AT1 Receptor Signaling Research Articles

AT1 receptor downregulation: A mechanism for improving glucose homeostasis.

There is a pathophysiological correlation between arterial hypertension and diabetes mellitus, established since the pre-diabetic state in the entity known as insulin resistance. It is known that high concentrations of angiotensin-II enable chronic activation of the AT1 receptor, promoting sustained vasoconstriction and the consequent development of high blood pressure. Furthermore, the chronic activation of the AT1 receptor has been associated with the development of insulin resistance. From a molecular outlook, the AT1 receptor signaling pathway can activate the JNK kinase. Once activated, this kinase can block the insulin signaling pathway, favoring the resistance to this hormone. In accordance with the previously mentioned mechanisms, the negative regulation of the AT1 receptor could have beneficial effects in treating metabolic syndrome and type 2 diabetes mellitus. This review explains the clinical correlation of the metabolic response that diabetic patients present when receiving negatively regulatory drugs of the AT1 receptor.

Quantitative Phosphoproteomics of the Angiotensin AT2-Receptor Signaling Network Identifies HDAC1 (Histone-Deacetylase-1) and p53 as Mediators of Antiproliferation and Apoptosis.

Angiotensin AT2-receptor signaling is atypical for a G-protein coupled receptor and incompletely understood. To obtain novel insights into AT2-receptor signaling, we mapped changes in the phosphorylation status of the entire proteome of human aortic endothelial cells in response to AT2-receptor stimulation. Phosphorylation status of human aortic endothelial cells after stimulation with C21 (1 µM; 0, 1, 3, 5, 20 minutes) was determined utilizing time-resolved quantitative phosphoproteomics. Specific changes in protein phosphorylation and acetylation were confirmed by Western Blotting. Functional tests included resazurin assay for cell proliferation, and caspase 3/7 luminescence assay or FACS analysis of annexin V expression for apoptosis. AT2-receptor stimulation significantly altered the phosphorylation status of 172 proteins (46% phosphorylations, 54% dephosphorylations). Bioinformatic analysis revealed a cluster of phospho-modified proteins involved in antiproliferation and apoptosis. Among these proteins, HDAC1 (histone-deacetylase-1) was dephosphorylated at serine421/423 involving serine/threonine phosphatases. Resulting HDAC1 inhibition led to p53 acetylation and activation. AT2-receptor stimulation induced antiproliferation and apoptosis, which were absent when cells were co-incubated with the p53 inhibitor pifithrin-α, thus indicating p53-dependence of these AT2-receptor mediated functions. Contrary to the prevailing view that AT2-receptor signaling largely involves phosphatases, our study revealed significant involvement of kinases. HDAC1 inhibition and resulting p53 activation were identified as novel, AT2-receptor coupled signaling mechanisms. Furthermore, the study created an openly available dataset of AT2-receptor induced phospho-modified proteins, which has the potential to be the basis for further discoveries of currently unknown, AT2-receptor coupled signaling mechanisms.

Deficiency of the kidney tubular angiotensin II type1 receptor–associated protein ATRAP exacerbates streptozotocin-induced diabetic glomerular injury via reducing protective macrophage polarization

Although activation of the renin-angiotensin system and of its glomerular components is implicated in the pathogenesis of diabetic nephropathy, the functional roles of the tubular renin-angiotensin system with AT1 receptor signaling in diabetic nephropathy are unclear. Tissue hyperactivity of the renin-angiotensin system is inhibited by the angiotensin II type 1 receptor-associated protein ATRAP, which negatively regulates receptor signaling. The highest expression of endogenous ATRAP occurs in the kidney, where it is mainly expressed by tubules but rarely in glomeruli. Here, we found that hyperactivation of angiotensin II type 1 receptor signaling in kidney tubules exacerbated diabetic glomerular injury in a mouse model of streptozotocin-induced diabetic nephropathy. These phenomena were accompanied by decreased expression of CD206, a marker of alternatively activated and tissue-reparative M2 macrophages, in the kidney tubulointerstitium. Additionally, adoptive transfer of M2- polarized macrophages into diabetic ATRAP-knockout mice ameliorated the glomerular injury. As a possible mechanism, the glomerular mRNA levels of tumor necrosis factor-α and oxidative stress components were increased in diabetic knockout mice compared to non-diabetic knockout mice, but these increases were ameliorated by adoptive transfer. Furthermore, proximal tubule-specific ATRAP downregulation reduced tubulointerstitial expression of CD206, the marker of M2 macrophages in diabetic mice. Thus, our findings indicate that tubular ATRAP-mediated functional modulation of angiotensin II type 1 receptor signaling modulates the accumulation of tubulointerstitial M2 macrophages, thus affecting glomerular manifestations of diabetic nephropathy via tubule-glomerular crosstalk.

