Alamandine but not angiotensin-(1-7) produces cardiovascular effects at the rostral insular cortex.

Experiments aimed to evaluate the tissue distribution of Mas-related G protein-coupled receptor D (MrgD) revealed the presence of immunoreactivity for the MrgD protein in the rostral insular cortex (rIC), an important area for autonomic and cardiovascular control. To investigate the relevance of this finding, we evaluated the cardiovascular effects produced by the endogenous ligand of MrgD, alamandine, in this brain region. Mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were recorded in urethane anesthetized rats. Unilateral microinjection of equimolar doses of alamandine (40 pmol/100 nL), angiotensin-(1-7), angiotensin II, angiotensin A, and Mas/MrgD antagonist d-Pro7-Ang-1-7 (50 pmol/100 nL), Mas antagonist A779 (100 pmol/100 nL), or vehicle (0.9% NaCl) were made in different rats (n = 4-6/group) into rIC. To verify the specificity of the region, a microinjection of alamandine was also performed into intermediate insular cortex (iIC). Microinjection of alamandine in rIC produced an increase in MAP (Δ = 15 ± 2 mmHg), HR (Δ = 36 ± 4 beats/min), and RSNA (Δ = 31 ± 4%), but was without effects at iIC. Strikingly, an equimolar dose of angiotensin-(1-7) at rIC did not produce any change in MAP, HR, and RSNA. Angiotensin II and angiotensin A produced only minor effects. Alamandine effects were not altered by A-779, a Mas antagonist, but were completely blocked by the Mas/MrgD antagonist d-Pro7-Ang-(1-7). Therefore, we have identified a brain region in which alamandine/MrgD receptor but not angiotensin-(1-7)/Mas could be involved in the modulation of cardiovascular-related neuronal activity. This observation also suggests that alamandine might possess unique effects unrelated to angiotensin-(1-7) in the brain.

Angiotensin (1-7) Attenuates the Nociceptive Behavior Induced by Substance P and NMDA via Spinal MAS1.

The intrathecal (i.t.) injection of substance P (SP) and N-methyl-D-aspartate (NMDA) induce transient nociceptive response by activating neurokinin (NK) 1 and NMDA receptors, respectively. We have recently reported that angiotensin (Ang) (1-7), an N-terminal fragment of Ang II, could alleviate several types of pain including neuropathic and inflammatory pain by activating spinal MAS1. Here, we investigated whether Ang (1-7) can inhibit the SP- and NMDA-induced nociceptive response. The nociceptive response induced by an i.t. injection of SP or NMDA was assessed by measuring the duration of hindlimb scratching directed toward the flank, biting and/or licking of the hindpaw or the tail for 5 min. Localization of MAS1 and either NK1 or NMDA receptors in the lumbar superficial dorsal horn was determined by immunohistochemical observation. The nociceptive response induced by SP and NMDA was attenuated by the i.t. co-administration of Ang (1-7) (0.03-3 pmol) in a dose-dependent manner. The inhibitory effects of Ang (1-7) (3 pmol) were attenuated by A779 (100 pmol), a MAS1 antagonist. Moreover, immunohistochemical analysis showed that spinal MAS1 co-localized with NK1 receptors and NMDA receptors on cells in the dorsal horn. Taken together, the i.t. injection of Ang (1-7) attenuated the nociceptive response induced by SP and NMDA via spinal MAS1, which co-localized with NK1 and NMDA receptors. Thus, the spinal Ang (1-7)/MAS1 pathway could represent a therapeutic target to effectively attenuate spinal pain transmission caused by the activation of NK1 or NMDA receptors.

Mas receptor activation attenuates allergic airway inflammation via inhibiting JNK/CCL2-induced macrophage recruitment.

