1. This study was performed to clarify whether the endothelin (ET) receptor subtypes mediating two pharmacologically heterogeneous response to ETH receptor agonists in normal mice are the product(s) of a single ETB receptor gene. 2. Vasodilator responses to sarafotoxin S6c (S6c) in the thoracic aorta and contractile responses to ET-1 and IRL1620 in the stomach were examined in tissues from normal and ETB receptor gene knockout mice, in the absence and presence of an ETA receptor antagonist, BQ-123, or an ETA/ETB receptor antagonist, PD142893. 3. In the normal mouse aorta precontracted with phenylephrine, S6c (0.1-100 nM) caused concentration-dependent relaxations (pD2 = 8.4). BQ-123 had no effect on these responses. However, PD142893 almost abolished the relaxations induced by 0.1-300 nM S6c. 4. In aortae taken from ETB receptor gene knockout mice, S6c up to 1 microM failed to cause relaxations, confirming that ETB receptors are involved in mediating this response. 5. In normal mouse gastric fundus, 0.1 nM-1 microM ET-1, S6c or IRL1620 caused dose-dependent, BQ-123-insensitive contractions, which were much more resistant to PD142893 than S6c-induced relaxations of the aorta. The pD2 values for S6c in the absence and presence of PD142893 (10 microM) were 8.12 +/- 0.11 and 7.70 +/- 0.11, respectively. 6. In the gastric fundus of the ETB receptor gene knockout mouse, S6c and IRL1620 caused no contractions. ET-1 (0.1 nM-1 microM) caused contractions sensitive to both BQ-123 and PD142893, indicating that only ETA receptors mediate ET-1-induced contractions of the knockout mouse gastric fundus. 7. Since both the PD142893-sensitive vasodilator response of the aorta and the PD142893-resistant contractile response of the gastric fundus to S6c were completely absent in the ETB receptor gene knockout mouse, we conclude that the two pharmacologically heterogeneous responses to S6c are mediated by receptors derived from the same ETB receptor gene.

Neutrophil recruitment and activation are principal events in inflammation. Upon activation neutrophils release myeloperoxidase (MPO), a heme enzyme, which binds to and transcytoses endothelial cells. Whereas the significance of the subendothelial deposition of MPO has evolved as a critical prerequisite for the enzyme's suppression of nitric oxide (NO⋅) bioavailability, the functional consequences of MPO binding to and interaction with endothelial and smooth muscle cells remain poorly understood. Cultured human endothelial cells (HUVECs) were exposed to MPO. Gene expression of the endothelin receptor type B (ETRB), which is critically involved not only in endothelin-1 clearance, but also in endothelin-mediated vasoconstriction, was significantly increased. Real time PCR, Western blotting and immunofluorescence confirmed up-regulation of ETRB in MPO-treated endothelial cells. Inhibition of MPO's enzymatic activity blunted the increase in ETRB protein expression. Treatment of the cells with the MAP kinase inhibitors PD98059 or SB203580 indicates that MPO activates ETRB expression via MAP kinase pathways. On human smooth muscle cells (HAoSMCs), which not only express the endothelin receptor type B (ETRB) but also express the endothelin receptor type A (ETRA), MPO also stimulated ETRB expression as opposed to ETRA expression, which remained unchanged. Functional ex vivo organ bath chamber studies with MPO-incubated rat femoral artery sections revealed increased ETRB agonist dependent constriction. Binding of MPO to endothelial and vascular smooth muscle cells increases expression of the endothelin receptor type B (ETRB) via classical MAP kinase pathways. This suggests that MPO not only affects vasomotion by reducing the bioavailability of vasodilating molecules but also by increasing responsiveness to vasoconstrictors, further advocating for MPO as a central, leukocyte-derived regulator of vascular tone.

