Irbesartan increased PPARγ activity in vivo in white adipose tissue of atherosclerotic mice and improved adipose tissue dysfunction

The effect of the PPARγ agonistic action of an AT₁ receptor blocker, irbesartan, on adipose tissue dysfunction was explored using atherosclerotic model mice. Adult male apolipoprotein E-deficient (ApoEKO) mice at 9 weeks of age were treated with a high-cholesterol diet (HCD) with or without irbesartan at a dose of 50 mg/kg/day for 4 weeks. The weight of epididymal and retroperitoneal adipose tissue was decreased by irbesartan without changing food intake or body weight. Treatment with irbesartan increased the expression of PPARγ in white adipose tissue and the DNA-binding activity of PPARγ in nuclear extract prepared from adipose tissue. The expression of adiponectin, leptin and insulin receptor was also increased by irbesartan. These results suggest that irbesartan induced activation of PPARγ and improved adipose tissue dysfunction including insulin resistance.     

Down-regulation of ether-a-go-go-related gene potassium channel protein through sustained stimulation of AT1 receptor by angiotensin II

We investigated the effects of AT₁ receptor stimulation by angiotensin II (Ang II) on human ether-a-go-go-related gene (hERG) potassium channel protein in a heterogeneous expression system with the human embryonic kidney (HEK) 293 cells which stably expressed hERG channel protein and were transiently transfected with the human AT₁ receptors (HEK293/hERG). Western-blot analysis showed that Ang II significantly decreased the expression of mature hERG channel protein (155-kDa band) in a time- and dose-dependent manner without affecting the level of immature hERG channel protein (135-kDa band). The relative intensity of 155-kDa band was 64.7 ± 6.8% of control (P < 0.01) after treatment of Ang II at 100 nM for 24 h. To investigate the effect of Ang II on the degradation of mature hERG channel protein, we blocked forward trafficking from ER to Golgi with a Golgi transit inhibitor brefeldin A (10 μM). Ang II significantly enhanced the time-dependent reduction of mature hERG channel protein. In addition, the proteasomal inhibitor lactacystin (5 μM) inhibited Ang II-mediated the reduction of mature hERG channel protein, but the lysosomal inhibitor bafilomycin A1 (1 μM) had no effect on the protein. The protein kinase C (PKC) inhibitor bisindolylmaleimide 1 (1 μM) antagonized the reduction of mature hERG channel protein induced by Ang II. The results indicate that sustained stimulation of AT₁ receptors by Ang II reduces the mature hERG channel protein via accelerating channel proteasomal degradation involving the PKC pathway.     

Synthesis and evaluation of the novel 2-[18F]fluoro-3-propoxy-triazole-pyridine-substituted losartan for imaging AT1 receptors

The 2-[¹⁸F]fluoro-3-pent-4-yn-1-yloxypyridine ([¹⁸F]FPyKYNE) analog of the potent non-peptide angiotensin II type 1 receptor (AT₁R) blocker losartan was produced via click chemistry linking [¹⁸F]FPyKYNE to azide-modified tetrazole-protected losartan followed by TFA deprotection. Preliminary small animal imaging with positron emission tomography (PET) in rats displayed high uptake in the kidneys with good contrast to surrounding tissue. Rat metabolism displayed the presence of 23% unchanged tracer in plasma at 30 min. Upon co-administration with AT₁R blocker candesartan (2.5, 5 and 10 mg/kg), a dose-dependent reduction (47–65%) in tracer uptake was observed in the kidney, while no difference was observed following AT₂R blocker PD123,319 (5 mg/kg), indicating binding selectivity for AT₁R over AT₂R and potential for imaging AT₁R using PET.     

