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Angiotensin II Type 2 Receptor Inhibits Cell Proliferation and Activates Tyrosine Phosphatase
Abstract
The angiotensin II type 2 (AT2) receptor inhibits basic fibroblast growth factor-induced proliferation of R3T3 fibroblast cells and transiently stimulates a vanadate-sensitive phosphotyrosine phosphatase, strongly suggesting that AT2 is a mitogen inhibitor. We generated AT2 gene-null mice that showed increased blood pressure, indicating the hypotensive action of AT2. However, inhibition of renomedullary AT2 by selective antagonists, as reported by Sassard and associates, show that AT2 suppresses pressure natriuresis. Thus, both AT1 and AT2 work in the direction of sodium retention, suggesting a unique role for angiotensin II in the kidney in terms of blood pressure regulation and sodium metabolism.
... AT 2 R-mediated antagonism of TNF-␣ 13 or of other non-RAS stimuli (eg, growth factors) has already been shown earlier by us and others. 10,23,24 Thus, AT 2 R stimulation seems to be able to interfere with signaling cascades coupled to detrimental stimuli, which do not necessarily have to be considered part of the RAS. "Conventional" ARBs seem not to be able to repress cytokine-induced IL6 expression, as shown in this study and very recently by Tian et al. 25 Apparently, additional peroxisome proliferator-activated receptor-␥-agonistic properties are necessary for a direct anti-inflammatory action of ARBs by a peroxisome proliferator-activated receptor-␥/ NF-B cross-talk. ...
... Such an interference has already been shown for extracellular regulated kinase and signal transducer and activator of transcription signaling and for the deactivation of NF-B. 13,18,23 Reduced NF-B activity resulting in decelerated IL6 transcription has also been observed in our study. The reduction of NF-B dependent IL6 promoter activity by direct AT 2 R stimulation was comparable in strength to inhibition by the classic NF-B inhibitor, hydrocortisone. ...
Angiotensin II type 2 (AT(2)) receptors can be regarded as an endogenous repair system, because the AT(2) receptor is upregulated in tissue damage and mediates tissue protection. A potential therapeutic use of this system has only recently come within reach through synthesis of the first selective, orally active, nonpeptide AT(2) receptor agonist, compound 21 (C21; dissociation constant for AT(2) receptor: 0.4 nM; dissociation constant for angiotensin II type 1 receptor: >10,000 nM). This study tested AT(2) receptor stimulation with C21 as a potential future therapeutic approach for the inhibition of proinflammatory cytokines and of nuclear factor kappaB. C21 dose-dependently (1 nM to 1 micromol/L) reduced tumor necrosis factor-alpha-induced interleukin 6 levels in primary human and murine dermal fibroblasts. AT(2) receptor specificity was controlled for by inhibition with the AT(2) receptor antagonist PD123319 and by the absence of effects in AT(2) receptor-deficient cells. AT(2) receptor-coupled signaling leading to reduced interleukin 6 levels involved inhibition of nuclear factor kappaB, activation of protein phosphatases, and synthesis of epoxyeicosatrienoic acid. Inhibition of interleukin 6 promoter activity by C21 was comparable in strength to inhibition by hydrocortisone. C21 also reduced monocyte chemoattractant protein 1 and tumor necrosis factor-alpha in vitro and in bleomycin-induced toxic cutaneous inflammation in vivo. This study is the first to show the anti-inflammatory effects of direct AT(2) receptor stimulation in vitro and in vivo by the orally active, nonpeptide AT(2) receptor agonist C21. These data suggest that pharmacological AT(2) receptor stimulation may be an orally applicable future therapeutic approach in pathological settings requiring the reduction of interleukin 6 or inhibition of nuclear factor kappaB.
... Several signaling pathways involve activation of protein phosphatases, whose function is to dephosphorylate and thus inactivate MAP kinases such as ERK-1 and ERK-2, resulting in inhibition of both AT 1 Rand other 'classical' growth factor-mediated pathways involved in cellular growth and differentiation (Stoll et al., 1995;Tsuzuki et al., 1996). Increased PTPase activity following AT 2 R stimulation has been demonstrated in PC12W cells (Bottari et al., 1992;Brechler et al., 1994), N1E-115 neuroblastoma cells (Nahmias et al., 1995) and R3T3 fibroblasts (Tsuzuki et al., 1996). ...
... Several signaling pathways involve activation of protein phosphatases, whose function is to dephosphorylate and thus inactivate MAP kinases such as ERK-1 and ERK-2, resulting in inhibition of both AT 1 Rand other 'classical' growth factor-mediated pathways involved in cellular growth and differentiation (Stoll et al., 1995;Tsuzuki et al., 1996). Increased PTPase activity following AT 2 R stimulation has been demonstrated in PC12W cells (Bottari et al., 1992;Brechler et al., 1994), N1E-115 neuroblastoma cells (Nahmias et al., 1995) and R3T3 fibroblasts (Tsuzuki et al., 1996). In PC12W cells and adult rat ventricular myocytes, induction of the PTPase MKP-1 is G-proteindependent Fischer et al., 1998). ...
The renin angiotensin system (RAS) is intricately involved in normal cardiovascular homeostasis. Excessive stimulation by the octapeptide angiotensin II contributes to a range of cardiovascular pathologies and diseases via angiotensin type 1 receptor (AT1R) activation. On the other hand, tElsevier Inc.he angiotensin type 2 receptor (AT2R) is thought to counter-regulate AT1R function. In this review, we describe the enhanced expression and function of AT2R in various cardiovascular disease settings. In addition, we illustrate that the RAS consists of a family of angiotensin peptides that exert cardiovascular effects that are often distinct from those of Ang II. During cardiovascular disease, there is likely to be an increased functional importance of AT2R, stimulated by Ang II, or even shorter angiotensin peptide fragments, to limit AT1R-mediated overactivity and cardiovascular pathologies.
... These anti-inflammatory effects do not counteract Ang II-induced, AT1R-mediated pro-inflammatory actions. AT2R-stimulation antagonizes the effects of TNF-α or other non-RAS stimuli, such as growth factors and has been reported previously [54][55][56]. The Ang II signaling through AT2R activation seems to interfere with other non-RAS signaling cascades coupled with harmful stimuli, and ARBs seem unable to block cytokine-induced IL-6 expression [57]. ...
Through the ACE2, a main enzyme of the renin–angiotensin system (RAS), SARS-CoV-2 gains access into the cell, resulting in different complications which may extend beyond the RAS and impact the Arachidonic Acid (ArA) pathway. The contribution of the RAS through ArA pathways metabolites in the pathogenesis of COVID-19 is unknown. We investigated whether RAS components and ArA metabolites can be considered biomarkers of COVID-19. We measured the plasma levels of RAS and ArA metabolites using an LC-MS/MS. Results indicate that Ang 1–7 levels were significantly lower, whereas Ang II levels were higher in the COVID-19 patients than in healthy control individuals. The ratio of Ang 1–7/Ang II as an indicator of the RAS classical and protective arms balance was dramatically lower in COVID-19 patients. There was no significant increase in inflammatory 19-HETE and 20-HETE levels. The concentration of EETs was significantly increased in COVID-19 patients, whereas the DHETs concentration was repressed. Their plasma levels were correlated with Ang II concentration in COVID-19 patients. In conclusion, evaluating the RAS and ArA pathway biomarkers could provide helpful information for the early detection of high-risk groups, avoid delayed medical attention, facilitate resource allocation, and improve patient clinical outcomes to prevent long COVID incidence.
... These antiinflammatory effects do not counteract Ang II-induced, AT1R-mediated pro-inflammatory actions. AT2R-stimulation antagonizes the effects of TNF-α or other non-RAS stimuli, such as growth factors and has been reported previously [42][43][44]. The Ang II signaling through AT2R activation seems to interfere with other non-RAS signaling cascades coupled with harmful stimuli, and ARBs seem unable to block cytokine-induced IL-6 expression [45]. ...
Through the ACE2, a main enzyme of the renin-angiotensin system (RAS), the SARS-CoV2 gains access into the cell, resulting in different complications which may extend beyond the RAS and impact the Arachidonic Acid (ArA) pathway. The contribution of the RAS through ArA pathways metabolites in the pathogenesis of COVID-19 is unknown. We investigated whether RAS components and ArA metabolites can be considered biomarkers of COVID-19. We measured the plasma levels of RAS and ArA metabolites using an LC-MS/MS. Results indicate that Ang1-7 levels were significantly lower, whereas Ang II levels were higher in the COVID-19 patients than healthy control individuals. The ratio of Ang1-7/Ang II as an indicator of the RAS classical and protective arms balance was dramatically lower in COVID-19 patients. There was no significant increase in inflammatory 19-HETE and 20-HETE lev-els. The concentration of EETs was significantly increased in COVID-19 patients, whereas the DHETs concentration was repressed. Their plasma levels were correlated with Ang II concentration in COVID-19 patients. In conclusion, evaluating the RAS and ArA pathway biomarkers could provide helpful information for the early detection of high-risk groups, avoid delayed medical attention, facilitate resource allocation and improve patient clinical outcomes to prevent long COVID incidence.
... AT 2 R is another functional Ang II receptor in RAS, and it is similar in molecular structure to the G protein-coupled receptor superfamily, which consists of 7 transmembrane regions [77]. The cDNA for AT 2 R encodes a protein of 363 amino acids with molecular weight of 41,220 Da; 34% of the amino acid sequence of AT 2 R is homologous with AT 1 R [78]. Although AT 2 R mRNA is not expressed in adult animals, the AT 2 R protein has been detected in fetal and newborn rat kidneys by using 3 Journal of the Renin-Angiotensin-Aldosterone System immunohistochemistry and Western blot analysis [79]. ...
Renal ischemia-reperfusion injury (RIRI) is a sequence of complicated events that is defined as a reduction of the blood supply followed by reperfusion. RIRI is the leading cause of acute kidney injury (AKI). Among the diverse mediators that take part in RIRI-induced AKI, the renin-angiotensin system (RAS) plays an important role via conventional (angiotensinogen, renin, angiotensin-converting enzyme (ACE), angiotensin (Ang) II, and Ang II type 1 receptor (AT1R)) and nonconventional (ACE2, Ang 1-7, Ang 1-9, AT2 receptor (AT2R), and Mas receptor (MasR)) axes. RIRI alters the balance of both axes so that RAS can affect RIRI-induced AKI. In overall, the alteration of Ang II/AT1R and AKI by RIRI is important to consider. This review has looked for the effects and interactions of RAS activities during RIRI conditions.
... As the extracellular signal-regulated kinase 1/2 (ERK1/2) has been shown to be involved in the signaling cascades mediated by AT1R [47], AT2R [48], and ACE [27], we measured the short-term phosphorylation status of ERK1/2 after 10 minutes of drug administration (Fig. 8). Compared to a 10 min exposure to ATII alone, exposure to losartan in the presence of ATII led to a significant increase in the p(hosphorylated)ERK/t(otal)ERK ratio by about 50%. ...
Background/aims:
Hypertension is treated primarily with angiotensin II (ATII) receptor blockers (ARBs) and angiotensin converting enzyme (ACE) inhibitors (ACEIs). Both ATII and ACEIs can trigger signal transduction via ACE, and a possible correlation between ARB/ACEI therapy and an increased risk of cancer is highly controversial. The question of whether or not ACE as a potential signal transducer affects human melanoma (MV3) cell behavior prompted the present study.
Methods:
Expression of ACE, ATII receptor types 1, 2 (AT1R, AT2R), COX2 and MMP2 in MV3 cells was examined by qPCR. AT1R, AT2R and ACE were inhibited with losartan, EMA401 and lisinopril, respectively. Adhesion, migration and invasiveness of MV3 cells seeded on a hepatocyte (Huh7) monolayer or a reconstituted collagen type I matrix were analyzed using video microscopy and Boyden chambers. Integrity of the Huh7 cell layer was confirmed by measuring transepithelial electrical resistance (TEER). ERK1/2 phosphorylation and MMP2 secretion were evaluated by Western blotting. MMP2 activity was inhibited with ARP-100.
