Endothelin-1 down-regulates nuclear factor erythroid 2-related factor-2 and contributes to perivascular adipose tissue dysfunction in obesity.

Perivascular adipose tissue (PVAT) negatively regulates vascular muscle contraction. However, in the context of obesity, the PVAT releases vasoconstrictor substances that detrimentally affect vascular function. A pivotal player in this scenario is the peptide endothelin-1 (ET-1), which induces oxidative stress and disrupts vascular function. The present study postulates that obesity augments ET-1 production in the PVAT, decreases the function of the nuclear factor erythroid 2-related factor-2 (Nrf2) transcription factor, further increasing reactive oxygen species (ROS) generation, culminating in PVAT dysfunction. Male C57BL/6 mice were fed either a standard or a high-fat diet for 16 weeks. Mice were also treated with saline or a daily dose of 100 mg·kg-1 of the ETA and ETB receptor antagonist Bosentan, for 7 days. Vascular function was evaluated in thoracic aortic rings, with and without PVAT. Mechanistic studies utilized PVAT from all groups and cultured WT-1 mouse brown adipocytes. PVAT from obese mice exhibited increased ET-1 production, increased ECE1 and ETA gene expression, loss of the anticontractile effect, as well as increased ROS production, decreased Nrf2 activity, and downregulated expression of Nrf2-targeted antioxidant genes. PVAT of obese mice also exhibited increased expression of Tyr216-phosphorylated-GSK3ß and KEAP1, but not BACH1 - negative Nrf2 regulators. Bosentan treatment reversed all these effects. Similarly, ET-1 increased ROS generation and decreased Nrf2 activity in brown adipocytes, events mitigated by BQ123 (ETA receptor antagonist). These findings place ET-1 as a major contributor to PVAT dysfunction in obesity and highlight that pharmacological control of ET-1 effects restores PVAT's cardiovascular protective role.

Role of Brain Endothelin Receptor Type B (ETB) in the Regulation of Tyrosine Hydroxylase in the Olfactory Bulb of DOCA-Salt Hypertensive Rats.

BACKGROUND: We previously reported that endothelins (ETs) regulate tyrosine hydroxylase (TH) activity and expression in the olfactory bulb (OB) of normotensive and hypertensive animals. Applying an ET receptor type A (ETA) antagonist to the brain suggested that endogenous ETs bind to ET receptor type B (ETB) to elicit effects. OBJECTIVE: The aim of the present work was to evaluate the role of central ETB stimulation on the regulation of blood pressure (BP) and the catecholaminergic system in the OB of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. METHODS: DOCA-salt hypertensive rats were infused for 7 days with cerebrospinal fluid or IRL-1620 (ETB receptor agonist) through a cannula placed in the lateral brain ventricle. Systolic BP (SBP) and heart rate were recorded by plethysmography. The expression of TH and its phosphorylated forms in the OB were determined by immunoblotting, TH activity by a radioenzymatic assay, and TH mRNA by quantitative real-time polymerase chain reaction. RESULTS: Chronic administration of IRL-1620 decreased SBP in hypertensive rats but not in normotensive animals. Furthermore, the blockade of ETB receptors also decreased TH-mRNA in DOCA-salt rats, but it did not modify TH activity or protein expression. CONCLUSION: These findings suggest that brain ETs through the activation of ETB receptors contribute to SBP regulation in DOCA-salt hypertension. However, the catecholaminergic system in the OB does not appear to be conclusively involved although mRNA TH was reduced. Present and previous findings suggest that in this salt-sensitive animal model of hypertension, the OB contributes to chronic BP elevation.

Endothelial and vascular smooth muscle dysfunction in hypertension.

The development of essential hypertension involves several factors. Vascular dysfunction, characterized by endothelial dysfunction, low-grade inflammation and structural remodeling, plays an important role in the initiation and maintenance of essential hypertension. Although the mechanistic pathways by which essential hypertension develops are poorly understood, several pharmacological classes available on the clinical settings improve blood pressure by interfering in the cardiac output and/or vascular function. This review is divided in two major sections. The first section depicts the major molecular pathways as renin angiotensin aldosterone system (RAAS), endothelin, nitric oxide signalling pathway and oxidative stress in the development of vascular dysfunction. The second section describes the role of some pharmacological classes such as i) RAAS inhibitors, ii) dual angiotensin receptor-neprilysin inhibitors, iii) endothelin-1 receptor antagonists, iv) soluble guanylate cyclase modulators, v) phosphodiesterase type 5 inhibitors and vi) sodium-glucose cotransporter 2 inhibitors in the context of hypertension. Some classes are already approved in the treatment of hypertension, but others are not yet approved. However, due to their potential benefits these classes were included.

