Aprocitentan (a Dual Endothelin-Receptor Antagonist) for Treatment-Resistant Hypertension.

ABSTRACT: Treatment-resistant hypertension (TRH) is associated with increased cardiovascular risks and progression of chronic kidney disease. The pathophysiology of TRH is multifactorial, including overactivity of the renin-angiotensin-aldosterone system and sympathetic nervous system, endothelial dysfunction, and volume overload. Endothelin-1 is a vasoconstrictive peptide that causes neurohormonal and sympathetic activation, increased aldosterone synthesis and secretion, endothelial dysfunction, vascular hypertrophy and remodeling, and fibrosis. Endothelin-1 acts through 2 receptors, ETA and ETB. Activation of ETA receptors in vascular smooth muscle cells results in vasoconstriction, whereas ETB receptor activation results in vasoconstriction in the vascular smooth muscle cells and vasodilation through nitric oxide release in endothelial cells. Aprocitentan is novel, oral, dual endothelin-receptor antagonist that has demonstrated a more favorable tolerability and safety profile in early clinical trials compared with other endothelin-receptor antagonists studied. Phase 2 trial data support a significant reduction in blood pressure compared to placebo and similar blood pressure reduction compared to a moderately dosed angiotensin-converting enzyme inhibitor in patients with essential hypertension. An ongoing phase 3 randomized clinical trial is evaluating aprocitentan's efficacy and safety in patients with TRH receiving multiple antihypertensives. Additional research is needed to determine aprocitentan's role in therapy, but this agent may be a suitable treatment option for TRH.

Ovarian hormones modulate endothelin-1 receptor responses in young women.

OBJECTIVE: We recently demonstrated ETBR mediate vasodilation in young but not postmenopausal women; it is unclear if this is related to age or a decline in ovarian hormones. The purpose of this study was to test the hypothesis that ETBR responses are modulated by ovarian hormones. METHODS: We measured cutaneous vasodilatory responses in 12 young women (22 ± 1 years, 23 ± 1 kg/m2 ) during the ML (days 20-25) and EF (days 2-5) phases of the menstrual cycle. Cutaneous microdialysis perfusions of lactated Ringer (control), ETBR antagonist (BQ-788, 300 nmol/L), and ETAR antagonist (BQ-123, 500 nmol/L) were performed, followed by local heating to 42°C. RESULTS: Serum estradiol (ML: 118 ± 16 vs EF: 44 ± 9 pg/mL, P < 0.05) and progesterone (ML: 8.3 ± 1.0 vs EF: 0.7 ± 0.2 ng/mL, P < 0.05) were higher during ML vs EF phase. ETBR blockade decreased vasodilation during ML (control: 91 ± 2 vs BQ-788: 83 ± 2%CVCmax, P < 0.05) but not EF (control: 89 ± 2 vs BQ-788: 89 ± 1%CVCmax). ETAR blockade also decreased vasodilation during ML (control: 91 ± 2 vs BQ-123: 87 ± 2%CVCmax, P < 0.05) but not EF (control: 89 ± 2 vs BQ-123: 92 ± 2%CVCmax). CONCLUSIONS: These data suggest that fluctuations in ovarian hormones modulate ETBR and ETAR responses in young women.

Homocysteine up-regulates ETB receptors via suppression of autophagy in vascular smooth muscle cells.

The change of autophagy is implicated in cardiovascular diseases (CVDs). Homocysteine (Hcy) up-regulates endothelin type B (ETB) receptors in vascular smooth muscle cells (VSMCs). However, it is unclear whether autophagy is involved in Hcy-induced-up-regulation of ETB receptors in VSMCs. The present study was designed to examine the hypothesis that Hcy up-regulates ETB receptors by inhibiting autophagy in VSMCs. Hcy treated the rat superior mesenteric artery (SMA) without endothelium in the presence and absence of AICAR, rapamycin or MHY1485 for 24 h. The contractile responses to sarafotoxin 6c (S6c) (an ETB receptor agonist) were studied using a sensitive myograph. Levels of protein expression were determined using Western blot analysis. Punctate staining of LC3B was exanimated by immunofluorescence using confocal microscopy. The results showed that Hcy inhibited AMPK, and activated mTOR, followed by impairing autophagy, and increased the levels of ETB receptor protein expression and the ETB receptor-mediated contractile responses to S6c in SMA without endothelium. However, these effects were reversed by AICAR or rapamycin. Additionally, MHY1485 up-regulated the AICAR-inhibited ETB receptor-mediated contractile response and the levels of ETB receptor protein expression in presence of Hcy. In conclusion, this suggested that Hcy up-regulated ETB receptors by inhibiting autophagy in VSMCs via AMPK/mTOR signaling pathway.