BACKGROUND: Defective absorption of acute allergic airway inflammation is involved in the initiation and development of chronic asthma. After allergen exposure, there is a rapid recruitment of macrophages around the airways, which promote acute inflammatory responses. The Ang-(1-7)/Mas receptor axis reportedly plays protective roles in various tissue inflammation and remodeling processes in vivo. However, the exact role of Mas receptor and their underlying mechanisms during the pathology of acute allergic airway inflammation remains unclear. OBJECTIVE: We investigated the role of Mas receptor in acute allergic asthma and explored its underlying mechanisms in vitro, aiming to find critical molecules and signal pathways. METHODS: Mas receptor expression was assessed in ovalbumin (OVA)-induced acute asthmatic murine model. Then we estimated the anti-inflammatory role of Mas receptor in vivo and explored expressions of several known inflammatory cytokines as well as phosphorylation levels of MAPK pathways. Mas receptor functions and underlying mechanisms were studied further in the human bronchial epithelial cell line (16HBE). RESULTS: Mas receptor expression decreased in acute allergic airway inflammation. Multiplex immunofluorescence co-localized Mas receptor and EpCAM, indicated that Mas receptor may function in the bronchial epithelium. Activating Mas receptor through AVE0991 significantly alleviated macrophage infiltration in airway inflammation, accompanied with down-regulation of CCL2 and phosphorylation levels of MAPK pathways. Further studies in 16HBE showed that AVE0991 pre-treatment inhibited LPS-induced or anisomycin-induced CCL2 increase and THP-1 macrophages migration via JNK pathways. CONCLUSION: Our findings suggested that Mas receptor activation significantly attenuated CCL2 dependent macrophage recruitments in acute allergic airway inflammation through JNK pathways, which indicated that Mas receptor, CCL2 and phospho-JNK could be potential targets against allergic airway inflammation.

Developing new drugs that activate the protective arm of the renin-angiotensin system as a potential treatment for respiratory failure in COVID-19 patients.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has reached pandemic proportions with negative impacts on global health, the world economy and human society. The clinical picture of COVID-19, and the fact that Angiotensin converting enzyme 2 (ACE2) is a receptor of SARS-CoV-2, suggests that SARS-CoV-2 infection induces an imbalance in the renin-angiotensin system (RAS). We review clinical strategies that are attempting to rebalance the RAS in COVID-19 patients by using ACE inhibitors, angiotensin receptor blockers, or agonists of angiotensin-II receptor type 2 or Mas receptor (MasR). We also propose that the new MasR activator BIO101, a pharmaceutical grade formulation of 20-hydroxyecdysone that has anti-inflammatory, anti-fibrotic and cardioprotective properties, could restore RAS balance and improve the health of COVID-19 patients who have severe pneumonia.

Synergism between Angiotensin receptors ligands: Role of Angiotensin-(1-7) in modulating AT2 R agonist response on nitric oxide in kidney cells.

Angiotensin-(1-7), an endogenous agonist for the MasR, has been shown to interact with ang-II AT1 R and AT2 R. Earlier we showed a physical and functional interaction between MasR and AT2 R in response to their respective agonists ang-(1-7) and C21. Moreover, ang-(1-7) is cardio-protective via AT1 R and alters ang-II function. Such complex nature of ang-(1-7) function is not clearly understood, particularly in relation to its functional interaction with these receptors. We tested how ang-(1-7) affects AT2 R function by utilizing HK-2 cells. The HK-2 cells were treated with a wide range of concentrations of angiotensin receptor agonists. The generation of NO• in response to agonists was determined as a readout and subjected to Bliss definition (δ score) to assess the nature of functional interaction between these receptors. Preincubation with ang-(1-7) followed by incubation with endogenous AT1 R/AT2 R agonist ang-II (δ = 162) or selective AT2 R agonist C21 (δ = 304) synergized NO• formation. The synergism was also observed when the order of incubation with ang-(1-7)/C21 was reversed (δ = 484), but not when the cells were simultaneously incubated with a mixture of ang-(1-7) and C21 (δ = 76). The synergism with nonpeptidic MasR agonist AVE0991 followed by C21 (δ = 45) was minimal. Ligand binding experiment suggested the binding of ang-(1-7) with these three receptors. However, the synergism observed with ang-(1-7) and ang-II/C21 was sensitive to the antagonists of AT2 R (PD123319) and AT1 R (candesartan), but not MasR (A779). Ang-(1-7) at lower concentrations synergies the AT2 R function in an AT1 R-dependent but MasR-independent manner. This phenomenon may have a physiological significance.