Signal Transduction Profiling of Angiotensin II Type 1 Receptor With Mutations Associated to Atrial Fibrillation in Humans.

The AT1 receptor (AT1R) has a major role in the Renin-Angiotensin System, being involved in several physiological events including blood pressure control and electrolyte balance. The AT1R is a member of the G protein coupled receptors (GPCR) family, classically known to couple Gαq and engage β-arrestin recruitment. Both G protein and arrestin signaling pathways are involved in modulation of different downstream kinases. A previous study reported that mutations in the AT1R (A244S and I103T-A244S) were positively correlated with higher risk of atrial fibrillation in men. Based on that report, we aimed to investigate if these mutations, including I103T only, could affect AT1R signal transduction profile, and consequently, implicate in atrial fibrillation outcome. To address that, we engineered an AT1R carrying the above-mentioned mutations, and functionally evaluated different signaling pathways. Phosphokinase profiler array to assess the mutations downstream effects on kinases and kinase substrates phosphorylation levels was used. Our results show that the I103T-A244S mutant receptor presents decreased β-arrestin 2 recruitment, which could lead to a harmful condition of sustained Gαq signaling. Moreover, the phosphokinase profiler array revealed that the same mutation led to downstream modulation of kinase pathways that are linked to physiological responses such as fibrous tissue formation, apoptosis and cell proliferation.

SARS-CoV-2 spike protein promotes IL-6 trans-signaling by activation of angiotensin II receptor signaling in epithelial cells.

Cytokine storm is suggested as one of the major pathological characteristics of SARS-CoV-2 infection, although the mechanism for initiation of a hyper-inflammatory response, and multi-organ damage from viral infection is poorly understood. In this virus-cell interaction study, we observed that SARS-CoV-2 infection or viral spike protein expression alone inhibited angiotensin converting enzyme-2 (ACE2) receptor protein expression. The spike protein promoted an angiotensin II type 1 receptor (AT1) mediated signaling cascade, induced the transcriptional regulatory molecules NF-κB and AP-1/c-Fos via MAPK activation, and increased IL-6 release. SARS-CoV-2 infected patient sera contained elevated levels of IL-6 and soluble IL-6R. Up-regulated AT1 receptor signaling also influenced the release of extracellular soluble IL-6R by the induction of the ADAM-17 protease. Use of the AT1 receptor antagonist, Candesartan cilexetil, resulted in down-regulation of IL-6/soluble IL-6R release in spike expressing cells. Phosphorylation of STAT3 at the Tyr705 residue plays an important role as a transcriptional inducer for SOCS3 and MCP-1 expression. Further study indicated that inhibition of STAT3 Tyr705 phosphorylation in SARS-CoV-2 infected and viral spike protein expressing epithelial cells did not induce SOCS3 and MCP-1 expression. Introduction of culture supernatant from SARS-CoV-2 spike expressing cells on a model human liver endothelial Cell line (TMNK-1), where transmembrane IL-6R is poorly expressed, resulted in the induction of STAT3 Tyr705 phosphorylation as well as MCP-1 expression. In conclusion, our results indicated that the presence of SARS-CoV-2 spike protein in epithelial cells promotes IL-6 trans-signaling by activation of the AT1 axis to initiate coordination of a hyper-inflammatory response.

7 Oral - Impact of angiotensin II pathway inhibition on tumor response to anti PD(L)1 based therapy

Background: Angiotensin II (Ang II) has been shown in preclinical work to increase TGF-β production through AT1 receptor signaling and to decrease TGF-β through AT2 receptor signaling. Thus, the ang II pathway through overlap with the TGF-β pathway may have a critical role in carcinogenesis as well as immune evasion and inhibiting AT1 may enhance clinical responses in combination with PD(L)1 blockade. Here we report efficacy of anti PD(L)1 based therapy in pts with advanced solid tumors concomitantly taking angiotensin receptor blockers (ARBs).

Central AT1 receptor signaling by circulating angiotensin II is permissive to acute intermittent hypoxia-induced sympathetic neuroplasticity.