The peptide hormone endothelin, produced by the vascular endothelium, is involved in several physiological functions, including maintenance of vascular tone and humoral homeostasis. Endothelin transmits signals through the endothelin receptor, a G-protein-coupled receptor. Structural studies of the endothelin type B receptor (ETBR) have been unsuccessful due to its structural flexibility and instability in detergent-solubilized solution. To overcome these problems, we explored thermostabilization of human ETBR by establishing an ETBR expression system in Escherichia coli, followed by systematic alanine scanning mutagenesis. Among 297 point mutations, 11 thermostabilizing residues were selected and further mutated to other amino acids. The thermostability indices of these residues, represented by the ratios of endothelin-1 (ET-1) binding activities with or without heat treatment at 27°C for 30min in a ligand-free form, were compared. The ligand affinity and apparent melting temperature (Tm) of the five most thermostable mutants, R124Y, D154A, K270A, S342A, and I381A, were then examined. The apparent Tm of three single mutants, R124Y, D154A, and K270A, was approximately 7°C higher than that of the wild type. The apparent Tm value of a combination of the five residues, named the Y5 ETBR mutant, was 17°C higher than that of the wild type. The Y5 ETBR mutant exhibited an affinity for ET-1 and activated Gq similar to the wild type. Further investigation of the pharmacological properties affected by combinatorial mutations of ET-1, ET-3, TxET-1, and K8794 suggested that Y5 ETBR is highly suitable for representing a ligand-free form of ETBR and is potentially applicable for studying an ET-1-bound form.

Endothelial cell (EC) insulin resistance and dysfunction, caused by diabetes, accelerates atherosclerosis. It is unknown whether specifically enhancing EC-targeted insulin action can decrease atherosclerosis in diabetes. Accordingly, overexpressing insulin receptor substrate-1 (IRS1) in the endothelia of Apoe-/- mice (Irs1/Apoe-/-) increased insulin signaling and function in the aorta. Atherosclerosis was significantly reduced in Irs1/ApoE-/- mice on diet-induced hyperinsulinemia and hyperglycemia. The mechanism of insulin's enhanced antiatherogenic actions in EC was related to remarkable induction of NO action, which increases endothelin receptor B (EDNRB) expression and intracellular [Ca2+]. Using the mice with knockin mutation of eNOS, which had Ser1176 mutated to alanine (AKI), deleting the only known mechanism for insulin to activate eNOS/NO pathway, we observed that IRS1 overexpression in the endothelia of Aki/ApoE-/- mice significantly decreased atherosclerosis. Interestingly, endothelial EDNRB expression was selectively reduced in intima of arteries from diabetic patients and rodents. However, endothelial EDNRB expression was upregulated by insulin via P13K/Akt pathway. Finally EDNRB deletion in EC of Ldlr-/- and Irs1/Ldlr-/- mice decreased NO production and accelerated atherosclerosis, compared with Ldlr-/- mice. Accelerated atherosclerosis in diabetes may be reduced by improving insulin signaling selectively via IRS1/Akt in the EC by inducing EDNRB expression and NO production.

We determined the expression profile of ∼300 G protein-coupled receptors (GPCRs) in embryonic cortical neural progenitor cells (NPCs) and identified a number of highly expressed GPCRs, among which endothelin-B receptor (ET(B)-R) was expressed at the highest level. We also revealed that endothelins (ETs) were predominantly expressed in CD31-positive endothelial cells of the embryonic cerebral cortex. Activation of ET(B)-R induced NPC assembly in vitro by promoting fibronectin-dependent-motility and N-cadherin-associated cell contact. NPC assembly also required a Rho-family GTPase(s) and phosphatidylinositol-3-kinase. In the embryonic cerebral cortex, a specific ET(B)-R agonist, IRL-1620, accelerated interkinetic nuclear migration (INM) of NPCs toward the ventricular wall (VW) ex vivo. Conversely, a specific ET(B)-R antagonist, BQ788, slowed INM, thereby inducing mislocalization of phospho-histone H3-positive M-phase nuclei in the ventricular zone (VZ) and decreasing the number of Tuj1-positive newborn neurons. Our results suggest that ET(B)-R-mediated assembly signals drive INM that precedes neurogenesis.

The five-year survival rate of lung adenocarcinoma patients (LUAD) is very low,and the methods of predicting survival are a great obstacle for LUAD therapies. Endothelin receptor type B (EDNRB) gene is associated with tumorigenesis. In this study, we aimed to evaluate the predictive value of EDNRB on LUAD.

Endothelin receptor antagonists inhibit the progression of many cancers, but research into their influence on glioma has been limited.

