Endothelin ETA Research Articles

Cerebral ischemia induces micro vascular pro-inflammatory cytokine expression via the MEK/ERK pathway

BackgroundCerebral ischemia from middle cerebral artery wall (MCA) occlusion results in increased expression of cerebrovascular endothelin and angiotensin receptors and activation of the mitogen-activated protein kinase (MAPK) pathway, as well as reduced local cerebral blood flow and increased levels of pro-inflammatory mediators in the infarct region. In this study, we hypothesised that inhibition of the cerebrovascular inflammatory reaction with a specific MEK1/2 inhibitor (U0126) to block transcription or a combined receptor blockade would reduce infarct size and improve neurological score.MethodsRats were subjected to a 2-hours middle cerebral artery occlusion (MCAO) followed by reperfusion for 48 hours. Two groups of treated animals were studied; (i) one group received intraperitoneal administration of a specific MEK1/2 inhibitor (U0126) starting at 0, 6, or 12 hours after the occlusion, and (ii) a second group received two specific receptor antagonists (a combination of the angiotensin AT1 receptor inhibitor Candesartan and the endothelin ETA receptor antagonist ZD1611), given immediately after occlusion. The middle cerebral arteries, microvessels and brain tissue were harvested; and the expressions of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and phosphorylated ERK1/2, p38 and JNK were analysed using immunohistochemistry.ResultsWe observed an infarct volume of 25 ± 2% of total brain volume, and reduced neurological function 2 days after MCAO followed by 48 hours of recirculation. Immunohistochemistry revealed enhanced expression of TNF-α, IL-1ß, IL-6 and iNOS, as well as elevated levels of phosphorylated ERK1/2 in smooth muscle cells of ischemic MCA and in associated intracerebral microvessels. U0126, given intraperitoneal at zero or 6 hours after the ischemic event, but not at 12 hours, reduced the infarct volume (11.7 ± 2% and 15 ± 3%, respectively), normalized pERK1/2, and prevented elevation of the expressions of TNF-α IL-1ß, IL-6 and iNOS. Combined inhibition of angiotensin AT1 and endothelin ETA receptors decreased the volume of brain damaged (12.3 ± 3; P < 0.05) but only slightly reduced MCAO-induced enhanced expression of iNOS and cytokinesConclusionThe present study shows elevated microvascular expression of TNF-α, IL-1ß, IL-6 and iNOS following focal ischemia, and shows that this expression is transcriptionally regulated via the MEK/ERK pathway.

Increased MAPK Activation and Impaired Insulin Signaling in Subcutaneous Microvascular Endothelial Cells in Type 2 Diabetes: The Role of Endothelin-1

OBJECTIVETo establish a method for isolation and culture of subcutaneous microvascular endothelial cells (MVEC) from small human tissue biopsies to compare gene and protein expression of insulin signaling molecules in MVEC from insulin-resistant and healthy control subjects.RESEARCH DESIGN AND METHODSStromavascular cells from subcutaneous needle biopsies of type 2 diabetic and control subjects were expanded in culture and the endothelial cells selected with magnetic immune separation. Western blots and RT-PCR were used for protein and gene expression assays.RESULTSAt least 99% of the expanded primary MVEC could be characterized as endothelial cells. The expression of insulin receptors was low, but insulin increased tyrosine phosphorylation of both the insulin receptor and insulin receptor substrate (IRS)-1 and activated protein kinase B (PKB). The IRS-1 protein expression was reduced and the serine phosphorylation of PKB in response to insulin attenuated whereas basal and insulin-stimulated phosphorylation of extracellular signal–related kinase (ERK)1/2 was increased in type 2 diabetes MVEC. Endothelin (ET)-1 mRNA levels were significantly higher in type 2 diabetes cells. The addition of ET-1 increased the phosphorylation of mitogen-activated protein kinase (MAPK), an effect antagonized by the MEK-1 inhibitor PD98059. Furthermore, the endothelin ETA and ETB receptor antagonists BQ123 and BQ788 decreased basal MAPK activity in type 2 diabetes MVEC and prevented the ET-1–induced activation.CONCLUSIONSWe developed a system for isolation and culture of human MVEC from small needle biopsies. Our observations support the concept of “selective” insulin resistance, involving IRS-1 and the PI3kinase pathway, as an underlying factor for a dysregulated microvascular endothelium in type 2 diabetes. Our data also support a role of ET-1 for the increased MAPK activity seen in nonstimulated type 2 diabetes MVEC.