Results:
Losartan, EMA401 and lisinopril stimulated MV3 melanoma cell migration and invasion in a coculture model with Huh7 cells while leaving proliferation and adhesion largely unaffected. The drugs did not interfere with TEER of the hepatocyte monolayer nor with MV3 cell proliferation, but tended to increase the phosphorylation of ERK1/2 and the expression of both COX2 and MMP2. Lisinopril caused a significant increase in MV3 cells' MMP2 secretion and an accelerated MV3 cell-mediated TEER breakdown. The MMP2 inhibitor ARP-100 could antagonize the lisinopril-stimulated invasion of the hepatocyte layer.
Conclusion:
Lisinopril stimulates MV3 cell invasion by increasing the expression and secretion of MMP2.
... Furthermore, depending on the model used, stimulation of the AT2R was shown to activate different tyrosine and serine/threonine phosphatases such as SHP-1, MKP-1, and PP2A, which can lead to transient inhibition of ERK1/2 and insulin signaling [130][131][132][133][134][135][136]. On the other hand, it was shown that AT2R can also induce sustained activation of ERK1/2 through the activation of Rap1/B-Raf [137,138]. ...
Polycystic ovary syndrome (PCOS) is a common and significant condition associated with hyperandrogenism, infertility, low quality of life, and metabolic comorbidities. One possible explanation of PCOS development is cellular dysfunction induced by nonesterified fatty acids (NEFAs), that is, lipotoxicity, which could explain both the hyperandrogenemia and insulin resistance that characterize women with PCOS. The literature suggests that androgen biosynthesis may be induced by overexposure of androgen-secreting tissues to NEFA and/or defective NEFA metabolism, leading to lipotoxic effects. Indeed, lipotoxicity could trigger androgenic hyperresponsiveness to insulin, LH, and ACTH. In most PCOS women, lipotoxicity also causes insulin resistance, inducing compensatory hyperinsulinemia, and may thus further increase hyperandrogenemia. Many approaches aimed at insulin sensitization also reduce lipotoxicity and have been shown to treat PCOS hyperandrogenemia. Furthermore, our group and others found that angiotensin II type 2 receptor (AT2R) activation is able to improve lipotoxicity. We provided evidence, using C21/M24, that AT2R activation improves adipocytes’ size and insulin sensitivity in an insulin-resistant rat model, as well as androgen levels in a PCOS obese rat model. Taken together, these findings point toward the important role of lipotoxicity in PCOS development and of the RAS system as a new target for the treatment of PCOS.
... AT2R stimulation results in the activation of signaling cascades that counteract many of the events mediated by AT1R 269 . AT2R stimulation activates several phosphatases, such as MAP Kinase Phosphatase-1 (MPK-1), SHP-1, and PP2A 131,270 , which serve to deactivate ERK1/2, STAT, JAK, and several other AT1R signaling molecules. ...
... Nevertheless, it has been reported that activation of the AT 2 receptor counteracts the effects triggered by the AT 1 receptor. Accordingly, it has been reported that the AT 2 receptor can activate phosphatases with consequent inhibition of MAPKs [51][52][53]. Activation of the AT 2 receptor has also been reported to regulate cGMP and NO levels [54,55]. A study from Lara et al. [56] has shown that, in the nephron proximal tubule, Ang-(1-7) inhibits the Na + -ATPase acting via the AT 2 receptor with subsequent activation of the G i /cGMP/PKG (protein kinase G) pathway. ...
GPCRs (G-protein-coupled receptors) are among the most important targets for drug discovery due to their ubiquitous expression and participation in cellular events under both healthy and disease conditions. These receptors can be activated by a plethora of ligands, such as ions, odorants, small ligands and peptides, including angiotensins and kinins, which are vasoactive peptides that are classically involved in the pathophysiology of cardiovascular events. These peptides and their corresponding GPCRs have been reported to play roles in other systems and under pathophysiological conditions, such as cancer, central nervous system disorders, metabolic dysfunction and bone resorption. More recently, new mechanisms have been described for the functional regulation of GPCRs, including the transactivation of other signal transduction receptors and the activation of G-protein-independent pathways. The existence of such alternative mechanisms for signal transduction and the discovery of agonists that can preferentially trigger one signalling pathway over other pathways (called biased agonists) have opened new perspectives for the discovery and development of drugs with a higher specificity of action and, therefore, fewer side effects. The present review summarizes the current knowledge on the non-canonical signalling and roles of angiotensins and kinins.
... No es necesario reajustar la dosis en pacientes con insuficiencia renal o hepática moderada. El losartan se absorbe bien por vía oral, pero sufre un importante efecto de primer paso hepático 13 . Se biotransforma rápidamente en el hígado por el citocromo P450 convirtiéndose en el metabolito que es 13 veces más potente que el losartan y el responsable de la acción antihipertensiva 14 . ...
Introduction
Standardization of the therapeutic interchange process in the hospital setting by the establishment and spreading of standard criteria has been defined as an activity conducent to increased health care quality, and hence improved patient care.
Objective
To establish standardized therapeutic swapping for angiotensin II receptor antagonists (ARA-II) in the treatment of blood hypertension, and to evaluate the suitability of therapeutic interchange in an integrated individualized drug dispensation system.
Material and methods
Standardized therapeutic interchange was performed based on therapeutic equivalence criteria for ARA-IIs such as candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, and valsartan, according to the pharmacodynamic characteristics, dosage recommendations, pharmacokinetic characteristics and interactions of each one of them. The suitability of therapeutic interchange was assessed in terms of standardization or adaptation to this practice developed by using percentage adherence in the previous 12 months (period A) and during the 12 months following its implementation and spread (period B).
Results
The only ARA-II included in the hospital's pharmacotherapeutic guide is losartan, on which standardized therapeutic interchange for initial and maintenance doses of any drug within this class was based. The overall number of interchanges performed was 417-216 during period A and 201 during period B. Implementing therapeutic swapping has significantly increased adherence to explicitly established criteria by 35.2% (95% CI: 25.9 to 44.5%). Similarly, during period B a reduction in the variability of therapeutic interchanges among various pharmacists was observed regarding losartan dosage.
Discussion
The standardization of therapeutic interchange procedures for ARA-IIs allowed a reduction of the variability seen in this process, and hence the potential for medication-related problems. Another benefit of the spread of therapeutic swapping has been an increased adjustment of medical prescriptions to the hospital's pharmacotherapeutic guide.
... Phosphatase activation has been one of the first signals associated with AT2 receptor activation. After the earlier studies in PC12W cells (Bottari et al., 1992b; Brechler et al., 1994), results have been confirmed in other cell lines, including N1E-115 cells (Nahmias et al., 1995), NG108-15 cells (Buisson et al., 1995), and R3T3 fibroblasts (Tsuzuki et al., 1996a,b). This phosphatase activation by the AT2 receptor is essential for its anti-proliferative and pro-apoptotic effects (for reviews, see Nouet and Nahmias, 2000; Steckelings et al., 2005; Porrello et al., 2009; Verdonk et al., 2012). ...
The angiotensin type 2 (AT2) receptor of angiotensin II has long been thought to be limited to few tissues, with the primary effect of counteracting the angiotensin type 1 (AT1) receptor. Functional studies in neuronal cells have demonstrated AT2 receptor capability to modulate neuronal excitability, neurite elongation, and neuronal migration, suggesting that it may be an important regulator of brain functions. The observation that the AT2 receptor was expressed in brain areas implicated in learning and memory led to the hypothesis that it may also be implicated in cognitive functions. However, linking signaling pathways to physiological effects has always proven challenging since information relative to its physiological functions has mainly emerged from indirect observations, either from the blockade of the AT1 receptor or through the use of transgenic animals. From a mechanistic standpoint, the main intracellular pathways linked to AT2 receptor stimulation include modulation of phosphorylation by activation of kinases and phosphatases or the production of nitric oxide and cGMP, some of which are associated with the Gi-coupling protein. The receptor can also interact with other receptors, either G protein-coupled such as bradykinin, or growth factor receptors such as nerve growth factor or platelet-derived growth factor receptors. More recently, new advances have also led to identification of various partner proteins, thus providing new insights into this receptor’s mechanism of action. This review summarizes the recent advances regarding the signaling pathways induced by the AT2 receptor in neuronal cells, and discussed the potential therapeutic relevance of central actions of this enigmatic receptor. In particular, we highlight the possibility that selective AT2 receptor activation by non-peptide and selective agonists could represent new pharmacological tools that may help to improve impaired cognitive performance in Alzheimer’s disease and other neurological cognitive disorders.
... In addition, T 3 -induced cardiomyocyte hypertrophy was totally blocked by the AT 1 R silencing or the AT 1 R blockade, suggesting that even the AT 1 R expression has not been altered by T 3 after 24 h of treatment, this receptor participates in T 3 -induced hypertrophy. The AT 2 R has a growth inhibitory effect [28,57]. However, some recent studies have demonstrated that this receptor is required in some models of cardiac hypertrophy [11,45]. ...
Several studies have implicated the renin angiotensin system in the cardiac hypertrophy induced by thyroid hormone. However,
whether Angiotensin type 1 receptor (AT1R) is critically required to the development of T3-induced cardiomyocyte hypertrophy as well as whether the intracellular mechanisms that are triggered by AT1R are able to contribute to this hypertrophy model is unknown. To address these questions, we employed a selective small interfering
RNA (siRNA, 50nM) or an AT1R blocker (Losartan, 1μM) to evaluate the specific role of this receptor in primary cultures of neonatal cardiomyocytes submitted
to T3 (10nM) treatment. The cardiomyocytes transfected with the AT1R siRNA presented reduced mRNA (90%, P<0.001) and protein (70%, P<0.001) expression of AT1R. The AT1R silencing and the AT1R blockade totally prevented the T3-induced cardiomyocyte hypertrophy, as evidenced by lower mRNA expression of atrial natriuretic factor (66%, P<0.01) and skeletal α-actin (170%, P<0.01) as well as by reduction in protein synthesis (85%, P<0.001). The cardiomyocytes treated with T3 demonstrated a rapid activation of Akt/GSK-3β/mTOR signaling pathway, which was completely inhibited by the use of PI3K inhibitors
(LY294002, 10μM and Wortmannin, 200nM). In addition, we demonstrated that the AT1R mediated the T3-induced activation of Akt/GSK-3β/mTOR signaling pathway, since the AT1R silencing and the AT1R blockade attenuated or totally prevented the activation of this signaling pathway. We also reported that local Angiotensin
I/II (Ang I/II) levels (120%, P<0.05) and the AT1R expression (180%, P<0.05) were rapidly increased by T3 treatment. These data demonstrate for the first time that the AT1R is a critical mediator to the T3-induced cardiomyocyte hypertrophy as well as to the activation of Akt/GSK-3β/mTOR signaling pathway. These results represent
a new insight into the mechanism of T3-induced cardiomyocyte hypertrophy, indicating that the Ang I/II-AT1R-Akt/GSK-3β/mTOR pathway corresponds to a potential mediator of the trophic effect exerted by T3 in cardiomyocytes.
... A-II has the ability to promote fibrosis through a number of mechanisms, including induction of collagen synthesis, inhibition of collagen-cleaving proteases, stimulation of the secretion of platelet-derived growth factor, and, most interestingly, direct stimulation of TGF-β receptor type II(Luft, 2003;Wolf, 2000). AT-1 stimulation also has the ability to promote fibrosis by up-regulating expression and synthesis of NF-κB and thus TGF-β, as well as a number of other cytokines (Tsuzuki, 1996). Downstream, aldosterone, in addition to its hypertensive effects mediated through the mineralocorticoid receptor, also up-regulates the expression of TGF-β (Juknevicius et al., 2000). ...
... Wirkung: Die selektive Stimulation des AT2-R senkt laut einiger Befunde den Blutdruck (Barber et al., 1999; Ichiki et al., 1995) und hemmt beispielsweise die Zellproliferation (Munzenmaier et Greene, 1996; Stoll et al., 1995; Tsuzuki et al., 1996). In Vorarbeiten der eigenen Arbeitsgruppe konnte eine signifikante Steigerung der O 2 ...
