Effect of AT(1) receptor blockade on cardiac apoptosis in angiotensin II-induced hypertension

Angiotensin II (ANG II) via AT(1) receptors induces apoptosis in cardiomyocytes in vitro. We tested the hypothesis that in vivo AT(1) receptor stimulation is accompanied by cardiac apoptosis and attempted to elucidate the molecular mechanisms involved in the death signaling pathway. Male Sprague-Dawley rats received ANG II (120 ng x kg(-1) x min(-1) sc) for 7 days with or without the AT(1) receptor antagonist losartan (10 mg x kg(-1) x day(-1) orally). Cardiac function was assessed by echocardiography. Apoptosis in the heart was detected and quantified by in situ TdT-mediated dUTP nick-end labeling (TUNEL) and radiolabeled DNA laddering. Expression of bax, bcl-2, caspase 3, and AT(1) and AT(2) receptors was examined by Western blot analysis. Activity of caspase 3 was also measured by a fluorometric immunosorbent enzyme assay. Tail cuff systolic blood pressure was elevated (P < 0.01, n = 6) in ANG II-infused rats (173 +/- 3 mmHg) versus controls (111 +/- 2 mmHg) and reduced by losartan (134 +/- 4 mmHg). Cardiac function was essentially unchanged in ANG II-infused rats. Increased internucleosomal DNA cleavage by TUNEL assay and radiolabeled DNA laddering showed results compatible with enhanced cardiomyocyte apoptosis in the hearts of ANG-II infused rats. The bax-to-bcl-2 ratio, expression of the active form of caspase 3 (17 kDa), and activity of caspase 3 in the hearts of the ANG II group increased more than twofold above controls. Protein expression of AT(1) and AT(2) receptors was significantly increased in ANG II-infused rats compared with control rats. Losartan-treated ANG II-infused rats exhibited normalized apoptosis, bax, caspase 3 activity, and AT(1) receptors. ANG II stimulation of AT(1) receptors in the heart in vivo is associated with an increased rate of apoptosis without major hemodynamic consequences. Bax and caspase 3 are involved in the apoptotic signaling pathway in this experimental paradigm.     

Mechanisms and functions of AT(1) angiotensin receptor internalization

The type 1 (AT(1)) angiotensin receptor, which mediates the known physiological and pharmacological actions of angiotensin II, activates numerous intracellular signaling pathways and undergoes rapid internalization upon agonist binding. Morphological and biochemical studies have shown that agonist-induced endocytosis of the AT(1) receptor occurs via clathrin-coated pits, and is dependent on two regions in the cytoplasmic tail of the receptor. However, it is independent of G protein activation and signaling, and does not require the conserved NPXXY motif in the seventh transmembrane helix. The dependence of internalization of the AT(1) receptor on a cytoplasmic serine-threonine-rich region that is phosphorylated during agonist stimulation suggests that endocytosis is regulated by phosphorylation of the AT(1) receptor tail. beta-Arrestins have been implicated in the desensitization and endocytosis of several G protein-coupled receptors, but the exact nature of the adaptor protein required for association of the AT(1) receptor with clathrin-coated pits, and the role of dynamin in the internalization process, are still controversial. There is increasing evidence for a role of internalization in sustained signal generation from the AT(1) receptor. Several aspects of the mechanisms and specific function of AT(1) receptor internalization, including its precise mode and route of endocytosis, and the potential roles of cytoplasmic and nuclear receptors, remain to be elucidated.     

CoMFA and CoMSIA studies of angiotensin (AT1) receptor antagonists

Journal of Molecular Modeling -     

CoMFA and CoMSIA studies of angiotensin (AT1) receptor antagonists

Two 3D-QSAR methods--CoMFA and CoMSIA--were applied to a set of 38 angiotensin receptor (AT1) antagonists. The conformation and alignment of molecules were obtained by a novel method - consensus dynamics. The representation of biological activity, partial charge formalism, absolute orientation of the molecules in the grid, and grid spacing were also studied for their effect on the CoMFA models. The models were thoroughly validated through trials using scrambled activities and bootstrapping. The best CoMFA model had a cross-validated correlation coefficient ( q2) of 0.632, which improved with "region focusing" to 0.680. This model had a "predictive" r2 of 0.436 on a test series that was unique and with little representation in the training set. Although the "predictive" r2 of the best CoMSIA model, which included steric, electrostatic, and hydrogen bond acceptor fields was higher than that of the best CoMFA model, the other statistical parameters like q2, r2, F value, and s were unsatisfactory. The contour maps generated using the best CoMFA model were used to identify the structural features important for biological activity in these compounds.     

