Reverse Induced Fit-Driven MAS-Downstream Transduction: Looking for Metabotropic Agonists.

BACKGROUND: Protective effects of MAS activation have spurred clinical interests in developing MAS agonists. However, current bases that drive this process preclude that physiological concentrations of peptide MAS agonists induce an atypical signaling that does not reach the metabotropic efficacy of constitutive activation. Canonical activation of MAS-coupled G proteins is only achieved by supraphysiological concentrations of peptide MAS agonists or physiological concentrations of chemically modified analogues. These pleiotropic differences are because of two overlapped binding domains: one non-metabotropic site that recognizes peptide agonists and one metabotropic domain that recognizes modified analogues. OBJECTIVE: It is feasible that supraphysiological concentrations of peptide MAS agonists undergo to chemical modifications required for binding to metabotropic domain. Receptor oligomerization enhances pharmacological parameters coupled to metabotropic signaling. The formation of receptor-signalosome complex makes the transduction of agonists more adaptive. Considering the recent identification of MAS-signalosome, we aimed to postulate the reverse induced fit hypothesis in which MAS-signalosome would trigger chemical modifications required for agonists bind to MAS metabotropic domain. METHODS: Here we cover rational perspectives for developing novel metabotropic MAS agonists in the view of the reverse induced-fit hypothesis. RESULTS: Predicting a 3D model of MAS metabotropic domain may guide the screening of chemical modifications required for metabotropic efficacy. Pharmacophore-based virtual screening would select potential metabotropic MAS agonists from virtual libraries from human proteome. CONCLUSIONS: Rational perspectives that consider reverse induced fit hypothesis during MAS activation for developing metabotropic MAS agonists represents the best approach in providing MAS ligands with constitutive efficacy at physiological concentrations.

Chronic restraint stress increases angiotensin II potency in the rat carotid: role of cyclooxygenases and reactive oxygen species.

OBJECTIVES: To investigate the mechanisms underlying the effects of chronic restraint stress on the vascular contractile response induced by angiotensin (Ang) II in rat carotid. METHODS: Concentration-response curves for AngII were obtained in endothelium-intact or endothelium-denuded carotid rings, in the absence or presence of SC-560 (COX-1 inhibitor), SC-236 (COX-2 inhibitor), wortmannin (PI3 K-Akt inhibitor), ML171 (NOX-1 inhibitor), VAS2870 (NOX-4 inhibitor), tiron (O2- scavenger) or PEG-catalase (H2 O2 scavenger). 6-ketoPGF1α , TXB2 , O2- or H2 O2 levels and superoxide dismutase and catalase activity or expression were also measured in rat carotid. KEY FINDINGS: Stress increased AngII potency in rat carotid. Muscular COX-1 or COX-2-derived metabolites negatively modulated AngII-induced contraction in control rat carotid. Endothelial COX-1 or COX-2-derived metabolites positively modulated AngII-induced contraction in stressed rat carotid. PI3 K-Akt, NOX-1, NOX-4, O2- and H2 O2 positively modulated AngII-induced contraction in stressed rat carotid. Stress increased 6-ketoPGF1α or H2 O2 generation and reduced catalase activity in rat carotid. Protein expression of COX-1, NOX-4 or p-Akt was increased in stressed rat carotid. CONCLUSIONS: Stress increases AngII potency in rat carotid by a mechanism that involves the increased generation of PGI2 and H2 O2 and the activation of Akt pathway. Such mechanism could play a pathophysiological role in cardiovascular diseases correlated with stress.

Endothelinergic Contractile Hyperreactivity in Rat Contralateral Carotid to Balloon Injury: Integrated Role for ETB Receptors and Superoxide Anion.