Reaktive Sauerstoffspezies und ihre Interaktionen mit NO und vaskulären Signalmechanismen spielen eine fundamentale Rolle bei der Entstehung der endothelialen Dysfunktion und damit von pathophysiologischen Veränderungen innerhalb der Blutgefäße. Als Hauptquelle von reaktiven Sauerstoffspezies in Endothelzellen ist die NAD(P)H-Oxidase bekannt. Diese Arbeit klärt zugrunde liegende Signalmechanismen zur Aktivierung der NAD(P)H-Oxidase auf: Die Ergebnisse zeigen, dass die Tyrosinphosphatase SHP-1 eine wichtige Rolle bei Regulation der NAD(P)H-Oxidase spielt. Das Signal der SHP-1 wird mittels Tyrosinde- bzw. Phosphorylierungen über die Phosphatidylinositol-3-Kinase vermittelt, welche via Rac1 die NAD(P)H-Oxidase kontrolliert. Aufgrund dieser Ergebnisse könnte der SHP-1 in Endothelzellen eine zentrale Bedeutung bei der Entstehung der Endotheldysfunktion zukommen, denn ihre Aktivität ist mitverantwortlich bei der Kontrolle der NAD(P)H-Oxidase. Als physiologischen Faktor, der diesen Signalweg beeinflusst fanden wir den bekannten Wachstumsfaktor VEGF. Darüber hinaus konnten wir zeigen, dass Estradiol über eine Aktivierung der SHP-1 die NAD(P)H-Oxidase Aktivität reduziert. Insgesamt stellt unsere Arbeit die SHP-1 als molekularen Schalter erstmals in Zusammenhang mit der Regulierung der NADP)H-Oxidase und klärt die zugrunde liegenden Signalmechanismen auf.
... In the heart, Ang II affects cardiac remodelling, contractility, and cell growth, most of which can be attributed to activation of AT 1 R [2][3][4]. In contrast, mediated by the activation of phosphotyrosine phosphatases that inactivate mitogen-activated protein kinases, AT 2 R has a significant growth inhibitory effect [20,47,49]. However, although in most of cases the effects associated to AT 2 R are opposite to those mediated by AT 1 R, several studies of literature have demonstrated that in some tissues that express both receptors, as occur in the cardiac tissue [4], AT 1 R and AT 2 R share, at least in part, a common signalling pathway [22,35,46], which could explain why similar effects may also be observed after stimulation of these receptors [42]. ...
Although most of effects of Angiotensin II (Ang II) related to cardiac remodelling can be attributed to type 1 Ang II receptor (AT(1)R), the type 2 receptor (AT(2)R) has been shown to be involved in the development of some cardiac hypertrophy models. In the present study, we investigated whether the thyroid hormone (TH) action leading to cardiac hypertrophy is also mediated by increased Ang II levels or by change on AT(1)R and AT(2)R expression, which could contribute to this effect. In addition, we also evaluated the possible contribution of AT(2)R in the activation of Akt and in the development of TH-induced cardiac hypertrophy. To address these questions, Wistar rats were treated with thyroxine (T(4), 0.1 mg/kg BW/day, i.p.), with or without AT(2)R blocker (PD123319), for 14 days. Cardiac hypertrophy was identified based on heart/body weight ratio and confirmed by analysis of atrial natriuretic factor mRNA expression. Cardiomyocyte cultures were used to exclude the influence of TH-related hemodynamic effects. Our results demonstrate that the cardiac Ang II levels were significantly increased (80%, P < 0.001) as well as the AT(2)R expression (50%, P < 0.05) in TH-induced cardiac hypertrophy. The critical involvement of AT(2)R to the development of this cardiac hypertrophy in vivo was evidenced after administration of AT(2) blocker, which was able to prevent in 40% (P < 0.01) the cardiac mass gain and the Akt activation induced by TH. The role of AT(2)R to the TH-induced cardiomyocyte hypertrophy was also confirmed after using PD123319 in the in vitro studies. These findings improve understanding of the cardiac hypertrophy observed in hyperthyroidism and provide new insights into the generation of future therapeutic strategies.
... f Ang II through the activation of its AT2 receptors first came from the study in coronary endothelial cells ( CEC ) ( Stoll et al . , 1995 ) and have been confirmed later , in NG108 - 15 cells , PC12W cells , R3T3 cells , renal mesangial cells ( Goto et al . , 1997 ; Laflamme et al . , 1996 ; Meffert et al . , 1996 ; Munzenmaier & Greene , 1996 ; Tsuzuki et al . , 1996 ) . ...
... There is a growing body of evidence showing that AT2 receptors are involved in the control of cell proliferation and differentiation in various cell types. For example, previous observations have shown that the AT2 receptor, in contrast to the AT1 receptor, can exert antiproliferative effects and inhibit cell growth in microvascular endothelial cells [23], vascular smooth muscle cells [24], and fibroblasts [25,26]. Antiproliferation can be a cellular program of its own, for example, in cases where excessive growth induced by growth factors needs to be controlled in the development. ...
The expression pattern of angiotensin AT2 receptors with predominance during fetal life and upregulation under pathological conditions during tissue injury/repair process suggests that AT2 receptors may exert an important action in injury/repair adaptive mechanisms. Less is known about AT2 receptors in acute ischemia-induced cardiac injury. We aimed here to elucidate the role of AT2 receptors after acute myocardial infarction. Double immunofluorescence staining showed that cardiac AT2 receptors were mainly detected in clusters of small c-kit+ cells accumulating in peri-infarct zone and c-kit+AT2+ cells increased in response to acute cardiac injury. Further, we isolated cardiac c-kit+AT2+ cell population by modified magnetic activated cell sorting and fluorescence activated cell sorting. These cardiac c-kit+AT2+ cells, represented approximately 0.19% of total cardiac cells in infarcted heart, were characterized by upregulated transcription factors implicated in cardiogenic differentiation (Gata-4, Notch-2, Nkx-2.5) and genes required for self-renewal (Tbx-3, c-Myc, Akt). When adult cardiomyocytes and cardiac c-kit+AT2+ cells isolated from infarcted rat hearts were cocultured, AT2 receptor stimulation in vitro inhibited apoptosis of these cocultured cardiomyocytes. Moreover, in vivo AT2 receptor stimulation led to an increased c-kit+AT2+ cell population in the infarcted myocardium and reduced apoptosis of cardiomyocytes in rats with acute myocardial infarction. These data suggest that cardiac c-kit+AT2+ cell population exists and increases after acute ischemic injury. AT2 receptor activation supports performance of cardiomyocytes, thus contributing to cardioprotection via cardiac c-kit+AT2+ cell population.
... It has been suggested that losartan can increase the production of NO (3), and it is possible that the chronic administration of losartan results in a higher production of NO than after its acute injection. Alternatively, it is also possible that the chronic blockade of AT 1 ANG II receptors produces a vasodilating effect mediated by the activation of AT 2 by circulating ANG II (29). ...
Nitric oxide (NO) is a vasodilator substance controlling renal papillary blood flow (PBF) in the rat. In this study we have evaluated the role of AT1 angiotensin II receptors as modulators of the whole kidney and papillary vasoconstrictor effects induced by the acute or chronic inhibition of NO synthesis. Experiments have been performed in anesthetized, euvolemic Munich-Wistar rats prepared for the study of renal blood flow (RBF) and PBF. In normal rats, acute administration of the NO synthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) increased mean arterial pressure (MAP) and decreased RBF and PBF. Either acute or chronic treatment with the AT1 receptor blocker losartan did not modify the decreases in RBF or PBF secondary to L-NAME. In animals made hypertensive by chronic inhibition of NO, basal MAP was higher, whereas RBF and PBF were lower than in the controls. In these animals, acute or chronic administration of losartan decreased MAP and increased both RBF and PBF significantly. These results indicate that, under normal conditions, the decreases in RBF or PBF induced by the acute inhibition of NO synthesis are not modulated by AT1-receptor stimulation. However, the arterial hypertension, renal vasoconstriction, and reduced PBF present in chronic NO-deficient hypertensive rats is partially due to the effects of angiotensin II, via stimulation of AT1-receptors.
... Moreover, antiproliferative effects of AT 2 receptor were also shown in mouse fibroblast R3T3 cells and in PC12W cells (rat pheochromocytoma cell line). 19,20 In contrast, Otsuka et al 21 observed very recently that mRNA expression for both AT 1 and AT 2 receptors was enhanced in the aorta of SHR and demonstrated that treatment with PD123319 reduced the media cross-sectional area of the aorta, whereas losartan reduced the arterial systolic blood pressure and the collagen concentration. They suggest that AT 1 receptor, but not AT 2 receptor, plays a crucial role in the remodeling of matrix tissue, while AT 2 receptor plays a role in the development of hypertrophy of smooth muscle in aorta in SHR. ...
Angiotensin II (Ang II) plays an important role in regulating cardiovascular hemodynamics and structure. Multiple lines of evidence have suggested the existence of Ang II receptor subtypes, and at least 2 distinct receptor subtypes have been defined on the basis of their differential pharmacological and biochemical properties and designated as type 1 (AT1) and type 2 (AT2) receptors. To date, most of the known effects of Ang II in adult tissues are attributable to the AT1 receptor. Recent cloning of the AT2 receptor contributes to reveal its physiological functions, but many functions of the AT2 receptor are still an enigma. AT1 and AT2 receptors belong to the 7-transmembrane, G protein-coupled receptor family. However, accumulating evidence demonstrates that the function and signaling mechanisms of these receptor subtypes are quite different, and these receptors may exert opposite effects in terms of cell growth and blood pressure regulation. We will review the role of the AT2 receptor in the cardiovascular system and the molecular and cellular mechanisms of AT2 receptor action.
... 7,11,14,16 PTPase activation by the AT 2 receptor has also been demonstrated in rat adrenal glomerulosa and PC12W cells, 18 neuronal NG 108-15 cells, 19 and R3T3 cells. 20 These results have led us to examine the possibility that AT 2 receptor stimulation dephosphorylates the tyrosine residues of Jak and/or STATs. ...
Angiotensin II type 2 (AT2) receptor exerts an inhibitory action on cell growth. In the present study, we report that the stimulation of AT2 receptor in AT2 receptor cDNA-transfected rat adult vascular smooth muscle cells (VSMCs) inhibited angiotensin II type 1 (AT1) receptor-mediated tyrosine phosphorylation of STAT (signal transducers and activators of transcription) 1alpha/beta, STAT2, and STAT3 without influence on Janus kinase. AT2 receptor activation also inhibited the tyrosine phosphorylation of STAT1alpha/beta induced by interferon-gamma, epidermal growth factor, and platelet-derived growth factor. Similar effects of AT2 receptor were observed in R3T3 fibroblast and mouse fetal VSMCs, which express endogenous AT2 receptor. Moreover, AT2 receptor inhibited serine phosphorylation of STAT1alpha and STAT3 via the inhibition of extracellular signal-regulated kinase (ERK) activation. Stimulation of AT2 receptor inhibited the binding of STATs with sis-inducing element in c-fos promoter, resulting in decreased c-fos expression. Taken together, our results suggest that AT2 receptor can crosstalk negatively with multiple families of growth receptors by inhibiting ERK and STAT activation.
... The growth-inhibitory effects of the AT 2 receptor are at least partially mediated by the activation of phosphotyrosine phosphatase, resulting in the inactivation of mitogen-activated protein (MAP) kinase, especially p 42 and p 44 MAP kinases, termed extracellular signal-regulated kinases (ERK). 8,[21][22][23][24] With respect to G-protein coupling, the data remain incomplete. Pertussis toxin attenuated AT 2 receptor-mediated inhibition of ERK activity, which suggests that G i is involved in AT 2 receptor cell signaling. ...
The renin-angiotensin system is a major physiological regulator of body fluid volume, electrolyte balance, and arterial pressure. Virtually all of the biological actions of the principle effector peptide angiotensin II (ANG II) have been attributed to an action at the type 1 (AT(1)) ANG receptor. Until recently, the functional role of the type 2 (AT(2)) receptor, if any, has been unknown, possibly because the AT(2) receptor has a low degree of expression compared with that of the AT(1) receptor. Evidence has now accumulated that the AT(2) receptor opposes functions mediated by the AT(1) receptor. Whereas the AT(1) receptor stimulates cell proliferation, the AT(2) receptor inhibits proliferation and promotes cell differentiation. These differences in growth responses have been ascribed to different cell signaling pathways in which the AT(1) receptor stimulates protein phosphorylation and the AT(2) receptor dephosphorylation. During the past 5 years, studies have demonstrated that the AT(2) receptor is responsible for vasodilation and natriuresis, thus opposing the vasoconstrictor and antinatriuretic effects of ANG II mediated through the AT(1) receptor. Work from our laboratory and others indicates that the AT(2) receptor stimulates vasodilation and natriuresis by an autocrine cascade including bradykinin, nitric oxide, and cyclic GMP. The AT(2) receptor also has been found to control vasodilator prostaglandins, which have a role in blood pressure regulation. The AT(2) receptor appears to play a counterregulatory protective role in the regulation of blood pressure and sodium excretion that opposes the AT(1) receptor.