Dual endothelin receptor antagonists contrast the effects induced by endothelin-1 on cultured human microvascular endothelial cells.

OBJECTIVES: To evaluate the ability of dual endothelin (ET) receptor antagonists (ETA/ETB -ETA/BRAs) to contrast the ET-1-induced effects on cultured human microvascular endothelial cells (HMVECs). METHODS: Some cultured HMVECs were untreated, or treated with ET-1 (100nM) or transforming growth factor ß1 (TGFß1, 10ng/mL) alone for 6 days, in order to induce the endothelial-to-mesenchymal transition (EndoMT). Other cultured HMVECs were pre-treated for 1hr with ETA/BRAs bosentan (10µM) or macitentan (1µM, 10µM) before the stimulation with ET-1 for 6 days. At the end of treatments, a mechanical injury was induced to cultured HMVECs (by scratching the cell monolayer with a sterile tip), and then the cell ability to re-fill the damaged area was determined after 24hrs. EndoMT phenotype markers and monocyte chemoattractant protein-1 (MCP-1) were evaluated by qRT-PCR and Western blotting. Statistical analysis was performed using Mann-Whitney-U non-parametric test. RESULTS: Both ET-1 and TGFß1 induced EndoMT and the MCP-1 over-expression in cultured HMVECs, as well as reduced the process of endothelial cell damage repair. Pre-treatment with ETA/BRAs let cultured HMVECs to significantly restore the in vitro damage of the cell monolayer and antagonised the EndoMT process as well as the MCP-1 over-expression (range p<0.05 - p<0.001). Conversely, untreated or TGFß1-treated HMVECs were found unaffected by the ETA/BRAs treatments. CONCLUSIONS: The treatment with dual ETA/BRAs seems to partially restore the altered cell function induced by ET-1 in cultured endothelial cells, and might justify their therapeutic efficiency in clinical conditions characterised by increased concentrations of ET-1.

Renin-angiotensin system blockade alone or combined with ETA receptor blockade: effects on the course of chronic kidney disease in 5/6 nephrectomized Ren-2 transgenic hypertensive rats.

BACKGROUND: Early addition of endothelin (ET) type A (ETA) receptor blockade to complex renin-angiotensin system (RAS) blockade has previously been shown to provide better renoprotection against progression of chronic kidney disease (CKD) in Ren-2 transgenic hypertensive rats (TGR) after 5/6 renal ablation (5/6 NX). In this study, we examined if additional protection is provided when ETA blockade is applied in rats with already developed CKD. METHODS: For complex RAS inhibition, an angiotensin-converting enzyme inhibitor along with angiotensin II type 1 receptor blocker was used. Alternatively, ETA receptor blocker was added to the RAS blockade. The treatments were initiated 6 weeks after 5/6 NX and the follow-up period was 50 weeks. RESULTS: When applied in established CKD, addition of ETA receptor blockade to the complex RAS blockade brought no further improvement of the survival rate (30% in both groups); surprisingly, aggravated albuminuria (588 ± 47 vs. 245 ± 38 mg/24 h, p < 0.05) did not reduce renal glomerular injury index (1.25 ± 0.29 vs. 1.44 ± 0.26), did not prevent the decrease in creatinine clearance (203 ± 21 vs. 253 ± 17 µl/min/100 g body weight), and did not attenuate cardiac hypertrophy to a greater extent than observed in 5/6 NX TGR treated with complex RAS blockade alone. CONCLUSIONS: When applied in the advanced phase of CKD, addition of ETA receptor blockade to the complex RAS blockade brings no further beneficial renoprotective effects on the CKD progression in 5/6 NX TGR, in addition to those seen with RAS blockade alone.

Sulfenic Acid Modification of Endothelin B Receptor is Responsible for the Benefit of a Nonsteroidal Mineralocorticoid Receptor Antagonist in Renal Ischemia.