Acute intermittent hypoxia (AIH) triggers sympathetic long-term facilitation (sLTF), a progressive increase in sympathetic nerve activity (SNA) linked to central AT1 receptor (AT1R) activation by circulating angiotensin II (ANG II). Here, we investigated AIH activation of the peripheral renin-angiotensin system (RAS) and the extent to which the magnitude of RAS activation predicts the magnitude of AIH-induced sLTF. In anesthetized male Sprague-Dawley rats, plasma renin activity (PRA) increased in a linear fashion in response to 5 (P = 0.0342) and 10 (P < 0.0001) cycles of AIH, with PRA remaining at the 10th cycle level 1 h later, a period over which SNA progressively increased. On average, SNA ramping began at the AIH cycle 4.6 ± 0.9 (n = 12) and was similar in magnitude 1 h later whether AIH consisted of 5 or 10 cycles (n = 6/group). Necessity of central AT1R in post-AIH sLTF was affirmed by intracerebroventricular (icv) losartan (40 nmol, 2 µL; n = 5), which strongly attenuated both splanchnic (P = 0.0469) and renal (P = 0.0018) sLTF compared with vehicle [artificial cerebrospinal fluid (aCSF), 2 µL; n = 5]. Bilateral nephrectomy largely prevented sLTF, affirming the necessity of peripheral RAS activation. Sufficiency of central ANG II signaling was assessed in nephrectomized rats. Whereas ICV ANG II (0.5 ng/0.5 µL, 30 min) in nephrectomized rats exposed to sham AIH (n = 4) failed to cause SNA ramping, it rescued sLTF in nephrectomized rats exposed to five cycles of AIH [splanchnic SNA (SSNA), P = 0.0227; renal SNA (RSNA), P = 0.0390; n = 5]. Findings indicate that AIH causes progressive peripheral RAS activation, which stimulates an apparent threshold level of central AT1R signaling that plays a permissive role in triggering sLTF.NEW & NOTEWORTHY Acute intermittent hypoxia (AIH) triggers sympathetic long-term facilitation (sLTF) that relies on peripheral renin-angiotensin system (RAS) activation. Here, increasing AIH cycles from 5 to 10 proportionally increased RAS activity, but not the magnitude of post-AIH sLTF. Brain angiotensin II (ANG II) receptor blockade and nephrectomy each largely prevented sLTF, whereas central ANG II rescued it following nephrectomy. Peripheral RAS activation by AIH induces time-dependent neuroplasticity at an apparent central ANG II signaling threshold, triggering a stereotyped sLTF response.

SAT-033 Studying Cell-Context Specific Effects on the Function of the AT1R in iPSCs and Their Differentiated Derivatives

Cardiovascular homeostasis is tightly regulated by numerous neurohormonal mediators such as the renin-angiotensin system which plays an important role in the maintenance of blood pressure. Central to this system is the peptide hormone angiotensin II (Ang II) whose signals are transduced via the AT1 receptor (AT1R), an important member of the superfamily of G protein-coupled receptors (GPCRs). Ang II binding results in receptor activation characterized by structural re-arrangements within the receptor structure and the subsequent activation of its cognate G protein partners. GPCRs are allosteric in nature and their biological activity is highly dependent on the cell context in which they are expressed1. Changes in the cellular background such as the differential availability of G proteins and effector molecules including putative dimer partners can affect receptor conformation and function. As such, we are interested in understanding how AT1R conformation and signaling are modulated by the cell context in which it is expressed1. In the past, studies that aimed at understanding signaling downstream of GPCRs mostly relied on heterologous expression systems such as HEK 293 cells because of their ease of culture. Such studies led to a ‘one size fits all’ notion that our findings could be reasonably extrapolated to guide drug discovery platforms relevant for human disease. However, it is clear that with the high rate of drug attrition, we need more physiologically relevant cellular models for studies of molecular signal transduction events to be translatable. With this in mind, we are generating iPSCs that stably express a panel of conformation-sensitive biosensors that reliably report on the conformational changes in the AT1R1,2. Our biosensors use resonance energy transfer between a bioluminescent donor and a fluorescent acceptor (FlAsH) where agonist-mediated conformational changes can be recorded1. Here, we will investigate how the conformation of the AT1R changes when expressed in AT1R-relevant cell types such as iPSC-derived cardiomyocytes and vascular smooth muscle cells. We will investigate how our conformational profiles differ in different iPSC-derived cell types in response to AT1R-specific agonists. Our goal is to gain a better mechanistic understanding of how cells are differentially wired leading to cell-specific conformational and signaling responses. We hope our results can guide rational drug design to better target the AT1R and other GPCRs. 1Devost D., et al (2017). Journal of Biological Chemistry, jbc-M116. 2Pei Y., et al (2015). Scientific reports, 5, 9205.