... This effect was abolished by inhibition of phosphotyrosine phosphatase (PTPase), indicating a role for this class of en-zymes (8,11). Similar rapid and transient increases in PTPase activity after AT 2 receptor activation have been observed in R3T3 fibroblasts (146,148) and NIE-115 cells (6,104). In the latter case, ANG II was shown to stimulate the activity of SHP-1, a soluble PTPase. ...
Since it was discovered ten years ago, the angiotensin II (ANG II) type 2 (AT(2)) receptor has been an enigma. This receptor binds ANG II with a high affinity but is not responsible for mediating any of the classical physiological actions of this peptide, all of which involve the ANG II type 1 (AT(1)) receptor. Furthermore, the AT(2) receptor exhibits dramatic differences in biochemical and functional properties and in patterns of expression compared with the AT(1) receptor. During the past decade, much information has been gathered about the AT(2) receptor, and the steadily increasing number of publications indicates a growing interest in this new and independent area of research. A number of studies suggest a role of AT(2) receptors in brain, renal, and cardiovascular functions and in the processes of apoptosis and tissue regeneration. Despite these advances, nothing stands out as the major singular function of these receptors. The study of AT(2) receptors has reached a crossroads, and innovative approaches must be considered so that unifying mechanisms as to the function of these unique receptors can be put forward. In this review we will discuss the advances that have been made in understanding the biology of the AT(2) receptor. Furthermore, we will consider how these discoveries, along with newer experimental approaches, may eventually lead to the elusive physiological and pathophysiological functions of these receptors.
... The AT 2 receptor also has been demonstrated to mediate inhibition of ANG II induced-hypertrophy in cultured myocytes (4). Furthermore, the AT 2 receptor is involved in the induction of apoptosis (49) and activation of tyrosine phosphatase (41). These observations support the hypothesis that the AT 2 receptor is coupled to an antigrowth process that counteracts the growthpromoting program initiated by AT 1 receptor activation (4). ...
The cellular localization of the AT(2) receptor and the regulation of its expression in hypertrophied left ventricle are not well known. We compared the expression of the cardiac AT(1) and AT(2) receptor in spontaneously hypertensive rats/Izumo strain (SHR/Izm) and Wistar Kyoto rats/Izumo strain (WKY/Izm), ages 4, 12, and 20 wk, by means of immunohistochemistry and Western blot analysis. In SHR/Izm, compared with WKY/Izm, blood pressure (161 +/- 2 vs. 120 +/- 2 mmHg at 12 wk, P </= 0.01, and 199 +/- 3 vs. 123 +/- 3 mmHg at 20 wk, P </= 0.01) and heart-to-body weight ratio (3.76 +/- 0.07 vs. 3.06 +/- 0.06 mg/g at 12 wk, P </= 0.01, and 3.90 +/- 0.08 vs. 3.01 +/- 0.12 mg/g at 20 wk, P </= 0.01) were significantly elevated. There was no difference in these values between the two strains at 4 wk of age. Histologically, 20-wk-old SHR/Izm demonstrated myocardial hypertrophy, a thickening of the smooth muscle layer of the intracardiac arteries, and perivascular fibrosis. By immunohistochemistry, the AT(2) receptor was localized to cardiomyocytes and vascular endothelial cells, but not in the vascular smooth muscle cells. No major AT(2) receptor signal was observed in perivascular fibrosis at any age in either strain of rats. No difference was detected in this localization between the two strains. By Western blotting, a single 44-kDa band for the AT(2) receptor and a single 60-kDa band for the AT(1) receptor were detected in ventricles from both strains of rats at all ages. Densitometric analysis demonstrated that the AT(2) receptor 44-kDa band was decreased by 20% at 12 wk and 32% at 20 wk (P < 0.01) in SHR/Izm compared with WKY/Izm. The intensity of the AT(1) receptor 60-kDa band was increased by 57% in 20-wk-old SHR/Izm compared with WKY/Izm (P < 0.05). There was no significant difference in the intensity of the 44- or 60-kDa bands in 4-wk-old animals of either strain. We demonstrated a decrease in the AT(2) receptor and an increase in the AT(1) receptor protein with no change in their localizations in hypertrophied left ventricular myocytes of SHR/Izm.
Angiotensin (Ang) II is one of the key hormones involved in cardiovascular homeostasis. It has potent vasoconstrictor effects and is directly involved in the vascular and cardiac remodeling observed in response to chronic or acute hypertension. Two major Ang II receptor subtypes, AT1 and AT2, have been described, and their roles in vivo and in vitro have now been investigated using nonpeptidic antagonists, losartan and PD123319, which block AT1 and AT2 receptors respectively. The aim of this review is to focus on in vivo data to better define the respective functions of the AT1 and AT2 receptor subtypes. In adult rats, the AT1 receptor subtype does not trigger trophic effects directly in cardiomyocytes, whereas the AT2 receptor may have a major trophic role in smooth muscle cells.
The renin angiotensin system (RAS) produced hormone peptides regulate many vital body functions. Dysfunctional signaling by receptors for RAS peptides leads to pathologic states. Nearly half of humanity today would likely benefit from modern drugs targeting these receptors. The receptors for RAS peptides consist of three G-protein-coupled receptors-the angiotensin II type 1 receptor (AT1 receptor), the angiotensin II type 2 receptor (AT2 receptor), the MAS receptor-and a type II trans-membrane zinc protein-the candidate angiotensin IV receptor (AngIV binding site). The prorenin receptor is a relatively new contender for consideration, but is not included here because the role of prorenin receptor as an independent endocrine mediator is presently unclear. The full spectrum of biologic characteristics of these receptors is still evolving, but there is evidence establishing unique roles of each receptor in cardiovascular, hemodynamic, neurologic, renal, and endothelial functions, as well as in cell proliferation, survival, matrix-cell interaction, and inflammation. Therapeutic agents targeted to these receptors are either in active use in clinical intervention of major common diseases or under evaluation for repurposing in many other disorders. Broad-spectrum influence these receptors produce in complex pathophysiological context in our body highlights their role as precise interpreters of distinctive angiotensinergic peptide cues. This review article summarizes findings published in the last 15 years on the structure, pharmacology, signaling, physiology, and disease states related to angiotensin receptors. We also discuss the challenges the pharmacologist presently faces in formally accepting newer members as established angiotensin receptors and emphasize necessary future developments.
Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.
The renin–angiotensin system (RAS) plays a pivotal role in the regulation of fluid, electrolyte balance and blood pressure, and is a modulator of cellular growth and proliferation. Biological actions of RAS are linked to the binding of the effector molecule, angiotensin II (AngII), to specific membrane receptors, mostly the AT1 subtype and, to a lesser extent, other subtypes. Following the identification and characterization of the AT2 subtype receptor, it has been proposed that a complex interaction between AngII and its receptors may play an important role in the effects of RAS. In this paper current information on AngII subtype receptors – their structure, regulation and intracellular signalling – are reviewed, with a particular emphasis on the potential relevance for cardiovascular pathophysiology. In addition, we discuss modulation of expression of the AT2 receptor and its interaction with the AT1 receptor subtype, as well as the potential effects of this receptor on blood pressure regulation. A better understanding of the integrated effects of the AngII subtype receptors may help to elucidate the function of the RAS, as well as their participation in the mechanisms of cardiovascular disease and attendant therapeutic implications.
The cause of essential hypertension is unknown and the pathogenesis is far from being under-stood completely. In the framework of this thesis, some unknown substances with strong ef-fects on the vasoregulatory system were isolated, identified and characterised. The basis of the thesis were the changes of the chromatographic and mass-spectrometric methods during the last ten years, which offered the possibility to isolate and to identify un-known biomolecules, and to clarify hereby unknown pathogenetic mechanisms. In the first part of the thesis, diadenosine polyphosphates (with 3-6 phosphates) were isolated, identified and quantified from different tissues and body fluids like adrenal glands, heart and platelets and plasma by using these innovative chromatographic and mass-spectrometric methods. Moreover, a potent vasoconstrictive dinucleoside polyphosphate with a purine and pyrimidine base was isolated from supernatants of stimulated endothelial cells. The concen-tration of this uridine adenosine tetraphosphate (Up4A) in plasma is sufficient to affect the vascular tone. The second part of the thesis deals with the isolation and characterisation of hypertensive agents in chronic renal failure (CRF) patients. In the secretome of mononuclear leukocytes, both Angiotensin II (Ang II) and des[Asp1]-[Ala1]-Ang II (named as “Ang A”) were isolated. The ratio Ang A / Ang II was significantly increased in plasma of CRF patients. Furthermore, p-hydroxy-hippuric acid was identified as a potent inhibitor of the Ca2+-ATPase, and phenylacetic acid was described as an inhibitor of the inducible NO synthase (iNOS). Finally, the effects of different hemofiltration membranes on the filtration rate of some identified sub-stances were analysed. In summary, some unknown substances with strong effects on the vasoregulatory system were isolated, identified and characterised. The thesis corroborates the hypothesis that hypertension is a disease caused by many yet unknown different factors and regulatory systems. It is very likely that there are still many other unknown factors. The identification of each unknown fac-tor offers the possibility to develop new and more appropriate therapeutic approaches.
Recent developments in our knowledge of the renin-angiotensin system (RAS) necessitate an update of the classical view on this system. These developments pertain to the pathways leading to formation of angiotensin II and other active metabolites, their receptors, biological functions and the presence of renin-angiotensin systems in tissues. The implications of the above new developments for the current interest in tissue renin-angiotensin systems as potential targets for drug therapy in cardiovascular disease are discussed in this review.
(Jpn Circ J 1997; 61: 807 - 813)
1. The active peptide hormone angiotensin II (AngII) is formed from its prohormone angiotensinogen by way of inactive angiotensin I. The highly specific protease, renin, responsible for the initiation of this system was elusive and considered unstable. We isolated it in a pure and stable form from the kidney of the pig, human, rat, and land submandibular glands of the mouse. It was shown that there is only one type of renin with highly stringent substrate specificity, except certain strains of the mouse which have two gene products.
2. The well‐known diversity of action of AngII can be attributed to the presence of more than two subtypes, AT 1 and AT 2 , as well as multiple signalling pathways for both of them.
3. The first subtype AT 1 was shown to mediate most of the traditionally recognized AngII functions such as vasoconstriction, electrolyte homeostasis etc.
4. Although the identification of the signalling modes of the second subtype AT 2 still remains elusive, we and others have shown evidence that its action is generally antagonistic to that of AT 1 . AT 2 inhibits AT 1 (growth factor‐stimulated cell growth), AT 2 attenuates the vasoconstriction induced by AT 1 . Since AT 2 seems to mediate nitric oxide formation in the renal cells, it may initiate a natriuretic pathway in contrast to the sodium‐retaining action of AT 1 ‐mediated AngII action.
5. Newer mechanisms and functions of these and other receptors will be clarified by the combination of molecular, cellular and integrated physiological studies.
Besides its involvement in reproductive functions, estrogen protects against the development of cardiovascular diseases. The guanylate cyclase/cGMP system is known to exert potent effects on the regulation of blood pressure and electrolyte balance. We examined whether 17β-estradiol can affect soluble guanylate cyclase in PC12 cells. The results indicate that 17β-estradiol decreases cGMP levels in PC12 cells. 17β-Estradiol decreases sodium nitroprusside (SNP)-stimulated, but not atrial natriuretic factor-stimulated cGMP formation in PC12 cells, indicating that 17β-estradiol decreases cGMP levels by inhibiting the activity of soluble guanylate cyclase. 17β-Estradiol also stimulates protein tyrosine phosphatase activities in PC12 cells and dephosphorylates at least three proteins. Addition of sodium vanadate, a protein tyrosine phosphatase inhibitor, blocks the inhibitory effects of 17β-estradiol on soluble guanylate cyclase activity in PC12 cells. Furthermore, transfection of SHP-1, a protein tyrosine phosphatase, into PC12 cells inhibits both basal and SNP-stimulated guanylate cyclase activity. Amino acid analysis also reveals that the 70-kDa subunit of soluble guanylate cyclase contains the SHP-1 substrate consensus sequence. These results suggest that 17β-estradiol inhibits soluble guanylate cyclase activity through SHP-1.