Endothelin receptor blockade does not affect blood pressure or angiotensin II levels in CYP1A1-Ren-2 transgenic rats with acutely induced hypertension

We found previously that selective blockade of endothelin ETA receptors is superior to nonselective ET A/ET B in attenuating hypertension and survival rate in Ren-2 transgenic rats (TGR). In the present pilot study, we were interested in whether similar effects will be found in TGR with inducible malignant hypertension (iTGR; official strain name Cyp1A1-Ren-2rats), which were derived from the original Ren-2 transgenic rat strain. Studies were performed in three-month old male iTGR. Treatment with either bosentan, a non-selective ET A/ET B, or with atrasentan, a selective ET A receptor blocker, was started on day 2 of the experiment. Feeding with indole-3-carbinole (I3C; 0.3% in rat chow), a natural xenobiotic which activates the Cyp1a1 promoter of the mouse Ren-2 gene, began on day 3 and lasted for 4 days until day 6. Systolic BP, body weight, plasma ANG II and tissue ANG II and ET-1 concentrations were determined daily. Severe hypertension developed as early as 1 day after beginning of I3C feeding which was accompanied by a significant reduction in body weight and by increases in plasma and tissue ANG II and left ventricle ET-1 concentrations. Atrasentan or bosentan had no effects on the rise in BP or plasma and tissue ANG II concentrations but prevented the rise in heart ventricle ET-1 concentration. Our data show that blockade of the ET system does not prevent or attenuate the rapid development of severe hypertension in iTGR; a long-term protective effect of ET blockade on cardiac (and renal) damage, however, cannot be excluded and awaits further investigations.

Mechanisms operated by endothelin ETA and ETB receptors in the trigeminal ganglion contribute to orofacial thermal hyperalgesia induced by infraorbital nerve constriction in rats

Endothelins, acting through specific endothelin ET(A) and/or ET(B) receptors, participate in nociceptive processing in models of cancer, inflammatory and neuropathic pain. The present study investigated which cell types express endothelin receptors in the trigeminal ganglion, and the contribution of mechanisms mediated by endothelin ET(A) and ET(B) receptors to orofacial heat hyperalgesia induced by unilateral constriction of the infraorbital nerve (CION). Both receptor types were identified by immunohistochemistry in the trigeminal ganglion, ET(A) receptors on small-sized non-myelinated and myelinated A-fibers and ET(B) receptors on both satellite glial cells and small-sized non-myelinated neuronal cells. CION promoted ipsilateral orofacial heat hyperalgesia which lasted from Day 2 until Day 10 after surgery. Ongoing CION-induced heat hyperalgesia (on Day 4) was reduced transiently, but significantly, by systemic or local treatment with antagonists of endothelin ET(A) receptors (atrasentan, 10 mg/kg, i.v.; or BQ-123, 10 nmol/lip), endothelin ET(B) receptors (A-192621, 20 mg/kg, i.v.; or BQ-788, 10 nmol/ lip), or of both ET(A)/ET(B) receptors (bosentan, 10 mg/kg, i.v.; or BQ-123 plus BQ-788, each at 10 nmol/lip). On the other hand, CION-induced heat hyperalgesia was transiently abolished over the first 90 min following i.p. injection of morphine hydrochloride (2.5 mg/kg), but fully resistant to reversal by indomethacin (4 mg/kg, i.p.) or celecoxib (10 mg/kg, i.p.). Thus, heat hyperalgesia induced by CION is maintained, in part, by peripheral signaling mechanisms operated by ET(A) and ET(B) receptors. Endothelin receptors might represent promising therapeutic targets for the control of trigeminal neuropathic pain.

