20-Hydroxyecdysone activates the protective arm of the RAAS via the MAS receptor.

20-Hydroxyecdysone (20E) is a steroid hormone that plays a key role in insect development through nuclear ecdysteroid receptors (EcR/RXR complex) and at least one membrane GPCR receptor (DopEcR). It also displays numerous pharmacological effects in mammals, where its mechanism of action is still debated, involving either an unidentified GPCR or the estrogen ERß receptor. The goal of this study was to better understand 20E mechanism of action in mammals. A mouse myoblast cell line (C2C12) and the gene expression of myostatin (a negative regulator of muscle growth) were used as a reporter system of anabolic activity. Experiments using protein-bound 20E established the involvement of a membrane receptor. 20E-like effects were also observed with angiotensin(1-7), the endogenous ligand of MAS. Additionally, the effect on myostatin gene expression was abolished by Mas receptor knock-down using siRNA or pharmacological inhibitors. 17ß-Estradiol (E2) also inhibited myostatin gene expression, but protein-bound E2 was inactive, and E2 activity was not abolished by angiotensin(1-7) antagonists. A mechanism involving cooperation between the MAS receptor and a membrane-bound palmitoylated estrogen receptor is proposed. The possibility to activate the MAS receptor with a safe steroid molecule is consistent with the pleiotropic pharmacological effects of ecdysteroids in mammals and, indeed, the proposed mechanism may explain the close similarity between the effects of angiotensin(1-7) and 20E. Our findings open up many possible therapeutic developments involving stimulation of the protective arm of the renin-angiotensin-aldosterone system (RAAS) with 20E.

Angiotensin (1-7) Alleviates Postresuscitation Myocardial Dysfunction by Suppressing Oxidative Stress Through the Phosphoinositide 3-Kinase, Protein Kinase B, and Endothelial Nitric Oxide Synthase Signaling Pathway.

ABSTRACT: There is increasing evidence that angiotensin (1-7) [Ang (1-7)] is an endogenous biologically active component of the renin-angiotensin system. However, the role of the Ang (1-7)-MasR axis in postresuscitation myocardial dysfunction (PRMD) and its associated mechanism are still unclear. In this study, we investigated the effect of the Ang (1-7)-MasR axis on myocardial injury after cardiac arrest-cardiopulmonary resuscitation-restoration of spontaneous circulation. We established a model of oxygen/glucose deprivation-reperfusion in myocardial cells in vitro and a rat model of cardiac arrest-cardiopulmonary resuscitation-restoration of spontaneous circulation in vivo. The cell apoptosis rate and the expression of the superoxide anion 3-nitrotyrosine were decreased in the Ang (1-7) group in vitro and in vivo. The mean arterial pressure was decreased, whereas +LVdp/dtmax and -LVdp/dtmax were increased in rats in the Ang (1-7) group. The mRNA and protein levels of Ang II type 1 receptor, MasR, phosphoinositide 3-kinase, protein kinase B, and endothelial nitric oxide synthase were increased in the Ang (1-7) group in vivo. These results indicate that the Ang (1-7)-MasR axis can alleviate PRMD by reducing myocardial tissue damage and oxidative stress through activation of the phosphoinositide 3-kinase-protein kinase B-endothelial nitric oxide synthase signaling pathway and provide a new direction for the clinical treatment of PRMD.

Novel Interactions Involving the Mas Receptor Show Potential of the Renin-Angiotensin system in the Regulation of Microglia Activation: Altered Expression in Parkinsonism and Dyskinesia.

The renin-angiotensin system (RAS) not only plays an important role in controlling blood pressure but also participates in almost every process to maintain homeostasis in mammals. Interest has recently increased because SARS viruses use one RAS component (ACE2) as a target-cell receptor. The occurrence of RAS in the basal ganglia suggests that the system may be targeted to improve the therapy of neurodegenerative diseases. RAS-related data led to the hypothesis that RAS receptors may interact with each other. The aim of this paper was to find heteromers formed by Mas and angiotensin receptors and to address their functionality in neurons and microglia. Novel interactions were discovered by using resonance energy transfer techniques. The functionality of individual and interacting receptors was assayed by measuring levels of the second messengers cAMP and Ca2+ in transfected human embryonic kidney cells (HEK-293T) and primary cultures of striatal cells. Receptor complex expression was assayed by in situ proximity ligation assay. Functionality and expression were assayed in parallel in primary cultures of microglia treated or not with lipopolysaccharide and interferon-γ (IFN-γ). The proximity ligation assay was used to assess heteromer expression in parkinsonian and dyskinetic conditions. Complexes formed by Mas and the angiotensin AT1 or AT2 receptors were identified in both a heterologous expression system and in neural primary cultures. In the heterologous system, we showed that the three receptors-MasR, AT1R, and AT2R-can interact to form heterotrimers. The expression of receptor dimers (AT1R-MasR or AT2R-MasR) was higher in microglia than in neurons and was differentially affected upon microglial activation with lipopolysaccharide and IFN-γ. In all cases, agonist-induced signaling was reduced upon coactivation, and in some cases just by coexpression. Also, the blockade of signaling of two receptors in a complex by the action of a given (selective) receptor antagonist (cross-antagonism) was often observed. Differential expression of the complexes was observed in the striatum under parkinsonian conditions and especially in animals rendered dyskinetic by levodopa treatment. The negative modulation of calcium mobilization (mediated by AT1R activation), the multiplicity of possibilities on RAS affecting the MAPK pathway, and the disbalanced expression of heteromers in dyskinesia yield new insight into the operation of the RAS system, how it becomes unbalanced, and how a disbalanced RAS can be rebalanced. Furthermore, RAS components in activated microglia warrant attention in drug-development approaches to address neurodegeneration.