AKI is associated with high mortality rates and the development of CKD. Ischemia/reperfusion (IR) is an important cause of AKI. Unfortunately, there is no available pharmacologic approach to prevent or limit renal IR injury in common clinical practice. Renal IR is characterized by diminished nitric oxide bioavailability and reduced renal blood flow; however, the mechanisms leading to these alterations are poorly understood. In a rat model of renal IR, we investigated whether the administration of the novel nonsteroidal mineralocorticoid receptor (MR) antagonist BR-4628 can prevent or treat the renal dysfunction and tubular injury induced by IR. Renal injury induced by ischemia was associated with increased oxidant damage, which led to a cysteine sulfenic acid modification in endothelin B receptor and consequently decreased endothelial nitric oxide synthase activation. These modifications were efficiently prevented by nonsteroidal MR antagonism. Furthermore, we demonstrated that the protective effect of BR-4628 against IR was lost when a selective endothelin B receptor antagonist was coadministered. These data describe a new mechanism for reduced endothelial nitric oxide synthase activation during renal IR that can be blocked by MR antagonism with BR-4628.

Activation of purinergic receptors (P2) in the renal medulla promotes endothelin-dependent natriuresis in male rats.

Renal endothelin-1 (ET-1) and purinergic signaling systems regulate Na(+) reabsorption in the renal medulla. A link between the renal ET-1 and purinergic systems was demonstrated in vitro, however, the in vivo interaction between these systems has not been defined. To test whether renal medullary activation of purinergic (P2) receptors promotes ET-dependent natriuresis, we determined the effect of increased medullary NaCl loading on Na(+) excretion and inner medullary ET-1 mRNA expression in anesthetized adult male Sprague-Dawley rats in the presence and absence of purinergic receptor antagonism. Isosmotic saline (NaCl; 284 mosmol/kgH2O) was infused into the medullary interstitium (500 µl/h) during a 30-min baseline urine collection period, followed by isosmotic or hyperosmotic saline (1,800 mosmol/kgH2O) for two further 30-min urine collection periods. Na(+) excretion was significantly increased during intramedullary infusion of hyperosmotic saline. Compared with isosmotic saline, hyperosmotic saline infused into the renal medulla caused significant increases in inner medullary ET-1 mRNA expression. Renal intramedullary infusion of the P2 receptor antagonist suramin inhibited the increase in Na(+) excretion and inner medullary ET-1 mRNA expression induced by NaCl loading in the renal medulla. Activation of medullary P2Y2/4 receptors by infusion of UTP increased urinary Na(+) excretion. Combined ETA and ETB receptor blockade abolished the natriuretic response to intramedullary infusion of UTP. These data demonstrate that activation of medullary P2 receptors promotes ET-dependent natriuresis in male rats, suggesting that the renal ET-1 and purinergic signaling systems interact to efficiently facilitate excretion of a NaCl load.

Endothelin therapeutics in cancer: Where are we?

In human cancers, the autocrine and paracrine loop mediated by the aberrantly activation of endothelin-1 (ET-1) receptor (ET-1R) elicits pleiotropic effects, preferentially mediated by the scaffold protein ß-arrestin 1 (ß-arr1), on tumor cells and on the host microenvironment, providing a strong rationale for targeting ET-1 receptors. This review describes the most up-to-date preclinical and clinical results obtained by using ET-1 therapeutics. The previous negative clinical results of ET-1 therapeutics should not prevent us from setting the standard of this class of drugs for future well-designed clinical trials. The preclinical data obtained with the dual ETAR and ETBR antagonist macitentan indicate that this molecule, which targets cancer cells and tumor-associated microenvironmental elements, could be a cancer therapeutic option. The field of ET-1 therapeutics will be improved in the next decade, facilitated by the new knowledge on the genomic landscape of the human stroma and tumor, and by the low invasive approaches based on liquid biopsies for the discovery of predictive biomarkers. The information obtained from preclinical studies in patient-derived models and from the Cancer Genome Atlas will set the scene of precision medicine for cancer. Results from these studies are expected to open the possibility that ET-1R antagonists might be more efficacious as molecular cancer therapeutics, able to hamper the functional ß-arr1-dependent signaling complexes, either alone or coupled with new targeted approaches.

The effects of MEK1/2 inhibition on cigarette smoke exposure-induced ET receptor upregulation in rat cerebral arteries.