Response to Endothelin-1 in Arteries from Human Colorectal Tumours: Role of Endothelin Receptors

To examine the reaction of tumour arteries to endothelin-1, we obtained arteries supplying blood flow to colorectal tumours from patients, as well as mesenteric arteries supplying the normal colon tissue from the same patients and mesenteric arteries from patients without a colorectal tumour pathology. The contraction in response to endothelin-1 and the relaxation produced by bradykinin was recorded in each of these arteries. Accordingly, the sensitivity to endothelin-1 but not the maximal response, was higher in the arteries supplying colorectal tumours than in mesenteric arteries supplying normal colon or in mesenteric arteries from patients with no tumour pathology. The contraction produced by endothelin-1 was not modified by exposure to L-NAME or meclofenamate in arteries supplying both the tumour and the normal colon. The endothelin ET(A) andET(B) receptors were expressed similarly in arteries supplying the tumour or normal colon. However, the antagonist of the endothelin ET(B) receptors BQ788 (10(-6) M) decreased the contractions in the arteries supplying the tumour but not in those supplying the normal colon. By contrast, the antagonist of endothelin ET(A) receptors BQ123 (10(-6) M) reduced the contraction equally in both these types of arteries. Likewise, in arteries precontracted with U46619, the relaxation in response to bradykinin was similar in all three types of arteries. Together, these results suggest that the arteries supplying human colorectal tumours are more sensitive to endothelin-1, which could be due to the enhanced activity of endothelin ET(B) receptors in the absence of any change in the modulatory effect of nitric oxide or prostanoids in the arterial response to this peptide.

Targeting endothelin ETA and ETB receptors inhibits antigen-induced neutrophil migration and mechanical hypernociception in mice

Endothelin may contribute to the development of inflammatory events such as leukocyte recruitment and nociception. Herein, we investigated whether endothelin-mediated mechanical hypernociception (decreased nociceptive threshold, evaluated by electronic pressure-meter) and neutrophil migration (myeloperoxidase activity) are inter-dependent in antigen challenge-induced Th1-driven hind-paw inflammation. In antigen challenge-induced inflammation, endothelin (ET) ET(A) and ET(B) receptor antagonism inhibited both hypernociception and neutrophil migration. Interestingly, ET-1 peptide-induced hypernociception was not altered by inhibiting neutrophil migration or endothelin ET(B) receptor antagonism, but rather by endothelin ET(A) receptor antagonism. Furthermore, endothelin ET(A), but not ET(B), receptor antagonism inhibited antigen-induced PGE(2) production, whereas either selective or combined blockade of endothelin ET(A) and/or ET(B) receptors reduced hypernociception and neutrophil recruitment caused by antigen challenge. Concluding, this study advances knowledge into the role for endothelin in inflammatory mechanisms and further supports the potential of endothelin receptor antagonists in controlling inflammation.

Enhanced expressions of microvascular smooth muscle receptors after focal cerebral ischemia occur via the MAPK MEK/ERK pathway

BackgroundMEK1/2 is a serine/threonine protein that phosphorylates extracellular signal-regulated kinase (ERK1/2). Cerebral ischemia results in enhanced expression of cerebrovascular contractile receptors in the middle cerebral artery (MCA) leading to the ischemic region. Here we explored the role of the MEK/ERK pathway in receptor expression following ischemic brain injury using the specific MEK1 inhibitor U0126.Methods and resultRats were subjected to a 2-h middle cerebral artery occlusion (MCAO) followed by reperfusion for 48-h and the ischemic area was calculated. The expression of phosphorylated ERK1/2 and Elk-1, and of endothelin ETA and ETB, angiotensin AT1, and 5-hydroxytryptamine 5-HT1B receptors were analyzed with immunohistochemistry using confocal microscopy in cerebral arteries, microvessels and in brain tissue. The expression of endothelin ETB receptor was analyzed by quantitative Western blot. We demonstrate that there is an increase in the number of contractile smooth muscle receptors in the MCA and in micro- vessels within the ischemic region. The enhanced expression occurs in the smooth muscle cells as verified by co-localization studies. This receptor upregulation is furthermore associated with enhanced expression of pERK1/2 and of transcription factor pElk-1 in the vascular smooth muscle cells. Blockade of transcription with the MEK1 inhibitor U0126, given at the onset of reperfusion or as late as 6 hours after the insult, reduced transcription (pERK1/2 and pElk-1), the enhanced vascular receptor expression, and attenuated the cerebral infarct and improved neurology score.ConclusionOur results show that MCAO results in upregulation of cerebrovascular ETB, AT1 and 5-HT1B receptors. Blockade of this event with a MEK1 inhibitor as late as 6 h after the insult reduced the enhanced vascular receptor expression and the associated cerebral infarction.