Endothelin-1 induces changes in the expression levels of steroidogenic enzymes and increases androgen receptor and testosterone production in the PC3 prostate cancer cell line.

Endothelin­1 (ET­1) is involved in the regulation of steroidogenesis. Additionally, patients with castration­resistant prostate cancer (PCa) have a higher ET­1 plasma concentration than those with localized PCa and healthy individuals. The aim of the present study was to evaluate the effect of ET­1 on steroidogenesis enzymes, androgen receptor (AR) and testosterone (T) production in PCa cells. The expression levels of endothelin receptors in prostate tissue from patients with localized PCa by immunohistochemistry, and those in LNCaP and PC3 cells were determined reverse transcription­quantitative PCR (RT­qPCR) and western blotting. Furthermore, the expression levels of ET­1 were determined in LNCaP and PC3 cells by RT­qPCR and western blotting. The ET­1 receptor activation was evaluated by intracellular calcium measurement, the expression levels of AR and enzymes participating in steroidogenesis [cytochrome P450 family 11 subfamily A member 1 (CyP11A1), cytochrome P450 family 17 subfamily A member 1, aldo­keto reductase family member C2 and 3ß­hydroxysteroid dehydrogenase/isomerase 2 (3ß HSD2)] were determined by western blotting and T concentration was determined by ELISA using PC3 cells. The present results revealed higher expression levels of endothelin A receptor (ETAR) in tissues obtained from samples of patients with PCa with a low Gleason Score. No changes were identified for endothelin B receptor (ETBR). PC3 cells expressed higher levels of ET­1 and ETAR, while LNCaP cells exhibited higher expression levels of ETBR. Blocking of ETAR and endothelin B receptor decreased the expression levels of CyP11A1 and 3ß HSD2 enzymes and AR in PC3 cells, as well as T secretion. These findings suggested that ET­1 has a potential role in modulating the intratumoral steroidogenesis pathway and might have relevance as a possible therapeutic target.

ETA receptors are involved in the febrile response induced by high dose of bacterial endotoxin.

Previous studies have demonstrated that endothelin-1 (ET-1) is involved in the febrile response induced by lipopolysaccharide (LPS) in male and female rats. This peptide induces fever acting on ETB receptors in the central nervous system. However, during sepsis, endothelinergic ETA receptors in the brain also exert an important role reducing the mortality of the animals. The present study evaluated the participation of ETA receptors in the febrile response induced by different doses LPS in rats. Male Wistar rats were treated with the ETA receptor antagonist BQ123 before or after the injection of a low dose (10 µg/kg) or a high dose (200 µg/kg) of LPS intraperitoneally. The febrile response was evaluated. The treatment with BQ123, in both protocols did not change the febrile response induced by the lower dose of LPS. The pre-treatment with BQ123 also did not significantly change the febrile response induced by a higher dose of LPS but the post-treatment with the antagonist abolished the febrile response induced by this dose of LPS. These results suggest that even though ETA receptors are not recruited in the febrile response induced by lower doses of LPS, they are involved in the febrile response induced by high doses of this stimulus.

Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells.

The lateral mobility of membrane receptors provides insights into the molecular interactions of protein binding and the complex dynamic plasma membrane. The image mean square displacement (iMSD) analysis is a method used to extract qualitative and quantitative information of the protein diffusion law and infers how diffusion dynamic processes may change when the cellular environment is modified. The aim of the study was to describe the membrane diffusing properties of two G-protein-coupled receptors namely Angiotensin II type 1 (AT1 ) and Endothelin 1 type A (ETA ) receptors and their corresponding receptor-ligand complexes in living cells using total internal reflection fluorescent microscopy and iMSD analysis. This study showed that both AT1 and ETA receptors displayed a mix of three modes of diffusion: free, confined, and partially confined. The confined mode was the predominant at the plasma membrane of living cells and was not affected by ligand binding. However, the local diffusivity and the confinement zone of AT1 receptors were reduced by the binding of its antagonist losartan, and the long-range diffusion with the local diffusivity coefficient of ETA receptors was reduced upon exposure to its antagonist BQ123. To the best of our knowledge, this is the first study addressing the protein diffusion laws of these two receptors on living cells using total internal reflection fluorescence microscopy and iMSD.