Cerebroprotective effects of angiotensin II (AT 1) receptor antagonists?

The results of the Acute Candesartan Cilexetil Therapy in Stroke Survivors (ACCESS) study show that treatment with an angiotensin receptor blocker (ARB) in the acute phase of a stroke improves mortality and cardiovascular morbidity. In addition, direct comparative antihypertensive trials have demonstrated beneficial effects of ARBs in preventing stroke. These possible cerebro-protective effects of ARBs are supported by animal studies, demonstrating that stimulation of the AT2 receptor was related to a reduction in both cerebral infarct size and mortality. In the present report, we review both pathophysiological and clinical evidence for possible cerebroprotective effects of ARBs, independent of their effect on blood pressure.     

Characterization of a polyclonal anti-peptide antibody to the angiotensin II type-1 (AT1) receptor.

A polyclonal antibody has been prepared against a synthetic peptide corresponding to amino acids 14-23 of the angiotensin II type-1 (AT1) receptor. The antibody is of high titer and mono-specific. Western blot analysis of membranes from rat liver, kidney, and adrenal gland showed that the antibody specifically recognizes a protein band of MW 70,000 whose amounts are highest in the liver, followed by kidney and adrenals. In addition, a relatively less prominent band of MW 95,000 was also detected. The relative distribution of this protein correlates well with the values obtained for [3H]-DuP753 binding and AT1 receptor mRNA.     

The angiotensin II AT2 receptor is an AT1 receptor antagonist

The vasopressor angiotensin II activates AT(1) and AT(2) receptors. Most of the known in vivo effects of angiotensin II are mediated by AT(1) receptors while the biological functions of AT(2) receptors are less clear. We report here that the AT(2) receptor binds directly to the AT(1) receptor and thereby antagonizes the function of the AT(1) receptor. The AT(1)-specific antagonism of the AT(2) receptor was independent of AT(2) receptor activation and signaling, and it was effective on different cells and on human myometrial biopsies with AT(1)/AT(2) receptor expression. Thus, the AT(2) receptor is the first identified example of a G-protein-coupled receptor which acts as a receptor-specific antagonist.     

Interaction of angiotensin II with the C-terminal 300-320 fragment of the rat angiotensin II receptor AT1a monitored by NMR

Interaction between angiotensin II (Ang II) and the fragment peptide 300-320 (fCT300-320) of the rat angiotensin II receptor AT1a was demonstrated by relaxation measurements, NOE effects, chemical shift variations, and CD measurements. The correlation times modulating dipolar interactions for the bound and free forms of Ang II were estimated by the ratio of the nonselective and single-selective longitudinal relaxation rates. The intermolecular NOEs observed in NOESY spectra between HN protons of 9Lys(fCT) and 6His(ang), 10Phe(fCT) and 8Phe(ang), HN proton of 3Tyr(fCT) and Halpha of 4Tyr(ang), 5Phe(fCT)Hdelta and Halpha of 4Tyr(ang) indicated that Ang II aromatic residues are directly involved in the interaction, as also verified by relaxation data. Some fCT300-320 backbone features were inferred by the CSI method and CD experiments revealing that the presence of Ang II enhances the existential probability of helical conformations in the fCT fragment. Restrained molecular dynamics using the simulated annealing protocol was performed with intermolecular NOEs as constraints, imposing an alpha-helix backbone structure to fCT300-320 fragment. In the built model, one strongly preferred interaction was found that allows intermolecular stacking between aromatic rings and forces the peptide to wrap around the 6Leu side chain of the receptor fragment.     

Expression of angiotensin II type 1 (AT(1)) receptor in the rostral ventrolateral medulla in rats.

