Angiotensin II type 2 receptor-mediated duodenal mucosal alkaline secretion in the rat

The aims of this study were to elucidate the distribution of angiotensin receptors (AT(1) and AT(2)) in the duodenal wall and to investigate whether AT(2) receptors are involved in the regulation of duodenal mucosal alkaline secretion, which is of importance for the mucosal defense against gastric acid. Immunohistochemistry was used to locate AT(1) and AT(2) receptors in chloralose-anesthetized rats. Duodenal mucosal alkaline output was measured by use of in situ pH-stat titration. Immunohistochemistry demonstrated a distinct staining for both AT(1) and AT(2) receptors in the lamina propria of the villi and also for AT(1) receptors in the muscularis interna. When angiotensin II was infused in the presence of the AT(1) receptor antagonist losartan, mucosal alkaline secretion increased by ~50%. This response was inhibited by the AT(2) receptor antagonist PD-123319. The AT(2) receptor agonist CGP-42112A increased mucosal alkaline secretion by ~50%. This increase was absent in the presence of PD-123319 but not in the presence of losartan or the local anesthetic lidocaine. We conclude that angiotensin II stimulates duodenal mucosal alkaline secretion by activation of AT(2) receptors located in the duodenal mucosa/submucosa.     

The angiotensin II type 1 receptor antagonist Losartan binds and activates bradykinin B2 receptor signaling

The angiotensin II type 1 receptor (AT1R) blocker (ARB) Losartan has cardioprotective effects during ischemia-reperfusion injury and inhibits reperfusion arrhythmias -effects that go beyond the benefits of lowering blood pressure. The renin-angiotensin and kallikrein-kinin systems are intricately connected and some of the cardioprotective effects of Losartan are abolished by blocking the bradykinin B2 receptor (B2R) signaling. In this study, we investigated the ability of six clinically available ARBs to specifically bind and activate the B2R. First, we investigated their ability to activate phosphoinositide (PI) hydrolysis in COS-7 cells transiently expressing the B2R. We found that only Losartan activated the B2R, working as a partial agonist compared to the endogenous ligand bradykinin. This effect was blocked by the B2R antagonist HOE 140. A competitive binding analysis revealed that Losartan does not significantly compete with bradykinin and does not change the binding affinity of bradykinin on the B2R. Furthermore, Losartan but not Candesartan mimicked the ability of bradykinin to increase the recovery of contractile force after metabolic stress in rat atrial tissue strips. In conclusion, Losartan is a partial agonist of the B2R through direct binding and activation, suggesting that B2R agonism could partly explain the beneficial effects of Losartan.     

EP24.15 interacts with the angiotensin II type I receptor and bradykinin B2 receptor

The carboxyl-terminal cytoplasmic domain of the angiotensin II type 1 receptor (AT1) is known to interact with several classes of intracellular proteins that may modulate receptor function. Employing yeast two-hybrid screening of a human embryonic kidney cDNA library with the carboxyl-terminal cytoplasmic domain of the AT1 receptor as a bait, we have isolated EP24.15 (EC 3.4.24.15, thimet oligopeptidase) as a potentially interacting protein. EP24.15 is widely distributed and is known to degrade bioactive peptides such as angiotensin I and II and bradykinin. In addition, EP24.15 was previously identified as a putative soluble angiotensin II binding protein. Two-hybrid screening also determined that EP24.15 can interact with the B2 bradykinin receptor. Transient expression of EP24.15 in a porcine kidney epithelial cell line stably expressing full length AT1 and full length B2 followed by affinity chromatography and co-immunoprecipitation confirmed EP24.15 association with both AT1 and B2 receptors. EP24.15 was also co-immunoprecipitated with AT1 and B2 in rat kidney brush border membranes (BBM) and basolateral membranes (BLM). Both AT1 and B2 undergo ligand-induced endocytosis. Analysis of endosomal fractions following immunoprecipitation with AT1 or B2 antibodies detected strong association of EP24.15 with the receptors in both light and heavy endosomal populations. Therefore, the present study indicates that EP24.15 associates with AT1 and B2 receptors both at the plasma membrane and after receptor internalization and suggests a possible mechanism for endosomal disposition of ligand that may facilitate receptor recycling.     