Urgent need for evaluating agonists of angiotensin-(1-7)/Mas receptor axis for treating patients with COVID-19.

ACE2 is a receptor of entry of SARS-CoV-2 into the host cells, and its upregulation has been implicated in increasing susceptibility of individuals to this infection. The clinical picture of COVID-19 suggests a role of ACE2 blockade, rather than its overexpression, in causing the pathogenesis. ACE2 blockade results in increased angiotensin II activity with simultaneous hampering of functions of angiotensin-(1-7)/MasR axis. Acute respiratory distress due to interstitial pulmonary fibrosis, cardiomyopathy and shock reported in COVID-19 patients can be explained by imbalanced angiotensin II and angiotensin-(1-7) activities. Failure of angiotensin II type 1 receptor blockers to control the severity of SARS-CoV-2 infections indicates the importance of simultaneous induction of angiotensin-(1-7)/MasR axis for correcting pathological conditions in COVID-19 through its anti-fibrotic, anti-inflammatory, vasodilatory, and cardioprotective roles. MasR agonists have also shown organ protective effects in a number of animal studies. Unfortunately, these agonists have not been tested in clinical studies. Their evaluation in seriously ill COVID-19 patients is urgently warranted to reduce mortality due to infection.

BET inhibitor I-BET151 sensitizes GBM cells to temozolomide via PUMA induction.

A significant roadblock in treatment of GBM multiforme (GBM) is resistance to temozolomide (TMZ). In this study, we investigated whether I-BET151, a specific BET inhibitor, could sensitize GBM cells to TMZ. Our findings showed that the action of I-BET151 could augment the effect of TMZ on cancer cells U251 and U87 cells. In U251 cells, administration of I-BET151 increased the TMZ-induced apoptosis GBM cells. I-BET151 remarkably enhanced the activities of caspase-3. In addition, I-BET151 promoted TMZ-induced migration and invasion in GBM cells. Moreover, I-BET151 increased the amount of reactive oxygen species as well as superoxide anions with a decrease of activity of SOD and the anti-oxidative properties of GBM cells. I-BET151 also induced increased PUMA expression, which is required for the functions of I-BET151 and regulates the synergistic cytotoxic effects of i-BET151 and TMZ in GBM cells. I-BET151 with TMZ also showed synergistic cytotoxic effects in vivo. These point out to an approach to tackle GBM using TMZ along with BET inhibitors.

Mas receptor is translocated to the nucleus upon agonist stimulation in brainstem neurons from spontaneously hypertensive rats but not normotensive rats.

AIMS: Activation of the angiotensin (Ang)-(1-7)/Mas receptor (R) axis protects from sympathetic overactivity. Endocytic trafficking is an essential process that regulates receptor (R) function and its ultimate cellular responses. We investigated whether the blunted responses to Ang-(1-7) in hypertensive rats are associated to an alteration in MasR trafficking. METHODS AND RESULTS: Brainstem neurons from Wistar-Kyoto (WKY) or spontaneously hypertensive rats (SHRs) were investigated for (i) Ang-(1-7) levels and binding and MasR expression, (ii) Ang-(1-7) responses (arachidonic acid and nitric oxide release and Akt and ERK1/2 phosphorylation), and (iii) MasR trafficking. Ang-(1-7) was determined by radioimmunoassay. MasR expression and functionality were evaluated by western blot and binding assays. MasR trafficking was evaluated by immunofluorescence. Ang-(1-7) treatment induced an increase in nitric oxide and arachidonic acid release and ERK1/2 and Akt phosphorylation in WKY neurons but did not have an effect in SHR neurons. Although SHR neurons showed greater MasR expression, Ang-(1-7)-elicited responses were substantially diminished presumably due to decreased Ang-(1-7) endogenous levels concomitant with impaired binding to its receptor. Through immunocolocalization studies, we evidenced that upon Ang-(1-7) stimulation MasRs were internalized through clathrin-coated pits and caveolae into early endosomes and slowly recycled back to the plasma membrane. However, the fraction of internalized MasRs into early endosomes was larger and the fraction of MasRs recycled back to the plasma membrane was smaller in SHR than in WKY neurons. Surprisingly, in SHR neurons but not in WKY neurons, Ang-(1-7) induced MasR translocation to the nucleus. Nuclear MasR expression and Ang-(1-7) levels were significantly greater in the nuclei of Ang-(1-7)-stimulated SHR neurons, indicating that the MasR is translocated with its ligand bound to it. CONCLUSION: MasRs display differential trafficking in brainstem neurons from SHRs, which may contribute to the impaired responses to Ang-(1-7).