Role of endothelin receptor type A on catecholamine regulation in the olfactory bulb of DOCA-salt hypertensive rats: Hemodynamic implications.

Increasing evidence shows that the olfactory bulb is involved in blood pressure regulation in health and disease. Enhanced noradrenergic transmission in the olfactory bulb was reported in hypertension. Given that endothelins modulate catecholamines and are involved in the pathogenesis of hypertension, in the present study we sought to establish the role of the endothelin receptor type A on tyrosine hydroxylase, the rate limiting enzyme in catecholamine biosynthesis, in the olfactory bulb of DOCA-salt hypertensive rats. Sprague-Dawley male rats, randomly divided into Control and DOCA-Salt hypertensive groups, were used to assess endothelin receptors by Western blot and confocal microscopy, and their co-localization with tyrosine hydroxylase in the olfactory bulb. Blood pressure and heart rate as well as tyrosine hydroxylase expression and activity were assessed following BQ610 (ETA antagonist) applied to the brain. DOCA-Salt hypertensive rats showed enhanced ETA and decreased ETB expression. ETA co-localized with tyrosine hydroxylase positive neurons. Acute ETA blockade reduced blood pressure and heart rate and decreased the expression of total tyrosine hydroxylase and its phosphorylated forms. Furthermore, it also diminished mRNA tyrosine hydroxylase expression and accelerated the enzyme degradation through the proteasome pathway as shown by pretreatment with MG132, (20s proteasome inhibitor) intracerebroventricularly applied. Present findings support that the brain endothelinergic system plays a major role through ETA activation in the increase of catecholaminergic activity in the olfactory bulb of DOCA-Salt hypertensive rats. They provide rationale evidence that this telencephalic structure contributes in a direct or indirect way to the hemodynamic regulation in salt dependent hypertension.

Involvement of Endothelin Receptors in Peripheral Sensory Neuropathy Induced by Oxaliplatin in Mice.

The aim of this study was to evaluate the participation of the endothelin ETA and ETB receptors and the effects of bosentan in oxaliplatin-induced peripheral sensory neuropathy (OIN) in mice. Adult male Swiss mice received 1 mg/kg of oxaliplatin intravenously, twice a week for 5 weeks. Dorsal root ganglia (DRG) and spinal cords were removed for evaluation of the endothelin ETA and ETB receptor expression. Afterwards, selective (BQ-123 and BQ-788; 10 nmol in 30 µL, intraplantarly) and non-selective (bosentan, 100 mg/kg, orally) antagonists were administered in order to evaluate the involvement of the endothelin receptors in OIN. Mechanical and thermal nociception tests were performed once a week for 56 days. Oxaliplatin induced mechanical and thermal hypersensitivity and increased the endothelin ETA receptor expression in both the DRG and spinal cord (P < 0.05). Endothelin ETB receptor expression was increased in the DRG (P < 0.05) but not in the spinal cord. Both endothelin ETA and ETB receptor selective antagonists partially prevented mechanical hyperalgesia in mice with OIN (P < 0.05). Moreover, bosentan prevented mechanical and thermal hypersensitivity in oxaliplatin-treated mice (P < 0.05). In conclusion, both endothelin ETA and ETB receptors seem to be involved in the OIN in mice and they should be considered possible targets for the management of this clinical feature.

Subverting bradykinin-evoked inflammation by co-opting the contact system: lessons from survival strategies of Trypanosoma cruzi.