Renin-angiotensin II-aldosterone axis has long been known as a regulator of blood pressure and fluid homeostasis. Yet, local renin-angiotensin II systems have been discovered and novel actions of angiotensin II (AngII) have emerged among which its ability to act as a immunomodulator and profibrotic molecule. The enzyme responsible for its synthesis, Angiotensin-converting-enzyme (ACE), is present in high concentrations in lung tissue. In the present paper, we review data from studies of the past decade that implicate AngII and functional polymorphisms of the ACE gene that increase ACE activity with increased susceptibility for asthma and chronic obstructive pulmonary disease (COPD) and for pulmonary hypertension. Moreover, drugs that inhibit the synthesis of AngII (ACE inhibitors) or that antagonize its actions on its receptors (Angiotensin II receptor blockers -ARBs) have been shown to provide beneficial effects. Another recent discovery reviewed is the presence of a homologue of ACE, ACE2, which cleaves a single amino acid from AngII and forms a heptapeptide with vasodilatory actions, Ang 1-7. The balance between ACE and ACE2 is crucial for controlling AngII levels. ACE and ACE2 also appear to modify the severity of Acute Respiratory Distress Syndrome (ARDS), with ACE2 playing a protective role. Finally, mention is made to the recent discovery of ACE2 as a receptor for the SARS Corona Virus.
Severe sepsis is characterized by relative hypotension associated with a high cardiac output, peripheral vasodilation, and organ dysfunction. The renin-angiotensin-aldosterone system (RAAS) is primarily activated to increase blood pressure, but recently potential pro-inflammatory effects of angiotensin II have attracted interest because of the reported association between angiotensin II levels and organ failure and mortality in sepsis. RAAS antagonists could represent a new therapeutic option in this setting.
The role of RAAS activation in severe sepsis and septic shock, and the potential benefits (and risks) of using RAAS antagonists.
Insight into RAAS function in severe sepsis and the potential for RAAS inhibitors to be used as an adjunctive therapy in patients with severe sepsis, with discussion of promising results from animal models of sepsis.
Use of RAAS antagonists is an emerging therapeutic option in severe sepsis because these agents may reduce endothelial damage, organ failure, and mortality. However, timing of administration of RAAS antagonists is important because reduced RAAS function may contribute to refractive hypotension later on in septic shock and benefits of RAAS antagonists seem to be restricted to the early phases of sepsis.
Angiotensin (Ang) II plays an important role in fibrogenesis in various organs, including the lung. The aim of this study is to elucidate (i) the effects of Ang II on the expression of cytokines, growth factors or matrix proteins in normal human lung fibroblasts, and (ii) the inhibitory effects of an Ang II type 1 (AT1) receptor blocker, candesartan.
Normal human adult lung fibroblasts were cultured. Candesartan was added and the cells were incubated. All the cells in culture dishes were collected at day 0 and 2, and the cell numbers were counted using a Neubauer haemocytometer (Clay-Adams, Parsippany, NJ, USA). The cell proliferation rates at day 2 were calculated in comparison to those at day 0. Total cellular RNA was extracted for real-time quantitative PCR, or the culture supernatant was collected for either a Sircol assay or enzyme-linked immunosorbent assay (ELISA). Laser scanning confocal microscopy was used for analyzing the cells with and without prior exposure to candesartan. Comparisons between the means of multiple groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's test or Games-Howell's test. Values of P < 0*05 were considered to be statistically significant.
Among the 12 fibrosis-associated cytokines and growth factors, mRNA expressions of interleukin (IL)-4, IL-7, and platelet-derived growth factor-D were significantly modulated by Ang II, and suppressed by candesartan. Soluble collagen and elastin levels were significantly elevated by Ang II, and suppressed by candesartan. Under confocal microscopy, the intracellular distribution of elastin was significantly increased by Ang II, and suppressed by candesartan.
These data indicate that Ang II promotes lung fibrosis by increasing the matrix formation, which was suppressed by AT1 receptor blocker.
In the past decade evidence has accumulated for local tissue renin-angiotensin systems and for direct trophic effects of angiotensin II. In the present update we review new evidence for the independent generation of cardiac angiotensin II and its relevance for cardiac pathophysiology. A major role of angiotensin II generated by cardiac tissue has become apparent for the development of volume overload-induced cardiac hypertrophy. In contrast, evidence that cardiac pressure overload is associated with increased generation of angiotensin II by cardiac tissue is missing, and a role for angiotensin II produced by cardiac tissue in pressure overload-induced cardiac hypertrophy remains to be addressed. Similarly, although evidence has accumulated for increased gene expression and production of components of the renin-angiotensin system in the area adjacent to a myocardial infarction, data on changes in cardiac angiotensin II in infarcted or remnant myocardium are very limited. Without these, one can not assess whether blockade of angiotensin II generated by cardiac tissue contributes to the beneficial effects of blockers of the renin-angiotensin system on cardiac remodeling postmyocardial infarction.
A series of pharmaceutical successes in the treatment of not only essential hypertension but also vascular hypertrophic and hyperplastic diseases, congestive heart failure, and renal degenerative diseases, with angiotensin-converting enzyme inhibitors and angiotensin (Ang) II receptor antagonists indicates that angiotensin may play a pivotal role in the genesis and maintenance of high blood pressure and resultant stroke, atherosclerosis, and heart and kidney diseases. There is more than one form of Ang II receptors. Using expression cloning, we isolated the AT1 cDNA from bovine adrenocortical cells from the kidney of spontaneously hypertensive rats and AT2 cDNA from rat PC12W cells and we showed that it was not the mas oncogene product. Further, we showed that in rodents, AT1 consists of two subtypes, AT1a and AT1b, which share a high degree of sequence homology in their coding regions, although mechanisms of their respective transcriptional control seemed to be different. By computer-assisted modeling and site-directed mutagenesis, we have delineated the docking site of Ang II. AT1a (and AT1b) serves most of the commonly recognized actions of Ang II. In addition, this G protein-coupled receptor (GPCR) also activates a tyrosine kinase mechanism that may be an underlying cause of Ang II-mediated hypertrophic and hyperplastic changes of cardiovascular tissues. In the vascular system, the phospholipase C (PLC) activated by Ang II seems to be PLC-β rather than PLC-γ1.
Although angiotensin II type 2 (AT2) receptor has recently been cloned, its functional role is not well understood. We tested the hypothesis that selective activation of AT2 receptor causes vasodilation in the preglomerular afferent arteriole (Af-Art), a vascular segment that accounts for most of the preglomerular resistance. We microperfused rabbit Af-Arts at 60 mmHg in vitro, and examined the effect of angiotensin II (Ang II; 10(-11)-10(-8) M) on the luminal diameter in the presence or absence of the Ang II type 1 receptor antagonist CV11974 (CV; 10(-8) M). Ang II was added to both the bath and lumen of preconstricted Af-Arts. Ang II further constricted Af-Arts without CV (by 74+/-7% over the preconstricted level at 10(-8) M; P < 0.01, n = 7). In contrast, in the presence of CV, Ang II caused dose-dependent dilation; Ang II at 10(-8) M increased the diameter by 29+/-2% (n = 7, P < 0.01). This dilation was completely abolished by pretreatment with an AT2 receptor antagonist PD123319 (10(-7) M, n = 6), suggesting that activation of AT2 receptor causes vasodilation in Af-Arts. The dilation was unaffected by inhibiting either nitric oxide synthase (n = 7) or cyclooxygenase (n = 7), however, it was abolished by either disrupting the endothelium (n = 10) or inhibiting the cytochrome P-450 pathway, particularly the synthesis of epoxyeicosatrienoic acids (EETs, n = 7). These results suggest that in the Af-Art activation of the AT2 receptor may cause endothelium-dependent vasodilation via a cytochrome P-450 pathway, possibly by EETs.
1. Previous work has shown that enalaprilat, an inhibitor of angiotensin-converting enzyme (ACE), potentiated the actions of ai-adrenoceptor antagonists; it was hypothesized that angiotensin II (Angll) modulated the activity of α1-adrenoceptors. This hypothesis was tested in Sprague-Dawley rat isolated perfused tail arteries using the AT1 receptor antagonist losartan and the AT2 receptor antagonist PD123319.
2. Losartan had no oti-adrenoceptor antagonist effects at concentrations below 1 μmol/L. Similarly, losartan (0.1 μmol/L) had no effect on the α1-adrenoceptor antagonist action of doxazosin (1,10 nmol/L) nor on the potentiation of doxazosin by enalaprilat (1μmol/L).
3. PD123319 (0.1 μmol/L) had no α1-adrenoceptor antagonist effect but altered the mode of action of the α1 -adrenoceptor antagonist doxazosin: PD123319 changed doxazosin from a competitive to a non-competitive antagonist, as evidenced by the reduced slope of the dose-response curve for the α1-adrenoceptor agonist phenylephrine.
4. These results suggest that Angll can modulate α1-adrenoceptor function in rat tail arteries via an indirect action at AT2 receptors. However, the present results do not rule out the involvement of bradykinin, endothelin or prostaglandin in the modulation of α1 -adrenoceptor function by angiotensin II.
ACE inhibitors are very effective in chronic heart failure at improving symptoms and reducing mortality, although they do produce occasional side effects such as a cough. Since this strategy of blocking the renin- angiotensin system has proven effective, the question naturally arises as to whether even more therapeutic mileage could be made from blocking the renin- angiotensin system even more effectively than with ACE inhibitors. Drugs liable to achieve this are the angiotensin II receptor antagonists, but the pharmacological issues are complex and it cannot be guaranteed even on basic considerations that angiotensin II antagonists will be better than ACE inhibitors.
Angiotensin II receptors are essential components of the renin-angiotensin system transducing angiotensin II mediated signals across the plasma membrane of many cell types in the cardiovascular system. To date, three subtypes of angiotensin II receptors have been identified by molecular cloning, termed angiotensin II type 1 (AT1A, AT1B) and type 2 (AT2) receptors. This review focuses on recent transgenic animal models which have been generated to study the in vivo significance of angiotensin receptor diversity. AT1A receptors are the major blood pressure regulators and have a potent growth-stimulatory effect on cardiac myocytes in vivo. The AT1B receptor subtype may participate in the control of vascular tone if AT1A receptors are absent. AT2 receptors are abundantly expressed during embryonic development and may also play a role in blood pressure regulation by influencing vascular development and differentiation.
Since the discovery of nonpeptidic ligands, the receptors for angiotensin (Ang) II have been classified into 2 subtypes (Ang II type 1 receptor [AT1-R] and Ang II type 2 receptor [AT2-R]). AT1-R mediates most of the cardiovascular actions of Ang II. AT2-R is expressed at very high levels in the developing fetus. Its expression is very low in the cardiovascular system of the adult. The expression of AT2-R can be modulated by pathological states associated with tissue remodeling or inflammation. In failing hearts or neointima formation after vascular injury, AT2-R is reexpressed in cells proliferating in interstitial regions or neointima and exerts an inhibitory effect on Ang II-induced mitogen signals or synthesis of extracellular matrix proteins, resulting in attenuation of the tissue remodeling. An extreme form of cell growth inhibition ends in programmed cell death, and this process, which is initiated by the withdrawal of growth factors, is also enhanced by AT2-R. Cardiac myocyte- or vascular smooth muscle-specific mice that overexpress AT2-R display an inhibition of Ang II-induced chronotropic or pressor actions, suggesting the role of AT2-R on the activity of cardiac pacemaker cells and the maintenance of vascular resistance. AT2-R also activates the kinin/nitric oxide/cGMP system in the cardiovascular and renal systems, resulting in AT2-R-mediated cardioprotection, vasodilation, and pressure natriuresis. These effects, transmitted by AT2-R, are mainly exerted by stimulation of protein tyrosine or serine/threonine phosphatases in a Gi protein-dependent manner. The expression level of AT2-R is much higher in human hearts than in rodent hearts, and the AT2-R-mediated actions are likely enhanced, especially by clinical application of AT1-R antagonists. Thus, in this review, the regulation of AT2-R expression, its cellular localization, its pathological role in cardiovascular and kidney diseases, and pharmacotherapeutic effects of AT2-R stimulation are discussed.