Overexpression of angiotensin II type 2 receptor promotes apoptosis and impairs insulin secretion in rat insulinoma cells

Proper follicular development is crucial for cumulus-oocyte complex (COC) maturation, ovulation and luteinisation. All these ovarian processes are regulated by finely tuned rapid tissue remodeling that involves hyaluronan and interconnecting hyaladherins-rich extracellular matrix synthesis and its breakdown by various proteinase systems like matrix metalloproteinase (MMP). Disrupted tissue remodeling machinery can result into pathophysiologies like atretic follicular cysts formation in polycystic ovary syndrome (PCOS). In present study, we employ superovulated (SO) and polycystic ovary (PCO) rat models and demonstrate that on contrary to SO, PCO rat ovary illustrates abnormal follicular morphology with differential levels of various ovarian factors [like HA (hyaluronan), TSG-6 (TNF-α-stimulated gene/protein 6), PTX-3 (pentraxin-3), HABP1 (hyaluronan binding protein 1), MMP2 (matrix metalloproteinase), MT1-MMP (membrane type 1-matrix metalloproteinase) and COX2 (Cyclooxygenase-2)] along with hyperactivities of gelatinases (like MMP9 and -2). Besides cultured COC expansion is blocked by anti-HABP1 antibody treatment showing reduced HABP1 expression. Overall, as MT1-MMP has inverse relation with HABP1 level and direct effect on MMP2 activity, the observations from current in vivo and in vitro studies indicate that disrupted ovarian HABP1 along with concurrent altered expression and hyperactivation of related MMPs can lead to abnormal follicular maturation resulting into ovarian dysfunction in PCO rat.     

Intracellular angiotensin II inhibits heterologous receptor stimulated Ca2+ entry.

Recent studies show that angiotensin II (AngII) can act from within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane AngII receptors. The role of this intracellular AngII (AngIIi) is unclear. Besides direct effects of AngIIi on cellular processes one could hypothesise a possible role of AngIIi in modulation of cellular responses induced after heterologous receptor stimulation. We therefore examined if AngIIi influences [Ca+]i in A7r5 smooth muscle cells after serotonin (5HT) or UTP receptor stimulation. Application of AngIIi using liposomes, markedly inhibited 45Ca2+ influx after receptor stimulation with 5HT or UTP. This inhibition was reversible by intracellular administration of the AT1-antagonist losartan and not influenced by the AT2-antagonist PD123319. Similar results were obtained in single cell [Ca2+]i measurements, showing that AngIIi predominantly influences Ca2+ influx and not Ca2+ release via AT1-like receptors. It is concluded that AngIIi modulates signal transduction activated by heterologous receptor stimulation.     

The angiotensin II type 2 receptor: an enigma with multiple variations

Since it was discovered ten years ago, the angiotensin II (ANG II) type 2 (AT(2)) receptor has been an enigma. This receptor binds ANG II with a high affinity but is not responsible for mediating any of the classical physiological actions of this peptide, all of which involve the ANG II type 1 (AT(1)) receptor. Furthermore, the AT(2) receptor exhibits dramatic differences in biochemical and functional properties and in patterns of expression compared with the AT(1) receptor. During the past decade, much information has been gathered about the AT(2) receptor, and the steadily increasing number of publications indicates a growing interest in this new and independent area of research. A number of studies suggest a role of AT(2) receptors in brain, renal, and cardiovascular functions and in the processes of apoptosis and tissue regeneration. Despite these advances, nothing stands out as the major singular function of these receptors. The study of AT(2) receptors has reached a crossroads, and innovative approaches must be considered so that unifying mechanisms as to the function of these unique receptors can be put forward. In this review we will discuss the advances that have been made in understanding the biology of the AT(2) receptor. Furthermore, we will consider how these discoveries, along with newer experimental approaches, may eventually lead to the elusive physiological and pathophysiological functions of these receptors.     