Identification of dual ligands targeting angiotensin II type 1 receptor and peroxisome proliferator-activated receptor-γ by core hopping of telmisartan

It has been reported previously that some angiotensin II receptor blockers not only antagonize angiotensin II type 1 receptor (AT₁R), but also exert stimulation in peroxisome proliferator-activated receptor γ (PPARγ) partial activation, among which telmisartan displays the best. Telmisartan has been tested as a bifunctional ligand with antihypertensive and hypoglycemic activity. Aiming at more potent leads with selective AT₁R antagonism and PPARγ partial agonism, the three parts of telmisartan including the distal benzimidazole ring, the biphenyl moiety, and the carboxylic acid group experienced modification by core hopping method in our study. The central benzimidazole ring, however, remained intact considering its great affinity toward AT₁R and PPARγ. We utilized computational techniques for the sake of details on the binding interactions and conformational stability. Standard precision docking analysis and absorption, distribution, metabolism, excretion, and toxicity prediction received 10 molecules with higher Glide scores, similar interactions, and improved pharmacokinetic profiles compared to telmisartan. Comp#91 with highest scores for AT₁R (?11.92 kcal/mol) and PPARγ (?13.88 kcal/mol) exhibited excellent binding modes and pharmacokinetic parameters. Molecular dynamics trajectories on best docking pose of comp#91 confirmed the docking results and verified the conformational stability with both receptors throughout the course of 20-ns simulations. Thus, comp#91 could be identified as a promising lead in the development of dual AT₁R antagonist and PPARγ partial agonist against hypertension and type 2 diabetes.     

Mechanical stress-evoked but angiotensin II-independent activation of angiotensin II type 1 receptor induces cardiac hypertrophy through calcineurin pathway

Mechanical stress can induce cardiac hypertrophy through angiotensin II (AngII) type 1 (AT₁) receptor independently of AngII, however, the intracellular mechanisms remain largely indeterminate. Since calcineurin, a Ca²⁺-dependent phosphatase, plays a critical role in pressure overload-induced cardiac hypertrophy, we therefore, asked whether calcineurin is involved in the AT₁ receptor-mediated but AngII-independent cardiac hypertrophy. Mechanical stretch failed to elicit hypertrophic responses in COS7 cells co-transfected with plasmid of AT₁ receptor and siRNA of calcineurin. Mechanical stresses for 2 weeks in vivo and for 24 h in vitro significantly induced upregulation of calcineurin expression and hypertrophic responses, such as the increases in cardiomyocytes size and specific gene expressions, in cardiomyocytes of angiotensinogen gene knockout (ATG^−/−) mice, both of which were significantly suppressed by a specific calcineurin inhibitor FK506, suggesting a critical role of calcineurin in mechanical stress-induced cardiac hypertrophy in the ATG^−/− mice. Furthermore, an AT₁ receptor blocker Losartan not only attenuated cardiac hypertrophy but also abrogated upregulation of cardiac calcineurin expression induced by mechanical stresses in the AngII-lacking mice, indicating that calcineurin expression is regulated by AT₁ receptor without the involvement of AngII after mechanical stress. These findings collectively suggest that mechanical stress-evoked but AngII-independent activation of AT₁ receptor induces cardiac hypertrophy through calcineurin pathway.     

Angiotensin II enhances endothelin-1-induced vasoconstriction through upregulating endothelin type A receptor

Endothelin-1 (ET-1) is the most potent vasoconstrictor by binding to endothelin receptors (ET_AR) in vascular smooth muscle cells (VSMCs). The complex of angiotensin II (Ang II) and Ang II type one receptor (AT₁R) acts as a transient constrictor of VSMCs. The synergistic effect of ET-1 and Ang II on blood pressure has been observed in rats; however, the underlying mechanism remains unclear. We hypothesize that Ang II leads to enhancing ET-1-mediated vasoconstriction through the activation of endothelin receptor in VSMCs. The ET-1-induced vasoconstriction, ET-1 binding, and endothelin receptor expression were explored in the isolated endothelium-denuded aortae and A-10 VSMCs. Ang II pretreatment enhanced ET-1-induced vasoconstriction and ET-1 binding to the aorta. Ang II enhanced ET_AR expression, but not ET_BR, in aorta and increased ET-1 binding, mainly to ET_AR in A-10 VSMCs. Moreover, Ang II-enhanced ET_AR expression was blunted and ET-1 binding was reduced by AT₁R antagonism or by inhibitors of PKC or ERK individually. In conclusion, Ang II enhances ET-1-induced vasoconstriction by upregulating ET_AR expression and ET-1/ET_AR binding, which may be because of the AngII/Ang II receptor pathways and the activation of PKC or ERK. These findings suggest the synergistic effect of Ang II and ET-1 on the pathogenic development of hypertension.     