Temporal consequences of neurocompensation to balloon injury on endothelinergic functionality in rat contralateral carotid were evaluated. Rats underwent balloon injury in left carotid and were treated with CP-96345 (NK1 antagonist). Concentration-response curves for endothelin-1 were obtained in contralateral (right) carotid at 2, 8, 16, 30, or 45 days after surgery in the absence or presence of BQ-123 (ETA antagonist), BQ-788 (ETB antagonist), or Tempol (superoxide-dismutase mimic). Endothelin-1-induced calcium mobilization was evaluated in functional assays carried out with BQ-123, BQ-788, or Tempol. Endothelin-1-induced NADPH oxidase-driven superoxide generation was measured by lucigenin chemiluminescence assays performed with BQ-123 or BQ-788. Endothelin-1-induced contraction was increased in contralateral carotid from the sixteenth day after surgery. This response was restored in CP-96345-treated rats. Endothelium removal or BQ-123 did not change endothelin-1-induced contraction in contralateral carotid. This response was restored by BQ-788 or Tempol. Contralateral carotid exhibited an increased endothelin-1-induced calcium mobilization, which was restored by BQ-788 or Tempol. Contralateral carotid exhibited an increased endothelin-1-induced lucigenin chemiluminescence, which was restored by BQ-788. We conclude that the NK1-mediated neurocompensatory response to balloon injury elicits a contractile hyperreactivity to endothelin-1 in rat contralateral carotid by enhancing the muscular ETB-mediated NADPH oxidase-driven generation of superoxide, which activates calcium channels.

Enhanced nitric oxide generation from nitric oxide synthases as the cause of increased peroxynitrite formation during acute restraint stress: Effects on carotid responsiveness to angiotensinergic stimuli in type-1 diabetic rats.

Diabetes mellitus is associated with reactive oxygen and nitrogen species accumulation. Behavioral stress increases nitric oxide production, which may trigger a massive impact on vascular cells and accelerate cardiovascular complications under oxidative stress conditions such as Diabetes. For this study, type-1 Diabetes mellitus was induced in Wistar rats by intraperitoneal injection of streptozotocin. After 28 days, cumulative concentration-response curves for angiotensin II were obtained in endothelium-intact carotid rings from diabetic rats that underwent to acute restraint stress for 3h. The contractile response evoked by angiotensin II was increased in carotid arteries from diabetic rats. Acute restraint stress did not alter angiotensin II-induced contraction in carotid arteries from normoglycaemic rats. However acute stress combined with Diabetes increased angiotensin II-induced contraction in carotid rings. Western blot experiments and the inhibition of nitric oxide synthases in functional assays showed that neuronal, endothelial and inducible nitric oxide synthase isoforms contribute to the increased formation of peroxynitrite and contractile hyperreactivity to angiotensin II in carotid rings from stressed diabetic rats. In summary, these findings suggest that the increased superoxide anion generation in carotid arteries from diabetic rats associated to the increased local nitric oxide synthases expression and activity induced by acute restrain stress were responsible for exacerbating the local formation of peroxynitrite and the contraction induced by angiotensin II.

Consequence of hyperhomocysteinaemia on α1-adrenoceptor-mediated contraction in the rat corpus cavernosum: the role of reactive oxygen species.

OBJECTIVES: Our main objective was to investigate the mechanisms underlying the effects of hyperhomocysteinaemia (HHcy) on contractile response mediated by α1-adrenoceptors in the rat corpus cavernosum. METHODS: Concentration-response curves for phenylephrine (PE) were obtained in strips of corpus cavernosum, in absence or after incubation with tiron, tempol or polyethylene glycol (PEG)-catalase combined or not with tempol. We also measured the superoxide anion (O2(-)) and hydrogen peroxide (H2O2) generation, superoxide dismutase (SOD) and catalase activity and α-actin expression in rat corpus cavernosum from both groups. KEY FINDINGS: HHcy increased PE-induced contraction in cavernosal strips. Tiron, PEG-catalase or tempol increased PE-induced contraction in strips from control rats, but it was not altered by tiron or PEG-catalase in HHcy rats, whereas tempol reduced this response. The combination of PEG-catalase and tempol did not alter the contractile response to PE in both groups. HHcy increased O2(-) generation and SOD activity, whereas H2O2 concentration was reduced. Finally, HHcy did not alter catalase activity or expression of α-actin. CONCLUSIONS: The major new finding from this study is that HHcy induced a marked increase in PE-induced contraction in rat corpus cavernosum by a mechanism that involves increased O2(-) generation and it could play a role in the pathogenesis of erectile dysfunction associated with HHcy.