Apparently normal kidney development in mice with conditional disruption of ANG II-AT1receptor genes in FoxD1-positive stroma cell precursors

An intact renin-angiotensin system involving ANG II type 1 (AT1) receptors is crucial for normal kidney development. It is still unclear in which cell types AT1 receptor signaling is required for normal kidney development, maturation, and function. Because all kidney cells deriving from stroma progenitor cells express AT1 receptors and because stromal cells fundamentally influence nephrogenesis and tubular maturation, we investigated the relevance of AT1 receptors in stromal progenitors and their descendants for renal development and function. For this aim, we generated and analyzed mice with conditional deletion of AT1A receptor in the FoxD1 cell lineage in combination with global disruption of the AT1B receptor gene. These FoxD1-AT1ko mice developed normally. Their kidneys showed neither structural nor functional abnormalities compared with wild-type mice, whereas in isolated perfused FoxD1-AT1ko kidneys, the vasoconstrictor and renin inhibitory effects of ANG II were absent. In vivo, however, plasma renin concentration and renal renin expression were normal in FoxD1-AT1ko mice, as were blood pressure and glomerular filtration rate. These findings suggest that a strong reduction of AT1 receptors in renal stromal progenitors and their descendants does not disturb normal kidney development.

Sensitization of the Angiotensin II AT1 Receptor Contributes to RKIP-Induced Symptoms of Heart Failure.

Inhibition of the G-protein-coupled receptor kinase 2 (GRK2) is an emerging treatment approach for heart failure. Therefore, cardio-protective mechanisms induced by GRK2 inhibition are under investigation. We compared two different GRK2 inhibitors, i.e., (i) the dual-specific GRK2 and raf kinase inhibitor protein, RKIP, and (ii) the dominant-negative GRK2-K220R mutant. We found that RKIP induced a strong sensitization of Gq/11-dependent, heart failure-promoting angiotensin II AT1 receptor signaling. The AT1-sensitizing function of RKIP was mediated by the RKIP-GRK2 interaction because the RKIP-S153V mutant, which does not interact with GRK2, had no effect on AT1-stimulated signaling. In contrast, GRK2-K220R significantly inhibited the AT1-stimulated signal. The in vivo relevance of these major differences between two different approaches of GRK2 inhibition was analyzed by generation of transgenic mice with myocardium-specific expression of RKIP and GRK2-K220R. Our results showed that a moderately increased cardiac protein level of RKIP was sufficient to induce major symptoms of heart failure in aged, 8-months-old RKIP-transgenic mice in two different genetic backgrounds. In contrast, GRK2-K220R protected against chronic pressure overload-induced cardiac dysfunction. The AT1 receptor contributed to RKIP-induced heart failure because treatment with the AT1 receptor antagonist, losartan, retarded symptoms of heart failure in RKIP-transgenic mice. Thus, sensitization of the heart failure-promoting AT1 receptor by the RKIP-GRK2 interaction contributes to heart failure whereas dominant-negative GRK2-K220R is cardioprotective. Because RKIP is up-regulated on cardiac biopsy specimens of heart failure patients, the deduced heart failure-promoting mechanism of RKIP could also be relevant for the human disease.

A NOVEL REGULATORY EFFECT OF AT1 RECEPTOR-INTERACTING MOLECULE ON VASCULAR SMOOTH MUSCLE CELLS

Objective: Previous studies reported that the production of superoxide is modulated by many factors including Ang II - AT1 receptor signaling. One of the major sources of superoxide in the aorta is NADPH oxidase located in the smooth muscle cells. The NADPH oxidase complex in the vascular smooth muscle cells consists of p22phox, Rac1, and Nox1. Previous studies showed that the carboxy-terminal cytoplasmic domain of AT1 receptor is involved in the control of receptor internalization and in linking receptor-mediated signal transduction to the specific biological response. Design and method: We previousy cloned a novel molecule interacting with carboxy-terminal domain of AT1 receptor, which we named ATRAP (for AT1 receptor-associated protein), using the yeast two-hybrid strategy. In this study, we tested the hypothesis that vascular smooth muscle cells express ATRAP and that ATRAP modulates Ang II-induced proliferative activity and oxidative stress in vascular smooth muscle cells. We identified that the ATRAP mRNA and protein were endogeneously expressed in VSMC, and found a colocalization of ATRAP and AT1 receptor in Ang II-stimulated VSMC. Results: The results of gain-of-function studies by adenoviral gene transfer demonstrated that overexpression of ATRAP significantly inhibited Ang II-mediated increases in c-fos gene transcription, BrdU incorpoaration, and mRNAs expression of NADPH oxidase complex (p < 0.05, n = 6). Conclusions: These results indicate that ATRAP significantly attenuates Ang II-mediated proliferative activity and oxidative stress in vascular smooth muscle cells, and may suggest a novel strategy to inhibit cardiovascular disease such as arteriosclerosis and hypertension.

