate compounds is high and is often associated with cardiovascular toxicity. Using the pesticide mevinphos as our tool, we investigated the roles of oxidative stress and nitrosative stress at the rostral ventrolateral medulla (RVLM), the brain stem site that maintains arterial pressure (AP) and sympathetic vasomotor tone, in the cardiovascular depressive effects of organophosphate poisons. Microinjection of mevinphos (10 nmol) into the RVLM of anesthetized Sprague-Dawley rats induced progressive hypotension that was accompanied by an increase (phase I), followed by a decrease (phase II) of an experimental index of baroreflex-mediated sympathetic vasomotor tone, with a fatality rate of 35%. During phase I, there was a preferential upregulation of angiotensin type I receptor (AT1R) messenger RNA (mRNA) and protein that leads to activation of NADPH oxidase (Nox) and increase in superoxide at the RVLM. Pharmacological antagonism of these signals exacerbated fatality and shorted survival time by eliminating baroreflex-mediated sympathetic vasomotor tone, AP, and heart rate. During phase II, there was a progressive upregulation of angiotensin type II receptor (AT2R) mRNA and protein that leads to increase in peroxynitrite in the RVLM, blockade of both sustained brain stem cardiovascular regulation and improved survival. We further found that AT1R and AT2R cross-interacted at transcriptional and signaling levels in the RVLM. We conclude that a transition from AT1R-mediated oxidative stress to AT2R-mediated nitrosative stress in the RVLM underlies the shift from sustained to impaired brain stem cardiovascular regulation that underpins cardiovascular fatality during mevinphos intoxication.

Commercially available angiotensin II At2 receptor antibodies are nonspecific.

Commercially available angiotensin II At2 receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, we characterized three commercially available At2 receptor antibodies: 2818-1 from Epitomics, sc-9040 from Santa Cruz Biotechnology, Inc., and AAR-012 from Alomone Labs. Using western blot analysis the immunostaining patterns observed were different for every antibody tested, and in most cases consisted of multiple immunoreactive bands. Identical immunoreactive patterns were present in wild-type and At2 receptor knockout mice not expressing the target protein. In the mouse brain, immunocytochemical studies revealed very different cellular immunoreactivity for each antibody tested. While the 2818-1 antibody reacted only with endothelial cells in small parenchymal arteries, the sc-9040 antibody reacted only with ependymal cells lining the cerebral ventricles, and the AAR-012 antibody reacted only with multiple neuronal cell bodies in the cerebral cortex. Moreover, the immunoreactivities were identical in brain tissue from wild-type or At2 receptor knockout mice. Furthermore, in both mice and rat tissue extracts, there was no correlation between the observed immunoreactivity and the presence or absence of At2 receptor binding or gene expression. We conclude that none of these commercially available At2 receptor antibodies tested met the criteria for specificity. In the absence of full antibody characterization, competitive radioligand binding and determination of mRNA expression remain the only reliable approaches to study At2 receptor expression.

Regulation of angiotensin II receptor expression.

The biological functions of angiotensin (Ang) II are mediated by two Ang II receptors, designated type 1 receptor (AT1R) and type 2 receptor (AT2R). Most of the cardiovascular effects of Ang II are mediated by AT1R that is expressed widely in the body. The expression of AT1R is up-regulated in cardiovascular lesions, and is regulated by many endogenous bioactive substances and drugs. The AT2R is generally considered to antagonize the effects of AT1R, but its precise function remains enigmatic and controversial, particularly in humans. The expression of AT2R is low in normal adult animals, but AT2R expression is up-regulated in cardiovascular lesions. The dynamic regulation of AT1R and AT2R expression suggests an active involvement of Ang II receptors in the development of cardiovascular diseases such as atherosclerosis, heart failure, chronic kidney diseases and cerebrovascular diseases. Further clarification of gene regulatory mechanisms of Ang II receptors may identify potential targets for the development of novel therapeutics for cardiovascular diseases.

Ang II and Ang IV: unraveling the mechanism of action on synaptic plasticity, memory, and epilepsy.

The central angiotensin system plays a crucial role in cardiovascular regulation. More recently, angiotensin peptides have been implicated in stress, anxiety, depression, cognition, and epilepsy. Angiotensin II (Ang II) exerts its actions through AT(1) and AT(2) receptors, while most actions of its metabolite Ang IV were believed to be independent of AT(1) or AT(2) receptor activation. A specific binding site with high affinity for Ang IV was discovered and denominated "AT(4) receptor". The beneficiary effects of AT(4) ligands in animal models for cognitive impairment and epileptic seizures initiated the search for their mechanism of action. This proved to be a challenging task, and after 20 years of research, the nature of the "AT(4) receptor" remains controversial. Insulin-regulated aminopeptidase (IRAP) was first identified as the high-affinity binding site for AT(4) ligands. Recently, the hepatocyte growth factor receptor c-MET was also proposed as a receptor for AT(4) ligands. The present review focuses on the effects of Ang II and Ang IV on synaptic transmission and plasticity, learning, memory, and epileptic seizure activity. Possible interactions of Ang IV with the classical AT(1) and AT(2) receptor subtypes are evaluated, and other potential mechanisms by which AT(4) ligands may exert their effects are discussed. Identification of these mechanisms may provide a valuable target in the development in novel drugs for the treatment of cognitive disorders and epilepsy.

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.

MAPK and angiotensin II receptor in kidney of newborn rats from losartan-treated dams.