Role of Angiotensin-(1-7) via MAS receptor in human sperm motility and acrosome reaction.

Rennin-angiotensin system (RAS) has been involved in sperm function, even so, little is known about the implication of one of the RAS axis formed by Ang-(1-7) (angiotensin-(1-7)) and MAS receptor. Hence, in the present work, we focused on elucidating the function of the MAS receptor in human spermatozoa. We analyzed the expression and localization of MAS receptor in human spermatozoa and we observed if its activation is able to modulate the sperm motility of normal motility and/or asthenozoospermic patients, as well as, the acrosome reaction of the spermatozoa. MAS receptor is present in human mature spermatozoa, not only at the mRNA level but also at protein level. MAS is localized at the acrosome region, as well as, in the tail of spermatozoa. The sperm incubation with MAS agonist Ang-(1-7) activates at dose-dependent manner the PI3K/AKT pathway (P < 0.01 vs control) and improves the motility of asthenozoospermic patients (P < 0.01 vs control), which is blocked by the specific antagonist (A779) (P < 0.01), but it do not modulate the acrosome reaction. These findings suggest that the ACE2/Ang-(1-7)/Mas axis may be a useful biochemical tool for the treatment of male infertility related to sperm mobility.

Ang-(1-7) treatment attenuates lipopolysaccharide-induced early pulmonary fibrosis.

Early pulmonary fibrosis is the leading cause of poor prognosis in patients with acute respiratory distress syndrome (ARDS). However, whether the renin-angiotensin system (RAS) can serve as a therapeutic target is unknown. In this study, an animal model of early pulmonary fibrosis was established via the LPS three-hit regimen. Afterwards, the animals were treated with intraperitoneal injections of Ang-(1-7), AVE0991, or A779 once per day for 20 days. The plasma and BALF AngII levels of the animals were increased, while there were no significant changes in Ang-(1-7) levels in lung tissue after LPS treatment. Furthermore, the AT1R protein levels were significantly increased and the Mas levels were significantly decreased on days 14 and 21. Administration of Ang-(1-7) downregulated LPS-induced AT1R mRNA expression, which was upregulated by A779. The expression of Mas mRNA responded in the opposite direction relative to AT1R. Moreover, LPS caused decreased levels of Mas and E-cadherin and increased AT1R, Vimentin, and Src phosphorylation levels. Ang-(1-7) or AVE0991 blocked these effects but was counteracted by A779 treatment. Our findings suggested that AngII and AT1R levels exhibit opposite dynamic trends during LPS-induced early pulmonary fibrosis, as do Ang-(1-7) and Mas. Ang-(1-7) exerts protective effects against early pulmonary fibrosis, mainly by regulating the balance between AngII and AT1R and between Ang-(1-7) and Mas and by inhibiting Src kinase activation.

Angiotensin-(1-7) inhibits sodium transport via Mas receptor by increasing nitric oxide production in thick ascending limb.

