AT1 Receptor Signaling Research Articles

Angiotensin II AT2 receptor regulates ureteric bud morphogenesis.

ANG II AT2 receptor (AT2R)-deficient mice exhibit abnormal ureteric bud (UB) budding, increased incidence of double ureters, and vesicoureteral reflux. However, the role of the AT2R during UB morphogenesis and the mechanisms by which aberrant AT2R signaling disrupts renal collecting system development have not been fully defined. In this study, we mapped the expression of the AT2R during mouse metanephric development, examined the impact of disrupted AT2R signaling on UB branching, cell proliferation, and survival, and investigated the cross talk of the AT2R with the glial-derived neurotrophic factor (GDNF)/c-Ret/Wnt11 signaling pathway. Embryonic mouse kidneys express AT2R in the branching UB and the mesenchyme. Treatment of embryonic day 12.5 (E12.5) metanephroi with the AT2R antagonist PD123319 or genetic inactivation of the AT2R in mice inhibits UB branching, decreasing the number of UB tips compared with control (34 +/- 1.0 vs. 43 +/- 0.6, P < 0.01; 36 +/- 1.8 vs. 48 +/- 1.3, P < 0.01, respectively). In contrast, treatment of metanephroi with the AT2R agonist CGP42112 increases the number of UB tips compared with control (48 +/- 1.8 vs. 39 +/- 12.3, P < 0.05). Using real-time quantitative RT-PCR and whole mount in situ hybridization, we demonstrate that PD123319 downregulates the expression of GDNF, c-Ret, Wnt11, and Spry1 mRNA levels in E12.5 metanephroi grown ex vivo. AT(2)R blockade or genetic inactivation of AT2R stimulates apoptosis and inhibits proliferation of the UB cells in vivo. We conclude that AT2R performs essential functions during UB branching morphogenesis via control of the GDNF/c-Ret/Wnt11 signaling pathway, UB cell proliferation, and survival.

Inhibition of angiotensin II receptor 1 limits tumor-associated angiogenesis and attenuates growth of murine melanoma

We evaluated the involvement of angiotensin II (AngII)-dependent pathways in melanoma growth, through the pharmacological blockage of AT1 receptor by the anti-hypertensive drug losartan (LOS). We showed immunolabeling for both AngII and the AT1 receptor within the human melanoma microenvironment. Like human melanomas, we showed that murine melanomas also express the AT1 receptor. Growth of murine melanoma, both locally and at distant sites, was limited in mice treated with LOS. The reduction in tumor growth was accompanied by a twofold decrease in tumor-associated microvessel density and by a decrease in CD31 mRNA levels. While no differences were found in the VEGF expression levels in tumors from treated animals, reduction in the expression of the VEGFR1 (Flt-1) at the mRNA and protein levels was observed. We also showed downregulation of mRNA levels of both Flt-4 and its ligand, VEGF-C. Together, these results show that blockage of AT1 receptor signaling may be a promising anti-tumor strategy, interfering with angiogenesis by decreasing the expression of angiogenic factor receptors.

Cross‐talk between NAD(P)H oxidase and AT1 receptors in the proximal tubules of obese Zucker rats

Abnormal regulation of renin‐angiotensin system and increased level of oxidative stress have been implicated in renal/cardiovascular diseases associated with obesity. Earlier, we have shown that Angiotensin II AT1 receptor activity causes greater antinatriuresis via enhanced stimulation of tubular Na,K‐ATPase (NKA) in obese Zucker rats. The present study investigated the role of NAD(P)H oxidase in the enhanced AT1 receptor function in obese Zucker rats. Lean and obese Zucker rats were given NAD(P)H oxidase inhibitor, apocynin (3.5 μg/kg/min; i.v infusion) and/or AT1 receptor antagonist, candesartan (100 μg/kg; i.v bolus). Pretreatment with apocynin completely abolished candesartan‐induced diuresis/natriuresis in obese Zucker rats. In lean Zucker rats, apocynin alone increased diuresis/natriuresis and had an additive effect on diuresis/natriuresis when given along with candesartan. The blood pressure and GFR remained unchanged during drug treatment indicating a tubular effect. Apocynin treatment abolished and brought the AngII‐stimulated NKA activity below the basal activity in the isolated proximal tubules of obese Zucker rats. The data suggest NAD(P)H oxidase as an antinatriuretic molecule that switches to AT1 receptor signaling in the proximal tubules of obese Zucker rats and thereby contributes to the enhanced antinatriuresis. (NIH‐R01‐DK‐61578; GEAR‐UH)