Understanding Angiotensin II Type 1 Receptor Signaling in Vascular Pathophysiology.

Angiotensin II is the most important endocrine ligand in the renin–angiotensin system (RAS), contributing to the development of several cardiovascular diseases including hypertension.1 Angiotensin II mediates its signal transduction and functions via the angiotensin II receptors.2 Historically, the presence of 2 subtypes of angiotensin II receptors were pharmacologically recognized based on the sensitivity to the first orally active nonpeptide angiotensin II receptor antagonist, losartan. The losartan-sensitive receptor was termed AT1 (angiotensin II receptor type 1) receptor. It was assumed to be a heterotrimeric GPCR (G protein–coupled receptor) because it generates inositol triphosphate and diacylglycerol, leading to intracellular Ca2+ elevation and protein kinase C activation, respectively. Most known physiological and pathophysiological functions of angiotensin II, including stimulation of vasoconstriction and salt and water reabsorption, are mediated through the AT1 receptor. The losartan-insensitive receptor was termed AT2 receptor, whereas its G protein coupling remains unclear.1,3,4 In 1991, 2 research groups in the United States independently isolated cDNA (termed AGTR1) encoding the mammalian AT1 receptor.5,6 Subsequently, rat AT2 receptor cDNA (AGTR2) was cloned in 1993.7,8 This pioneer work revealed complete amino acid sequences of the angiotensin II receptor subtypes belonging to the 7-transmembrane GPCR superfamily. In the early 1990s, several studies reported that AT1 receptor elicits tyrosine phosphorylation of multiple proteins and activation of MAPK (mitogen-activated protein kinase; eg, p42/p44 MAPK)/ERK (extracellular signal–regulated kinase; eg, ERK1/2) in various cell types including vascular smooth muscle cells (VSMC). The early 1990s also saw the establishment of the concept that angiotensin II via the AT1 receptor has a direct action on cardiac myocytes, fibroblasts, and VSMCs causing hypertrophic and fibrotic cardiovascular remodeling.9,10 The cardiovascular remodeling caused by angiotensin II seemed to be …

Abstract WP259: AT2 Receptor-interacting Protein Enhances Protective Effect of AT2 Receptor Stimulation in Ischemic Brain Damage

Objective: Accumulating evidences and previous our research suggest that angiotensin II type 2 (AT 2 ) receptor stimulation could contribute to protection against ischemic brain damage. We have cloned ATIP (AT 2 receptor interacting protein) as a protein interacting specifically with the C-terminal tail of the AT 2 receptor, and reported that ATIP might play key roles in diverse mechanisms of AT 2 receptor signaling. However, the effect of ATIP on ischemic brain damage is still unclear. Therefore, we investigated the effects of the ATIP and compound 21 (C21), a selective non-peptidic AT2 receptor agonist, on focal cerebral ischemia. Method: Ten-week-old male ATIP-transgenic (ATIP-Tg) and littermate (WT) mice were subjected to middle cerebral artery occlusion (MCAO) with silicon-coated micro-filament. C21 (10 μg/kg/day) was administered 2 weeks before MCAO. Twenty-four hours after MCAO, ischemic area was determined. Cerebral blood flow (CBF) before and after MCAO was measured by laser speckle flowmetry. Collateral circulation was evaluated by the perfusion of India ink. Expression of mRNA was determined by real-time RT-PCR. Results: There was no significant difference in ischemic size without C21 treatment between two strains. Treatment with C21 decreased ischemic size in both strains. Interestingly, this protective effect of C21 was more marked in ATIP-Tg compared with WT mice. In CBF of core region of ischemic area, there were no significant differences among all groups. However, the reduction of CBF in penumbra region just after MCA occlusion was attenuated in ATIP-Tg mice with C21 administration. Treatment with C21 tended to increase the cerebral collateral number before MCA occlusion in ATIP-Tg mice. Expression of vascular endothelial growth factor (VEGF) mRNA in the cortex before MCA occlusion did not differ among all groups. Expression of methyl methanesulfonate sensitive 2 (MMS2) as a neuroprotective factor increased in ipsilateral hemisphere of ATIP-Tg mice compared with contralateral hemisphere. Conclusions: These results suggested that ATIP could enhance the cerebral protective effects of AT 2 receptor stimulation at least in part due to the attenuation of CBF reduction and increase of MMS2 expression after ischemia.