Endothelin A receptor antagonist modulates lymphocyte and eosinophil infiltration, hyperreactivity and mucus in murine asthma

Levels of endothelins are particularly high in the lung, and there is evidence that these peptides are involved in asthma. Asthma is a chronic inflammatory disease associated with lymphocyte infiltration. In the present study, we used a murine model of asthma to investigate the role of endothelins in lymphocyte and eosinophil infiltration into the airway hyperreactivity and mucus secretion. Sensitized C57Bl/6 mice were treated with endothelin ETA receptor antagonist (BQ123) or endothelin ETB receptor antagonist (BQ788) 30 min before an antigen aerosol challenge. After 24 h, dose response curves to methacholine were performed in isolated lungs, FACS analysis of lymphocytes and eosinophil counts were performed in bronchoalveolar lavage fluid and mucus index was determined by histopathology. In sensitized and antigen-challenged mice there is a marked increase in the T CD4+, T CD8+, B220+, Tγδ+ and NK1.1+ lymphocyte subsets. Treatment with BQ123 further increased these cell populations. The number of eosinophils, airway hyperreactivity and mucus were all reduced by BQ123 treatment. The BQ 788 had no significant effect on the parameters analyzed. Treatment with BQ123 reduced the endothelin concentration in lung homogenates, suggesting that endothelins exert a positive feedback on their synthesis. We show here that in murine asthma the ETA receptor antagonist up-regulates lymphocyte infiltration and reduces eosinophils, hyperreactivity and mucus.

Up-regulation of endothelin type B receptors in the human internal mammary artery in culture is dependent on protein kinase C and mitogen-activated kinase signaling pathways

BackgroundUp-regulation of vascular endothelin type B (ETB) receptors is implicated in the pathogenesis of cardiovascular disease. Culture of intact arteries has been shown to induce similar receptor alterations and has therefore been suggested as a suitable method for, ex vivo, in detail delineation of the regulation of endothelin receptors. We hypothesize that mitogen-activated kinases (MAPK) and protein kinase C (PKC) are involved in the regulation of endothelin ETB receptors in human internal mammary arteries.MethodsHuman internal mammary arteries were obtained during coronary artery bypass graft surgery and were studied before and after 24 hours of organ culture, using in vitro pharmacology, real time PCR and Western blot techniques. Sarafotoxin 6c and endothelin-1 were used to examine the endothelin ETA and ETB receptor effects, respectively. The involvement of PKC and MAPK in the endothelin receptor regulation was examined by culture in the presence of antagonists.ResultsThe endohtelin-1-induced contraction (after endothelin ETB receptor desensitization) and the endothelin ETA receptor mRNA expression levels were not altered by culture. The sarafotoxin 6c contraction, endothelin ETB receptor protein and mRNA expression levels were increased after organ culture. This increase was antagonized by; (1) PKC inhibitors (10 μM bisindolylmaleimide I and 10 μM Ro-32-0432), and (2) inhibitors of the p38, extracellular signal related kinases 1 and 2 (ERK1/2) and C-jun terminal kinase (JNK) MAPK pathways (10 μM SB203580, 10 μM PD98059 and 10 μM SP600125, respectively).ConclusionIn conclusion, PKC and MAPK seem to be involved in the up-regulation of endothelin ETB receptor expression in human internal mammary arteries. Inhibiting these intracellular signal transduction pathways may provide a future therapeutic target for hindering the development of vascular endothelin ETB receptor changes in cardiovascular disease.