PURPOSE OF REVIEW: During Chagas disease, Trypanosoma cruzi alternates between intracellular and extracellular developmental forms. After presenting an overview about the roles of the contact system in immunity, I will review experimental studies showing that activation of the kallikrein-kinin system (KKS) translates into mutual benefits to the host/parasite relationship. RECENT FINDINGS: T. cruzi trypomastigotes initiate inflammation by activating tissue-resident innate sentinel cells via the TLR2/CXCR2 pathway. Following neutrophil-evoked microvascular leakage, the parasite's major cysteine protease (cruzipain) cleaves plasma-borne kininogens and complement C5. Tightly regulated by angiotensin-converting enzyme (ACE), kinins and C5a in turn further propagate inflammation via iterative cycles of mast cell degranulation, contact system activation, bradykinin release and activation of endothelial bradykinin B2 receptors (B2R). Recently, studies in the intracardiac model of infection revealed a dichotomic role for bradykinin and endothelin-1: generated upon contact activation (mast cell/KKS pathway), these pro-oedematogenic peptides reciprocally stimulate trypomastigote invasion of heart cells that naturally overexpress B2R and endothelin receptors (ETaR/ETbR). SUMMARY: Studies focusing on the immunopathogenesis of Chagas disease revealed that the contact system plays a dual role in host/parasite balance: T. cruzi co-opts bradykinin-induced plasma leakage as a strategy to increment heart parasitism and increase immune resistance by upregulating type-1 effector T-cell production in secondary lymphoid tissues.

Expression profile of endothelin receptors (ETA and ETB) and microRNAs-155 and -199 in the corpus cavernosum of rats submitted to chronic alcoholism and diabetes mellitus.

Recent evidence shows that chronic ethanol consumption increases endothelin (ET)-1 induced sustained contraction of trabecular smooth muscle cells of the corpora cavernosa in corpus cavernosum of rats by a mechanism that involves increased expression of ETA and ETB receptors. Our goal was to evaluate the effects of alcohol and diabetes and their relationship to miRNA-155, miRNA-199 and endothelin receptors in the corpus cavernosum and blood of rats submitted to the experimental model of diabetes mellitus and chronic alcoholism. Forty-eight male Wistar rats were divided into four groups: control (C), alcoholic (A), diabetic (D), and alcoholic-diabetic (AD). Samples of the corpus cavernosum were prepared to study the protein expression of endothelin receptors by immunohistochemistry and expression of miRNAs-155 and -199 in serum and the cavernous tissue. Immunostaining for endothelin receptors was markedly higher in the A, D, and AD groups than in the C group. Moreover, a significant hypoexpression of the miRNA-199 in the corpus cavernosum tissue from the AD group was observed, compared to the C group. When analyzing the microRNA profile in blood, a significant hypoexpression of miRNA-155 in the AD group was observed compared to the C group. The miRNA-199 analysis demonstrated significant hypoexpression in D and AD groups compared to the C group. Our findings in corpus cavernosum showed downregulated miRNA-155 and miRNA-199 levels associated with upregulated protein expression and unaltered mRNA expression of ET receptors suggesting decreased ET receptor turnover, which can contribute to erectile dysfunction in diabetic rats exposed to high alcohol levels.

Chronic Blockade of Brain Endothelin Receptor Type-A (ETA) Reduces Blood Pressure and Prevents Catecholaminergic Overactivity in the Right Olfactory Bulb of DOCA-Salt Hypertensive Rats.

Overactivity of the sympathetic nervous system and central endothelins (ETs) are involved in the development of hypertension. Besides the well-known brain structures involved in the regulation of blood pressure like the hypothalamus or locus coeruleus, evidence suggests that the olfactory bulb (OB) also modulates cardiovascular function. In the present study, we evaluated the interaction between the endothelinergic and catecholaminergic systems in the OB of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Following brain ET receptor type A (ETA) blockade by BQ610 (selective antagonist), transcriptional, traductional, and post-traductional changes in tyrosine hydroxylase (TH) were assessed in the OB of normotensive and DOCA-salt hypertensive rats. Time course variations in systolic blood pressure and heart rate were also registered. Results showed that ETA blockade dose dependently reduced blood pressure in hypertensive rats, but it did not change heart rate. It also prevented the increase in TH activity and expression (mRNA and protein) in the right OB of hypertensive animals. However, ETA blockade did not affect hemodynamics or TH in normotensive animals. Present results support that brain ETA are not involved in blood pressure regulation in normal rats, but they significantly contribute to chronic blood pressure elevation in hypertensive animals. Changes in TH activity and expression were observed in the right but not in the left OB, supporting functional asymmetry, in line with previous studies regarding cardiovascular regulation. Present findings provide further evidence on the role of ETs in the regulation of catecholaminergic activity and the contribution of the right OB to DOCA-salt hypertension.

Facial hyperalgesia due to direct action of endothelin-1 in the trigeminal ganglion of mice.