In the present study, the changes of amino acids release in the spinal cord after the application of angiotensin II (ANG II) in the rostral ventrolateral medulla (RVLM) and the distribution of ANG receptors on neurons of the RVLM were investigated. A microdialysis experiment showed that microinjection of angiotensin II into the RVLM significantly (P < 0.01) increased the release of aspartate and glutamate in the intermediolateral column of the spinal cord. Immunofluorescence technique combined with confocal microscopy demonstrated that most of the glutamatergic and GABAergic neurons in the RVLM of both Wistar and spontaneously hypertensive rats (SHR) were double labeled with ANG type 1 (AT1) receptor. Immunocytochemical studies demonstrated that the mean optic density of AT1 receptor of the cell surface as well as the whole cell was higher (P < 0.05) in SHR than that in Wistar rats, indicating that the higher expression of AT1 receptors in the RVLM may contribute to the higher responsiveness of SHR to ANG II stimulation. Immunogold staining and electronmicroscopic study demonstrated that AT1 receptor in the RVLM was distributed on the rough endoplasmic reticulum, cell membrane, and nerve processes. The results suggest that effects evoked by ANG II in the RVLM are closely related to glutamatergic and GABAergic pathways. These results indirectly support the hypothesis that ANG II in the RVLM may activate vasomotor sympathetic glutamatergic neurons, leading to an increase in sympathetic nerve activity and arterial blood pressure.     

Increased AT(1) receptor heterodimers in preeclampsia mediate enhanced angiotensin II responsiveness

Several examples of functional G-protein-coupled receptor heterodimers have been identified. However, it is not known whether receptor heterodimerization is involved in the pathogenesis of human disorders. Here we show that in preeclamptic hypertensive women, a significant increase in heterodimerization occurs between the AT(1)-receptor for the vasopressor angiotensin II and the B(2)-receptor for the vasodepressor bradykinin. AT(1)-B(2)-receptor heterodimerization in preeclampsia correlated with a 4-5-fold increase in B(2)-receptor protein levels. Expression of the AT(1)-B(2) heterodimer increased the responsiveness to angiotensin II and conferred resistance in AT(1)-receptors to inactivation by reactive oxygen species raised in normotensive and preeclamptic pregnancies. We suggest that AT(1)-B(2) heterodimers contribute to angiotensin II hypersensitivity in preeclampsia. Moreover, we identify preeclampsia as the first disorder associated with altered G-protein-coupled receptor heterodimerization.     

125I[Sar(1)Ile(8)] angiotensin II has a different affinity for AT(1) and AT(2) receptor subtypes in ovine tissues

Iodinated angiotensin II (Ang II) and its analogues are often assumed to have equal affinities for AT(1) and AT(2) receptor subtypes. However, using saturation and competition binding assays in several tissues from pregnant, nonpregnant, and fetal sheep, we found the affinity of 125I[Sar(1)Ile(8)] Ang II for Ang II receptors was different (P<0.05) between tissue types. The dissociation constants (Kd) and half maximal displacements of [Sar(1)Ile(8)] Ang II (Sar IC(50)) were directly related (P<0.05) to proportions of AT(1) receptors, and inversely related (P<0.05) to proportions of AT(2) receptors in tissues from all groups combined, in tissues from individual groups (pregnant, nonpregnant or fetal), and in some individual tissues (uterine arteries and aortae). This suggests that 125I[Sar(1)Ile(8)] Ang II has a different affinity for AT(1) and AT(2) receptors in ovine tissues. The Kds of 125I[Sar(1)Ile(8)] Ang II for "pure" populations of AT(1) and AT(2) receptors were 1.2 and 0.3 nM, respectively, i.e. affinity was four-fold higher for AT(2) receptors. We corrected the measured proportions of the receptor subtypes using their fractional occupancies. In tissues which contained at least 10% of each receptor subtype, the corrected proportions were significantly altered (P<0.05), even in some tissues, to the extent of being reversed.     