Cigarette smoking, a major stroke risk factor, upregulates endothelin receptors in cerebral arteries. The present study examined the effects of MEK1/2 pathway inhibition on cigarette smoke exposure-induced ET receptor upregulation. Rats were exposed to the secondhand smoke (SHS) for 8weeks followed by intraperitoneal injection of MEK1/2 inhibitor, U0126 for another 4weeks. The urine cotinine levels were assessed with high-performance liquid chromatography. Contractile responses of isolated cerebral arteries were recorded by a sensitive wire myograph. The mRNA and protein expression levels of receptor and MEK/ERK1/2 pathway molecules were examined by real-time PCR and Western blotting, respectively. Cerebral artery receptor localization was determined with immunohistochemistry. The results showed the urine cotinine levels from SHS exposure group were significantly higher than those from the fresh group. In addition, the MEK1/2 inhibitor, U0126 significantly reduced SHS exposure-increased ETA receptor mRNA and protein levels as well as contractile responses mediated by ETA receptors. The immunoreactivity of increased ETA receptor expression was primarily cytoplasmic in smooth muscle cells. In contrast, ETB receptor was noted in endothelial cells. However, the SHS-induced decrease in endothelium-dependent relaxation was unchanged after U0126 treatment. Furthermore, SHS increased the phosphorylation of MEK1/2 and ERK1/2 protein in cerebral arteries. By using U0126 could inhibit the phosphorylated ERK1/2 protein but not MEK1/2. Taken together, our data show that treatment with MEK1/2 pathway inhibitor offsets SHS exposure-induced ETA receptor upregulation in rat cerebral arteries.

Discovery of Dual ETA/ETB Receptor Antagonists from Traditional Chinese Herbs through in Silico and in Vitro Screening.

Endothelin-1 receptors (ETAR and ETBR) act as a pivotal regulator in the biological effects of ET-1 and represent a potential drug target for the treatment of multiple cardiovascular diseases. The purpose of the study is to discover dual ETA/ETB receptor antagonists from traditional Chinese herbs. Ligand- and structure-based virtual screening was performed to screen an in-house database of traditional Chinese herbs, followed by a series of in vitro bioassay evaluation. Aristolochic acid A (AAA) was first confirmed to be a dual ETA/ETB receptor antagonist based intracellular calcium influx assay and impedance-based assay. Dose-response curves showed that AAA can block both ETAR and ETBR with IC50 of 7.91 and 7.40 µM, respectively. Target specificity and cytotoxicity bioassay proved that AAA is a selective dual ETA/ETB receptor antagonist and has no significant cytotoxicity on HEK293/ETAR and HEK293/ETBR cells within 24 h. It is a feasible and effective approach to discover bioactive compounds from traditional Chinese herbs using in silico screening combined with in vitro bioassay evaluation. The structural characteristic of AAA for its activity was especially interpreted, which could provide valuable reference for the further structural modification of AAA.

Endothelin receptor B protects granulocyte macrophage colony-stimulating factor mRNA from degradation.

Evidence is lacking on the differential effects of the two therapeutic concepts of endothelin receptor antagonists (ERAs): the blockade of only the endothelin receptor A (ETAR; selective antagonism) versus both ETAR and endothelin receptor B (ETBR; dual blockade). Ambrisentan, a selective ERA, and bosentan, a dual blocker, are both available for therapy. We hypothesized that there are differences in the potential of ERAs to ameliorate inflammatory processes in human airway smooth muscle cells (HASMCs) and aimed to unravel underlying mechanisms. We used HASMC culture, enzyme-linked immunosorbent assay, and quantitative reverse-transcription polymerase chain reaction. Tumor necrosis factor α (TNFα) induced transcription and expression of chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 3 (CXCL3), granulocyte macrophage colony-stimulating factor (GM-CSF), and matrix metalloproteinase 12 (MMP12) in HASMCs. In concentration-response experiments, bosentan led to a significantly greater reduction of GM-CSF and MMP12 protein release than ambrisentan, whereas there was no significant difference in their effect on GM-CSF and MMP12 mRNA. Both ERAs reduced CXCL3 protein and mRNA equally but had no effect on CXCL2. Blocking mitogen-activated protein kinases revealed that both ETAR and ETBR signal through p38 mitogen-activated protein kinase, but ETBR also signals through extracellular signal-regulated kinase (ERK) 1/2 to induce GM-CSF expression. In the presence of the transcription inhibitor actinomycin D, bosentan, but not ambrisentan, reduced GM-CSF but not MMP12 or CXCL3 mRNA. In conclusion, blockade of each endothelin receptor subtype reduces GM-CSF transcription, but blocking ETBR additionally protects GM-CSF mRNA from degradation via ERK-1/2. Accordingly, blocking both ETAR and ETBR leads to a stronger reduction of TNFα-induced GM-CSF protein expression. This mechanism might be specific to GM-CSF. Our data stress the anti-inflammatory potential of ERA and warrant further investigation of their utility in chronic inflammatory airway diseases.