OBJECTIVE: This study assessed the ability of endothelin-1 (ET-1) to evoke heat hyperalgesia when injected directly into the trigeminal ganglia (TG) of mice and determined the receptors implicated in this effect. The effects of TG ETA and ETB receptor blockade on alleviation of heat hyperalgesia in a model of trigeminal neuropathic pain induced by infraorbital nerve constriction (CION) were also examined. METHODS: Naive mice received an intraganglionar (i.g.) injection of ET-1 (0.3-3 pmol) or the selective ETB R agonist sarafotoxin S6c (3-30 pmol), and response latencies to ipsilateral heat stimulation were assessed before the treatment and at 1-h intervals up to 5 h after the treatment. Heat hyperalgesia induced by i.g. ET-1 or CION was assessed after i.g. injections of ETA R and ETB R antagonists (BQ-123 and BQ-788, respectively, each at 0.5 nmol). KEY FINDINGS: Intraganglionar ET-1 or sarafotoxin S6c injection induced heat hyperalgesia lasting 4 and 2 h, respectively. Heat hyperalgesia induced by ET-1 was attenuated by i.g. BQ-123 or BQ-788. On day 5 after CION, i.g. BQ-788 injection produced a more robust antihyperalgesic effect compared with BQ-123. CONCLUSIONS: ET-1 injection into the TG promotes ETA R/ETB R-mediated facial heat hyperalgesia, and both receptors are clearly implicated in CION-induced hyperalgesia in the murine TG system.

Expression profile of endothelin receptors (ETA and ETB) and microRNAs-155 and -199 in the corpus cavernosum of rats submitted to chronic alcoholism and diabetes mellitus

Recent evidence shows that chronic ethanol consumption increases endothelin (ET)-1 induced sustained contraction of trabecular smooth muscle cells of the corpora cavernosa in corpus cavernosum of rats by a mechanism that involves increased expression of ETA and ETB receptors. Our goal was to evaluate the effects of alcohol and diabetes and their relationship to miRNA-155, miRNA-199 and endothelin receptors in the corpus cavernosum and blood of rats submitted to the experimental model of diabetes mellitus and chronic alcoholism. Forty-eight male Wistar rats were divided into four groups: control (C), alcoholic (A), diabetic (D), and alcoholic-diabetic (AD). Samples of the corpus cavernosum were prepared to study the protein expression of endothelin receptors by immunohistochemistry and expression of miRNAs-155 and -199 in serum and the cavernous tissue. Immunostaining for endothelin receptors was markedly higher in the A, D, and AD groups than in the C group. Moreover, a significant hypoexpression of the miRNA-199 in the corpus cavernosum tissue from the AD group was observed, compared to the C group. When analyzing the microRNA profile in blood, a significant hypoexpression of miRNA-155 in the AD group was observed compared to the C group. The miRNA-199 analysis demonstrated significant hypoexpression in D and AD groups compared to the C group. Our findings in corpus cavernosum showed downregulated miRNA-155 and miRNA-199 levels associated with upregulated protein expression and unaltered mRNA expression of ET receptors suggesting decreased ET receptor turnover, which can contribute to erectile dysfunction in diabetic rats exposed to high alcohol levels.

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.

Potential Role of Endothelin in Early Vascular Aging.

Early vascular aging is a process associated with gradual alterations in the vessels, regarding their structure and function, taking a more rapid course than normal biological aging in the arteries. In the presence of cardiovascular disease, these age-associated alterations are accelerated, contributing in the appearance or the progression of cardiovascular disease, such as high blood pressure, dyslipidemia, smoking and diabetes. Endothelin-1 (ET-1) is the most abundant and important endothelin produced by vascular cells. ET-1 exerts its biological actions through the activation of two receptors: ETA and ETB. Many important functions are mediated by the activation of these receptors, such as cardiovascular remodeling, vasoconstriction, cell proliferation and differentiation, production of extracellular matrix, and water and sodium secretion control. ETA receptor seems to participate in the pathogenesis and development of diseases, such as diabetes, atherosclerosis, systemic and pulmonary hypertension, and cardiac remodeling after myocardial ischemia, whereas ETB receptor seems to prevent the overstimulation of ETA receptor, acting as a clearance receptor. Increased ET-1 system activity may contribute to vascular dysfunction in aging via multiple pathways, such as direct hemodynamic effects, vascular oxidative stress, inflammatory activity, mitogenic stimulation of the vascular smooth muscle cells and fibrotic processes. Endothelin receptor antagonists were considered to be used for the treatment of some diseases like hypertension, diabetes and chronic kidney disease. However, besides pulmonary hypertension, this class is not in clinical use because of the side effects and the availability of safer drugs for the treatment of these diseases.