The present work examined the effects of the subtype 2 of angiotensin II (AT2) receptors on the pressure-natriuresis using a new peptide agonist, and the possible involvement of cyclic guanosine 3′, 5′ monophosphate (cyclic GMP) in these effects.
In adult anaesthetized rats (Inactin, 100 mg kg−1, i.p.) deprived of endogenous angiotensin II by angiotensin converting enzyme inhibition (quinapril, 10 mg kg−1, i.v.), T2-(Ang II 4–8)2 (TA), a highly specific AT2 receptor agonist (5, 10 and 30 μg kg−1 min−1, i.v.) or its solvent was infused in four groups. Renal functions were studied at renal perfusion pressures (RPP) of 90, 110 and 130 mmHg and urinary cyclic GMP excretion when RPP was at 130 mmHg. The effects of TA (10 μg kg−1 min−1) were reassessed in animals pretreated with PD 123319 (PD, 50 μg kg−1 min−1, i.v.), an AT2 receptor antagonist and the action of the same dose of PD alone was also determined.
Increases in RPP from 90 to 130 mmHg did not change renal blood flow (RBF) but induced 8 and 15 fold increases in urinary flow and sodium excretion respectively. The 5 μg kg−1 min−1 dose of TA was devoid of action. The 10 and 30 μg kg−1 min−1 doses did not alter total RBF and glomerular filtration rate, but blunted pressure-diuresis and natriuresis relationships. These effects were abolished by PD.
TA decreased urinary cyclic GMP excretion. After pretreatment with PD, this decrease was reversed to an increase which was also observed in animals receiving PD alone.
In conclusion, renal AT2 receptors oppose the sodium and water excretion induced by acute increases in blood pressure and this action cannot be directly explained by changes in cyclic GMP.
British Journal of Pharmacology (1999) 126, 826–832; doi:10.1038/sj.bjp.0702362
This study investigates the growth effects and associated signaling pathways of angiotensin II (Ang II) in human vascular smooth muscle cells.
Cultured vascular smooth muscle cells derived from resistance arteries (< 300 microm diameter) from subcutaneous gluteal biopsies of healthy subjects (n = 6) and human aortic vascular smooth muscle cells were used. Cells were studied between passages 3 and 6. Both 3H-thymidine and 3H-leucine incorporation were measured as indices of vascular smooth muscle cell hyperplasia (DNA synthesis) and cell hypertrophy (protein synthesis), respectively. Growth effects of Ang II (10(-12) - 10(-6) mol/l), in the absence and presence of 10(-5) mol/l losartan (AT1 antagonist) and PD123319 (AT2 antagonist), were determined. Ang II-induced effects were compared to those of endothelin-1. To determine whether extracellular signal-regulated kinase (ERK)-dependent pathways play a role in Ang II-mediated growth, cells were pretreated with the selective ERK kinase (MEK) inhibitor, PD98059 (10(-5) mol/l). ERK activation was determined by Western blot in the absence and presence of PD98059.
Ang II dose-dependently increased 3H-thymidine incorporation in cells from aorta (Emax = 276 +/- 10.4% of control) and resistance arteries (Emax = 284 +/- 5.1% of control). Ang II also stimulated 3H-leucine incorporation in cells from aorta (Emax = 162 +/- 11.6 of control) and resistance arteries (Emax 175 +/- 10% of control). Unlike Ang II, endothelin-1 failed to significantly alter cellular growth, except at high concentrations (> 10(-7) mol/l), where it had a weak stimulatory effect Losartan, but not PD123319, blocked Ang II-stimulated growth responses. Ang II significantly increased phosphorylation of ERK-1 and ERK-2, with maximum responses obtained at 5 min. PD98059 inhibited Ang II-stimulated ERK activity and abrogated agonist-induced DNA and protein synthesis. Losartan, but not PD123319 inhibited Ang II-induced phosphorylation of ERK-1 and ERK-2.
Ang II stimulates both hyperplasia and hypertrophy in vascular smooth muscle cells from human arteries. These growth effects are mediated via Ang II receptors of the AT1 subtype that are linked to ERK-dependent signaling pathways.
All the components of the renin-angiotensin system (RAS), including the AT(1)receptor, have previously been shown to be present in the human term placenta. However, the presence of the RAS components has not been fully investigated in the human placenta throughout pregnancy. The aim of this study was to examine the localization of the angiotensin receptors AT(1)and AT(2)using immunocytochemistry and the expression of prorenin, angiotensinogen and the AT(1)and AT(2)receptor mRNA using RT-PCR in the human placenta in the first, second and third trimesters of pregnancy. Localization of the AT(1)receptor was shown throughout gestation in the syncytiotrophoblast, cytotrophoblast, Hofbauer cells and the fetal vascular endothelium. Expression of mRNA for prorenin, angiotensinogen and the AT(1)receptor was shown in the placenta throughout gestation. However, localization or mRNA expression of the AT(2)receptor was not detected in any of the placental samples studied. These results clearly show the expression of a majority of the components of the RAS in the placenta from early gestation onwards. Therefore, these results suggest that the RAS may have a role in the human placenta throughout gestation.
Selective blockade of the angiotensin II AT1 receptor represents a novel mechanism for interrupting the renin-angiotensin system without altering the potential benefits of AT2 receptor stimulation. This selective inhibition produces none of the disadvantages associated with reduced bradykinin metabolism and angiotensin II generated by non-angiotensin-converting enzyme pathways. Eprosartan is a potent (1.4 nmol/L) AT1 receptor antagonist that competitively blocks angiotensin II-induced vascular contraction. In various animal models of disease, including hypertension and stroke, eprosartan is effective in reducing disease progression. Eprosartan also has sympathoinhibitory activity, as demonstrated by an inhibition of the pressor responses induced by activation of sympathetic outflow through spinal cord stimulation in pithed rats. In contrast, some of the other angiotensin II receptor antagonists, such as losartan, at equivalent angiotensin II blocking doses, have no effect on sympathetic nervous system activity. Because eprosartan can inhibit both the direct effects of angiotensin II as well as the indirect effects that are mediated by enhanced sympathetic neurotransmission, this may represent an important advance in the treatment of elevated systolic blood pressure.
Angiotensin II (ANG II) is increasingly recognised as a growth factor, both in its own right and through interactions with other growth factors. There is a high density of ANG II receptors in the rat fetus, especially the AT2 receptor, the function of which is still uncertain. We have now studied the effects of ANG II on growth and development in the rat embryo in vitro between d 9.5 and 11.5, and characterised the receptor subtype mediating these effects. Embryos were cultured in whole rat serum, a high molecular weight retenate after ultrafiltration of whole rat serum, retenate with angiotensin II and retenate with ANG II and AT1 or AT2 receptor blockers. Growth and development were scored using conventional methods. Culture in retenate was associated with a marked reduction in growth and development by comparison with whole rat serum. This was partly, and significantly (P < 0.001), reversed by angiotensin II. The optimum concentration of angiotensin II was found to be angiotensin II 10(-11) M, within the physiological range. Angiotensin II had highly significant effects on both somatic (P < 0.001) and yolk sac/allantoic (P < 0.005) development. The latter effects suggest a role for angiotensin II in placentation. The effects of angiotensin II were blocked by PD123319, an AT2 blocker, but not by GR117289, an AT1 blocker. Interestingly, culture in retenate with GR117289 without added angiotensin II was also associated with some increase in growth (P < 0.05). Angiotensin II in low concentrations was measurable in the retenate, presumably arising from the action of endogenous renin on angiotensinogen. We therefore postulate that this effect of GR117289 was due to the action of endogenous angiotensin II on 'uncovered' AT2 receptors. This study has thus demonstrated a direct growth promoting effect of angiotensin II during organogenesis in the whole rat embryo in vitro. This effect is mediated through the AT2 receptors.
Angiotensin II (AngII) generation in response to vascular injury has long been assumed to influence neointimal proliferation contributing to restenosis. This concept has been supported by evidence that ACE inhibition and AT1 receptor blockade effectively limits restenosis in the rat. On the other hand, ACE inhibition has proven ineffective in clinical trails. The present study examines the response of the porcine coronary artery after angioplasty in vitro and compares the actions of an ACE inhibitor to AngII receptor antagonists.
Captopril, an ACE inhibitor, and the AngII receptor antagonists, losartan and PD123319, were evaluated for their ability to attenuate neointimal proliferation in a porcine organ culture model of coronary restenosis. The neointima was significantly increased by 300% after angioplasty compared to non-angioplasty controls. The AT1 receptor antagonist, losartan, produced a significant reduction in neointimal index at 10(-5) mol/l, while its in vivo metabolite, EXP3174, reduced neointimal proliferation at 10(-6) mol/l. PD123319, a selective antagonist of the AT2 receptor, also restricted neointimal proliferation at 10(-5) mol/l. Treatment with captopril (10(-6) mol/l) increased the neointimal proliferation by approximately 200% after angioplasty.
Direct blockade of AngII receptors effectively inhibits cell proliferation and restenosis post-angioplasty in vitro. ACE inhibition, exclusive of flow, does not attenuate proliferative restenosis. These data suggest that AngII contributes to neointimal proliferation and validates the concept that receptor antagonists could contribute to the therapeutic management of restenosis.
The treatment of essential hypertension continues to be carried out by drugs, combined with the adaptation of life style. The development of various types of antihypertensive drugs has not only greatly improved the management of hypertension, but also offered significant methodological sophistication of the pharmacological and pathophysiological sciences. Antihypertensive drugs and related experimental agents have been widely used in pharmaco-logical and pathophysiological research. The beneficial effects of such agents will be illustrated by means of several examples, emphasizing the sympathetic nervous system, the renin-angiotensin-aldosterone system, and calcium homeostasis as major targets. As pharmacological tools, which are also antihypertensives, we discuss various types of centrally acting antihypertensives, ganglionic and peripheral neuronal blocking agents, alpha- and beta-adrenoceptor antagonists, angiotensin converting enzyme (ACE)-inhibitors, renin-inhibitors, angiotensin II-receptor antagonists (AT1-blockers) and calcium antagonists. Finally, a few remarks will be made concerning the beneficial therapeutic effects of classic and newer antihypertensive drugs, such as beta-blockers, diuretics, calcium antagonists, ACE-inhibitors and AT1-blockers.
The human heart expresses type 2 angiotensin (AT(2)) receptor, but the function is poorly defined.
In the present study, we investigated (1) the cellular localization of the AT(2) receptor and (2) the relationship between the AT(2) receptor protein expression and the cardiac function of patients with ischemic heart disease. The receptor localization was assessed by immunohistochemistry and the protein expression was quantified by Western blotting in atrial tissues freshly obtained from 22 patients undergoing coronary artery bypass graft surgery (63.0+/-11.0 years old; male ratio, 85%). Prior to the surgery, blood was drawn for determination of atrial-natriuretic hormone level and the left ventricular function was assessed by ultrasound cardiography.
The results of immunohistochemistry showed that the AT(2) receptor was localized to cardiomyocytes and was not present in fibroblasts, vascular smooth muscles, or vascular endothelium. Atrial tissues showed various degrees of structural remodeling, but the localization of the AT(2) receptor was not altered in any tissue sections. The amount of the AT(2) receptor was negatively correlated with end-diastolic left ventricular diastolic dimension (r=-0.56, P<0.01), calculated left ventricular mass index (r=-0.51, P<0.02) and the plasma atrial natriuretic peptide (ANP) concentration (r=-0. 62, P<0.01) and positively correlated with left ventricular ejection fraction (r=0.48, P<0.05).
(1) The AT(2) receptor is localized to cardiomyocytes independently of the cardiac function. (2) Left ventricular dysfunction is associated with decreased expression of myocardial AT(2) receptor protein.