Celecoxib, but not indomethacin, ameliorates the hypertensive and perivascular fibrotic actions of cyclosporine in rats: role of endothelin signaling.

The immunosuppressant drug cyclosporine (CSA) is used with nonsteroidal antiinflammatory drugs (NSAIDs) in arthritic conditions. In this study, we investigated whether NSAIDs modify the deleterious hypertensive action of CSA and the role of endothelin (ET) receptors in this interaction. Pharmacologic, protein expression, and histopathologic studies were performed in rats to investigate the roles of endothelin receptors (ETA/ETB) in the hemodynamic interaction between CSA and two NSAIDs, indomethacin and celecoxib. Tail-cuff plethysmography measurements showed that CSA (20 mg kg(-1) day(-1), 10 days) increased systolic blood pressure (SBP) and heart rate (HR). CSA hypertension was associated with renal perivascular fibrosis and divergent changes in immunohistochemical signals of renal arteriolar ETA (increases) and ETB (decreases) receptors. While these effects of CSA were preserved in rats treated concomitantly with indomethacin (5 mg kg(-1) day(-1)), celecoxib (10 mg kg(-1) day(-1)) abolished the pressor, tachycardic, and fibrotic effects of CSA and normalized the altered renal ETA/ETB receptor expressions. Selective blockade of ETA receptors by atrasentan (5 mg kg(-1) day(-1)) abolished the pressor response elicited by CSA or CSA plus indomethacin. Alternatively, BQ788 (ETB receptor blocker, 0.1 mg kg(-1) day(-1)) caused celecoxib-sensitive elevations in SBP and potentiated the pressor response evoked by CSA. Together, the improved renovascular fibrotic and endothelin receptor profile (ETA downregulation and ETB upregulation) mediate, at least partly, the protective effect of celecoxib against the hypertensive effect of CSA. Clinically, the use of celecoxib along with CSA in the management of arthritic conditions might provide hypertension-free regimen.

Involvement of endothelins in deoxycorticosterone acetate-salt hypertension through the modulation of noradrenergic transmission in the rat posterior hypothalamus.

NEW FINDINGS: What is the central question of this study? Does ex vivo administration of endothelin-1 and endothelin-3 regulate noradrenergic transmission in the posterior hypothalamus of deoxycorticosterone acetate-salt hypertensive rats compared with normotensive rats? What is the main finding and its importance? Endothelin-1 and endothelin-3 enhanced diverse mechanisms leading to increased noradrenergic transmission in the posterior hypothalamus of deoxycorticosterone acetate-salt hypertensive rats. Unveiling the role of brain endothelins in hypertension would probably favour the development of new therapeutic targets for the treatment of essential hypertension, which still represents a challenging disease with high mortality. Brain catecholamines participate in diverse biological functions regulated by the hypothalamus. We have previously reported that endothelin-1 and endothelin-3 (ET-1 and ET-3) modulate catecholaminergic activity in the anterior and posterior hypothalamus of normotensive rats. The aim of the present study was to evaluate the interaction between endothelins and noradrenergic transmission in the posterior hypothalamus of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. We assessed the effects of ET-1 and ET-3 on tyrosine hydroxylase activity and expression, neuronal noradrenaline (NA) release, neuronal NA transporter (NAT) activity and expression, monoamine oxidase activity and NA endogenous content and utilization (as a marker of turnover) in the posterior hypothalamus of DOCA-salt hypertensive rats. In addition, levels of ETA and ETB receptors were assayed in normotensive and hypertensive rats. Results showed that tyrosine hydroxylase activity and total and phosphorylated levels, NAT activity and content, NA release, monoamine oxidase activity and NA utilization were increased in DOCA-salt rats. Both ET-1 and ET-3 further enhanced all noradrenergic parameters except for total tyrosine hydroxylase level and NA endogenous content and utilization. The expression of ETA receptors was increased in the posterior hypothalamus of DOCA-salt rats, but ETB receptors showed no changes. These results show that ET-1 and ET-3 upregulate noradrenergic activity in the posterior hypothalamus of DOCA-salt hypertensive rats. Our findings suggest that the interaction between noradrenergic transmission and the endothelinergic system in the posterior hypothalamus may be involved in the development and/or maintenance of hypertension in this animal model.