Sodium transport in the thick ascending loop of Henle (TAL) is tightly regulated by numerous factors, especially angiotensin II (Ang II), a key end-product of the renin-angiotensin system (RAS). However, an alternative end-product of the RAS, angiotensin-(1-7) [Ang-(1-7)], may counter some of the Ang II actions. Indeed, it causes vasodilation and promotes natriuresis through its effects in the proximal and distal tubule. However, its effects on the TAL are unknown. Because the TAL expresses the Mas receptor, an Ang-(1-7) ligand, which in turn may increase NO and inhibit Na+ transport, we hypothesized that Ang-(1-7) inhibits Na transport in the TAL, via a Mas receptor/NO-dependent mechanism. We tested this by measuring transport-dependent oxygen consumption (VO2 ) in TAL suspensions. Administering Ang-(1-7) decreased VO2 ; an effect prevented by dimethyl amiloride and furosemide, signifying that Ang-(1-7) inhibits transport-dependent VO2 in TAL. Ang-(1-7) also increased NO levels, known inhibitors of Na+ transport in the TAL. The effects of Ang-(1-7) on VO2 , as well as on NO levels, were ameliorated by the Mas receptor antagonist, D-Ala, in effect suggesting that Ang-(1-7) may inhibit transport-dependent VO2 in TAL via Mas receptor-dependent activation of the NO pathway. Indeed, blocking NO synthesis with L-NAME prevented the inhibitory actions of Ang-(1-7) on VO2 . Our data suggest that Ang-(1-7) may modulate TAL Na+ transport via Mas receptor-dependent increases in NO leading to the inhibition of transport activity.

A Novel Angiotensin-(1-7) Glycosylated Mas Receptor Agonist for Treating Vascular Cognitive Impairment and Inflammation-Related Memory Dysfunction.

Increasing evidence indicates that decreased brain blood flow, increased reactive oxygen species (ROS) production, and proinflammatory mechanisms accelerate neurodegenerative disease progression such as that seen in vascular contributions to cognitive impairment and dementia (VCID) and Alzheimer's disease and related dementias. There is a critical clinical need for safe and effective therapies for the treatment and prevention of cognitive impairment known to occur in patients with VCID and chronic inflammatory diseases such as heart failure (HF), hypertension, and diabetes. This study used our mouse model of VCID/HF to test our novel glycosylated angiotensin-(1-7) peptide Ang-1-6-O-Ser-Glc-NH2 (PNA5) as a therapy to treat VCID and to investigate circulating inflammatory biomarkers that may be involved. We demonstrate that PNA5 has greater brain penetration compared with the native angiotensin-(1-7) peptide. Moreover, after treatment with 1.0/mg/kg, s.c., for 21 days, PNA5 exhibits up to 10 days of sustained cognitive protective effects in our VCID/HF mice that last beyond the peptide half-life. PNA5 reversed object recognition impairment in VCID/HF mice and rescued spatial memory impairment. PNA5 activation of the Mas receptor results in a dose-dependent inhibition of ROS in human endothelial cells. Last, PNA5 treatment decreased VCID/HF-induced activation of brain microglia/macrophages and inhibited circulating tumor necrosis factor α, interleukin (IL)-7, and granulocyte cell-stimulating factor serum levels while increasing that of the anti-inflammatory cytokine IL-10. These results suggest that PNA5 is an excellent candidate and "first-in-class" therapy for treating VCID and other inflammation-related brain diseases.

Reversal of Aortic Enlargement Induced by Increased Biomechanical Forces Requires AT1R Inhibition in Conjunction With AT2R Activation.

Objective- Pharmacological inhibition of the AT1R (angiotensin II type 1 receptor) with losartan can attenuate ascending aortic remodeling induced by transverse aortic constriction (TAC). In this study, we investigated the role of the AT2R (angiotensin II type 2 receptor) and MasR (Mas receptor) in TAC-induced ascending aortic dilation and remodeling. Approach and Results- Wild-type C57BL/6J mice were subjected to sham or TAC surgeries in the presence and absence of various drugs. Aortic diameters were assessed by echocardiography, central blood pressure was measured in the ascending aorta 2 weeks post-operation, and histology and gene expression analyses completed. An angiotensin-converting enzyme inhibitor, captopril, decreased systolic blood pressure to the same level as losartan but did not attenuate aortic dilation, adventitial inflammation, medial collagen deposition, elastin breakage, or Mmp9 (matrix metalloproteinase-9) expression when compared with TAC mice. In contrast, co-administration of captopril with an AT2R agonist, compound 21, attenuated aortic dilation, medial collagen content, elastin breaks, and Mmp9 expression, whereas co-administration of captopril with a MasR agonist (AVE0991) did not reverse aortic dilation and led to aberrant aortic remodeling. An AT2R antagonist, PD123319, reversed the protective effects of losartan in TAC mice. Treatment with compound 21 alone showed no effect on TAC-induced aortic enlargement, blood pressure, elastin breakage, or Mmp9 expression. Conclusions- Our data indicate that when AT1R signaling is blocked, AT2R activation is a key modulator to prevent aortic dilation that occurs with TAC. These data suggest that angiotensin-converting enzyme inhibitor may not be as effective as losartan for slowing aneurysm growth because losartan requires intact AT2R signaling to prevent aortic enlargement.