Phosphorylation of tyrosine 319 of the angiotensin II type 1 receptor mediates angiotensin II-induced trans-activation of the epidermal growth factor receptor

Although tyrosine kinases are critically involved in the angiotensin II (Ang II) type 1 (AT1) receptor signaling, how AT1 receptors activate tyrosine kinases is not fully understood. We examined the structural requirements of the AT1 receptor for transactivation of the epidermal growth factor (EGF) receptor (EGFR). Studies using carboxyl terminal-truncated AT1 receptors indicated that the amino acid sequence between 312 and 337 is required for activation of EGFR. The role of the conserved YIPP motif in this sequence in transactivation of EGFR was investigated by mutating tyrosine 319. Ang II failed to activate EGFR in cells expressing AT1-Y319F, whereas EGFR was activated even without Ang II in cells expressing AT1-Y319E, which mimics the AT1 receptor phosphorylated at Tyr-319. Immunoblot analyses using anti-phospho Tyr-319-specific antibody showed that Ang II increased phosphorylation of Tyr-319. EGFR interacted with the AT1 receptor but not with AT1-Y319F in response to Ang II stimulation, whereas the EGFR-AT1 receptor interaction was inhibited in the presence of dominant negative SHP-2. The requirement of Tyr-319 seems specific for EGFR because Ang II-induced activation of other tyrosine kinases, including Src and JAK2, was preserved in cells expressing AT1-Y319F. Extracellular signal-regulated kinase activation was also maintained in AT1-Y319F through activation of Src. Overexpression of wild type AT1 receptor in cardiac fibroblasts enhanced Ang II-induced proliferation. By contrast, overexpression of AT1-Y319F failed to enhance cell proliferation. In summary, Tyr-319 of the AT1 receptor is phosphorylated in response to Ang II and plays a key role in mediating Ang II-induced transactivation of EGFR and cell proliferation, possibly through its interaction with SHP-2 and EGFR.     

The angiotensin Ⅱ type 1 receptor and receptor－associated proteins

The mechanisms of regulation, activation and signal transduction of the angiotensin II (Ang II) type 1 (AT1) receptor have been studied extensively in the decade after its cloning. The AT1 receptor is a major component of the renin-angiotensin system (RAS). It mediates the classical biological actions of Ang II. Among the structures required for regulation and activation of the receptor, its carboxyl-terminal region plays crucial roles in receptor internalization, desensitization and phosphorylation. The mechanisms involved in heterotrimeric G-protein coupling to the receptor, activation of the downstream signaling pathway by G proteins and the Ang II signal transduction pathways leading to specific cellular responses are discussed. In addition, recent work on the identification and characterization of novel proteins associated with carboxyl-terminus of the AT1 receptor is presented. These novel proteins will advance our understanding of how the receptor is internalized and recycled as they provide molecular mechanisms for the activation and regulation of G-protein-coupled receptors.     

Ligand-independent signals from angiotensin II type 2 receptor induce apoptosis

Conventional models of ligand-receptor regulation predict that agonists enhance the tone of signals generated by the receptor in the absence of ligand. Contrary to this paradigm, stimulation of the type 2 (AT(2)) receptor by angiotensin II (Ang II) is not required for induction of apoptosis but the level of receptor protein expression is critical. We compared Ang II-dependent and -independent AT(2) receptor signals involved in regulating apoptosis of cultured fibroblasts, epithelial cells and vascular smooth muscle cells. We found that induction of apoptosis-blocked by pharmacological inhibition of p38 mitogen-activated protein kinase and caspase 3-is a constitutive function of the AT(2) receptor. Biochemical and genetic studies suggest that the level of AT(2) receptor expression is critical for physiological ontogenesis and its expression is restricted postnatally, coinciding with cessation of developmental apoptosis. Re-expression of the AT(2) receptor in remodeling tissues in the adult is linked to control of tissue growth and regeneration. Therefore, we propose that overexpression of the AT(2) receptor itself is a signal for apoptosis that does not require the renin-angiotensin system hormone Ang II.     

Interaction between bradykinin subtype 2 and angiotensin II type 2 receptors during post-MI left ventricular remodeling