Endothelin-1 increases the expression of VEGF-R1/Flt-1 receptors in rat cultured astrocytes through ETB receptors.

Expressions of vascular endothelial growth factor (VEGF) receptors in astrocytes are increased in damaged brains. To clarify the regulatory mechanisms of VEGF receptors, the effects of endothelin-1 (ET-1) were examined in rat cultured astrocytes. Expressions of VEGF-R1 and -R2 receptor mRNA were at similar levels, whereas the mRNA expressions of VEGF-R3 and Tie-2, a receptor for angiopoietins, were lower. Placenta growth factor, a selective agonist of the VEGF-R1 receptor, induced phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). Phosphorylations of FAK and ERK 1/2 were also stimulated by VEGF-E, a selective VEGF-R2 agonist. Increased phosphorylations of FAK and ERK1/2 by VEGF165 were reduced by selective antagonists for VEGF-R1 and -R2. Treatment with ET-1 increased VEGF-R1 mRNA and protein levels. The effects of ET-1 on VEGF-R1 mRNA were mimicked by Ala(1,3,11,15) -ET-1, a selective agonist for ETB receptors, and inhibited by BQ788, an ETB antagonist. ET-1 did not affect the mRNA levels of VEGF-R2, -R3, and Tie-2. Pre-treatment with ET-1 potentiated the effects of placenta growth factor on phosphorylations of FAK and ERK1/2. These findings suggest that ET-1 induces up-regulation of VEGF-R1 receptors in astrocytes, and potentiates VEGF signals in damaged nerve tissues. To clarify the regulatory mechanisms of vascular endothelial growth factor (VEGF) receptors, the effects of endothelin-1 (ET-1) were examined in rat cultured astrocytes. Effects of selective VEGF-R1 and R2 agonist showed that these receptors were linked to focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). Treatment with ET-1 increased expression of VEGF-R1, which was mediated by ETB receptors. Pre-treatment with ET-1 potentiated the VEGF-R1-mediated activations of FAK and ERK1/2. These findings suggest that ET-1 induces up-regulation of VEGF-R1 receptors in astrocytes.

Cardiopulmonary bypass is associated with altered vascular reactivity of isolated pulmonary artery in a porcine model: therapeutic potential of inhaled tezosentan.

OBJECTIVE: Whereas it is established that endothelin-1 elicits sustained deleterious effects on the cardiovascular system during cardiopulmonary bypass (CPB), presently it remains unknown whether the inhaled administration of the dual ETA and ETB antagonist tezosentan prevents the development of pulmonary endothelial dysfunction. DESIGN: A prospective, randomized laboratory investigation. SETTING: University research laboratory. PARTICIPANTS: Landrace swine. INTERVENTIONS: Three groups of animals underwent a 90-minute period of full bypass followed by a 60-minute period of reperfusion. Among treated groups, one received tezosentan through inhalation prior to CPB, whereas the other one received it intravenously at weaning from CPB; the third group remained untreated. Pulmonary vascular reactivity studies, realized on a total of 285 rings, were performed in all groups, including 1 sham. MEASUREMENTS AND MAIN RESULTS: The contractility of pulmonary arteries to prostaglandin F2α and to the thromboxane A2 mimetic U46619 was preserved in animals submitted to CPB. By contrast, there were significant increases both in the maximal contraction to endothelin-1 and in the plasma levels of the peptide 60 minutes after reperfusion. Tezosentan administered by inhalation or intravenously did not prevent the development of pulmonary CPB-associated endothelial dysfunction. However, while hemodynamic disturbances were improved with both routes, the inhaled administration had a beneficial effect on oxygen parameters over intravenous administration. CONCLUSIONS: Despite the blockade of the endothelin-1 pathway with tezosentan, the development of the pulmonary endothelial dysfunction associated with CPB still occurred. However, only the inhalation route had a significant impact on gas exchange during CPB.

Progressive endothelin-1 gene activation initiates chronic/end-stage renal disease following experimental ischemic/reperfusion injury.