Metabolic Reprogramming of Trophoblast Cells in Response to Hypoxia.

Hypoxia of trophoblast cells is an important regulator of normal development of the placenta. However, some pathological states associated with hypoxia, e.g. preeclampsia, impair the functions of placental cells. Oxyquinoline derivative inhibits HIF-prolyl hydroxylase by stabilizing HIF-1 transcription complex, thus modeling cell response to hypoxia. In human choriocarcinoma cells BeWo b30 (trophoblast model), oxyquinoline increased the expression of a core hypoxia response genes along with up-regulation of NOS3, PDK1, and BNIP3 genes and down-regulation of the PPARGC1B gene. These changes in the expression profile attest to activation of the metabolic cell reprogramming mechanisms aimed at reducing oxygen consumption by enabling the switch from aerobic to anaerobic glucose metabolism and the respective decrease in number of mitochondria. The possibility of practical use of the therapeutic properties of oxyquinoline derivatives is discussed.

Modulation of the Allosteric Communication between the Polo-Box Domain and the Catalytic Domain in Plk1 by Small Compounds.

The Polo-like kinases (Plks) are an evolutionary conserved family of Ser/Thr protein kinases that possess, in addition to the classical kinase domain at the N-terminus, a C-terminal polo-box domain (PBD) that binds to phosphorylated proteins and modulates the kinase activity and its localization. Plk1, which regulates the formation of the mitotic spindle, has emerged as a validated drug target for the treatment of cancer, because it is required for numerous types of cancer cells but not for the cell division in noncancer cells. Here, we employed chemical biology methods to investigate the allosteric communication between the PBD and the catalytic domain of Plk1. We identified small compounds that bind to the catalytic domain and inhibit or enhance the interaction of Plk1 with the phosphorylated peptide PoloBoxtide in vitro. In cells, two new allosteric Plk1 inhibitors affected the proliferation of cancer cells in culture and the cell cycle but had distinct phenotypic effects on spindle formation. Both compounds inhibited Plk1 signaling, indicating that they specifically act on Plk1 in cultured cells.

Common Co-activation of AXL and CDCP1 in EGFR-mutation-positive Non-smallcell Lung Cancer Associated With Poor Prognosis.

Epidermal growth factor receptor (EGFR)-mutation-positive non-smallcell lung cancer (NSCLC) is incurable, despite high rates of response to EGFR tyrosine kinase inhibitors (TKIs). We investigated receptor tyrosine kinases (RTKs), Src family kinases and focal adhesion kinase (FAK) as genetic modifiers of innate resistance in EGFR-mutation-positive NSCLC. We performed gene expression analysis in two cohorts (Cohort 1 and Cohort 2) of EGFR-mutation-positive NSCLC patients treated with EGFR TKI. We evaluated the efficacy of gefitinib or osimertinib with the Src/FAK/Janus kinase 2 (JAK2) inhibitor, TPX0005 in vitro and in vivo. In Cohort 1, CUB domain-containing protein-1 (CDCP1) was an independent negative prognostic factor for progression-free survival (hazard ratio of 1.79, p=0.0407) and overall survival (hazard ratio of 2.23, p=0.0192). A two-gene model based on AXL and CDCP1 expression was strongly associated with the clinical outcome to EGFR TKIs, in both cohorts of patients. Our preclinical experiments revealed that several RTKs and non-RTKs, were up-regulated at baseline or after treatment with gefitinib or osimertinib. TPX-0005 plus EGFR TKI suppressed expression and activation of RTKs and downstream signaling intermediates. Co-expression of CDCP1 and AXL is often observed in EGFR-mutation-positive tumors, limiting the efficacy of EGFR TKIs. Co-treatment with EGFR TKI and TPX-0005 warrants testing.