Ang-(1-7) is an endogenous \u03b2-arrestin-biased agonist of the AT1 receptor with protective action in cardiac hypertrophy

The renin-angiotensin system (RAS) plays a key role in the control of vasoconstriction as well as sodium and fluid retention mediated mainly by angiotensin (Ang) II acting at the AT1 receptor (AT1R). Ang-(1-7) is another RAS peptide, identified as the endogenous ligand of the Mas receptor and known to counterbalance many of the deleterious effects of AngII. AT1R signaling triggered by β-arrestin-biased agonists has been associated to cardioprotection. Because position 8 in AngII is important for G protein activation, we hypothesized that Ang-(1-7) could be an endogenous β-arrestin-biased agonist of the AT1R. Here we show that Ang-(1-7) binds to the AT1R without activating Gq, but triggering β-arrestins 1 and 2 recruitment and activation. Using an in vivo model of cardiac hypertrophy, we show that Ang-(1-7) significantly attenuates heart hypertrophy by reducing both heart weight and ventricular wall thickness and the increased end-diastolic pressure. Whereas neither the single blockade of AT1 or Mas receptors with their respective antagonists prevented the cardioprotective action of Ang1-7, combination of the two antagonists partially impaired the effect of Ang-(1-7). Taken together, these data indicate that Ang-(1-7) mediates at least part of its cardioprotective effects by acting as an endogenous β-arrestin-biased agonist at the AT1R.

Abstract P409: Defective Renal Ang III and AT2 Receptor Signaling in Pre-hypertensive Spontaneously Hypertensive Rats (SHR)

The intrarenal renin-angiotensin (RAS) system controls blood pressure and electrolyte balance. Angiotensin II (Ang II), the principal RAS effector peptide, is metabolized by aminopeptidase A to des-apartyl 1 -Ang II (Ang III). Our previous studies showed that Ang III, not Ang II, is the preferred endogenous agonist for Ang type-2 receptor (AT 2 R)-induced natriuresis in normal rats, and that hypertensive 12-week old SHR lack natriuretic responses to Ang III. The present study tested whether Ang III induced natriuresis in 4 week old pre-hypertensive SHR by activating/translocating intrarenal AT 2 Rs and internalizing the major renal proximal tubule (RPT) sodium transporter NHE-3. Female 4 week old SHR (N=6) and WKY (N=6) rats were studied after 24 h systemic AT 1 R blockade with candesartan. The left kidney received a 30 min renal interstitial (RI) infusion of vehicle followed by cumulative RI infusions of Ang III (3.5, 7.0, 14, and 28 nmol/kg/min; each dose for 30 min). The right kidney received vehicle RI infusions. In 4 week old WKY, RI Ang III increased urine sodium excretion (U Na V) dose dependently from control of 0.04 ± 0.01 to 0.08 ± 0.02 (P&lt;0.05), 0.09 ± 0.02 (P&lt;0.01), 0.07 ± 0.01 (P&lt;0.05), and 0.07 ± 0.01 (P&lt;0.05) μmol/min. In 4 week old pre-hypertensive SHR, RI Ang III failed to induce natriuresis at all doses except 28 nmol/kg/min (0.03 ± 0.01 vs. 0.05 ± 0.01 μmol/min; P&lt;0.05). There was no change in U Na V in right control kidneys of WKY or SHR. Also, Ang III had no effect on mean arterial pressure (MAP) in WKY or SHR. However, MAP was slightly higher, but normotensive, in SHR than WKY. Control and Ang III infused kidneys were processed for confocal microscopy from a separate group of rats. In WKY, RI Ang III induced translocation of AT 2 Rs from subapical to apical membranes of RPT cells [RPTC] (1767 ± 41 vs. 1313 ± 47 RFU; P&lt;0.001). Simultaneously, Ang III induced retraction of NHE-3 from in SHR, intrarenal Ang III failed to induce AT 2 R translocation, NHE-3 retraction, and pSer 552 -NHE-3 upregulation. These results apical to subapical membranes of RPTCs (1703 ± 90 vs. 1388 ± 91 RFU; P&lt;0.05). Consistent with NHE-3 retraction, Ang III increased pSer 552 -NHE-3 (1102 ± 57 vs. 830 ± 66 RFU; P&lt;0.01). In contrast,demonstrate defective Ang III and AT 2 R signaling in pre-hypertensive SHR.