The role of central AT1 receptors in age related changes of hypothalamic redox signaling and adrenomedullary tyrosine hydroxylase expression

The aim of this study was to test the role of angiotensin II (AngII)‐mediated mechanisms in age‐related changes of hypothalamic catecholamine biosynthesis, redox signaling and sympathetic nervous system (SNS) activation. Young (5 mo, n=6) and old (27 mo, n=6) F‐344xBN rats were infused intacerebroventricularly with either artificial CSF or losartan (15 µg/hour) for 3 days using osmotic minipumps. Level of hypothalamic tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine biosynthesis, decreased significantly with age, and in response to losartan both in young and old animals. In addition, MnSOD, catalase and NADPH oxidase‐p47 levels were decreased in the hypothalamus with age, and also with losartan treatment in both age groups. Adrenomedullary TH, an indicator of SNS activity, increased significantly with age, but decreased after losartan infusion in both young and old rats reversing the age‐related TH upregulation. These results suggest that central AT1 receptor signaling play an important role in the regulation of hypothalamic redox signaling and SNS regulation.

Protective role of angiotensin II type 2 receptor signaling in a mouse model of pancreatic fibrosis

The renin-angiotensin system contributes to pathological processes in a variety of organs. In the pancreas, blocking the angiotensin II (AII) type 1 receptor (AT1) attenuates pancreatic fibrogenesis in animal models of pancreatitis. Because the role of the AII type 2 receptor (AT2) in modulating pancreatic injury is unknown we investigated the role of AT2 in pancreatic injury and fibrosis. Pancreatic fibrosis was induced by repetitive cerulein administration in C57BL/6 wild-type (WT) or AT2-deficient (AT2-/-) mice and assessed by morphology and gene expression at 10 days. There was no difference between WT and AT2-/- mice in the degree of acute pancreatic injury as assessed by amylase release at 9 and 12 h and by histological examination of the pancreas at 12 h. In contrast, parenchymal atrophy and fibrosis were more pronounced in AT2-/- mice compared with WT mice at 10 days. Fibrosis was accompanied by activation of pancreatic stellate cells (PSC) evaluated by Western blot analysis for alpha-smooth muscle actin and by immunocytochemistry; PSC activation was further increased in AT2-/- mice compared with WT mice. The level of pancreatic transforming growth factor-beta1 mRNA and protein after repetitive cerulein treatment was higher in AT2-/- mice than in WT mice. Our results demonstrate that, in contrast to AT1 receptor signaling, AT2 receptor signaling modulates protective antifibrogenic effects in a mouse model of cerulein-induced pancreatic fibrogenesis. We propose that the effects of AII on injury-induced pancreatic fibrosis may be determined by the balance between AT1 and AT2 receptor signaling.

Deletion of Angiotensin II Type 2 Receptor Attenuates Protective Effects of Bone Marrow Stromal Cell Treatment on Ischemia–Reperfusion Brain Injury in Mice