Angiotensin II acts on at least two distinct receptor subtypes (AT1 and AT2). Most known effects of angiotensin II in adult tissues are attributable to the AT1 receptor. The function of AT2 receptor is undefined, but its abundant expressions in fetal tissues, immature brain, skin wound, and atretic ovarian follicles suggest a role in growth and development. Previous studies suggested that AT2 receptor may not be G protein-coupled. Here, from a rat fetus expression library, we cloned a cDNA encoding a unique 363-amino acid protein with pharmacological specificity, tissue distribution, and developmental pattern of the AT2 receptor. It is 34% identical in sequence to the AT1 receptor, sharing a seven-transmembrane domain topology. A review of prior data on other receptors suggests that this receptor may belong to a unique class of seven-transmembrane receptors (including somatostatin SSTR1, dopamine D3, and frizzled protein Fz) for which G protein coupling has not been demonstrated. All members of this class exhibit fetal and developmental and/or neuronal-specific expression. A conserved motif in the third intracellular loop, distinguishing this class from “classical” G protein-coupled receptors, may mediate novel intracellular effects.
The growth-inhibiting peptide hormone somatostatin stimulates phosphotyrosine phosphatase activity in the human pancreatic cell line MIA PaCa-2. This hormonal activation was mediated by a pertussis toxin-sensitive guanosine 5'-triphosphate-binding protein (G protein) in the membranes of these cells. Activation of this G protein by somatostatin stimulated the dephosphorylation of exogenous epidermal growth factor receptor prepared from A-431 cells in vitro. This pathway may mediate the antineoplastic action of somatostatin in these cells and in human tumors and could represent a general mechanism of G protein coupling that is utilized by normal cells in the hormonal control of cell growth.
Dopaminergic D2 receptor agonists, such as bromocriptine, are potent anti-proliferative agents in the treatment of human pituitary adenomas. We have reproduced the anti-proliferative effect of dopamine in an established pituitary cell line stably transfected with the rat D2 dopamine receptor cDNA. We found that dopaminergic inhibition of DNA synthesis parallels the stimulation of a phosphotyrosine phosphatase activity. Both actions are blocked by pertussis toxin and by the phosphotyrosine phosphatase inhibitor, vanadate. We suggest that the anti-proliferative action of dopamine is mediated, at least in part, by the dopaminergic stimulation of a phosphotyrosine phosphatase.
Angiotensin II is known primarily for its effects on blood pressure and electrolyte homeostasis, but recent studies suggest that angiotensin II may play a role in the regulation of cellular growth. This study was undertaken to identify the angiotensin II receptor subtypes expressed during fetal and neonatal development and to characterize their cellular localization. Using an in situ receptor binding assay on sagittal frozen sections of fetal and neonatal rats, bound 125I-[Sar1,Ile8]-angiotensin II was visualized by film and emulsion autoradiography. Bound radioligand was detected by E11 (embryonic day 11) and maximal binding occurred by E19-21. Radioligand binding remained unaltered 30 min after birth, whereas a noticeable and stable decrease was observed 12 h postparturition. The highly abundant angiotensin II receptors were shown to be AT2 by the marked reduction in radioligand binding achieved with PD123177 (10(-7)M), a specific AT2 receptor antagonist, whereas DuP 753 (10(-5)M), an AT1 receptor antagonist, had little effect. Emulsion autoradiography showed radioligand binding in the undifferentiated mesenchyme of the submucosal layers of the intestine and stomach, connective tissue and choroid surrounding the retina, subdermal mesenchyme adjacent to developing cartilage, diaphragm, and tongue. Residual AT2 receptors were found on the dorsal subdermal region of the tongue 72 h after birth. AT1 receptors were detected in the placenta at E13 and in the aorta, kidney, lung, liver, and adrenal gland at E19-21, consistent with an adult distribution. The transient expression of AT2 receptors in the mesenchyme of the fetus suggests a role of angiotensin II in fetal development.
The type 1 angiotensin II (AT1) receptor is well characterized but the type 2 (AT2) receptor remains an enigma. We tested the hypothesis that the AT2 receptor can modulate the growth of vascular smooth muscle cells by transfecting an AT2 receptor expression vector into the balloon-injured rat carotid artery and observed that overexpression of the AT2 receptor attenuated neointimal formation. In cultured smooth muscle cells, AT2 receptor transfection reduced proliferation and inhibited mitogen-activated protein kinase activity. Furthermore, we demonstrated that the AT2 receptor mediated the developmentally regulated decrease in aortic DNA synthesis at the latter stages of gestation. These results suggest that the AT2 receptor exerts an antiproliferative effect, counteracting the growth action of AT1 receptor.
Angiotensin II (ANG II) is known to be a potent growth promoting factor for vascular smooth muscle cells and fibroblasts but little is known about its influence on growth in endothelial cells. We studied the effects of ANG II on endothelial growth and the role of the angiotensin receptor subtypes involved. Proliferation of rat coronary endothelial cells (CEC) and rat vascular smooth muscle cells (VSMC) was determined by [3H]thymidine incorporation, the MTT-test and by directly counting cells in a coulter counter. Angiotensin AT1- and AT2-receptors were demonstrated by binding studies and by the presence of their respective mRNA through reverse transcription polymerase chain reaction (RT-PCR). In contrast to VSMC, which in culture only express the AT1-receptor, CEC express both, AT1- and AT2-receptors simultaneously up to the third passage. Whereas ANG II stimulated growth of quiescent VSMC, an effect abolished by pretreatment with the AT1-receptor antagonist, losartan, ANG II did not induce proliferation in quiescent CEC. However, after pretreatment of quiescent endothelial cells (< passage 4) with the AT2-receptor antagonist, PD 123177, ANG II induced proliferation. This effect was reversed by additional pretreatment with losartan. ANG II significantly inhibited the proliferation of bFGF-stimulated CEC in a dose-dependent manner by maximally 50%. This effect was prevented by PD 123177 while losartan was ineffective. The AT2-receptor agonist, CGP 42112, mimicked the antiproliferative actions of ANG II, confirming the specificity of the effect. Our results show that the growth modulating actions of ANG II depend on the type of angiotensin receptor present on a given cell. In coronary endothelial cells, the antiproliferative actions of the AT2-receptor offset the growth promoting effects mediated by the AT1-receptor.
The effects of somatostatin analogues RC-160 and SMS-201-995 on tyrosine phosphatase and cell proliferation were investigated in COS-7 and NIH 3T3 cells expressing human somatostatin receptor subtype 1 or 2 (SSTR1 or SSTR2). Binding experiments were performed on membranes from COS-7 cells expressing human SSTR1 or SSTR2 using 125I-labeled [Tyr11]S-14 or [Tyr3]SMS-201-995, respectively. The somatostatin analogues RC-160 and SMS-201-995 exhibited low affinity for SSTR1 (IC50 of 0.43 and 1.5 microM, respectively) and high affinity for SSTR2 (IC50 of 0.27 and 0.19 nM). Addition of these analogues to cells expressing either SSTR1 or SSTR2 did not result in an inhibition of adenylate cyclase activity. In SSTR2-expressing cells, both analogues induced a rapid stimulation of a tyrosine phosphatase activity (EC50: RC-160, 2 pM; SMS-201-995, 6 pM) and an inhibition of serum-stimulated proliferation (EC50: RC-160, 6.3 pM; SMS-201-995, 12 pM). In SSTR1-expressing cells, only RC-160 induced stimulation of a tyrosine phosphatase activity. Both analogues caused an inhibition of cell proliferation at a concentration higher than 10 nM in accordance with their affinities for the SSTR1 receptor subtype. A good correlation between the affinities of RC-160 and SMS-201-995 for each receptor subtype and their potencies to inhibit cell proliferation suggests the involvement of these receptors in cell growth regulation. Tyrosine phosphatase was stimulated by both these analogues in SSTR2 and by RC-160 in SSTR1 at affinities similar to their ability to inhibit growth and bind to receptors, implicating tyrosine phosphatase as a transducer of the growth inhibition signal. We also found that mRNAs of receptor subtypes were variably expressed in different pancreatic and colon cancer cell lines, indicating the necessity of a precise analysis of receptor subtypes in target tissues before therapy with analogues.
The gene of human angiotensin II type 2 (AT2) receptor was isolated from a genomic DNA library prepared from human placenta. The coding region of the human AT2 receptor gene was contained in a single exon coding segment of the gene indicating an intronless structure of the coding region. The amino acid sequence of human AT2 receptor deduced from its nucleotide sequence has 363 amino acids and shows a high degree of sequence identity to rat and mouse receptor sequences. Specific binding of [125I]Sar1Ile8-angiotensin II was demonstrated in COS-7 cells transfected with a plasmid containing the human AT2 sequence. Scatchard analysis and ligand displacement profile were typical of the AT2 receptor. Reverse transcription-polymerase chain reaction analysis showed that AT2 receptor mRNA was expressed in adult uterus and pheochromocytoma.
There are two major isoforms of the angiotensin II receptor, type 1 (AT1) and type 2 (AT2). AT2 is distinguished from AT1 with respect to its ligand selectivity, its insensitivity to non-hydrolyzable GTP analogues, and its as yet unidentified biological functions. In the present study we have expression-cloned AT2 cDNA from a cDNA library of a rat pheochromocytoma cell line (PC12w). Rat AT2 cDNA encodes a 363-amino acid protein that has seven transmembrane domains. AT1 is the closest in homology to AT2 but with only a 32% identity of amino acid sequence. Stably expressed in COS-7 cells, the receptor showed selective binding to AT2-specific ligands PD123319 and CGP42112A but not to the AT1-specific ligand, losartan. Northern blot analysis revealed that the mRNA of rat AT2 was expressed not only in PC12w cells but also in the adrenal glands and in the inferior olive of the brain, both of which are known to contain AT2 type binding sites. The expressed AT2 receptor mediated angiotensin II-induced inhibition of protein tyrosine phosphatase, an action that was dependent on a pertussis toxin-sensitive G-protein-coupled mechanism in COS-7 cells. The AT2-specific ligand CGP42112A was an agonist rather than antagonist in the inhibition of phosphotyrosine phosphatase. AT2 did not cause a decrease in cGMP in PC12w or COS-7 cells expressing AT2 stably. These results indicate that the AT2 receptor is structurally and functionally different from AT1 and suggest novel functional roles of the renin-angiotensin system in cross-talk with phosphotyrosine signaling by modulating protein phosphotyrosine levels.
THERE are two major angiotensin II receptor isoforms, AT1
and AT2. AT1 mediates the well-known pressor and mitogenic
effects of angiotensin II (refs 1-5), but the signalling mechanism and physiological
role of AT2 (refs 6-11) has not been established. Its abundant expression
in fetal tissues12 and certain brain nuclei13 suggest possible roles
in growth, development and neuronal functions. Here we report the unexpected finding
that the targeted disruption of the mouse AT
2 gene resulted in a
significant increase in blood pressure and increased sensitivity to the pressor
action of angiotensin II. Thus AT2 mediates a depressor effect and
antagonizes the AT1-mediated pressor action of angiotensin II. In
addition, disruption of the AT
2 gene attenuated exploratory
behaviour and lowered body temperature. Our results show that angiotensin II
activates AT1 and AT2, which have mutually counteracting
haemo-dynamic effects, and that AT2 regulates central nervous system
functions, including behaviour.
Micromolar concentrations of sodium orthovanadate stimulated the proliferation of bovine capillary endothelial cells, but not bovine aortic endothelial cells. Vanadate was equally potent at inducing protein tyrosine phosphorylation and changes in morphology in both types of cells. However, vanadate treatment lead to an inhibition of protein tyrosine kinase activity in the aortic endothelial cells, but not the capillary endothelial cells. In capillary endothelial cells, the effect of vanadate was additive with basic FGF (bFGF) at low concentrations of bFGF. There was no interaction between bFGF and vanadate in aortic endothelial cells. TGF-beta, which inhibits the induction of endothelial cell proliferation by bFGF, appeared to shift the dose response curve to vanadate in capillary endothelial cells, increasing the proliferative effect of vanadate at low vanadate concentrations, but decreasing the proliferative effect at higher vanadate concentrations.