Angiotensin II type 2 receptor (AT(2)R) overexpression (AT(2)TG) attenuates left ventricular remodeling in a mouse model of anterior myocardial infarction (MI). We hypothesized that the beneficial effects of cardiac AT(2)TG are mediated via the bradykinin subtype 2 receptor (B(2)R). Fourteen transgenic mice overexpressing the AT(2)R (AT(2)TG mice), 10 mice with a B(2)R deletion (B(2)KO mice), 13 AT(2)TG mice with B(2)R deletion (AT(2)TG/B(2)KO mice), and 11 wild-type (WT) mice were studied. All mice were on a C57BL/6 background. Mice were studied by cardiac magnetic resonance imaging at baseline and days 1, 7, and 28 after MI induced by 1 h of occlusion of the left anterior descending artery followed by reperfusion. Short-axis images from apex to base were used to compare ventricular volumes and ejection fraction (EF). At baseline, end-diastolic volume index (EDVI) and end-systolic volume index (ESVI) were lower and EF higher in AT(2)TG mice compared with the other three strains. Infarct size was similar between groups. No differences were observed in global remodeling parameters at day 28 between AT(2)TG and AT(2)TG/B(2)KO mice; however, EDVI and ESVI were lower and EF higher in both transgenic groups than in WT or B(2)KO mice. Both strains lacking B(2)R demonstrated increased collagen content and less hypertrophy in adjacent noninfarcted regions at day 28. Attenuation of postinfarct remodeling by overexpression of AT(2)R is not directly mediated via a B(2)R pathway. However, B(2)R does appear to have a role in the smaller cavity size and hyperdynamic function observed at baseline in AT(2)TG mice and in limiting collagen deposition during postinfarct remodeling.     

The scaffold protein CNK1 interacts with the angiotensin II type 2 receptor

The scaffold protein CNK1 mediates proliferative as well as antiproliferative responses including differentiation and apoptosis. The angiotensin II type 2 (AT2) receptor belongs to the class of G protein-coupled receptors and also promotes antiproliferative effects. Here we report that CNK1 binds through the sterile alpha motif (SAM) and the conserved region in CNK (CRIC) to the AT2 receptor. The exchange of a conserved leucine residue with arginine in the CRIC domain increases the binding affinity of CNK1 to the AT2 receptor. The insertion of a negatively charged amino acid stretch into the linker region between the N- and the C-terminal part of CNK1 strengthens the interaction between CNK1 and the AT2 receptor in a Ras-regulated manner. The biological significance of the interaction was supported by coprecipitation of CNK1 and the AT2 receptor in mouse heart extracts. Thus, CNK1 may play a role in the AT2 receptor-mediated signaling pathways.     

Angiotensin III stimulates high stretch-induced ANP secretion via angiotensin type 2 receptor

Angiotensin III (Ang III) is metabolized from Ang II by aminopeptidase (AP) A and in turn, Ang III is metabolized to Ang IV by APN. Ang III is known to have a similar effect to Ang II on aldosterone secretion, but the effect of Ang III on atrial natriuretic peptide (ANP) secretion from cardiac atria is not known. The aim of the present study is to define the effect of Ang III on ANP secretion and its receptor subtype using isolated perfused beating atria. The volume load was achieved by elevating the height of outflow catheter connected with isolated atria from 5 cmH₂O to 7.5 cmH₂O. Atrial stretch by volume load increased atrial contractility and ANP secretion. Ang III stimulated stretch-induced ANP secretion in a dose-dependent manner without change in atrial contractility. The stimulated effect of Ang III (1 μM) on stretch-induced ANP secretion was blocked by the pretreatment of Ang II type 2 (AT₂) receptor antagonist but not by AT₁ or Mas receptor antagonist. Pretreatment with inhibitor of phosphoinositide 3-kinase (PI3K), Akt, nitric oxide synthase, soluble guanylyl cyclase, or protein kinase G (PKG) attenuated Ang III-stimulated ANP secretion. When Ang III (40 nM) or Ang II (4 nM) was infused for 10 min into anesthetized rats, mean arterial pressure was increased about 10%. However, Ang III increased plasma ANP level by 35.81 ± 10.19% but Ang II decreased plasma ANP level by 30.41 ± 7.27%. Therefore, we suggest that Ang III, opposite to Ang II, stimulated stretch-induced ANP secretion through AT₂ receptor/PI3K/Akt/nitric oxide/PKG pathway.     

Identification of two subtypes in the rat type I angiotensin II receptor.

A rat adrenal cDNA library was screened by colony hybridization using a rat cDNA fragment of type I angiotensin II receptor (AT1A) previously isolated from the kidney. Two cDNA clones were identified, designated as AT1B, to have a nucleotide sequence highly homologous to and yet distinct from AT1A. The amino acid sequence of AT1B consists of 359 amino acid residues and has 96% identity with AT1A. No conspicuous difference in the ligand binding characteristics was observed between AT1A and AT1B. The mRNA for AT1B was expressed in many tissues as is the case with AT1A, and most abundantly expressed in the adrenal glands in the Sprague-Dawley rats. The existence of two subtypes in the rat type I angiotensin II receptor might explain the diverse actions of angiotensin II in various tissues.     