Protective effects of bone marrow stromal cells (MSCs) on ischemic brain damage have been highlighted. We examined the possibility that deletion of AT(2) receptor could attenuate the cerebroprotective effects of MSC using AT(2) receptor-deficient mice (Agtr2 (-)) and the effect of selective AT(1) receptor blocker. Wild-type mice (Agtr2 (+)) were subjected to 3 hours of focal brain ischemia followed by reperfusion (ischemia-reperfusion injury). Simultaneously, Agtr2 (+)-MSC, Agtr2 (-)-MSC, or saline was injected through the tail vein. Survival rates at 6 days after ischemia-reperfusion injury were as follows: approximately 50% in saline-injected mice, 80% in Agtr2 (+)-MSC-injected mice, and 20% in Agtr2 (-)-MSC-injected mice. Neurological deficit after ischemia-reperfusion injury was improved in Agtr2 (+)-MSC-injected mice, but not in Agtr2 (-)-MSC-injected mice. After 48 hours of ischemia-reperfusion injury, brain infarct size was reduced in Agtr2 (+)-MSC-injected mice, but not in Agtr2 (-)-MSC-injected mice. Moreover, brain edema was significantly ameliorated in Agtr2 (+)-MSC-treated mice but not in Agtr2 (-)-MSC-treated mice. Furthermore, the increase in mRNA expression of tumor necrosis factor-alpha and monocyte chemoattractant protein-1 in the ischemic brain was less in Agtr2 (+)-MSC-treated mice in the ipsilateral site, but was similar in the contralateral hemisphere. Tumor necrosis factor-alpha level was increased in both the contralateral hemisphere and ipsilateral hemisphere of Agtr2 (-)-MSC-treated mice. In contrast, monocyte chemoattractant protein-1 levels tended to increase Agtr2 (-)-MSC-treated mice without a significant difference. Treatment of MSC with an AT(1) receptor blocker, valsartan, significantly improved survival rates in Agtr2 (-)-MSC-injected mice. These results suggest that AT(2) receptor signaling in MSC attenuated brain damage and neurological deficit (deleted).

Oxidative stress-induced renal angiotensin AT1 receptor upregulation causes increased stimulation of sodium transporters and hypertension

Reactive oxygen species have emerged as important molecules in cardiovascular dysfunction such as diabetes and hypertension. Recent work has shown that oxidative stress and angiotensin II signaling mutually regulate each other by multiple mechanisms and contribute to the development of hypertension. Most of the known biological actions of angiotensin II can be attributed to AT1 receptors. The present study was carried out to investigate the role of renal AT1 receptor signaling in oxidative stress-mediated hypertension. Male Sprague-Dawley rats received tap water (control) or 30 mM L-buthionine sulfoximine (BSO), an oxidant, with and without 1 mM tempol (an antioxidant) for 2 wk. Compared with control rats, BSO-treated rats exhibited increased oxidative stress and reduced antioxidant levels and developed hypertension. BSO treatment also caused increased renal proximal tubular AT1 receptor protein abundance, message levels, and ligand binding. In these rats, angiotensin II caused significantly higher accumulation of inositol trisphosphate (IP3) and phospholipase C (PLC) activation which was sensitive to blockade by AT1 but not to AT2 antagonist. Also, angiotensin II-mediated, AT1-dependent MAP kinase, Na-K-ATPase, and Na/H exchanger 3 activation was higher in BSO-treated rats than in control rats. Tempol supplementation of BSO-treated rats restored redox status, normalized AT1 receptor expression, and decreased blood pressure. Tempol also normalized the angiotensin II-mediated, AT1-dependent IP3 accumulation and PLC, MAP kinase, Na-K-ATPase, and Na/H exchanger 3 stimulation. These data suggest that oxidative stress leads to AT1 receptor upregulation, which in turn causes overstimulation of sodium transporters and subsequently contributes to sodium retention and hypertension. Tempol, while reducing oxidative stress, normalizes AT1 receptor signaling and decreases blood pressure.

Triple Twist Theory of Rho Inhibition by the Angiotensin II Type 2 Receptor

Although there are some exceptions,1,2 numerous publications support the counterregulatory roles of the angiotensin II type 2 receptor (AT2) against the AT1 receptor functions, such as inhibition of vascular contraction and hypertrophy.3–5 However, it is still very uncertain as to how the AT2 receptor signals interfere with those of the AT1 receptor in the cardiovascular system.5,6 Past findings suggest that the signal transduction of AT1 inhibition by the AT2 receptor may involve multiple distinct mechanisms. Some of these mechanisms appear to be indirect, such as production of nitric oxide through bradykinin opposing the vasoconstrictor actions of the AT1 receptor.3 The direct inhibitory crosstalk of the 2 receptors occurs proximal to the receptor heterodimerization, as well as downstream from the receptors between AT1-activated protein kinases epidermal growth factor receptor kinase and extracellular signal-regulated kinase (ERK)1/2/p42/44 mitogen-activated protein kinase (MAPK), etc and AT2-activated protein phosphatases protein phosphatase 2A, SHP-1, and MAPK phosphatase-1.7,8 The activation of the protein phosphatases by the AT2 receptor may or may not require heterotrimeric G proteins (Gi or Gs) and/or the recently identified AT2 receptor C-terminal tail–interacting proteins.4–6 Given that induction …