[Single nucleotide polymorphisms in genes of endothelin-1 and receptor A associated to cardiovascular in essential hypertension]. / Polimorfismos de un solo nucleótido en genes de endotelina-1 y su receptor A asociados a daño cardiovascular en hipertensión arterial esencial.

INTRODUCTION: The endothelin system, for its vasoconstrictor action, is related to the development of essential hypertension (HTAe). The polymorphism analysis of their genes represents a new approach to the study of this disease. We propose to analyze the interaction between stages of essential hypertension (HTAe) and risk factors with polymorphisms 138ex1 ins/del A gene endothelin-1 (ET-1) and H323H receptor gene A ET-1 (ETRA). PATIENTS AND METHODS: We included 300 patients of both sexes, unrelated, who consecutively attended the clinic hypertension medical service. Each one underwent a complete physical examination, electrocardiogram, echocardiogram, and Rx thorax. The degree of severity of hypertension was determined in stages. The determination of polymorphisms was performed by amplification followed by cutting by specific restriction enzyme from DNA obtained from peripheral blood. RESULTS: The 46% of patients had HTAe controlled, 17.6% had organ damage or cardiovascular, brain or kidney disease. It was observed that the 4A/4A carriers showed lower frequency of cardiovascular disease, kidney and brain (P<.032; 95% CI: 11.1-21.4). For H323H polymorphism, the evaluation by images showed a higher frequency of the dilations of left auricular (P=.02) and auricular fibrillation (P=.03) between the T/T carrier, a higher frequency of cardiomegaly was detected in C/C patients (P=.04). CONCLUSION: The genotypes, 4A/4A of the ET-1 gene and the T/T from ETRA gene might be involved in worse outcome of cardiovascular damage. Their identification could help recognize subgroups of the hypertensive patients with different risk.

Differential regulation of oxidative stress and cytokine production by endothelin ETA and ETB receptors in superoxide anion-induced inflammation and pain in mice.

The present study investigated whether endothelin-1 acts via ETA or ETB receptors to mediate superoxide anion-induced pain and inflammation. Mice were treated with clazosentan (ETA receptor antagonist) or BQ-788 (ETB receptor antagonist) prior to stimulation with the superoxide anion donor, KO2. Intraplantar treatment with 30 nmol of clazosentan or BQ-788 reduced mechanical hyperalgesia (47% and 42%), thermal hyperalgesia (68% and 76%), oedema (50% and 30%); myeloperoxidase activity (64% and 32%), and overt-pain like behaviours, such as paw flinching (42% and 42%) and paw licking (38% and 62%), respectively. Similarly, intraperitoneal treatment with 30 nmol of clazosentan or BQ-788 reduced leukocyte recruitment to the peritoneal cavity (58% and 32%) and abdominal writhing (81% and 77%), respectively. Additionally, intraplantar treatment with clazosentan or BQ-788 decreased spinal (45% and 41%) and peripheral (47% and 47%) superoxide anion production as well as spinal (47% and 47%) and peripheral (33% and 54%) lipid peroxidation, respectively. Intraplantar treatment with clazosentan, but not BQ-788, reduced spinal (71%) and peripheral (51%) interleukin-1 beta as well as spinal (59%) and peripheral (50%) tumor necrosis factor-alpha production. Therefore, the present study unveils the differential mechanisms by which ET-1, acting on ETA or ETB receptors, regulates superoxide anion-induced inflammation and pain.

Potential role for ET-2 acting through ETA receptors in experimental colitis in mice.