Endothelin ETB receptor heterodimerization: beyond the ETA receptor

The idea that endothelin ETA and ETB receptors may form homodimers and heterodimers has gained increasing interest in recent years. The existence of such interactions between endothelin receptors has the potential to explain some puzzling results from receptor binding and functional studies. Zeng and colleagues take ETB receptor heterodimerization beyond the ETA receptor, reporting renal endothelin ETB-dopamine D(3) receptor interactions at the cellular level that appear to have functional consequences in vivo.

Venous endotelin-1 (ET-1) and brain natriuretic peptide (BNP) plasma levels during 6-month bosentan treatment for pulmonary arterial hypertension

Objective Bosentan, an endothelin (ET) ETA–ETB receptors antagonist, is an effective therapy for idiopathic pulmonary arterial hypertension (PAH) and for PAH related to connective tissue disease (CTD). The aim of this study was to evaluate the behaviour of ET-1 and brain natriuretic peptide (BNP) venous plasma levels during a 6-month dual ET-1 receptor blockade and the potential influence of baseline ET-1 venous plasma levels on the clinical efficacy of bosentan. Setting and patients Twenty-five patients with PAH (idiopathic n = 16, CTD n = 9) in WHO functional class II–III were included in this study. After initial evaluation, patients' WHO class, 6-minute walking-test (6MWT), ET-1 and BNP venous plasma levels were assessed at baseline and after 6-month bosentan therapy. To evaluate whether the ET-1 levels could influence the clinical response to bosentan, data were analyzed for the whole population which was stratified according to high and low ET-1 plasma levels (on the basis of the baseline median value of ET-1 plasma: Gr.1 < 18.7 pg/ml, Gr.2 > 18.7 pg/ml). Results Study population included patients with moderate–severe PAH. After 6-month of treatment we observed a significant increase in 6MWT distance (from 435 ± 85) m to 467 ± 77 m, p > 0.001) and an improvement in WHO class (from 2.4 ± 0.5 to 2 ± 0.6 p > 0.01), with a significant decrease in BNP (from 87 ± 33 pg/ml to 67 ± 41 pg/ml, p = 0.006) and a trend towards lower ET-1 plasma levels (from 17.7 ± 5 pg/ml to 16 ± 6 pg/ml, p = ns). Improvement in effort tolerance (Δ distance) was not correlated to modification in ET-1 (ΔET-1) and BNP (ΔBNP) plasma levels, while we found a significant correlation between ΔET-1 and ΔBNP ( r = 0.63, p = 0.0006). Analyzing the subpopulation, Gr.2 patients were older (Gr.1: 41 ± 10 years vs Gr.2: 50 ± 9 years, p = 0.04), had less effort capacity (6MWT distance, Gr.1: 469 ± 76 m, vs Gr.2: 398 ± 82 m, p = 0.03), and showed a trend towards higher BNP values (Gr.1: 82 ± 41 pg/ml vs Gr.2: 92 ± 23 pg/ml, p = 0.051), but no significant differences in pulmonary hemodynamics. After the 6-month treatment both groups showed a significant improvement in 6MWT (Gr.1: + 32 ± 24 m, Gr.2: + 32 ± 21 m p = 0.05) without differences between groups. WHO class had a trend towards lower class (Gr.1: − 0.5 ± 0.5, Gr.2: − 0.3 ± 0.4 p = 0.15) in both groups. BNP plasma levels showed a significant decrease only in Gr.2 (Gr.1: − 6 ± 41 pg/ml, Gr.2: − 34 ± 19 pg/ml p = 0.02); similarly ET-1 plasma levels showed a trend towards a decrease only in Gr.2 (Gr.1: 0.2 ± 4.6 pg/ml, Gr.2: − 3.8 ± 6.6 pg/ml p = 0.09). Conclusions Our data confirm that bosentan is an effective therapy for patients with PAH. Its clinical efficacy (effort tolerance and NYHA) seems to be independent from baseline venous ET1 plasma levels. Bosentan therapy seems to elicit different patterns in ET-1 and BNP plasma levels, with decrease of the peptides only in patients with higher activation of the systemic endothelin system. Further studies are warranted to explore the potential impact of baseline ET-1 levels on the long-term effects (clinical worsening) of bosentan therapy.