Inhibition of renin activity slows down the progression of HIV-associated nephropathy

In the present study, we evaluated the effect of inhibition of renin activity (aliskiren) on the progression of renal lesions in two different mouse models (Vpr and Tg26) of human immunodeficiency virus (HIV)-associated nephropathy (HIVAN). In protocol A, Vpr mice were fed either water (C-VprA) or doxycycline [Doxy (D-VprA)] in their drinking water for 6 wk. In protocols B and C, Vpr mice received either normal saline (C-VprB/C), Doxy + normal saline (D-VprB/C), or Doxy + aliskiren (AD-VprB/C) for 6 wk (protocol B) or 12 wk (protocol C). In protocols D and E, Vpr mice were fed Doxy for 6 wk followed by kidney biopsy. Subsequently, half of the mice were administered either normal saline (D-VprD/E) or aliskiren (AD-VprD/E) for 4 wk (protocol D) or 8 (protocol E) wk. All D-VprA mice showed renal lesions in the form of focal segmental glomerular sclerosis and dilatation of tubules. In protocols B and C, aliskiren diminished both progression of renal lesions and proteinuria. In protocol C, aliskiren also diminished (P < 0.01) the rise in blood urea. In all groups, Doxy-treated mice displayed increased serum ANG I levels (the product of plasma renin activity); on the other hand, all aliskiren-treated mice displayed diminished serum ANG I levels. Renal tissues of D-VprC displayed increased ANG II content; however, aliskiren attenuated renal tissue ANG II production in AD-VprC. In protocol D, AD-VprD showed a 24.2% increase in the number of sclerosed glomeruli compared with 139.2% increase in sclerosed glomeruli in D-VprD (P < 0.01) from their baseline. The attenuating effect of aliskiren on the progression of renal lesions continued in AD-VprE. Aliskiren also diminished blood pressure, proteinuria, and progression of renal lesions in Tg26 mice. These findings indicate that inhibition of renin activity has a potential to slow down the progression of HIVAN.     

Stimulation of ANP by angiotensin-(1-9) via the angiotensin type 2 receptor

Aims

Angiotensin-(1-9) [Ang-(1-9)] and Ang-(1-7) are cleaved by Ang converting enzyme 2 forming Ang I and Ang II, respectively, and the truncated Angs play a role in regulating atrial natriuretic peptide (ANP) secretion. Previously, we found that Ang-(1-7) stimulates ANP secretion via the Mas receptor. However, the effect of Ang-(1-9) on ANP secretion is still unknown. The aim of the present study is to determine whether Ang-(1-9) stimulates ANP secretion and to characterize the signaling pathway involved in stimulating secretion.

Main methods

We examined the effects of Ang-(1-9) on ANP secretion and atrial contractility with and without inhibitors in isolated perfused atria.

Key findings

Ang-(1-9) stimulated ANP secretion and concentration without change in atrial contractility. Ang-(1-9)-induced-ANP secretion was increased from 5% to 60% by 3 μM Ang-(1-9) during the low-stretch state of the atrium. This stimulatory effect of Ang-(1-9) on ANP secretion was attenuated by pretreatment with an Ang II type 2 receptor (AT₂R) antagonist but not by AT₁R or Mas receptor antagonist. In addition, pretreatment with inhibitors of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) blocked Ang-(1-9)-induced ANP secretion. In the high-stretch atrial state, Ang-(1-9)-induced ANP secretion was increased more than in the low-stretch state following addition of 1 μM Ang-(1-9) (from 108% to 170%). In an in vivo experiment, acute infusion of Ang-(1-9) increased plasma ANP level without altering arterial blood pressure. This effect was attenuated by pretreatment with AT₂R antagonist but not by Mas receptor antagonist.

Significance

These results suggest that Ang-(1-9) stimulates ANP secretion via the AT₂R-PI3K-Akt-NO-cGMP pathway.     