OBJECTIVE AND DESIGN: This study attempted to clarify the roles of endothelins and mechanisms associated with ETA/ETB receptors in mouse models of colitis. MATERIALS AND METHODS: Colitis was induced by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS, 1.5 mg/animal) or dextran sulfate sodium (DSS, 3%). After colitis establishment, mice received Atrasentan (ETA receptor antagonist, 10 mg/kg), A-192621 (ETB receptor antagonist, 20 mg/kg) or Dexamethasone (1 mg/kg) and several inflammatory parameters were assessed, as well as mRNA levels for ET-1, ET-2 and ET receptors. RESULTS: Atrasentan treatment ameliorates TNBS- and DSS-induced colitis. In the TNBS model was observed reduction in macroscopic and microscopic score, colon weight, neutrophil influx, IL-1ß, MIP-2 and keratinocyte chemoattractant (KC) levels, inhibition of adhesion molecules expression and restoration of IL-10 levels. However, A192621 treatment did not modify any parameter. ET-1 and ET-2 mRNA was decreased 24 h, but ET-2 mRNA was markedly increased at 48 h after TNBS. ET-2 was able to potentiate LPS-induced KC production in vitro. ETA and ETB receptors mRNA were increased at 24, 48 and 72 h after colitis induction. CONCLUSIONS: Atrasentan treatment was effective in reducing the severity of colitis in DSS- and TNBS-treated mice, suggesting that ETA receptors might be a potential target for inflammatory bowel diseases.

Involvement of Central Endothelin ETA and Cannabinoid CB1 Receptors and Arginine Vasopressin Release in Sepsis Induced by Cecal Ligation and Puncture in Rats.

We previously reported that endothelin-1 (ET-1) reduced the frequency of spontaneous excitatory currents in vasopressinergic magnocellular cells through the activation of endothelin ETA receptors in rat brain slices. This effect was abolished by a cannabinoid CB1 receptor antagonist, suggesting the involvement of endocannabinoids. The present study investigated whether the blockade of ETA or CB1 receptors during the phase of increased levels of ET-1 after severe sepsis increases the survival rate of animals concomitantly with an increase in plasma arginine vasopressin (AVP) levels. Sepsis was induced in male Wistar rats by cecal ligation and puncture (CLP). Treatment with the CB1 receptor antagonist rimonabant (Rim; 10 and 20 mg/kg, orally) 4 h after CLP (three punctures) significantly increased the survival rate compared with the CLP per vehicle group. Intracerebroventricular treatment with the ETA receptor antagonist BQ123 (100 pmol) or with Rim (2 µg) 4 and 8 h after CLP but not the ETB receptor antagonist BQ788 (100 pmol), also significantly improved the survival rate. Sham-operated and CLP animals that were treated with Rim had significantly lower core temperature than CLP animals. However, oral treatment with Rim did not change bacterial count in the peritoneal exudate, neutrophil migration to the peritoneal cavity, leucopenia or increased plasma interleukin-6 levels induced by CLP. Both Rim and BQ123 also increased AVP levels 12 h after CLP. The blockade of central CB1 and ETA receptors in the late phase of sepsis increased the survival rate, reduced body temperature and increased the circulating AVP levels.

Analysis of immunostaining and western blotting of endothelin 1 and its receptors in mitral stenosis.

INTRODUCTION: Rheumatic Fever represents a serious public health problem in developing countries, with thousands of new cases each year. It is an autoimmune disease, which occurs in response to infection by streptococcus A. OBJECTIVE: The aim of this study was to evaluate the immunolabeling and protein expression for endothelin-1 and 3 (ET-1, ET-3) and its receptors (ETA, ETB) in rheumatic mitral valves. METHODS: Immunohistochemistry was used to identify ET-1/ET-3 and ETA/ETB receptors in rheumatic and control mitral valves. Quantitative analysis of immunostaining for ET-1/ET-3 and ETA/ETB receptors was performed. In addition, western blot analysis was carried out to assess protein levels in tissue samples. RESULTS: ET-1 and ETA receptor immunostaining predominated in stenotic valves, mainly associated with fibrotic regions, inflammatory areas and neovascularization. Quantitative analysis showed that the average area with positive expression of ET-1 was 18.21 ± 14.96%. For ETA and ETB, the mean expressed areas were respectively 15.06 ± 13.13% and 9.20 ± 11.09%. ET-3 did not have a significant expression. The correlation between the expression of both endothelin receptors were strongly positive (R = 0.74, P = 0.02), but the correlation between ET-1 and its receptor were negative for both ETA (R = -0.37, P = 0.25), and ETB (R = -0.14, P = 0.39). This data was supported by western blot analysis. CONCLUSION: The strong correlation between ET-1 and its receptors suggests that both play a role in the pathophysiology of rheumatic mitral valve stenosis and may potentially act as biomarkers of this disease.