Angiotensin II regulates the activity of mouse suprachiasmatic nuclei neurons

Neuropeptide signaling plays key roles in coordinating cellular activity within the suprachiasmatic nuclei (SCN), site of the master circadian oscillator in mammals. The neuropeptide angiotensin II (ANGII) and its cognate receptor AT1, are both expressed by SCN cells, but unlike other SCN neurochemicals, very little is known about the cellular actions of ANGII within this circadian clock. We used multi-electrode, multiunit, extracellular electrophysiology, coupled with whole-cell voltage and current clamp techniques to investigate the actions of ANGII in mouse SCN slices. ANGII (0.001-10 microM) dose dependently stimulated and inhibited extracellularly recorded neuronal discharge in many SCN neurons ( approximately 60%). Both actions were blocked by pre-treatment with the AT1 receptor antagonist ZD7155 (0.03 microM), while suppressions but not activations were prevented by pre-treatment with the GABA A receptor antagonist bicuculline (20 microM). AT1 receptor blockade itself suppressed discharge in a subset ( approximately 30%) of SCN neurons, and this action was not blocked by bicuculline. In voltage-clamped SCN neurons (-70 mV), AT1 receptor activation dose-dependently enhanced the frequency of action potential-driven, GABA A receptor-mediated currents, but did not alter their responses to exogenously applied GABA. In current-clamped SCN neurons perfused with tetrodotoxin, ANGII induced a membrane depolarization with a concomitant decrease in input resistance. In conclusion we show that AT1 receptor activation by ANGII depolarizes SCN neurons and stimulates action potential firing, leading to increased GABA release in the mouse SCN. Additionally we provide the first evidence that endogenous AT1 receptor signaling tonically regulates the activities of some SCN neurons.

Genetic disruption of guanylyl cyclase/natriuretic peptide receptor-A upregulates ACE and AT1 receptor gene expression and signaling: role in cardiac hypertrophy

Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) signaling antagonizes the physiological effects mediated by the renin-angiotensin system (RAS). The objective of this study was to determine whether the targeted-disruption of Npr1 gene (coding for GC-A/NPRA) leads to the activation of cardiac RAS genes involved on the hypertrophic remodeling process. The Npr1 gene-knockout (Npr1(-/-)) mice showed 30-35 mmHg higher systolic blood pressure (SBP) and a 63% greater heart weight-to-body weight (HW/BW) ratio compared with wild-type (Npr1(+/+)) mice. The mRNA levels of both angiotensin-converting enzyme and angiotensin II type 1a receptor were increased by three- and fourfold, respectively, in Npr1(-/-) null mutant mice hearts compared with the wild-type Npr1(+/+) mice hearts. In parallel, the expression levels of interleukin-6 and tumor necrosis factor-alpha were increased by four- to fivefold, in Npr1(-/-) mice hearts compared with control animals. The NF-kappaB binding activity in nuclear extracts of Npr1(-/-) mice hearts was increased by fourfold compared with wild-type Npr1(+/+) mice hearts. Treatments with captopril or hydralazine equally attenuated SBP; however, only captopril significantly decreased the HW/BW ratio and suppressed cytokine gene expression in Npr1(-/-) mice hearts. The ventricular cGMP level was reduced by almost sixfold in Npr1(-/-) mice compared with wild-type control mice. The results of the present study indicate that disruption of NPRA/cGMP signaling leads to the augmented expression of cardiac RAS pathways that promote the development of cardiac hypertrophy and remodeling.