Alamandine acts via MrgD to induce AMPK/NO activation against ANG II hypertrophy in cardiomyocytes.

The renin-angiotensin system (RAS) plays a pivotal role in the pathogenesis of cardiovascular diseases. New members of this system have been characterized and shown to have biologically relevant actions. Alamandine and its receptor MrgD are recently identified components of RAS. In the cardiovascular system, alamandine actions included vasodilation, antihypertensive, and antifibrosis effects. Currently, the actions of alamandine on cardiomyocytes are unknown. Here our goal was twofold: 1) to unravel the signaling molecules activated by the alamandine/MrgD axis in cardiomyocytes; and 2) to evaluate the ability of this axis to prevent angiotensin II (ANG II)-induced hypertrophy. In cardiomyocytes from C57BL/6 mice, alamandine treatment induced an increase in nitric oxide (NO) production, which was blocked by d-Pro7-ANG-(1-7), a MrgD antagonist. This NO rise correlated with increased phosphorylation of AMPK. Alamandine-induced NO production was preserved in Mas-/- myocytes and lost in MrgD-/- cells. Binding of fluorescent-labeled alamandine was observed in wild-type cells, but it was dramatically reduced in MrgD-/- myocytes. We also assessed the consequences of prolonged alamandine exposure to cultured neonatal rat cardiomyocytes (NRCMs) treated with ANG II. Treatment of NRCMs with alamandine prevented ANG II-induced hypertrophy. Moreover, the antihypertrophic actions of alamandine were mediated via MrgD and NO, since they could be prevented by d-Pro7-ANG-(1-7) or inhibitors of NO synthase or AMPK. ß-Alanine, a MrgD agonist, recapitulated alamandine's cardioprotective effects in cardiomyocytes. Our data show that alamandine via MrgD induces AMPK/NO signaling to counterregulate ANG II-induced hypertrophy. These findings highlight the therapeutic potential of the alamandine/MrgD axis in the heart.

A novel autophagy inhibitor berbamine blocks SNARE-mediated autophagosome-lysosome fusion through upregulation of BNIP3.

Increasing evidences reveal that autophagy inhibitor could enhance the effect of chemotherapy to cancer. However, few autophagy inhibitors are currently approved for clinical application in humans. Berbamine (BBM) is a natural compound extracted from traditional Chinese medicine that is widely used for treatment of a variety of diseases without any obvious side effects. Here we found that BBM is a novel auophagy inhibitor, which potently induced the accumulation of autophagosomes by inhibiting autophagosome-lysosome fusion in human breast cancer cells. Mechanistically, we found that BBM blocked autophagosome-lysosome fusion by inhibiting the interaction of SNAP29 and VAMP8. Furthermore, BBM induced upregulation of BNIP3 and the interaction between SNAP29 and BNIP3. BNIP3 depletion or SNAP29 overexpression abrogated BBM-mediated blockade of autophagosome-lysosome fusion through the interaction between SNAP29 and VAMP8, whereas BNIP3 overexpression blocked autophagosome-lysosome fusion through inhibition of the interaction between SNAP29 and VAMP8. These findings suggest that upregulation of BNIP3 and interaction between BNIP3 and SNAP29 could be involved in BBM-mediated blockade of autophagosome-lysosome fusion through inhibition of the interaction between SNAP29 and VAMP8. Our findings identify the critical role of BNIP3 in blockade of autophagosome-lysosome fusion mediated by BBM, and suggest that BBM could potentially be further developed as a novel autophagy inhibitor, which could enhance the effect of chemotherapy to cancer.

Intrarenal Mas and AT1 receptors play a role in mediating the excretory actions of renal interstitial angiotensin-(1-7) infusion in anaesthetized rats.