Angiotensin II Type 2 Receptor Decreases Transforming Growth Factor-β Type II Receptor Expression and Function in Human Renal Proximal Tubule Cells

Transforming growth factor-β (TGF-β), via its receptors, induces epithelial-mesenchymal transition (EMT) and plays an important role in the development of renal tubulointersitial fibrosis. Angiotensin II type 2 receptor (AT₂R), which mediates beneficial renal physiological functions, has received attention as a prospective therapeutic target for renoprotection. In this study, we investigated the effect and underlying mechanism of AT₂R on the TGF-β receptor II (TGF-βRII) expression and function in human proximal tubular cells (HK-2). Here, we show that the AT₂R agonist CGP42112A decreased TGF-βRII protein expression in a concentration- and time-dependent manner in HK-2 cells. The inhibitory effect of the AT₂R on TGF-βRII expression was blocked by the AT₂R antagonists PD123319 or PD123177. Stimulation with TGF-β1 enhanced EMT in HK-2 cells, which was prevented by pre-treatment with CGP42112A. One of mechanisms in this regulation is associated with the increased TGF-βRII degradation after activation of AT₂R. Furthermore, laser confocal immunofluorescence microscopy showed that AT₂R and TGF-βRII colocalized in HK-2 cells. AT₂R and TGF-βRII coimmunoprecipitated and this interaction was increased after AT₂R agonist stimulation for 30 min. The inhibitory effect of the AT₂R on TGF-βRII expression was also blocked by the nitric oxide synthase inhibitor L-NAME, indicating that nitric oxide is involved in the signaling pathway. Taken together, our study indicates that the renal AT₂R regulates TGF-βRII expression and function via the nitric oxide pathway, which may be important in the control of renal tubulointerstitial fibrosis.     

METHIONINE PROXIMITY ASSAY,A NOVEL METHOD FOR EXPLORING PEPTIDE LIGAND–RECEPTOR INTERACTION

ABSTRACT

Probing G-protein coupled receptor (GPCR) structures is a priority in the functional and structural understanding of GPCRs. In the past, we have used several approaches around photoaffinity labeling in order to establish contact points between peptide ligands and their cognate receptors. Such contact points are helpful to build reality based molecular models of GPCRs and to elucidate their activation mechanisms. Most studies of peptidergic GPCRs have been done with photolabeling peptides containing the benzophenone moiety as a reputedly non-selective probe. However our recent results are now showing that p-benzoylphenylalanine (Bpa) has some selectivity for Met residues in the receptor protein, reducing the accuracy of this method. Turning a problem into an asset, modified analogues of Bpa, e.g. p,?p″-nitrobenzoylphenylalanine (NO₂Bpa), display increased selectivity for such Met residues. It means a photoprobe containing such modified benzophenone-moieties does not label a receptor protein unless a Met residue is in the immediate vicinity. This unique property allows us to propose and show the feasibility and utility of a new method for scanning the contact areas of peptidergic GPCRs, the Methionine Proximity Assay (MPA). Putative contact residues of the receptor are exchanged to Met residues by site-directed mutagenesis and are subjected to photoaffinity labeling with such modified benzophenone-containing peptides. Successful incorporation indicates physical proximity of those residues. This principle is established and explored with benzophenone-containing analogues of angiotensin II and the two known human angiotensin II receptors AT₁ and AT₂, determining contact points in both receptors. This approach has several important advantages over other scanning approaches, e.g., the SCAM procedure, since the MPA-method can be used in the hydrophobic core of receptors.     

New insights and perspectives on intrarenal renin-angiotensin system: focus on intracrine/intracellular angiotensin II

Although renin, the rate-limiting enzyme of the renin-angiotensin system (RAS), was first discovered by Robert Tigerstedt and Bergman more than a century ago, the research on the RAS still remains stronger than ever. The RAS, once considered to be an endocrine system, is now widely recognized as dual (circulating and local/tissue) or multiple hormonal systems (endocrine, paracrine and intracrine). In addition to the classical renin/angiotensin I-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II receptor (AT₁/AT₂) axis, the prorenin/(Pro)renin receptor (PRR)/MAP kinase axis, the ACE2/Ang (1-7)/Mas receptor axis, and the Ang IV/AT₄/insulin-regulated aminopeptidase (IRAP) axis have recently been discovered. Furthermore, the roles of the evolving RAS have been extended far beyond blood pressure control, aldosterone synthesis, and body fluid and electrolyte homeostasis. Indeed, novel actions and underlying signaling mechanisms for each member of the RAS in physiology and diseases are continuously uncovered. However, many challenges still remain in the RAS research field despite of more than one century's research effort. It is expected that the research on the expanded RAS will continue to play a prominent role in cardiovascular, renal and hypertension research. The purpose of this article is to review the progress recently being made in the RAS research, with special emphasis on the local RAS in the kidney and the newly discovered prorenin/PRR/MAP kinase axis, the ACE2/Ang (1-7)/Mas receptor axis, the Ang IV/AT₄/IRAP axis, and intracrine/intracellular Ang II. The improved knowledge of the expanded RAS will help us better understand how the classical renin/ACE/Ang II/AT₁ receptor axis, extracellular and/or intracellular origin, interacts with other novel RAS axes to regulate blood pressure and cardiovascular and kidney function in both physiological and diseased states.     