Participation of transmembrane proline 82 in angiotensin II AT 1 receptor signal transduction

Most of the classical physiological effects of the octapeptide angiotensin II (AngII) are produced by activating the AT 1 receptor which belongs to the G-protein coupled receptor family (GPCR). Peptidic GPCRs may be functionally divided in three regions: (i) extracellular domains involved in ligand binding; (ii) intracellular domains implicated in agonist-induced coupling to G protein and (iii) seven transmembrane domains (TM) involved in signal transduction. The TM regions of such receptors have peculiar characteristics such as the presence of proline residues. In this project we aimed to investigate the participation of two highly conserved proline residues (Pro82 and Pro162), located in TM II and TM IV, respectively, in AT1 receptor signal transduction. Both mutations did not cause major alterations in AngII affinity. Functional assays indicated that the P162A mutant did not influence the signal transduction. On the other hand, a potent deleterious effect of P82A mutation on signal transduction was observed. We believe that the Pro82 residue is crucial to signal transduction, although it is not possible to say yet if this is due to a direct participation or if due to a structural rearrangement of TM II. In this last hypothesis, the removal of proline residue might be correlated to a removal of a kink, which in turn can be involved in the correct positioning of residues involved in signal transduction.

Angiotensin II type 1 receptor signaling contributes to platelet-leukocyte-endothelial cell interactions in the cerebral microvasculature

Angiotensin II type 1 (AT(1)) receptor signaling has been implicated in cerebral microvascular alterations associated with ischemia, diabetes mellitus, hypercholesterolemia, and atherosclerosis. Platelets, which express AT(1) receptors, also appear to contribute to the thrombogenic and inflammatory responses that are elicited by these pathological conditions. This study assesses the role of AT(1) receptor activation on platelet-leukocyte-endothelial cell interactions elicited in cerebral microvasculature by ischemia and reperfusion. Intravital microscopy was used to monitor the adhesion of platelets and leukocytes that were labeled with different fluorochromes, whereas dihydrorhodamine-123 was used to quantify oxygen radical production in cerebral surface of mice that were either treated with the AT(1) receptor agonist Val-angiotensin II (ANG II) or subjected to bilateral common carotid artery occlusion (BCCAO) followed by reperfusion. ANG II elicited a dose- and time- dependent increase in platelet-leukocyte-endothelial cell interactions in cerebral venules that included rolling platelets, adherent platelets on the leukocytes and the endothelial cells, rolling leukocytes, and adherent leukocytes. All of these interactions were attenuated by treatment with either P-selectin or P-selectin glycoprotein ligand 1 (PSGL-1) antibody. The AT(1) receptor antagonist candesartan and losartan as well as diphenyleneiodonium, an inhibitor of flavoproteins including NAD(P)H oxidase, significantly reduced the platelet-leukocyte-endothelial cell interactions elicited by either ANG II administration or BCCAO/reperfusion. The increased oxygen radical generation elicited by BCCAO/reperfusion was also attenuated by candesartan. These findings are consistent with an AT(1) receptor signaling mechanism, which involves oxygen radical production and ultimately results in P-selectin- and PSGL-1-mediated platelet-leukocyte-endothelial cell interactions in the cerebral microcirculation.

Enhanced Na‐K‐ATPase (NKA) Affinity for Sodium and Reduced AT1 Receptor Function on NKA Activity in the Proximal Tubule of Streptozotocin‐Induced Diabetic Rats

Early type 1 diabetes is associated with enhanced renal Na reabsorption; however the mechanism is not known. Present study determined the kinetic properties of NKA in the proximal tubule (PTs) and NKA stimulation by Angiotensin II (Ang II) as a potential mechanism responsible for enhanced renal Na reabsorption. Isolated PTs from streptozotocin-induced diabetic and control rats were used. Ang II (10 pM) stimulated NKA activity in PTs of control rats (83±4 vs. 107±6 nmole Pi/mg/min), but not in diabetic rats (110±12 vs. 122±11 nmole Pi/mg/min). The AT1 receptor expression was similar in PTs of control and diabetic rats. The expression of G-proteins (Giα2 and Giα3), potentially associated with AT1 receptor signaling, was decreased by 25% in the diabetic rats. Kinetic parameters of NKA revealed an increased affinity, low K0.5, for Na in the diabetic rats (Cont, 4.7±0.5 vs. diab. 11.7±0.09 mM); the Vmax was similar in both rat groups (111±14 vs 127±14 nmole Pi/mg/min). The basal ser-phosphorylation of NKA α1 subunit was lower in diabetic compared with control rats. The data suggest that the higher basal NKA affinity for Na possibly due to lower Ser-phosphorylaion of α1-subunit, and not the AT1 receptor signaling, in the PTs may be responsible for increased renal Na reabsorption associated with early stage of diabetes. (supported by DK-061578)