Smoking is known to cause chronic inflammatory changes in the bronchi and to contribute to airway hyper-reactivity, such as in bronchial asthma. To study the effect of smoking on the endothelin system in rat airways, bronchial segments were exposed to DMSO-soluble smoking particles (DSP) from cigarette smoke, to nicotine and to DMSO, respectively.

Background.  Both endothelin receptor type B ([ETBR], a G protein-coupled receptor that mediates the vascular effects of the potent vasoconstrictor endothelin-1) and human cytomegalovirus ([HCMV], a ubiquitous herpesvirus) have been implicated in the pathogenesis of cardiovascular disease (CVD). The effects of HCMV infection on ETBR expression are unknown. We hypothesized that HCMV may contribute to the pathogenesis of CVD via ETBR modulation. Methods.  Human CMV effects on ETBR were studied in vitro in endothelial cells (ECs) and smooth muscle cells (SMCs) and ex vivo in human carotid plaque tissue specimens. Expression of ETBR and viral immediate-early were quantified using quantitative polymerase chain reaction. Functional consequences after ETBR blockade in ECs were examined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide proliferation, wound healing, tube formation, and flow adhesion assays. Results.  Human CMV is capable of upregulating both ETBR mRNA and protein expression in ECs and SMCs. The ETBR was also abundantly expressed in ECs, foam cells, and SMCs, and, more importantly, in HCMV-positive cells in human carotid plaques. Endothelin receptor type B blockade led to decreased proliferation and reduced tumor necrosis factor α-mediated leukocyte recruitment in both uninfected and HCMV-infected ECs. Direct HCMV infection was antimigratory and antiangiogenic in ECs. Conclusions.  Human CMV may contribute to CVD via ETBR induction.

The free-energy landscape of interaction between a medium-sized peptide, endothelin 1 (ET1), and its receptor, human endothelin type B receptor (hETB), was computed using multidimensional virtual-system coupled molecular dynamics, which controls the system's motions by introducing multiple reaction coordinates. The hETB embedded in lipid bilayer was immersed in explicit solvent. All molecules were expressed as all-atom models. The resultant free-energy landscape had five ranges with decreasing ET1-hETB distance: completely dissociative, outside-gate, gate, binding pocket, and genuine-bound ranges. In the completely dissociative range, no ET1-hETB interaction appeared. In the outside-gate range, an ET1-hETB attractive interaction was the fly-casting mechanism. In the gate range, the ET1 orientational variety decreased rapidly. In the binding pocket range, ET1 was in a narrow pathway with a steep free-energy slope. In the genuine-bound range, ET1 was in a stable free-energy basin. A G-protein-coupled receptor (GPCR) might capture its ligand from a distant place.

Preterm cerebral white matter injury (WMI), a major form of prenatal brain injury, may potentially be treated by oligodendrocyte (OL) precursor cell (OPC) transplantation. However, the defective differentiation of OPCs during WMI seriously hampers the clinical application of OPC transplantation. Thus, improving the ability of transplanted OPCs to differentiate is critical to OPC transplantation therapy for WMI. We established a hypoxia-ischemia-induced preterm WMI model in mice and screened the molecules affected by WMI using single-cell RNA sequencing. We revealed that endothelin (ET)-1 and endothelin receptor B (ETB) are a pair of signaling molecules responsible for the interaction between neurons and OPCs and that preterm WMI led to an increase in the number of ETB-positive OPCs and premyelinating OLs. Furthermore, the maturation of OLs was reduced by knocking out ETB but promoted by stimulating ET-1/ETB signaling. Our research reveals a new signaling module for neuron-OPC interaction and provides new insight for therapy targeting preterm WMI.

The proliferative and antiapoptotic actions of endothelin (ET)-1 in cancer cells have been documented and ET receptor antagonists have been exploited as potential anticancer drugs. Glioblastoma cell lines express both ETA and ETB receptors and previous works have shown that ETB receptors are involved in the proliferation of different cancer cell types. In this study we have investigated the effects of two structurally unrelated ETB receptor antagonists, BQ788 and A192621, on cell survival, proliferation and apoptosis in 1321-N1, U87 and IPDDCA2 glioma cell lines. BQ788 and A192621 reduced glioma cells viability and proliferation assessed by BrdU incorporation and cell cycle analysis by flow cytometry, while in contrast the ETA receptor antagonist BQ123 had no effect on cell survival. TUNEL assay and immunocytochemical experiments showed that BQ788 and A192621 trigger apoptotic processes mainly via activation of the intrinsic mitochondrial pathway involving caspase-9 activation, AIF release and cytochrome c translocation. Furthermore, treatment with ETB antagonists downregulates ERK- and p38MAPK-dependent pathways but does not affect VEGF mRNA levels. Our findings support the hypothesis that ETB antagonists represent a new promising therapeutic strategy for the treatment of high grade gliomas.