Characterization of AT2 Receptor Expression in NIH 3T3 Fibroblasts

1. A high expression of angiotensin II receptors and of angiotensin-converting enzyme (ACE) activity was detected in confluent NIH 3T3 fibroblasts.2. Characterization with selective ligands, dithiothreitol, and GTPS, indicated that only the AT₂ subtype was expressed.3. AT₂ receptors and ACE expression were strictly dependent on the cell density and growth phase of the cells, with AT₂ receptors being expressed earlier than ACE. In contrast, high expression of AT₂ receptors irrespective of their growth state was observed in NIH 3T3 cells lacking contact inhibition upon neoplastic transformation with ras.4. Our results imply a possible relation of AT₂ receptors to cell growth and cell–cell contact.     

Angiotensin II induces the production of MMP-3 and MMP-13 through the MAPK signaling pathways via the AT1 receptor in osteoblasts

Angiotensin II (Ang II) plays an important role in the maintenance of bone mass and integrity by activation of the mitogen-activated protein kinases (MAPKs) and by modulation of balance between resorption by osteoclasts and formation by osteoblasts. However, the role of Ang II in the turnover of extracellular matrix (ECM) in osteoid by osteoblasts remains unclear. Therefore, we examined the effect of Ang II on the expression of matrix metalloproteinases (MMPs), plasminogen activators (PAs), and their inhibitors [i.e., tissue inhibitors of metalloproteinases (TIMPs) and PA inhibitor-1 (PAI-1)] using osteoblastic ROS17/2.8 cells. Treatment with Ang II strikingly increased the expressions of MMP-3 and -13 and promoted cell proliferation associated with reduced alkaline phosphatase activity as well as enhanced phosphorylated expression of extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, and stress-activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) in ROS17/2.8 cells. However, Ang II had no effect on the expression of MMP-2, -9, -14, urokinase-type PA, tissue-type PA, TIMP-1, -2, -3, and PAI-1 in cells. Losartan (AT₁ receptor blocker) blocked Ang II-induced expression of MMP-3 and -13, whereas PD123319 (AT₂ receptor blocker) did not completely block these responses. Losartan also blocked the Ang II-induced phosphorylation of ERK1/2, p38 MAPK, and SAPK/JNK. MAPK kinase 1/2 inhibitor PD98059 and JNK inhibitor SP600125 suppressed Ang II-induced expression of MMP-3 and -13. These results suggested that Ang II stimulated the degradation process that occurs during ECM turnover in osteoid by increasing the production of MMP-3 and -13 through MAPK signaling pathways via the AT₁ receptor in osteoblasts. Furthermore, our findings suggest that Ang II does not influence the plasminogen/plasmin pathway in osteoblasts.     

Na-ATPase in spontaneous hypertensive rats: Possible AT1 receptor target in the development of hypertension

Clinical and experimental data show an increase in sodium reabsorption on the proximal tubule (PT) in essential hypertension. It is well known that there is a link between essential hypertension and renal angiotensin II (Ang II). The present study was designed to examine ouabain-insensitive Na⁺-ATPase activity and its regulation by Ang II in spontaneously hypertensive rats (SHR). We observed that Na⁺-ATPase activity was enhanced in 14-week-old but not in 6-week-old SHR. The addition of Ang II from 10^− 12 to 10^− 6 mol/L decreased the enzyme activity in SHR to a level similar to that obtained in WKY. The Ang II inhibitory effect was completely reversed by a specific antagonist of AT₂ receptor, PD123319 (10^− 8 mol/L) indicating that a system leading to activation of the enzyme in SHR is inhibited by AT₂-mediated Ang II. Treatment of SHR with losartan for 10 weeks (weeks 4-14) prevents the increase in Na⁺-ATPase activity observed in 14-week-old SHR. These results indicate a correlation between AT₁ receptor activation in SHR and increased ouabain-insensitive Na⁺-ATPase activity. Our results open new possibilities towards our understanding of the pathophysiological mechanisms involved in the increased sodium reabsorption in PT found in essential hypertension.     