Angiotensin type 1 receptor (AT1) double null mice (DKO) exhibit augmented renal renin and neuronal nitric oxide synthase (nNOS) protein expression

DKO mice display reduced blood pressure, renal vascular thickening, medullary atrophy, and tubulointerstitial injury. Juxtamedullary efferent arterioles of AT1A−/− mice, and afferent arterioles of DKO mice exhibit significantly larger baseline vessel diameters compared to wild type (WT). We propose that an imbalance of AngII and nitric oxide signaling produces renal microvascular dilation. Kidneys were harvested from WT, AT1B−/−, AT1A−/−, and DKO mice, fixed, sectioned, and immunostained for renin and nNOS protein localization. The percent of glomeruli positive for JGA renin and the density of JGA renin immunostaining were significantly higher in AT1A−/− and DKO than WT (60±1%, n=3; 56±9%, n=6; 22±3%, n=6 and 15±9; 15±7; 1±0 fold, respectively). Renin expression of AT1B−/− was not different from WT. AT1A−/− and DKO mice displayed significant recruitment of renin protein expression along the afferent arteriole and interlobular artery (15±7%, 7±3 vessels/kidney n=3; 9±3%, 3±1 vessels/kidney n=6, respectively) compared to WT. WT and AT1B−/− kidneys displayed only JGA renin. Percent of glomeruli with positive MD nNOS immunostaining was not different for WT, AT1A−/−, AT1B−/− and DKO kidneys (46±2, 33±4, 34±, 38±5%), however, AT1A−/− kidneys demonstrated a significant increase in the percent of glomeruli with nNOS positive JGA (19±5 WT, 52±3 AT1A−/−, 8±3 AT1B−/−, 33±10 DKO), afferent arterioles, and interlobular arteries. Density of MD nNOS immunostaining was 7.0±2.6 fold in DKO compared to WT. Density of vascular nNOS immunostaining was 20±7 fold in AT1A−/− relative to WT. Loss of AT1 receptor signaling is coupled to enhanced nNOS protein expression in renal tubular and microvascular structures and may lead to increases in nitric oxide generation. NIDDK-62003 & P20RR018766

Angiotensin II-Induced Neural Differentiation via Angiotensin II Type 2 (AT2) Receptor-MMS2 Cascade Involving Interaction between AT2Receptor-Interacting Protein and Src Homology 2 Domain-Containing Protein-Tyrosine Phosphatase 1

Angiotensin II (Ang II) type 2 (AT2) receptors are abundantly expressed not only in the fetal brain where they probably contribute to brain development, but also in pathological conditions to protect the brain against stroke; however, the detailed mechanisms are unclear. Here, we demonstrated that AT2 receptor signaling induced neural differentiation via an increase in MMS2, one of the ubiquitin-conjugating enzyme variants. The AT2 receptor, MMS2, Src homology 2 domain-containing protein-tyrosine phosphatase 1 (SHP-1), and newly cloned AT2 receptor-interacting protein (ATIP) were highly expressed in fetal rat neurons and declined after birth. Ang II induced MMS2 expression in a dose-dependent manner, reaching a peak after 4 h of stimulation, and this effect was enhanced with AT1 receptor blocker, valsartan, but inhibited by AT2 receptor blocker PD123319. Moreover, we observed that an AT2 receptor agonist, CGP42112A, alone enhanced MMS2 expression. Neurons treated with small interfering RNA of MMS2 failed to exhibit neurite outgrowth and synapse formation. Moreover, the increase in AT2 receptor-induced MMS2 mRNA expression was enhanced by overexpression of ATIP but inhibited by small interfering RNA of SHP-1 and overexpression of catalytically dominant-negative SHP-1 or a tyrosine phosphatase inhibitor, sodium orthovanadate. After AT2 receptor stimulation, ATIP and SHP-1 were translocated into the nucleus after formation of their complex. Furthermore, increased MMS2 expression mediates the inhibitor of DNA binding 1 proteolysis and promotes DNA repair. These results provide a new insight into the contribution of AT2 receptor stimulation to neural differentiation via transactivation of MMS2 expression involving the association of ATIP and SHP-1.