Vasoconstrictors such as angiotensin II (ang II) stimulate vascular smooth muscle cell growth and share many signal transduction mechanisms with growth factors. Recently, growth factors have been shown to stimulate mitogen-activated protein (MAP) kinases, a family of serine/threonine protein kinases which phosphorylate pp90rsk, a cytosolic kinase that phosphorylates ribosomal S6 protein. We examined the effect of ang II on MAP kinase activity and phosphorylation. Ang II stimulated MAP kinase activity by 4-fold after 5 min exposure and also increased tyrosine phosphorylation of 42 kDa (74 +/- 41%) and 44 kDa (263 +/- 85%) proteins, shown to be pp42mapk and pp44mapk by Western blot analysis using a MAP kinase antibody. These results suggest that ang II-stimulated protein synthesis is mediated by a MAP kinase dependent pathway.
We localized and characterized angiotensin II AT1 and AT2 receptors in the skin of 2-week-old rats during experimental wound healing. Both AT1 and AT2 were present in the skin. Three days after wounding, the expression of angiotensin II receptors was significantly enhanced in the dermis as well as in a localized band within the superficial dermis of the skin surrounding the wound. The major proportion of this increase was due to angiotensin II AT2 receptors. Our results suggest a physiological role for AT2 receptors in the process of tissue repair.
The role of angiotensin receptor subtypes 1 and 2 was assessed on neointima formation after injury in rat carotid artery. The effects of angiotensin converting enzyme inhibition by perindopril (3 mg.kg-1 x day-1 p.o.) and selective blockade of angiotensin subtype 1 receptors by DuP 753 (5 and 30 mg.kg-1 x day-1 p.o.) were compared on proliferative response to balloon injury. In rats treated 6 days before and for 14 days after injury, perindopril significantly reduced (-76%, p < 0.01) myointimal hyperplasia. In contrast, DuP 753 at 5 mg.kg-1 x day-1 did not modify the hyperplastic response to balloon catheterization. Only at 30 mg.kg-1 x day-1 was DuP 753 able to reduce neointima formation (-47%, p < 0.05). This dose was equipotent to perindopril on the renin-angiotensin system as assessed by the pressor response to angiotensin II and angiotensin I. Therefore, blockade of subtype 1 receptors was a less effective means of suppression of myointimal growth than angiotensin converting enzyme inhibition, suggesting that another angiotensin receptor subtype or converting enzyme substrates are involved in this process. For the determination of whether angiotensin subtype 2 receptors were implicated, the specific subtype 2 receptor antagonist CGP 42112A (1 mg.kg-1 x day-1) was continuously infused perivascularly for 14 days in the vicinity of the injured carotid artery. CGP 42112A was as effective in preventing neointima formation as perindopril (-73%, p < 0.01, versus -76%, p < 0.01, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)
Genetic and biochemical studies have indicated that the cdc25 protein controls the entry into mitosis by triggering tyrosine dephosphorylation of the cdc2 protein kinase. We show that the isolated cdc25 protein can catalyze dephosphorylation of several model phosphatase substrates, including p-nitrophenyl phosphate and two distinct tyrosine-phosphorylated peptides. The cdc25-dependent cleavage reaction closely resembles dephosphorylation by known tyrosine phosphatases: the reaction requires a reducing agent, shows high sensitivity to sodium vanadate, and proceeds efficiently in the presence of metal chelators. Moreover, the phosphatase activity of the cdc25 protein is eliminated by treatment with N-ethylmaleimide or by alteration of a single conserved cysteine residue by site-directed mutagenesis. These observations indicate that the cdc25 protein can function as a tyrosine phosphatase in the absence of any other protein.
Two angiotensin II receptor subtypes, A and B, have been described by means of specific and selective ligands (Biochem Biophys Res Commun 1989; 163:284-91). The present report describes the binding characteristics and the distribution of these subtypes in various rat tissues. Adrenal and uterus expressed both subtypes but in different proportions. Subtype B predominated in the adrenal glomerulosa (60%), whereas there was a greater proportion of subtype A in the medulla (70%). In uterus, subtype B was preferentially expressed (70%), and there was no difference in receptor distribution between muscle layers and serosa. Liver, kidney, and cultured aortic smooth muscle cells expressed only subtype B. In all of the tissues tested, the Ki values of various competing ligands were similar for each subtype expressed. It is proposed that subtype B is the vascular receptor. The function of subtype A, however, is still to be determined.
In cultured rat aortic smooth muscle cells, angiotensin II induced tyrosine phosphorylation of at least 9 proteins with molecular masses of 190, 117, 105, 82, 79, 77, 73, 45 and 40 kDa in time- and dose-dependent manners. Other vasoconstrictors such as [Arg]vasopressin, 5-hydroxytryptamine and norepinephrine induced the tyrosine phosphorylation of the same set of proteins as angiotensin II. The tyrosine phosphorylation of these proteins was mimicked by the protein kinase C-activating phorbol ester, phorbol 12 myristate 13-acetate, and the Ca2+ ionophore, ionomycin. These results demonstrate that the vasoconstrictors stimulate the tyrosine phosphorylation of several proteins in vascular smooth muscle cells and suggest that the tyrosine phosphorylation reactions are the events distal to the activation of protein kinase C and Ca2+ mobilization in the intracellular signalling pathways of the vasoconstrictors.
We have demonstrated the existence of two distinct subtypes of the angiotensin II receptor in the rat adrenal gland using radioligand binding and tissue section autoradiography. The identification of the subtypes was made possible by the discovery of two structurally dissimilar, nonpeptide compounds, DuP 753 and EXP655, that show reciprocal selectivity for the two subtypes. In the rat adrenal cortex, DuP 753 inhibited 80% of the total AII binding with an IC50 value on the sensitive sites of 2 x 10(-8) M, while EXP655 displaced only 20%. In the rat adrenal medulla, EXP655 gave 90% inhibition of AII binding with an IC50 value of 3.0 x 10(-8) M, while DuP 753 was essentially inactive. The combination of the two compounds completely inhibited AII binding in both tissues.
Two major isoforms of angiotensin II receptors, AT1 and AT2, have been defined on the basis of their ligand selectivity. While AT1 is known to mediate typical biological actions of angiotensin II as a cardiovascular regulator, the biological function of AT2 has not yet been established. In the present study using a rat pheochromocytoma cell line, which expresses AT2 exclusively, we found that angiotensin II inhibits phosphotyrosine phosphatase activity in vivo as measured by the inhibition of hydrolysis of [32P]-phosphate from the 32P-labeled synthetic peptide substrate, Raytide. This phosphotyrosine phosphatase inhibition was completely reversed by pertussis toxin, which indicates a G-protein coupled mechanism. In SDS-polyacrylamide gel electrophoresis we found that the phosphotyrosine group of an 85 kDa protein was a substrate mainly preserved, presumably as a consequence of the plausible intracellular phosphotyrosine phosphatase inhibition by angiotensin II.
Angiotensin II recognizes two receptor subtypes, AT1 and AT2, both of them having been recently cloned. Although AT2 receptors represent 5-10% of angiotensin II receptors in the kidneys of adult rats, their function remains unknown. In the present work, we examined the possible contribution of AT2 receptors to the regulation of pressure-natriuresis in anesthetized rats infused either with the specific AT2 antagonist PD 123319, or with CGP 42112B, an AT2 ligand with agonistic properties. The effects of PD 123319 were examined in a preparation with stable levels of angiotensin II, and in which AT1 receptors were blocked by the specific antagonist losartan. The effects of CGP 42112B were studied in rats deprived of endogenous angiotensin II. AT2 receptor blockade with PD 123319 did not change the renal blood flow while it increased the diuresis and natriuresis. These effects persisted even after full AT1 receptor blockade with losarfan. CGP 42112B did not modify the renal blood flow, but dose-dependently decreased urine flow and natriuresis. These results show that, contrary to AT1 receptors, renal AT2 receptors have no effect on total renal blood flow, but blunt the pressure-natriuresis, thus demonstrating that this receptor subtype is involved in a function of importance for body fluid and blood pressure regulation.
Most of angiotensin II's (Ang II) documented effects have been attributed to the interaction of this peptide with a G-protein coupled receptor termed AT1. The role and the signalling mechanisms of the more recently characterized AT2 receptor, which does not appear to interact with G-proteins, are however still unclear. We report here that this receptor mediates the rapid dephosphorylation of tyrosine residues of specific proteins in the 60 to 150 KDa range in PC12W cells which express only AT2 receptors. We further characterized this phosphatase activity using the synthetic substrate para-nitrophenyl phosphate. Dephosphorylation of this substrate in response to Ang II is not affected by Ser/Thr phosphatase inhibitors, but is completely prevented by the protein tyrosine phosphatase (PTPase) inhibitor sodium orthovanadate. This effect is mimicked by the AT2 selective agonist CGP42112 and is not affected by the AT1 antagonist losartan, In contrast to the recently reported PTPase stimulation by somatostatin and dopamine, PTPase stimulation by Ang II is not affected by the guanyl nucleotides GTP gamma S and GDP beta S. Moreover, depletion of solubilized membrane preparations from G-proteins by lectin affinity chromatography does not alter Ang II stimulation of the measured PTPase activity. These findings indicate that Ang II stimulates a PTPase activity through AT2 receptors via G-protein independent pathways. This signalling mechanism may be involved in AT2 receptor mediated actions of Ang II such as particulate guanylate cyclase inhibition, modulation of T-type Ca++ channels and regulation of cell proliferation and differentiation.
Despite some recent reports describing the effects of AT2 receptor selective ligands in vitro and in vivo, the physiological function of this receptor is still a matter of debate. This problem stems amongst others from the difficulty in interpreting results from in vivo experiments with drugs of which it is not known whether they act as agonists or antagonists. We reported earlier that angiotensin II inhibits basal and atrial natriuretic peptide stimulated particulate guanylate cyclase activity through AT2 receptors in PC12W cells. We have used this parameter in intact PC12W cells in order to determine the pharmacological properties of different widely used angiotensin receptor ligands. We found CGP 42112 to behave as a full agonist in this system, whereas PD 123319 and Sar Ile angiotensin II act as antagonists. As expected, the AT1 antagonist losartan did not affect this response.
R3T3 cells are a fibroblast cell line found to selectively express the AT2 subtype of angiotensin II binding sites. We have previously shown that in these cells, the AT2 sites do not appear to be coupled to G-proteins, do not modulate any of the common second messenger pathways associated with activation of angiotensin II receptors, and do not internalize bound ligand. Here we report that expression of AT2 sites in these cells are subject to modulation by a variety of conditions. In actively growing cells the expression of AT2 sites is very low, while in confluent, quiescent cells, the expression of AT2 sites is markedly increased. Addition of serum, or growth factors, to quiescent cells causes a rapid decrease in the number of cell-surface AT2 sites. Further, incubation of cells with ligands that bind to AT2 sites causes a marked increase in the number of these sites in a time and dose dependent manner indicating homologous up-regulation of expression. These results indicate that expression of AT2 sites in R3T3 cells is under sensitive and rapid control and further indicate that these cells may be an excellent model for studying the physiological regulation of expression of these sites.
The renal effects of angiotensin II(AII) are attributed to AT1 receptors. In contrast, the function of renal AT2 receptors in unknown. Using a microdialysis technique, we monitored changes in renal interstitial fluid (RIF) prostaglandin E2 (PGE2) and cyclic guanosine 3', 5'-monophosphate (cGMP) in response to dietary sodium (Na) depletion alone, or Na depletion or normal Na diet combined with the AT1 receptor blocker, Losartan, the AT2 receptor blocker, PD 123319 (PD), or angiotensin II, individually or combined in conscious rats. Na depletion significantly increased PGE2 and cGMP. During Na depletion, Losartan decreased PGE2 and did not change cGMP. In contrast, PD significantly increased PGE2 and decreased cGMP. Combined administration of Losartan and PD decreased PGE2 and cGMP. During normal Na diet, RIF PGE2 and cGMP increased in response to angiotensin II. Neither Losartan nor PD, individually or combined, changed RIF PGE2 or cGMP. Combined administration of angiotensin II and Losartan or PD produced a significant decrease in response of PGE2 and cGMP to angiotensin II, respectively. These data demonstrate that activation of the reninangiotensin system during Na depletion increases renal interstitial PGE2 and cGMP. The AT1 receptor mediates renal production of PGE2. The AT2 receptor mediates cGMP. AT2 blockade potentiates angiotensin-induced PGE2 production at the AT1 receptor.