The specificity of angiotensin II receptor binding in rat brain

125I-angiotensin II (125I-AII) binding was examined in the hypothalamic-thalamic-septal-midbrain (HTSM) region of HLA-Wistar rats in the presence of CNS-active agents. Angiotensin I, II, and III and saralasin competed for 125 I-AII binding, whereas structurally unrelated peptides such as arginine and lysine vasopressin, oxytocin, LHRH, TRH, bradykinin, and substance P did not. In contrast, ACTH and neurotensin exhibited a weak, dose-dependent competition for 125 I-AII binding. The relative potencies of AII, AI, neurotensin and ACTH were 100:1:0.1:0.05, respectively. Neurotensin and ACTH competition was not additive with AII suggesting interaction at shared binding sites. Most importantly, a wide variety of other CNS active agents such as methyldopa, naloxone, catecholamines, clondidine, and reserpine, failed to inhibit 125 I-AII binding, thus further defining the specificity of the CNS AII receptor.     

Impact of angiotensin II type 2 receptor blockade on experimental radiation nephropathy

In the rat, blockade of angiotensin II type 1 receptors diminishes the functional changes that occur after kidney irradiation. It has been hypothesized that some of the beneficial effects of angiotensin II type 1 blockers in renal disease are caused by a rise in angiotensin II that stimulates the angiotensin II type 2 receptor. If this hypothesis applied in this model, blockade of the type 2 receptor should exacerbate radiation nephropathy and/or counteract the beneficial effects of type 1 receptor blockade. To assess this hypothesis, rats were given total-body irradiation plus bone marrow transplantation and then treated for 12 weeks with a type 1 receptor blocker (L158,809), a type 2 blocker (PD123319), both blockers, or no blockers. Rats were assessed for renal function (proteinuria, hypertension, azotemia) and renal failure for up to 62 weeks. Contrary to the hypothesis, the type 2 blocker alone produced a temporary delay in the development of radiation nephropathy, and it substantially enhanced the efficacy of the type 1 blocker. This implies that both type 1 and type 2 angiotensin receptors need to be blocked to achieve the maximum level of prophylaxis of radiation nephropathy. We speculate that the beneficial effect of the angiotensin II type 2 receptor blocker is due to a reduction in radiation-induced renal cell proliferation or fibrosis.     

Upregulation of angiotensin II type 2 receptor expression in estrogen-induced pituitary hyperplasia

Recent evidence shows that reexpression and upregulation of angiotensin II (ANG II) type 2 (AT2) receptor in adult tissues occur during pathological conditions such as tissue hyperplasia, inflammation, and remodeling. In particular, expression of functional AT2 receptors in the pituitary and their physiological significance and regulation have not been described. In this study, we demonstrate that chronic in vivo estrogen treatment, which induces pituitary hyperplasia, enhances local AT2 expression (measured by Western blot and RT-PCR) concomitantly with downregulation of ANG II type 1 (AT1) receptors. In vivo progesterone treatment of estrogen-induced pituitary hyperplasia did not modify either the ANG II receptor subtype expression pattern or octapeptide-induced and AT1-mediated calcium signaling. Nevertheless, an unexpected potentiation of the ANG II prolactin-releasing effect was observed in this group, and this response was sensitive to both AT1 and AT2 receptor antagonists. These data are the first to document that ANG II can act at the pituitary level through the AT2 receptor subtype and that estrogens display a differential regulation of AT1 and AT2 receptors at this level.     

Mechanical stress activates angiotensin II type 1 receptor without the involvement of angiotensin II

The angiotensin II type 1 (AT1) receptor has a crucial role in load-induced cardiac hypertrophy. Here we show that the AT1 receptor can be activated by mechanical stress through an angiotensin-II-independent mechanism. Without the involvement of angiotensin II, mechanical stress not only activates extracellular-signal-regulated kinases and increases phosphoinositide production in vitro, but also induces cardiac hypertrophy in vivo. Mechanical stretch induces association of the AT1 receptor with Janus kinase 2, and translocation of G proteins into the cytosol. All of these events are inhibited by the AT1 receptor blocker candesartan. Thus, mechanical stress activates AT1 receptor independently of angiotensin II, and this activation can be inhibited by an inverse agonist of the AT1 receptor.