This study assessed whether endothelin-1 (ET-1) helps mediate postischemic acute kidney injury (AKI) progression to chronic kidney disease (CKD). The impact(s) of potent ETA or ETB receptor-specific antagonists (Atrasentan and BQ-788, respectively) on disease progression were assessed 24 h or 2 weeks following 30 min of unilateral ischemia in CD-1 mice. Unilateral ischemia caused progressive renal ET-1 protein/mRNA increases with concomitant ETA, but not ETB, mRNA elevations. Extensive histone remodeling consistent with gene activation and increased RNA polymerase II (Pol II) binding occurred at the ET-1 gene. Unilateral ischemia produced progressive renal injury as indicated by severe histologic injury and a 40% loss of renal mass. Pre- and post-ischemia or just postischemic treatment with Atrasentan conferred dramatic protective effects such as decreased tubule/microvascular injury, normalized tissue lactate, and total preservation of renal mass. Nuclear KI-67 staining was not increased by Atrasentan, implying that increased tubule proliferation was not involved. Conversely, ETB blockade had no protective effect. Thus, our findings provide the first evidence that ET-1 operating through ETA can have a critical role in ischemic AKI progression to CKD. Blockade of ETA provided dramatic protection, indicating the functional significance of these results.

DMSO-soluble cigarette smoke particles alter the expression of endothelin B receptor in rat coronary artery.

In coronary artery diseases, cigarette smoking is a risk factor and the endothelin system plays a key role in the pathogenesis. This study was to examine if dimethylsulfoxide-soluble smoke particles (DSP) upregulate endothelin type-B (ETB) receptors in the coronary artery and investigate the mechanism. The isolated rat coronary arteries were organ-cultured for 24 h. The contractile response of the coronary artery was recorded by myograph. The mRNA and protein expression of the ETB receptors was studied using quantitative real-time PCR and immunohistochemistry. Results showed that the ETB receptor agonist, sarafotoxin 6c, induced a weak contraction in the fresh coronary artery. After culture, the contraction curve mediated by ETB receptor was shifted towards the left with an increased Emax of 152 ± 12%. DSP of 0.2 and 0.4 µl/ml shifted the concentration-contractile curves towards the left with further increased Emax of 270 ± 26 and 280 ± 29%, respectively. The culture increased ETB receptor mRNA and protein levels from fresh arteries, which was further enhanced by DSP. PD98059 (ERK1/2 inhibitor), wedelolactone (NF-κB inhibitor), actinomycin D or cycloheximide significantly inhibited the DSP-enhanced contraction and expression of mRNA and protein of the ETB receptor. However, SB203580 (p38 inhibitor) further increased DSP-enhanced contraction and protein expression of the ETB receptor. The results indicate that DSP upregulates ETB receptors in rat coronary artery via ERK1/2 and the NF-κB pathway.

Impaired endothelin calcium signaling coupled to endothelin type B receptors in penile arteries from insulin-resistant obese Zucker rats.

INTRODUCTION: Erectile dysfunction is considered as an early sign of subclinical vascular disease and endothelial dysfunction and a highly prevalent condition in diabetic patients. AIM: The current study assessed whether impaired vascular effects of endothelin (ET)-1 may contribute to the vascular dysfunction of penile arteries from a rat model of insulin resistance. METHODS: The effect of ETA and ETB receptor antagonists was assessed on the intracellular Ca(2+) [Ca(2+) ]i and contractile responses to ET-1 in penile arteries from obese Zucker rats (OZR) and lean Zucker rats (LZR), and ET receptor expression in the arterial wall was assessed by immunohistochemistry. MAIN OUTCOME MEASURE: Changes in ET-1 [Ca(2+) ]i and vasoconstriction and ET receptor expression were evaluated in penile arteries from insulin-resistant rats. RESULTS: ET-1-induced vasoconstriction was associated with a higher increase in smooth muscle [Ca(2+) ]i in penile arteries from OZR compared with LZR. Removal of the endothelium inhibited and enhanced contractions to the lowest and highest doses of ET-1, respectively, mainly in OZR. The selective ETA receptor antagonist BQ-123 inhibited ET-1 vasoconstriction and [Ca(2+) ]i response in both LZR and OZR. The ETB receptor antagonist BQ-788 had little effect in healthy arteries but markedly inhibited ET-1-induced increases in [Ca(2+) ]i and vasoconstriction in arteries from OZR. ETA receptors were located on the smooth muscle and endothelium of penile arteries, whereas ETB receptors were found on the arterial endothelium in LZR and OZR, and also on the smooth muscle in OZR, immunostaining for both receptors being higher in OZR. CONCLUSION: Penile arteries from OZR exhibit an impaired ET-1 Ca(2+) signaling along with changes in the ET receptor profile. Thus, whereas ET-1 contraction and the associated [Ca(2+) ]i increase are mediated by smooth muscle ETA receptors in healthy arteries, ETB receptors contribute to contraction and are coupled to the augmented ET-1 [Ca(2+) ]i response under conditions of insulin resistance.