Pharmacological significance of the interplay between angiotensin receptors: MAS receptors as putative final mediators of the effects elicited by angiotensin AT1 receptors antagonists.

The interplay between angiotensin AT1 receptors and MAS receptors relies on several inward regulatory mechanisms from renin-angiotensin system (RAS) including the functional crosstalk between angiotensin II and angiotensin-(1-7), the competitive AT1 antagonism exhibited by angiotensin-(1-7), the antagonist feature assigned to AT1/MAS heterodimerization on AT1 signaling and the AT1-mediated downregulation of angiotensin-converting enzyme 2 (ACE2). Recently, such interplay has acquired an important significance to RAS Pharmacology since a few studies have supporting strong evidences that MAS receptors mediate the effects elicited by AT1 antagonists. The present Perspective provides an overview of the regulatory mechanisms involving AT1 and MAS receptors, their significance to RAS Pharmacology and the future directions on the interplay between angiotensin receptors.

Vasoprotective effects of neurocompensatory response to balloon injury during diabetes involve the improvement of Mas signaling by TGFß1 activation.

Balloon injury in diabetic rats triggers a sensory neurocompensatory response that restores the blood flow in contralateral carotid. These vasoprotective effects result from H2O2-mediated relaxation that counteracts AT1-dependent contractile hyperreactivity. The most important mechanism from the renin-angiotensin-system in counteracting AT1-mediated effects is that one is mediated by Mas receptors. Thus, we hypothesized that the vasoprotective effects of balloon neurocompensation in diabetic rats could result from the improvement of Mas signaling by H2O2-mediated sensory mechanisms. NK1 receptors are sensory components whose activation could lead to H2O2 generation upon TGFß1 release and ALK5-mediated Nox4 upregulation. Based on this, we aimed to investigate: (1) the role of the TGFß1/ALK5-Nox4-H2O2 pathway on modulating Mas signaling in diabetic rat contralateral carotid; and (2) the contribution of Mas signaling in the control of local blood flow. Our results showed that balloon neurocompensation restored diabetic rat contralateral carotid flow by improving Mas signaling through NK1-mediated TGFß1 release. TGFß1/ALK5 activation enhanced Nox4 expression and Nox4-driven generation of H2O2. In turn, H2O2 enhanced the local Mas-mediated relaxation. Since restenosis impairs diabetic rat ipsilateral carotid flow, the restoration of diabetic rat contralateral carotid flow may prevent further damages in cerebral irrigation by carotid pathways after angioplasty during diabetes.

MAS receptors mediate vasoprotective and atheroprotective effects of candesartan upon the recovery of vascular angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis functionality.

AT1 antagonists effectively prevent atherosclerosis since AT1 upregulation and angiotensin II-induced proinflammatory actions are critical to atherogenesis. Despite the classic mechanisms underlying the vasoprotective and atheroprotective actions of AT1 antagonists, the cross-talk between angiotensin-converting enzyme-angiotensin II-AT1 and angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axes suggests other mechanisms beyond AT1 blockage in such effects. For instance, angiotensin-converting enzyme 2 activity is inhibited by reactive oxygen species derived from AT1-mediated proinflammatory signaling. Since angiotensin-(1-7) promotes antiatherogenic effects, we hypothesized that the vasoprotective and atheroprotective effects of AT1 antagonists could result from their inhibitory effects on the AT1-mediated negative modulation of vascular angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis functionality. Interestingly, our results showed that early atherosclerosis triggered in thoracic aorta from high cholesterol fed-Apolipoprotein E-deficient mice impairs angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis functionality by a proinflammatory-redox AT1-mediated pathway. In such mechanism, AT1 activation leads to the aortic release of tumor necrosis factor-α, which stimulates NAD(P)H oxidase/Nox1-driven generation of superoxide and hydrogen peroxide. While hydrogen peroxide inhibits angiotensin-converting enzyme 2 activity, superoxide impairs MAS functionality. Candesartan treatment restored the functionality of angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis by inhibiting the proinflammatory-redox AT1-mediated mechanism. Candesartan also promoted vasoprotective and atheroprotective effects that were mediated by MAS since A779 (MAS antagonist) co-treatment inhibited them. The role of MAS receptors as the final mediators of the vasoprotective and atheroprotective effects of candesartan was supported by the vascular actions of angiotensin-(1-7) upon the recovery of the functionality of vascular angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis.