Several lines of evidence suggest that angiotensin II (A-II) participates in the postnatal development of the kidney in rats. Many effects of A-II are mediated by mitogen-activated protein kinase (MAPK) pathways. This study investigated the influence that treatment with losartan during lactation has on MAPKs and on A-II receptor types 1 (AT(1)) and 2 (AT(2)) expression in the renal cortices of the offspring of dams exposed to losartan during lactation. In addition, we evaluated the relationship between such expression and changes in renal function and structure. Rat pups from dams receiving 2% sucrose or losartan diluted in 2% sucrose (40 mg/dl) during lactation were killed 30 days after birth, and the kidneys were removed for histological, immunohistochemical, and Western blot analysis. AT(1) and AT(2) receptors and p-p38, c-Jun N-terminal kinases (p-JNK) and extracellular signal-regulated protein kinases (p-ERK) expression were evaluated using Western blot analysis. The study-group rats presented an increase in AT(2) receptor and MAPK expression. In addition, these rats also presented lower glomerular filtration rate (GFR), greater albuminuria, and changes in renal structure. In conclusion, newborn rats from dams exposed to losartan during lactation presented changes in renal structure and function, which were associated with AT(2) receptor and MAPK expression in the kidneys.

Distribution of the non-AT1, non-AT2 angiotensin-binding site in the rat brain: preliminary characterization.

The discovery of a novel, non-AT1, non-AT2 binding site for angiotensin II (Ang II) in the brain adds a new dimension to the brain angiotensin system. The distribution of the non-AT1, non-AT2 binding site in the rat brain was determined using radioligand-binding assays and in vitro receptor autoradiography. There is a marked rostral to caudal gradient of the density of this binding site from the olfactory bulbs to the cervical spinal cord, with a consistent binding affinity, K(d) approximately 1-3 nM. Binding is widespread throughout the brain, however, areas of very intense binding are present in a large number of brain regions. The olfactory nerve layer of the olfactory bulb has the highest binding site density. Very high binding site density is also seen in the cerebral cortex with highest binding density in pyriform, insular and entorhinal cortex. Very high binding occurs in brain regions associated with dopaminergic reward (nucleus accumbens, ventral tegmental area) and motor (substantia nigra, caudate/putamen) systems. Very high to high binding also occurs in brain regions associated with the development of Alzheimer's disease (nucleus basalis of Meynert, substantia innominata). Very high to high binding is also seen in brain regions associated with cardiovascular regulation (subfornical organ, median, medial and anteroventral preoptic nucleus, paraventricular nucleus of the hypothalamus, solitary tract nucleus), areas that harbor high densities of the AT1 Ang II receptor subtype. High non-AT1, non-AT2 binding site density is present in brain regions containing high levels of the AT2 Ang II receptor subtype (amygdala, several thalamic nuclei, superior colliculus). Very high binding is also present in the choroid plexus, peri-third ventricular ependyma, and the subcommissural organ. The widespread, yet discrete distribution of high levels of this binding site suggests that it could function as a component of the blood-brain barrier, as a highly specific angiotensinase, or as a receptor for Ang II that mediates known and novel functions of this peptide, or that it serves as a clearance receptor for Ang II.

Characterization of the brain-specific non-AT(1), non-AT(2) angiotensin binding site in the mouse.

In the present study the existence of a non-AT(1), non-AT(2) angiotensin (Ang) binding site unmasked by the organomercurial protease inhibitor p-chloromercuribenzoate (PCMB) was demonstrated in mouse brain membranes, consistent with observations previously reported in the rat (Karamyan and Speth, 2007b). The pharmacological specificity of the non-AT(1), non-AT(2) angiotensin binding site was similar to the rat brain: Sar(1)-Ile(8)-Ang II > Ang III >or= Ang II > Ang I> p-aminophenylalanine(6) Ang II> CGP42112 >> Ang IV > Ang 1-7 congruent with shorter angiotensin fragments. Neurotensin, bradykinin, and luteinizing hormone-releasing hormone showed K(i) values >10 microM, while substance P and VIP had K(i) values of approximately 2 microM. The non-AT(1), non-AT(2) angiotensin binding site was not present in adrenal, liver or kidney. Subcellular fractionation showed a higher density of [(125)I]Ang II binding in plasma membrane (P2) fractions of cerebral cortex and hypothalamus relative to debris (P1) fractions. The binding site is present in the brains of mice in which the AT(1a), AT(1b), AT(2), Mas, and neprilysin (EC 3.4.24.11, neutral endopeptidase) was knocked out confirming that the binding site is not a heretofore described angiotensin receptor or neprilysin. These observations confirm that this novel Ang binding site is distinct from classical AT(1), AT(2), AT(4) and Ang 1-7 receptors while retaining a high specificity for angiotensins that act on the known angiotensin receptors. Whether this binding site functions as a novel receptor for angiotensins or a specific angiotensinase with variable functionality at different redox states will require further study.

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.

Fat redistribution and adipocyte transformation in uninephrectomized rats.

Dyslipidemia complicates renal function leading to disturbances of major homeostatic organs in the body. Here we examined the effect of chronic renal dysfunction induced by uninephrectomy on fat redistribution and lipid peroxidation in rats treated with an angiotensin-converting enzyme (ACE) inhibitor (lisinopril) for up to 10 months. Uninephrectomized rats developed fat redistribution and hypercholesterolemia typical of chronic renal failure when compared with sham-operated rats or lisinopril-treated uninephrectomized rats. The weight of the peri-renal fat was significantly less in the untreated compared to the lisinopril-treated uninephrectomized rats or those rats with a sham operation. We also found that there was a shift of heat-protecting unilocular adipocytes to heat-producing multilocular fat cells in the untreated uninephrectomized rats. Similarly in these rats we found a shift of subcutaneous and visceral fat to ectopic fat with excessive lipid accumulation and lipofuscin pigmentation. Lisinopril treatment prevented fat redistribution or transformation and lipid peroxidation. This study shows that ACE inhibition may prevent the fat anomalies associated with chronic renal dysfunction.