Stable expression of a functional rat angiotensin II (AT1A) receptor in CHO-K1 cells: Rapid desensitization by angiotensin II

The octapeptide angiotensin II mediates the physiological actions of the renin-angiotensin system through activation of several angiotensin II receptor subtypes; in particular the AT₁. In many tissues, the presence of multiple angiotensin II receptor subtypes, together with a low number of receptors, makes it difficult to study biological responses to physiological concentrations (10^–11–10^–9 M) of angiotensin II. Also, cultured cells show diminished angiotensin II receptor binding with respect to time in culture and passage number. To address these problems, we expressed the recombinant AT_1A receptor in CHO-K1 cells. The stably transfected receptor was characterized using radioligand binding studies and functional coupling to cytosolic free calcium. Radioligand binding of [¹²⁵I] angiotensin II to the angiotensin II receptor was specific, saturable, reversible and modulated by guanine nucleotides. Like the endogenous AT_1A receptor, reported in a variety of tissues, the specific, noncompetitive, nonpeptide AII receptor antagonist, EXP3174, blocked binding of [¹²⁵I] angiotensin II to the transfected receptor. Scatchard analysis demonstrated that the transfected receptor had a dissociation constant of 1.9 nM with a density of 3.4 pmol/mg protein.An important feature of many of the responses to angiotensin II is the rapid desensitization that occurs following agonist occupancy and the development of tachyphylaxis. In AT_1A receptor transfected CHO-K1 cells, angiotensin II (10^–9 M) stimulated a rapid increase in cytosolic free calcium that was completely desensitized within 50 sec following receptor occupancy. Agonist induced desensitization was unaffected when receptor internalization was blocked by pretreatment with concanavalin A or incubation at 4°C, and no changes in AT_1A receptor affinity or number were observed. Receptor desensitization was also unaffected by inhibition or activation of protein kinase C. Thus, we have established a permanent, high-level transfectant of the AT_1A receptor in CHO-K1 cells and have shown that these receptors rapidly desensitize following exposure to physiological concentrations of agonist. The mechanism of rapid desensitization is not related to receptor sequestration, internalization or controlled by PKC phosphorylation. This provides an excellent model for studying AII actions mediated through a specific receptor subtype, at subnanomolar concentrations.     

The novel angiotensin II type 1 receptor (AT1R)-associated protein ATRAP downregulates AT1R and ameliorates cardiomyocyte hypertrophy

Activation of angiotensin II (Ang II) type 1 receptor (AT1R) signaling is reported to play an important role in cardiac hypertrophy. We previously cloned a novel molecule interacting with the AT1R, which we named ATRAP (for Ang II type 1 receptor-associated protein). Here, we report that overexpression of ATRAP significantly decreases the number of AT1R on the surface of cardiomyocytes, and also decreases the degree of p38 mitogen-activated protein kinase phosphorylation, the activity of the c-fos promoter and protein synthesis upon Ang II treatment. These results indicate that ATRAP significantly promotes downregulation of the AT1R and further attenuates certain Ang II-mediated hypertrophic responses in cardiomyocytes.     

Specific single chain variable fragment (ScFv) antibodies to angiotensin II AT2 receptor: evaluation of the angiotensin II receptor expression in normal and tumor-bearing mouse lung

To gain insight into the mechanism by which angiotensin II type 2 receptor (AT(2)) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT(2) single chain variable fragment (ScFv) antibodies using a rodent phage-displayed recombinant antibody library with various peptide fragments of the receptor protein, and investigated the expression of the AT(2) receptor protein. The specificity of the antibodies was verified using AT(2) over-expressing COS-7 cells and AT(2) naturally expressing PC12W cells. In control wild type mouse lung, a stronger immunoreactivity was observed in bronchial epithelial cells. A moderate immunoreactivity was detected in pulmonary vascular walls and vascular endothelial cells. In the lungs possessing tobacco-specific nitrosamine (NNK)-induced tumors, significantly increased AT(2) and AT(1 )immunostaining was observed in adenomatous lesions. These data suggest that the increase in both receptors' expression in the alveolar epithelial cells may be accompanied with the onset of NNK-induced tumorigenesis and hence play important roles in lung tumorigenesis.     