NEW FINDINGS: What is the central question of this study? Dietary sodium manipulation alters the magnitude of angiotensin-(1-7) [Ang-(1-7)]-induced natriuresis. The present study sought to determine whether this was related to relative changes in the activity of intrarenal Mas and/or AT1 receptors. What is the main finding and its importance? Angiotensin-(1-7)-induced diuresis and natriuresis is mediated by intrarenal Mas receptors. However, intrarenal AT1 receptor blockade also had an inhibitory effect on Ang-(1-7)-induced natriuresis and diuresis. Thus, Ang-(1-7)-induced increases in sodium and water excretion are dependent upon functional Mas and AT1 receptors. We investigated whether angiotensin-(1-7) [Ang-(1-7)]-induced renal haemodynamic and excretory actions were solely dependent upon intrarenal Mas receptor activation or required functional angiotensin II type 1 (AT1 ) receptors. The renin-angiotensin system was enhanced in anaesthetized rats by prior manipulation of dietary sodium intake. Angiotensin-(1-7) and AT1 and Mas receptor antagonists were infused into the kidney at the corticomedullary border. Mas receptor expression was measured in the kidney. Mean arterial pressure, urine flow and fractional sodium excretion were 93 ± 4 mmHg, 46.1 ± 15.7 µl min-1  kg-1 and 1.4 ± 0.3%, respectively, in the normal-sodium group and 91 ± 2 mmHg, 19.1 ± 3.3 µl min-1  kg-1 and 0.7 ± 0.2%, respectively, in the low-sodium group. Angiotensin-(1-7) infusion had no effect on mean arterial pressure in rats receiving a normal-sodium diet but decreased it by 4 ± 5% in rats receiving a low-sodium diet (P < 0.05). Interstitial Ang-(1-7) infusion increased urine flow twofold and fractional sodium excretion threefold (P < 0.05) in rats receiving a normal-sodium diet and to a greater extent, approximately three- and fourfold, respectively, in rats receiving the low-sodium diet (both P < 0.05). Angiotensin-(1-7)-induced increases in urine flow and fractional sodium excretion were absent in both dietary groups during intrarenal AT1 or Mas receptor inhibition after either losartan or A-779, respectively. Thus, AT1 receptor activation, as well as Mas receptor activation, plays an essential role in mediating Ang-(1-7)-induced natriuresis and diuresis. Whether this is because Ang-(1-7) partly antagonizes AT1 receptors or whether Ang-(1-7)-induced natriuresis is mediated through AT1 -Mas receptor dimerization remains unclear.

The effects of angiotensin-(1-7) on the exchanger NHE3 and on [Ca2+]i in the proximal tubules of spontaneously hypertensive rats.

The acute effects of angiotensin-1-7 [ANG-(1-7)] on the reabsorptive bicarbonate flow (J[Formula: see text]) were evaluated using stationary microperfusion in vivo in the proximal tubules of spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar-Kyoto (WKY) rats, using a microelectrode sensitive to H+ In WKY rats, the control J[Formula: see text] was 2.40 ± 0.10 nmol·cm-2·s-1 (n = 120); losartan (10-7 M) or A779 (10-6 M, a specific Mas antagonist), alone or in combination with losartan, decreased the J[Formula: see text] ANG-(1-7) had biphasic effects on J[Formula: see text]: at 10-9 M, it inhibited, and at 10-6, it stimulated the flow. S3226 [10-6 M, a specific Na+-H+ exchanger 3 (NHE3) antagonist] decreased J[Formula: see text] and changed the stimulatory effect of ANG-(1-7) to an inhibitory one but did not alter the inhibitory action of ANG-(1-7). In SHR, the control J[Formula: see text] was 2.04 ± 0.13 nmol·cm-2·s-1 (n = 56), and A779 and/or losartan reduced the flow. ANG-(1-7) at 10-9 M increased J[Formula: see text], and ANG-(1-7) at 10-6 M reduced it. The effects of A779, losartan, and S3226 on the J[Formula: see text] were similar to those found in WKY rats, which indicated that in SHR, the ANG-(1-7) action on the NHE3 was via Mas and ANG II type 1. The cytosolic calcium in the WKY or SHR rats was ~100 nM and was increased by ANG-(1-7) at 10-9 or 10-6 M. In hypertensive animals, a high plasma level of ANG-(1-7) inhibited NHE3 in the proximal tubule, which mitigated the hypertension caused by the high plasma level of ANG II.