Angiotensin AT2 receptor agonist stimulates high stretch induced- ANP secretion via PI3K/NO/sGC/PKG/pathway

Angiotensin II (Ang II) type 1 receptor (AT₁R) mediates the major cardiovascular effects of Ang II. However, the effects mediated via AT₂R are still controversial. The aim of the present study is to define the effect of AT₂R agonist CGP42112A (CGP) on high stretch-induced ANP secretion and its mechanism using in vitro and in vivo experiments. CGP (0.01, 0.1 and 1 μM) stimulated high stretch-induced ANP secretion and concentration from isolated perfused rat atria. However, atrial contractility and the translocation of extracellular fluid did not change. The augmented effect of CGP (0.1 μM) on high stretch-induced ANP secretion was attenuated by the pretreatment with AT₂R antagonist or inhibitor for phosphoinositol 3-kinase (PI3K), nitric oxide (NO), soluble guanylyl cyclase (sGC), or protein kinase G (PKG). However, antagonist for AT₁R or Mas receptor did not influence CGP-induced ANP secretion. In vivo study, acute infusion of CGP for 10 min increased plasma ANP level without blood pressure change. In renal hypertensive rat atria, AT₂R mRNA and protein levels were up-regulated and the response of plasma ANP level to CGP infusion in renal hypertensive rats augmented. The pretreatment with AT₂R antagonist for 10 min followed by CGP infusion attenuated an increased plasma ANP level induced by CGP. However, pretreatment with AT₁R or Mas receptor antagonist unaffected CGP-induced increase in plasma ANP level. Therefore, we suggest that AT₂R agonist CGP stimulates high stretch-induced ANP secretion through PI3K/NO/sGC/PKG pathway and these effects are augmented in renal hypertensive rats.     

Brain Angiotensin II: New Developments, Unanswered Questions and Therapeutic Opportunities