Correlation between VEGFR-2 receptor kinase domain-containing receptor (KDR) mRNA and angiotensin II receptor type 1 (AT1-R) mRNA in endometrial cancer

PurposeAngiogenesis, a multistep process that results in new blood vessel formation from preexisting vasculature is essential for both the growth of solid tumour and for metastasis. Stimulation of vascular endothelial growth factor receptor (VEGFR), a transmembrane glycoprotein, results in mitogenesis. Within this family of receptors, VEGFR 2/kinase-insert-domain containing receptor appears to be principally upregulated during tumorigenesis. The aim of this study was to determine the expression of VEGFR-2/kinase-insert-domain containing receptor (KDR) and its correlation with angiotensin receptor type 1 (AT1-R) and clinical factors in endometrial carcinoma.MethodsThe expression of KDR and AT1-R was studied in endometrial carcinoma and normal endometrium by Real-time RT-PCR and Western blot analysis in 136 samples. The expression profile was correlated with the clinicopathological characteristics of endometrial adenocarcinoma.ResultsWe noted a significant correlation between the expression of KDR and AT1-R in tumour grade G1, G2 and G3 (R_s = 0.50; p = 0.002, R_s = 0.69; p = 0.0001, R_s = 0.52; p = 0.005, respectively). In stage I and stage II carcinoma, a significant correlation was also found between the expression of KDR and AT1-R (R_s = 0.70, p = 0.0001, R_s = 0.67; p = 0.001, respectively). Moreover significant correlation was observed between both KDR and AT1-R in tissue with different myometrial invasion (R_s = 0.54, p = 0.0001, R_s = 0.68; p = 0.0001; respectively for tumours with invasion into the inner half and invasion into the outer half).ConclusionsBasing on received correlation between AT1-R and KDR expression and previous results we speculate that angiotensin through AT1-R modulates KDR expression and thus have influence on local VEGF level. However, further studies are required to clarify the biological interaction between KDR, AT1-R and other hormonal regulators in endometrial carcinoma.     

Identification and characterization of naturally occurring variants of the macrophage scavenger receptor (SR-A)

The scavenger receptor (SR) family comprises a group of cell surface proteins functionally defined by their ability to bind chemically modified lipoproteins. In macrophages, the class A Type I and Type II SRs (SR-AI/II) are thought to play a key role in adherence to and phagocytosis of infectious agents. Immunoprecipitation studies show that the rat anti-SR-AI/II monoclonal antibody 2F8 detects the mature, trimeric form of the receptor expressed in peritoneal macrophages from A/J, but not from C57Bl/6J (B6) mice. Subsequent sequencing of cDNA and genomic clones indicates that SR-AI and AII of A/J and B6 mice differ in sequence at nine positions, two in the cytoplasmic domain and seven in the extracellular spacer and α-helical coiled coil domains. These sequence polymorphisms are non-conservative and produce distinct receptor molecules that differ by four charged residues and alter recognition of the receptor by the monoclonal 2F8 antibody. The B6 SR-AI/II haplotype appears unique, since most inbred strains analyzed show the A/J-type haplotype. Interestingly, several of the B6 polymorphic variant residues are conserved in human and bovine receptors, suggesting a recent divergence of the A/J haplotype. Initial studies in CHO-derived cells expressing individual receptor isoforms indicate that the A/J and B6 receptors are stable and can mature into oligomers expressed in the membrane fractions of these cells. In these transfectants, no major functional differences were detected between receptors of the two haplotypes with respect to internalization and degradation of ¹²⁵I-labeled acetylated LDL. However, since SR-AI/II recognizes a large number of structurally unrelated anionic molecules, the possibility that different haplotypes may affect either binding and release of other ligands, or receptor recycling, cannot be excluded. Received: 11 January 2000 / Accepted: 30 March 2000     

Effects of captopril and angiotensin II receptor blockers (AT1, AT2) on myocardial ischemia-reperfusion induced infarct size