Acute restraint stress increases carotid reactivity in type-I diabetic rats by enhancing Nox4/NADPH oxidase functionality.

Hyperglycemia increases the generation of reactive oxygen species and affects systems that regulate the vascular tone including renin-angiotensin system. Stress could exacerbate intracellular oxidative stress during Diabetes upon the activation of angiotensin AT1/NADPH oxidase pathway, which contributes to the development of diabetic cardiovascular complications. For this study, type-I Diabetes was induced in Wistar rats by intraperitoneal injection of streptozotocin. 28 days after streptozotocin injection, the animals underwent to acute restraint stress for 3 h. Cumulative concentration-response curves for angiotensin II were obtained in carotid rings pre-treated or not with Nox or cyclooxygenase inhibitors. Nox1 or Nox4 expression and activity were assessed by Western blotting and lucigenin chemiluminescence, respectively. The role of Nox1 and Nox4 on reactive oxygen species generation was evaluated by flow cytometry and Amplex Red assays. Cyclooxygenases expression was assessed by real-time polymerase chain reaction. The contractile response evoked by angiotensin II was increased in diabetic rat carotid. Acute restraint stress increased this response in this vessel by mechanisms mediated by Nox4, whose local expression and activity in generating hydrogen peroxide are increased. The contractile hyperreactivity to angiotensin II in stressed diabetic rat carotid is also mediated by metabolites derived from cyclooxygenase-2, whose local expression is increased. Taken together, our findings suggest that acute restraint stress exacerbates the contractile hyperreactivity to angiotensin II in diabetic rat carotid by enhancing Nox4-driven generation of hydrogen peroxide, which evokes contractile tone by cyclooxygenases-dependent mechanisms. Finally, these findings highlight the harmful role played by acute stress in modulating diabetic vascular complications.

Diabetes confers a vasoprotective role to the neurocompensatory response elicited by carotid balloon injury: consequences on contralateral carotid tone and blood flow.

The purpose from this study was to investigate the consequences of sensory neurocompensation to carotid balloon injury in diabetic rats on angiotensin II-induced contraction and basal blood flow in contralateral carotid. Concentration-response curves for angiotensin II and blood flow were obtained in contralateral carotid from non-treated or capsaicin-treated streptozotocin-induced diabetic rats that underwent carotid balloon injury. Diabetes increased angiotensin II-induced contraction and impaired the blood flow in non-operated rat carotid. In diabetic rats, balloon injury led to neointima formation, which reduced the blood flow in ipsilateral carotid. Carotid balloon injury in diabetic rats reduced angiotensin II-induced contraction and restored the blood flow in contralateral carotid when compared to diabetic non-operated rat carotid. Capsaicin inhibited the effects evoked by carotid balloon injury on diabetic rat contralateral carotid. Endothelium removal, PEG-catalase (hydrogen peroxide scavenger) or l-NPA (neuronal nitric oxide synthase, nNOS, inhibitor) increased angiotensin II-induced contraction in contralateral carotid from diabetic operated rats to the levels observed in diabetic non-operated rat carotid. Our findings suggest that carotid balloon injury in diabetic rats elicits a neurocompensation that attenuates the diabetic hyperreactivity to angiotensin II in contralateral carotid by a sensory nerves-dependent mechanism mediated by hydrogen peroxide derived from endothelial nNOS. This sensory mechanism also restored the blood flow in this vessel, compensating the impaired blood flow in diabetic rat ipsilateral carotid. Thus, our major conclusions are that Diabetes confers a vasoprotective significance to the neurocompensation to carotid balloon injury in preventing further damage at carotid cerebral irrigation after angioplasty in diabetic subjects.