1. There are two Angiotensin II systems in the brain. The discovery of brain Angiotensin II receptors located in neurons inside the blood brain barrier confirmed the existence of an endogenous brain Angiotensin II system, responding to Angiotensin II generated in and/or transported into the brain. In addition, Angiotensin II receptors in circumventricular organs and in cerebrovascular endothelial cells respond to circulating Angiotensin II of peripheral origin. Thus, the brain responds to both circulating and tissue Angiotensin II, and the two systems are integrated. 2. The neuroanatomical location of Angiotensin II receptors and the regulation of the receptor number are most important to determine the level of activation of the brain Angiotensin II systems. 3. Classical, well-defined actions of Angiotensin II in the brain include the regulation of hormone formation and release, the control of the central and peripheral sympathoadrenal systems, and the regulation of water and sodium intake. As a consequence of changes in the hormone, sympathetic and electrolyte systems, feed back mechanisms in turn modulate the activity of the brain Angiotensin II systems. It is reasonable to hypothesize that brain Angiotensin II is involved in the regulation of multiple additional functions in the brain, including brain development, neuronal migration, process of sensory information, cognition, regulation of emotional responses, and cerebral blood flow. 4. Many of the classical and of the hypothetical functions of brain Angiotensin II are mediated by stimulation of Angiotensin II AT₁ receptors. 5. Brain AT₂ receptors are highly expressed during development. In the adult, AT₂ receptors are restricted to areas predominantly involved in the process of sensory information. However, the role of AT₂ receptors remains to be clarified. 6. Subcutaneous or oral administration of a selective and potent non-peptidic AT₁ receptor antagonist with very low affinity for AT₂ receptors and good bioavailability blocked AT₁ receptors not only outside but also inside the blood brain barrier. The blockade of the complete brain Angiotensin II AT₁ system allowed us to further clarify some of the central actions of the peptide and suggested some new potential therapeutic avenues for this class of compounds. 7. Pretreatment with peripherally administered AT₁ antagonists completely prevented the hormonal and sympathoadrenal response to isolation stress. A similar pretreatment prevented the development of stress-induced gastric ulcers. These findings strongly suggest that blockade of brain AT₁ receptors could be considered as a novel therapeutic approach in the treatment of stress-related disorders. 8. Peripheral administration of AT₁ receptor antagonists strongly affected brain circulation and normalized some of the profound alterations in cerebrovascular structure and function characteristic of chronic genetic hypertension. AT₁ receptor antagonists were capable of reversing the pathological cerebrovascular remodeling in hypertension and the shift to the right in the cerebral autoregulation, normalizing cerebrovascular compliance. In addition, AT₁ receptor antagonists normalized the expression of cerebrovascular nitric oxide synthase isoenzymes and reversed the inflammatory reaction characteristic of cerebral vessels in hypertension. As a consequence of the normalization of cerebrovascular compliance and the prevention of inflammation, there was, in genetically hypertensive rats a decreased vulnerability to brain ischemia. After pretreatment with AT₁ antagonists, there was a protection of cerebrovascular flow during experimental stroke, decreased neuronal death, and a substantial reduction in the size of infarct after occlusion of the middle cerebral artery. At least part of the protective effect of AT₁ receptor antagonists was related to the inhibition of the Angiotensin II system, and not to the normalization of blood pressure. These results indicate that treatment with AT₁ receptor antagonists appears to be a major therapeutic avenue for the prevention of ischemia and inflammatory diseases of the brain. 9. Thus, orally administered AT₁ receptor antagonists may be considered as novel therapeutic compounds for the treatment of diseases of the central nervous system when stress, inflammation and ischemia play major roles. 10. Many questions remain. How is brain Angiotensin II formed, metabolized, and distributed? What is the role of brain AT₂ receptors? What are the molecular mechanisms involved in the cerebrovascular remodeling and inflammation which are promoted by AT₁ receptor stimulation? How does Angiotensin II regulate the stress response at higher brain centers? Does the degree of activity of the brain Angiotensin II system predict vulnerability to stress and brain ischemia? We look forward to further studies in this exiting and expanding field.     

Involvement of the AT1 receptor in the venoconstriction induced by angiotensin II in both the inferior vena cava and femoral vein

Although angiotensin II-induced venoconstriction has been demonstrated in the rat vena cava and femoral vein, the angiotensin II receptor subtypes (AT₁ or AT₂) that mediate this phenomenon have not been precisely characterized. Therefore, the present study aimed to characterize the pharmacological receptors involved in the angiotensin II-induced constriction of rat venae cavae and femoral veins, as well as the opposing effects exerted by locally produced prostanoids and NO upon induction of these vasomotor responses. The obtained results suggest that both AT₁ and AT₂ angiotensin II receptors are expressed in both veins. Angiotensin II concentration-response curves were shifted toward the right by losartan but not by PD 123319 in both the vena cava and femoral vein. Moreover, it was observed that both 10⁻⁵ M indomethacin and 10⁻⁴ M L-NAME improve the angiotensin II responses in the vena cava and femoral vein. In conclusion, in the rat vena cava and femoral vein, angiotensin II stimulates AT₁ but not AT₂ to induce venoconstriction, which is blunted by vasodilator prostanoids and NO.     

AT4 receptor structure-binding relationship: N-terminal-modified angiotensin IV analogues

The effect of structural changes in the N-terminal amino acid of AIV, with respect to AT₄ receptor binding, was examined by competition with [¹²⁵I]AIV in bovine adrenal membranes. Analogues with modifications of the first residue α-amino group possessed lower affinities than the primary amine-containing parent compound. Peptides with a residue 1 α-carbon in the d conformation exhibited poor affinity for the AT₄ receptor. Modifications of the residue 1 R-group demonstrate that a straight chain aliphatic moiety containing four carbons is optimal for receptor-ligand binding, as evidenced by the extremely high affinity of [Nle¹]AIV (K_i = 3.59±0.51 pM). Replacement of the 1–2 peptide bond of AIV with the methylene bond isostere Ψ (CH₂-NH), increased the K_i approximately fivefold, indicating that the peptide bond may be replaced wihle maintaining relatively high-affinity receptor binding.