Glioblastoma (GBM) is the most common malignant brain tumor with median survival of 12-15 months. Owing to uncertainty in clinical outcome, additional prognostic marker(s) apart from existing markers are needed. Since overexpression of endothelin B receptor (ETBR) has been demonstrated in gliomas, we aimed to test whether ETBR is a useful prognostic marker in GBM and examine if the clinically available endothelin receptor antagonists (ERA) could be useful in the disease treatment.

The endothelins and their receptors are best known for their regulation of the vascular system. Their widespread expression in epithelial cells and their overexpression in some tumors has prompted investigation into their ability to regulate cancer progression. In this study, we assessed the mRNA expression of the major endothelin B receptor gene (EDNRB) isoforms and found differences in both mRNA and protein expression in normal breast cells and breast cancer cell lines. Knocking down the EDNRB gene in breast cancer cells altered invasiveness toward endothelin 3 (ET3), and we observed EDNRB isoform-specific regulation of breast cancer cell invasion and cell signaling, as well as isoform- and subtype-specific differences in breast cancer patient survival. The results reported in this study emphasize the importance of the endothelin B receptor in breast cancer. To our knowledge, this study is the first to clarify the differential expression and roles of specific EDNRB isoforms in breast cancer.

Individuals with Trisomy 21 (T21) exhibit numerous hematological abnormalities, including reductions in numbers of circulating B and T lymphocytes. To elucidate molecular mechanisms underlying these phenotypes, we differentiated human isogenic disomic and trisomic pluripotent cells, and observed that trisomic cells showed defects in B cell, but not T cell differentiation. Global gene expression of differentiated, trisomic B cells revealed reduced expression of genes encoding endothelin signaling components, namely the Endothelin Receptor B (EDNRB), and its ligand Endothelin1 (EDN1). Depletion of EDNRB mRNA in cord blood-derived CD34+ cells led to defective B cell differentiation, supporting a hypothesis that low EDNRB expression in T21 contributes to intrinsic lymphoid defects. Further evidence for the role of the EDNRB pathway in B cell differentiation was obtained through CRISPR/Cas9 gene targeting in disomic and trisomic iPS cells. Knockout of EDNRB in both cell backgrounds reduced the capacity for B cell differentiation. Collectively, this work identifies downregulation of EDNRB as a causative factor for impaired B lymphocyte generation in trisomic cells, which may contribute to defects in immune function associated with T21. Furthermore, a novel role for endothelin signaling in regulation of B cell development has been identified.

Does endothelin-1 (ET-1) regulate matrix metalloproteinase (MMP) 14 and 15 production and invasion of human first trimester trophoblasts?

Hepatic production and release of endothelin-1 (ET-1) binding to endothelin B (ETB) receptors, overexpressed in the lung microvasculature, is associated with accumulation of pro-angiogenic monocytes and vascular remodeling in experimental hepatopulmonary syndrome (HPS) after common bile duct ligation (CBDL). We have recently found that lung vascular monocyte adhesion and angiogenesis in HPS involve interaction of endothelial C-X3-C motif ligand 1 (CX3CL1) with monocyte CX3C chemokine receptor 1 (CX3CR1), although whether ET-1/ETB receptor activation influences these events is unknown. Our aim was to define if ET-1/ETB receptor activation modulates CX3CL1/CX3CR1 signaling and lung angiogenesis in experimental HPS. A selective ETB receptor antagonist, BQ788, was given for 2 weeks to 1-week CBDL rats. ET-1 (±BQ788) was given to cultured rat pulmonary microvascular endothelial cells overexpressing ETB receptors. BQ788 treatment significantly decreased lung angiogenesis, monocyte accumulation, and CX3CL1 levels after CBDL. ET-1 treatment significantly induced CX3CL1 production in lung microvascular endothelial cells, which was blocked by inhibitors of Ca(2+) and mitogen-activated protein kinase (MEK)/ERK pathways. ET-1-induced ERK activation was Ca(2+) independent. ET-1 administration also increased endothelial tube formation in vitro, which was inhibited by BQ788 or by blocking Ca(2+) and MEK/ERK activation. CX3CR1 neutralizing antibody partially inhibited ET-1 effects on tube formation. These findings identify a novel mechanistic interaction between the ET-1/ETB receptor axis and CX3CL1/CX3CR1 in mediating pulmonary angiogenesis and vascular monocyte accumulation in experimental HPS.