Fluorescence Correlation Spectroscopy (FCS) a powerful biophysic method for drug discovery: study of Alexa532-endothelin 1 binding on the endothelin ETA receptor on living cells

Fluorescence Correlation Spectroscopy (FCS) a powerful biophysic method for drug discovery: study of Alexa532-endothelin 1 binding on the endothelin ETA receptor on living cells

Nonselective ETA/ETB-receptor blockade increases systemic blood pressure of Bio 14.6 cardiomyopathic hamstersThis article is one of a selection of papers published in the special issue (part 1 of 2) on Forefronts in Endothelin.

To examine the role of endothelin ETA and ETB receptors in congestive heart failure due to cardiomyopathy, the effect of chronic treatment with selective ETA- and ETB-receptor antagonists (atrasentan and A-192621, respectively), alone and in combination, was assessed on functional and biochemical parameters of 52-week-old Bio 14.6 cardiomyopathic hamsters. Compared with control animals, cardiomyopathic hamsters treated for 9 weeks with atrasentan showed no variation in MAP; however, selective ETB- and combined nonselective ETA- and ETB-receptor antagonists increased systemic blood pressure. After selective ETB-receptor blockade, plasma endothelin levels were augmented. Importantly, this increase was highly enhanced (more than 8-fold) by concomitant ETA-receptor antagonism. Furthermore, the left ventricle:body weight ratio of cardiomyopathic hamsters treated with A-192621, alone or in combination with atrasentan, was significantly increased. On the other hand, decreased left ventricular end-diastolic pressure was observed in cardiomyopathic hamsters after selective ETA- or combined nonselective ETA/ETB-receptor antagonism, while only selective ETA-receptor blockade reduced left ventricular endothelin levels. Our results suggest that, in congestive heart failure, ETB receptors are essential to limit circulating endothelin levels, which may argue for improved cardiac benefits after long-term treatment with highly selective ETA-receptor antagonists.

In vitro and in vivo effects of endothelin-1 and YM598, a selective endothelin ETA receptor antagonist, on the lower urinary tract

We investigated the contractile response of the lower urinary tract to endothelin-1 in vitro (rabbits) and in vivo (dogs). We also assessed the effects of a selective endothelin ETA receptor antagonist, (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2, 2′-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598), on endothelin-1-induced contractile responses. In the in vitro study, endothelin-1 induced contractile responses in isolated rabbit bladder base, urethra, and prostate tissues. YM598 (10−7–10−5 M) antagonized these endothelin-1-induced contractile responses without affecting the maximal responses. In the in vivo study, endothelin-1 induced the elevation of non-prostatic urethral pressure as well as prostatic urethral pressure even in the presence of tamsulosin (10 μg/kg, i.v.) in anesthetized male dogs. YM598 (0.1–3 mg/kg, i.v.) inhibited these endothelin-1-induced contractile responses in a dose-dependent fashion. These results suggest that endothelin ETA receptors play an important role in the lower urinary tract contraction, and that the selective endothelin ETA receptor antagonist YM598 has ameliorating effects on various urinary dysfunctions, including benign prostatic hyperplasia.

Abstract 2301: Agonistic Autoantibodies Against the Endothelin 1 ETA - and α1-Adrenergic- Receptor in the Sera of Patients with Idiopathic Pulmonary Arterial Hypertension.