Aliskiren, exendin-4, and insulin: their impact on endothelin receptor subtype(s) regulation/binding in type 1 diabetic rat hearts.

This study focuses on the impact of aliskiren and (or) glucagon-like peptide-1 analogue on the binding affinity/regulation of endothelin-1 (ET-1) to its receptor subtypes A (ETAR) and B (ETBR) at the level of the coronary endothelium and the cardiomyocytes in a type-1 diabetic rat model. Seven groups were used: (i) normal rats, (ii) rats with induced diabetes, (iii) rats with induced diabetes that were treated with insulin, (iv) rats with induced diabetes that were treated with exendin-4, (v) rats with induced diabetes that were treated with aliskiren, (vi) rats with induced diabetes that were co-treated with insulin plus aliskiren, and (vii) rats with induced diabetes that were co-treated with exendin-4 plus aliskiren. Heart perfusion with [(125)I]-ET-1 was employed to estimate ET-1 binding affinity (τ = 1/K-n) to ETAR and ETBR at the level of the coronary endothelium and the cardiomyocytes. Plasma ET-1 levels were measured using enzyme immunoassay, whereas densities of ETAR and ETBR were detected using Western blot. No significance differences were detected in the τ of ETAR and ETBR between normal and diabetic in cardiomyocytes and the coronary endothelium. Exendin-4 normalized the τ value for ETAR and ETBR on coronary endothelium, while aliskiren normalized it on cardiomyocytes. Furthermore, ETAR and ETBR densities were normalized with monotreatments of aliskiren and exendin-4, compared with up-regulated ETAR and down-regulated ETBR band densities in the diabetic animals. Our data indicate that aliskiren alleviates diabetes-associated hypertrophy in type 1 diabetes mellitus.

Endothelin ET(B) receptors are involved in the relaxation to the pig urinary bladder neck.

AIMS: The involvement of endothelin receptors in the contraction of the lower urinary tract smooth muscle is well established. There is scarce information, however, about endothelin receptors mediating relaxation of the bladder outlet region. The current study investigates the possible existence of endothelin ET(B) receptors involved in the relaxation of pig bladder neck. METHODS: ET(B) receptor expression was determined by immunohistochemistry and urothelium-denuded bladder neck strips were mounted in organ baths for isometric force recording. RESULTS: ET(B) -immunoreactivity (ET(B) -IR) was observed within nerve fibers among smooth muscle bundles and urothelium. BQ3020 (0.01-300 nM), an ET(B) receptor agonist, produced concentration-dependent relaxations which were reduced by BQ788, an ET(B) receptor antagonist, and by inhibitors of protein kinase A (PKA) and large (BK(Ca) )- or small (SK(Ca) )-conductance Ca(2+) -activated K(+) channels. Pretreatment with BK(Ca) or SK(Ca) channel inhibitors plus PKA blocking did not cause further inhibition compared with that exerted by inhibiting BK(Ca) or SK(Ca) channels only. BQ3020-induced relaxation was not modified by blockade of either nitric oxide (NO) synthase, guanylyl cyclase, cyclooxygenase (COX) or of intermediate-conductance Ca(2+) -activated-(IK(Ca) ), ATP-dependent-(K(ATP) ), or voltage-gated-(K(v) ) K(+) channels. Under non-adrenergic non-cholinergic (NANC) conditions, electrical field stimulation (0.5-16 Hz) evoked frequency-dependent relaxations, which were reduced by BQ788 and potentiated by threshold concentrations of BQ3020. CONCLUSIONS: These results suggest that BQ3020 produces relaxation of the pig bladder neck via activation of muscle endothelin ET(B) receptors, NO/cGMP- and COX-independent-, cAMP-PKA pathway-dependent-mechanisms, and involving BK(Ca) and SK(Ca) channel activation. ET(B) receptors are also involved in the NANC inhibitory neurotransmission.