A bioluminescence resonance energy transfer 2 (BRET2) assay for monitoring seven transmembrane receptor and insulin receptor crosstalk

The angiotensin AT1 receptor is a seven transmembrane (7TM) receptor, which mediates the regulation of blood pressure. Activation of angiotensin AT1 receptor may lead to impaired insulin signaling indicating crosstalk between angiotensin AT1 receptor and insulin receptor signaling pathways. To elucidate the molecular mechanisms behind this crosstalk, we applied the BRET2 technique to monitor the effect of angiotensin II on the interaction between Rluc8 tagged insulin receptor and GFP2 tagged insulin receptor substrates 1, 4, 5 (IRS1, IRS4, IRS5) and Src homology 2 domain-containing protein (Shc). We demonstrate that angiotensin II reduces the interaction between insulin receptor and IRS1 and IRS4, respectively, while the interaction with Shc is unaffected, and this effect is dependent on Gαq activation. Activation of other Gαq-coupled 7TM receptors led to a similar reduction in insulin receptor and IRS4 interactions whereas Gαs- and Gαi-coupled 7TM receptors had no effect. Furthermore, we used a panel of kinase inhibitors to show that angiotensin II engages different pathways when regulating insulin receptor interactions with IRS1 and IRS4. Angiotensin II inhibited the interaction between insulin receptor and IRS1 through activation of ERK1/2, while the interaction between insulin receptor and IRS4 was partially inhibited through protein kinase C dependent mechanisms. We conclude that the crosstalk between angiotensin AT1 receptor and insulin receptor signaling shows a high degree of specificity, and involves Gαq protein, and activation of distinct kinases. Thus, the BRET2 technique can be used as a platform for studying molecular mechanisms of crosstalk between insulin receptor and 7TM receptors.

Abstract WP290: AT2 Receptor Stimulation Reduces Stroke Size in Concert With AT2 Receptor-Interacting Protein

Objective: We previously reported that the ischemic brain area was significantly larger in angiotensin II type 2 (AT 2 ) receptor-deficient mice after middle cerebral artery (MCA) occlusion compared to wild-type mice. We have cloned ATIP (AT 2 receptor interacting protein) as a protein interacting specifically with the C-terminal tail of the AT 2 receptor, and suggest that ATIP might play key roles in diverse mechanisms of AT 2 receptor signaling. However, the effect of ATIP on brain damage has not been clarified. We investigated the possibility that ATIP could enhance the protective effects of compound 21 (C21), a selective direct non-peptidic AT 2 receptor agonist, on focal cerebral ischemia. Method: Ten week-old male ATIP-transgenic (ATIP-Tg) and littermate (WT) mice were subjected to permanent MCA occlusion with silicon-coated micro-filament. C21 (10 μg/kg/day) was administered 2 weeks before MCA occlusion. Twenty-four hours after MCA occlusion, ischemic area and neurological deficit was assessed. Cerebral blood flow (CBF) was measured by laser speckle flowmetry. Expression of methyl methanesulfonate sensitive 2 (MMS2) as a neuroprotective factor were measured by real-time RT-PCR. Collateral circulation was evaluated by the perfusion of India ink. Results: Systolic blood pressure did not differ between ATIP-Tg and WT mice with or without C21. There was no significant difference in ischemic size without C21 treatment between two strains. Treatment with C21 decreased ischemic size and improved neurological deficit in both strains. These protective effects by C21 were more marked in ATIP-Tg mice compared with WT mice. Treatment of C21 did not affect CBF in the core region of ischemic area after MCA occlusion in both strains; however, the reduction of CBF in penumbra region was markedly attenuated in ATIP-Tg mice treated with C21. MMS2 expression increased in ipsilateral hemisphere of ATIP-Tg mice compared with contralateral hemisphere. C21 treatment tended to increase collateral number in ATIP-Tg mice. Conclusions: These results suggested that ATIP could enhance the cerebral protective effects of AT 2 receptor stimulation at least in part due to the improvement of CBF and increase of MMS2 expression.