Emerging Concept of Adipogenesis Regulation by the Renin–Angiotensin System

The renin–angiotensin system (RAS) has been the focus of attention not only for its classical action through circulating RAS but also its local actions in tissues such as the brain,1 liver,2 lung,3 and adipose tissue.4 The roles of the local RAS in adipose tissue have recently been highlighted, because obesity is one of the major risks for the metabolic syndrome with hypertension and glucose intolerance. However, there have been inconsistent reports on the role of the adipose tissue RAS. The role of local RAS in adipogenesis has been discussed but is not well elucidated. In this issue of Hypertension , Matsushita et al5 examined the role of local RAS during adipocyte differentiation using human mesenchymal stem cells (MSCs) instead of preadipocytes in an elegant fashion. They reported the influence of RAS on MSCs as follows (Figure 1): (1) Comparison of the expression of RAS components between MSCs and differentiated adipocytes revealed that mRNA of renin and the angiotensin II (Ang II) type-2 (AT2) receptor were highly expressed, and Ang II concentration was significantly increased in differentiated adipocytes; (2) administration of Ang II with or without an Ang II type-1 (AT1) receptor blocker (ARB), valsartan, in MSC during adipogenesis showed that Ang II inhibited adipogenesis, and AT2 receptor activation by valsartan further inhibited it; and (3) moreover, treatment with valsartan alone also inhibited adipocyte differentiation. These results suggest that the local RAS, especially AT2 receptor signaling, has a crucial role in adipogenesis. Figure 1. Effect of endogenous RAS on adipocyte differentiation from MSCs. RAS-related genes are expressed in MSCs, and Ang II secretion is increased during …

Role of Tyrosine Kinase Receptors in Angiotensin II AT2 Receptor Signaling: Involvement in Neurite Outgrowth and in p42/p44mapk Activation in NG108-15 Cells

NG108-15 cells, which have a rounding-up morphology when cultured in serum-supplemented medium, extend neurites when stimulated for 3 d with angiotensin II (Ang II). The aim of the present study was to investigate whether growth factor receptors are necessary for mediating the effects of Ang II. A 3-d treatment with AG879, an inhibitor of nerve growth factor receptor TrkA, strongly affected neurite outgrowth and phosphorylation of p42/p44(mapk) induced by Ang II. PD168393, an inhibitor of epidermal growth factor (EGF) receptor slightly decreased Ang II-induced neurite outgrowth, whereas AG213, an inhibitor of both platelet-derived growth factor receptor and EGF receptor, stimulated neurite outgrowth and p42/p44(mapk) phosphorylation on its own, without affecting further stimulation with Ang II. Moreover, Ang II induced the phosphorylation of TrkA (maximum at 5 min of incubation in the presence of serum or at 20 min in cells depleted in serum for 2 h) and a rapid increase in Rap1 activity, both effects abolished in cells preincubated with 10 microm AG879. In summary, the present results demonstrate that AT(2) receptor-induced sustained activation of p42/p44(mapk) and corresponding neurite outgrowth are mediated by phosphorylation of the nerve growth factor TrkA receptor. However, the results also point out that the presence of other growth factors, such as EGF or PDFG, may interfere with the effect of Ang II. Altogether, the current findings clearly indicate that the effects of the AT(2) receptor on neurite outgrowth dynamics are modulated by the presence of growth factors in the culture medium.