The aim of the present study was to determine whether local administration of endothelin induces the release of dopamine in the rat striatum and to characterize and localize endothelin receptors in this brain region. Local injection of endothelin-1 (10 pmol) into the ventral striatum of urethane-anaesthetized rats caused an increase of 8 microM in the extracellular concentration of dopamine as measured by in vivo chronoamperometry. The peak increase in dopamine concentration occurred within 5 min of endothelin injection. Injection of the selective endothelin-B receptor agonist [Ala1.3,11.15]endothelin-1 (10 pmol) also caused an increase in extracellular dopamine concentration, suggesting that endothelin is acting at the endothelin-B receptor to elicit its effect. In rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway, the response to local injection of endothelin-1 (10 pmol) was significantly inhibited on the lesioned side as compared to the non-lesioned side. In contrast, pretreatment of the rats with the N-methyl-D-aspartate receptor antagonist dizocilpine maleate (5 mg/kg, i.p.) or the nitric oxide synthase inhibitor NG-nitro-L-arginine (3 mg/kg, i.p.) did not alter the endothelin-induced release of dopamine. In binding studies, addition of endothelin-1 displaced [125I]endothelin-1 with a Ki of 220 pM. The endothelin-B receptor antagonist BQ788 displaced [125I]endothelin-1 with a Ki of 120 nM, whereas the endothelin-A receptor antagonist BQ123 produced only a 25% displacement at 10 microM, suggesting that endothelin receptors in the striatum are of the endothelin-B subtype. In rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal dopamine system, [125I]endothelin-1 binding was reduced by 53% in lesioned striatum compared to non-lesioned striatum, with no difference in the Kd. These data provide evidence that endothelin acts on a homogeneous population of endothelin-B receptors within the striatum to cause the release of dopamine and that a significant proportion of these receptors is located on dopaminergic neurons.

Renin is the rate-limiting step in the production of angiotensin II: a critical element in the regulation of blood pressure and in the pathogenesis of hypertension. Renin release from the juxtaglomerular (JG) cell is stimulated by the second messenger cAMP and inhibited by increases in calcium (Ca). Endothelins (ETs) inhibit renin release in a Ca-dependent manner. JG cells contain multiple isoforms of canonical transient receptor potential (TRPC) Ca-permeable channels. The proposed hypothesis is that endothelin inhibits renin release by activating TRPC store-operated Ca channels. RT-PCR and immunofluorescence revealed expression of both ETA and ETB receptors in mouse JG cells. Incubation of primary cultures of JG cells with ET-1 (10 nmol/L) decreased renin release by 28%. Addition of either an ETA or an ETB receptor blocker completely prevented the ET inhibition of renin release. Incubation with the TRPC blocker (SKF 96365, 50 μmol/L) completely reversed the Ca-mediated inhibition of renin release by ETs. These results suggest that endothelin inhibits renin release from JG cells via both ETA and ETB receptors, which leads to the activation of TRPC store-operated Ca channels.

Endothelin-1 (ET-1) is important in the pathogenesis of systemic sclerosis (SSc). ET-1 binds two receptors, endothelin type A (ETA) and endothelin type B (ETB). Dual ETA/ETB receptor antagonists and a selective ETA receptor antagonist are used clinically to treat SSc, and the effect of these antagonists on fibroblast activation has been described. However, the role of ETB receptor signaling in fibrogenesis is less clear. This study was conducted to evaluate the profibrotic function of ETB receptor signaling in a murine model of bleomycin (BLM)-induced scleroderma.