Abstract P301: Renal Expression of Collectrin (TMEM27) is Downregulated During Angiotensin II-induced Hypertension

Collectrin ( Tmem27 ) is transmembrane glycoprotein with homology to ACE-2, but lacks any catalytic domain. It plays a key role as a chaperone of amino acid transporters, and is abundantly expressed in the kidney in the proximal tubules and collecting duct. Deletion of collectrin in the mouse results in hypertension (HTN) at baseline and augmented salt-sensitivity that are associated with decreased renal nitric oxide and increased superoxide levels. During high salt diet, renal expression of collectrin is upregulated, suggesting an adaptive homeostatic response to salt loading. Here, we queried whether the expression of collectrin is regulated by angiotensin II (Ang II). Wild-type 129S6 mice were made hypertensive with Ang II osmotic minipump @ 600 ng/kg/min x 2 weeks, and were compared to age-matched untreated WT 129 mice. Shown in Fig. 1 , renal mRNA expression of collectrin is significantly reduced after 2 weeks of Ang II (Panel A). Immunostaining shows collectrin protein level is also significantly diminished to near undetectable level (Panel B). We show for the first time that Ang II regulates the expression of collectrin, suggesting that the action of Ang II on blood pressure may be mediated, in part, through the downregulation of collectrin. Further studies are needed to determine the effect of AT 1 and AT 2 receptor signaling on renal expression of collectrin during Ang II-HTN in vivo.

Interactions Between the Immune and the Renin-Angiotensin Systems in Hypertension.

The renin–angiotensin system (RAS) is an essential regulator of blood pressure homeostasis. The primary effector molecule of the RAS is angiotensin (Ang) II, an 8-amino acid peptide that initiates tissue-specific processes via binding to the type 1 or 2 angiotensin II receptors (AT1 or AT2). The AT1 receptor is a G-protein–coupled receptor expressed in most tissues throughout the body. Although the rodent expresses 2 AT1 receptor isoforms, AT1A and AT1B, the closest functional homolog to the human AT1 receptor in most rodent tissues is AT1A. The most widely appreciated functions of Ang II signaling via the AT1 receptor are to augment global vascular tone and to promote sodium retention in the kidney, thus playing a crucial role in acute and long-term maintenance of blood pressure.1,2 Ang II elicits these effects by increasing the contractility of vascular smooth muscle, promoting the release of aldosterone from the adrenal cortex, and directly increasing the expression or activity of solute transporters in the renal tubules that promote sodium reabsorption. As such, blockade of the RAS has been a mainstay in the treatment for hypertension and its complications, including cardiovascular and kidney disease.2,3 Although the cause of essential hypertension is unclear, inhibition of the RAS is effective in reducing blood pressure in a wide variety of patients, which suggests that inappropriate RAS activation is common in the pathogenesis of this idiopathic condition. Global RAS inhibitors ameliorate hypertension by blocking the production of Ang II or by preventing activation of the AT1 receptor. These medications are global inhibitors in the sense that they disrupt AT1 receptor signaling concomitantly in all tissues. The ongoing effort to uncover novel pathogenic mechanisms of hypertension has revealed a significant role for …

Potent inhibition of angiotensin AT1 receptor signaling by RGS8: importance of the C-terminal third exon part of its RGS domain

R4/B subfamily RGS (regulator of G protein signaling) proteins play roles in regulation of many GPCR-mediated responses. Multiple RGS proteins are usually expressed in a cell, and it is difficult to point out which RGS protein species are functionally important in the cell. To evaluate intrinsic potency of these RGS proteins, we compared inhibitory effects of RGS1, RGS2, RGS3, RGS4, RGS5, RGS8 and RGS16 on AT1 receptor signaling. Intracellular Ca2+ responses to angiotensin II were markedly attenuated by transiently expressed RGS2, RGS3 and RGS8, compared to weak inhibition by RGS1, RGS4, RGS5 and RGS16. N-terminally deleted RGS2 (RGS2 domain) lost this potent inhibitory effect, whereas RGS domains of RGS3 and RGS8 showed strong inhibition similar to those of the full-length proteins. To investigate key determinants that specify the differences in potency, we constructed chimeric domains by replacing one or two of three exon parts of RGS8 domain with the corresponding part of RGS5. The chimeric RGS8 domains containing the first or the second exon part of RGS5 showed strong inhibitory effects similar to that of wild type RGS8, but the chimeric domain with the third exon part of RGS5 lost its activity. On the contrary, replacement of the third exon part of RGS5 with the corresponding residues of RGS8 increased the inhibitory effect. The role of the third exon part of RGS8 domain was further confirmed with the chimeric RGS8/RGS4 domains. These results indicate the potent inhibitory activity of RGS8 among R4/B subfamily proteins and importance of the third exon.