Vascular Cell Locomotion

See related article, pages 1479–1489 Since the discovery of osteopontin (OPN) in the vasculature, vascular biologists have been intrigued by its potential role in vascular arteriosclerosis and vessel calcification during the aging process.1 Osteopontin is a phosphorylated acidic glycoprotein adhesion molecule, originally cloned from bone; and as in bone, its cardiovascular functions include cellular migration, adhesion, and spreading.2 The most salient features of this intriguing calcium-regulating molecule are its ability to interact with αvβ3 and other integrins on the surface of cells. Because this ligand–receptor complex is known to be involved in cell migration, it is not surprising that an antibody to the β3 subunit of this receptor effectively reduced restenosis in patients.3 Interestingly, angiotensin II and other growth factors, including platelet-derived growth factor and transforming growth factor-β, regulate OPN gene expression in vascular smooth muscle cell (VSMC) cultures and fibroblasts.1,4 Cardiac fibroblasts have been extensively reported to produce OPN in response to these growth factors, resulting in fibroblast proliferation. On the one hand, OPN is thought to cause vascular dystrophy by altering extracellular matrix formation; on the other hand, cellular motogenesis is a hallmark of this emerging vascular cytokine. Its cellular expression includes a variety of inflammatory cells, such as T cells and macrophages, as well as mesenchymal cells of the vasculature including adventitial myofibroblasts. Moreover, OPN has both pro- and antiinflammatory properties. As a proinflammatory agent it can attract and modulate the function of the above mentioned cell types. Of seemingly major importance, OPN interacts with intracellular signaling agents to promote cell migration and matrix metalloproteinases, both of which are required for cell tunneling through dense and elastin-rich vascular tissue. In fibroblasts these agents appear to include cell surface CD44 and ERM proteins located just below the plasma membrane …

Angiotensin II Type 2 Receptor Agonist Directly Inhibits Proximal Tubule Sodium Pump Activity in Obese But Not in Lean Zucker Rats

We have reported recently that the renal angiotensin II type 2 (AT2) receptors are upregulated and involved in promoting natriuresis/diuresis in obese but not in lean Zucker rats. In the present study, we tested the hypothesis that there is an enhanced AT2 receptor signaling via NO/cGMP pathway leading to greater inhibition of the Na(+), K(+)-ATPase (NKA) activity in the proximal tubules (PT) of obese rather than lean Zucker rats. The AT2 agonist CGP42112 (0.1 to 100 nmol/L) inhibited (33% at 100 nmol/L) the NKA activity in the PTs of obese but not in lean Zucker rats. The AT2 antagonist PD123319 (1 micromol/L), not the angiotensin II type 1 antagonist losartan (1 micromol/L), significantly diminished the CGP42112-induced inhibition of the NKA activity in obese rats. The AT2 agonist (10 nmol/L)-induced NKA inhibition was abolished by the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one (10 micromol/L), the NO synthase inhibitor NG-nitro-L-arginine methyl ester (100 micromol/L), and the protein kinase G inhibitor K1388 (2 micromole/L). CGP42112 (10 nmol/L) caused an increase in serine phosphorylation of NKA alpha1-subunit in PT of obese rats. Measurement of cGMP and NO revealed that CGP42112 (0.1 to 100 nmol/L) increased cGMP and NO accumulation in the PTs of obese but not lean rats. The CGP42112-induced stimulation of NO and cGMP was blocked by PD123319 (1 micromol/L), NG-nitro-L-arginine methyl ester (100 micromol/L), and 1H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one (10 micromol/L) but not by losartan (1 micromol/L). The data suggest that the AT2 receptor activation via stimulation of the NO/cGMP/protein kinase G pathway directly inhibits the tubular NKA activity that provides as a mechanism responsible for the AT2 receptor-mediated natriuresis in obese but not in lean Zucker rats.

The AT2 receptor—A matter of love and hate

In 1989, the development of specific angiotensin receptor antagonists which distinguish between two angiotensin receptor subtypes (AT1 and AT2) led to a breakthrough in angiotensin research. It turned out, that the AT1 receptor was almost entirely responsible for the “classical” actions of angiotensin II related to the regulation of blood pressure as well as volume and electrolyte balance. However, actions and signal transduction mechanisms coupled to the AT2 receptor remained enigmatic for a long time. The present review summarizes the current knowledge of AT2 receptor distribution, signaling and function with an emphasis on growth/anti-growth, differentiation and the regeneration of neuronal tissue.