The renin-angiotensin system (RAS) plays a major role in regulating the cardiovascular system, and disorders of the RAS contribute largely to the cardiac pathophysiology, including myocardial ischemia-reperfusion (MI/R) injury. Two subtypes of angiotensin II (Ang II) receptors have been defined on the basis of their differential pharmacological properties. The current study was undertaken to address the question as to whether the inhibition of the angiotensin converting enzyme (ACE) by captopril and the AT1 and AT2 receptor blockers losartan and PD123319 modulate MI/R-induced infarct size in an in vivo rat model. To produce necrosis, a branch of the descending left coronary artery was occluded for 30 min followed by two hours of reperfusion. ECG changes, blood pressure, and heart rate were measured during the experiment. Captopril (3 mg/kg), losartan (2 mg/kg), and PD123319 (20 μg/kg/min) were given in an IV 10 min before ischemia and were continued during the ischemic period. The infarcted area was measured by TTC staining. The volume of infarct and the risk zone was determined by planimetry. Compared to the control group (55.62±4.00%) both captopril and losartan significantly reduced the myocardial infarct size (30.50±3.26% and 37.75±4.44%), whereas neither PD123319 nor PD123319+losartan affected the infarct size volume (46.50±3.72% and 54.62±2.43%). Our data indicates that captopril and losartan exert cardioprotective activity after an MI/R injury. Also, infarct size reduction by losartan was halted by a blockade of the AT2 receptor. Therefore, the activation of AT2 receptors may be potentially protective and appear to oppose the effects mediated by the AT1 receptors.     

Interaction of a non-peptide agonist with angiotensin II AT1 receptor mutants

To identify residues of the rat AT1A angiotensin II receptor involved with signal transduction and binding of the non-peptide agonist L-162,313 (5,7-dimethyl-2-ethyl-3-[[4-[2(n-butyloxycarbonylsulfonamido)-5-isobutyl-3-thienyl]phenyl]methyl]imidazol[4,5,6]-pyridine) we have performed ligand binding and inositol phosphate turnover assays in COS-7 cells transiently transfected with the wild-type and mutant forms of the receptor. Mutant receptors bore modifications in the extracellular region: T88H, Y92H, G1961, G196W, and D278E. Compound L-162,313 displaced [125I]-Sar1,Leu8-AngII from the mutants G196I and G196W with IC50 values similar to that of the wild-type. The affinity was, however, slightly affected by the D278E mutation and more significantly by the T88H and Y92H mutations. In inositol phosphate turnover assays, the ability of L-162,313 to trigger the activation cascade was compared with that of angiotensin II. These assays showed that the G196W mutant reached a relative maximum activation exceeding that of the wild-type receptor; the efficacy was slightly reduced in the G1961 mutant and further reduced in the T88H, Y92H, and D278E mutants. Our data suggest that residues of the extracellular domain of the AT1 receptor are involved in the binding of the non-peptide ligand, or in a general receptor activation phenomenon that involves conformational modifications for a preferential binding of agonists or antagonists.     

Characterization of domain borders and of a naturally occurring major fragment of staphylococcal alpha-toxin

A naturally occurring staphylococcal alpha-toxin fragment with an apparent membrane-binding capacity but without toxic activities is shown to be derived from the C-terminal half of the intact polypeptide chain by cleavage between position 134 and 135 in the parent molecule. The resulting N-terminus is slightly ragged with a fragment start not only at position 135 but also at the adjacent position 136. Another naturally occurring fragment starts at position 9, derived from an original cleavage between position 8 and 9 in the parent molecule. Analysis of non-purified fragment mixtures confirmed these positions and established that only one further region, at positions 71-72, is partly sensitive to proteolysis under natural conditions. Trypsin treatment has limited effects on the native toxin molecule, giving essentially only two initial cleavages with resultant large fragments. One of these cleavages is at the peptide bond between position 131 and 132, thus only three residues away from the position of the major naturally occurring cleavage. The other bond sensitive to trypsin is between position 8 and 9, thus identically positioned to the cleavage occurring naturally. Together, all the cleavages define a region in a central segment of the polypeptide chain that has all the properties of an inter-domain segment. The C-terminal half appears to constitute a membrane-binding domain, and the N-terminal half a structure needed for full biological activity, functionally subdividing the parent polypeptide chain.