Background: Idiopathic pulmonary arterial hypertension (IPAH) is a progressive fatal disease of unknown cause. This disease is accompanied by an massive increase of pulmonary vascular resistance (remodelling and increase of vascular tonus) inducing right heart failure. It was shown that the synthesis of vasoconstrictors (endothelin 1, thromboxane A2, serotonine) is elevated in patients with IPAH. Methods: We analyzed sera of patients with PH with regards to functional autoantibodies (AABs) against G-protein coupled receptors using spontaneously beating rat cardiomyocytes as bioassay. Moreover, we investigated the effect of the AAB on the translocation of the transcription factors NFκB and AP-1. Results: The sera of patients with IPAH contain functional AABs against the α1-adrenergic (α1-AR) and the endothelin1 ETA receptor. We purified the AABs by affinity chromatography. AABs against the α1-AR exert a positive chronotropic effect. AABs against the ETA receptor induce like endothelin 1 a negative chronotropic effect. The agonistic effect of the AABs was dose-dependent and blocked by prazosine (α1-AR blocker) and BQ610 (ETA antagonist), respectively. Moreover, the agonistic effect of the AABs was neutralized by peptides corresponding to the second extracellular loop of both receptors. Therefore, the epitopes of the AABs are localized on the second extracellular loops of the receptors. The α1-AR AABs as well as the ETA-receptor AABs induce a permanent stimulation without desensitization of the receptor mediated signal cascade. The α1-adrenergic agonist phenylephrine and endothelin1 as ET receptor agonist cause a translocation of the transcription factor NFκB and AP-1 from the cytosol into the nucleus in cultured neonatal rat cardiomyocytes. The same translocation was observed using the AABs against the α1-AR and ETA receptor. Conclusion: The agonist-like AABs against the α1-AR and the ETA receptor influence in vitro the signalling of cultured cells. Moreover, the AABs prevent the desensitization of the receptor mediated signal cascade normally seen by ongoing receptor stimulation. Therefore, we assumed that the functional AABs against the α1-AR and the ETA receptor may be involved in the pathogenesis of IPAH.

The effects of TBC3214, a selective endothelin ETA receptor antagonist, on orthodontic tooth movement in rats

Many chemical messengers are involved in the process of alveolar bone and periodontal ligament remodelling during orthodontic tooth movement. Among them is probably endothelin-1 (ET-1). Its role in this process has been partly explained using tezosentan, which affects endothelin A (ET(A)) and endothelin B (ET(B)) receptors. Tezosentan enhances orthodontic tooth movement. The aim of this study was to determine the possible effects of a highly selective ET(A) antagonist on orthodontic tooth movement in rats. Thirty male Wistar rats, 11-12 weeks of age, divided into three equal groups. In group I, a closed-coil spring was used and they were treated daily with 15 mg/kg body weight of TBC3214, a highly selective ET(A) antagonist. A closed-coil spring was also used in group II and the animals were treated daily with a placebo. Group III were treated daily with a placebo. The coil spring delivered a force of 25 cN and was attached between the upper left first molar and upper left incisor. The distance between the teeth was measured with a digital calliper (accuracy +/- 0.01 mm) on days 0, 7, 14, 21, 24, 32, 37, and 40. The differences in the distance between the teeth were calculated to determine the amount of tooth movement. Statistical analysis was performed using two-way analysis of variance, Bonferroni's correction, and paired t-tests. The distance between the upper left first molar and the upper left incisor decreased in groups I and II. In group I, tooth movement was significantly less on days 32 and 37 (P < 0.01) and on day 40 (P < 0.001) compared with group II. In group III, the distance between the teeth increased during the study (P < 0.001). In animals treated daily with TBC3214, tooth movement was significantly less compared with the animals treated with a placebo. It is concluded that ET-1, which is the predominant form of endothelin isopeptides, is involved in orthodontic tooth movement in rats, probably by enhancing bone resorption via ET(A) receptors.

Altered endothelin receptor subtypes in colorectal cancer

The vasoactive peptide endothelin-1 (ET-1) acts via two endothelin receptor subtypes, ETA (ETAR) and ETB (ETBR). ET-1 and ETAR are overexpressed in colorectal cancer tissues. In vitro, ET-1 acting via ETAR, is a mitogen for colorectal cancer cells. To identify other potential stimulatory loops, we investigated the distribution and cell-specific localization of both ETAR and ETBR in tissue sections from patients with colorectal cancer. Frozen sections from specimens of colorectal cancer (n=9) and normal colon (n=9) were cut and subjected to either (i) autoradiography or (ii) a combination of cell type-specific immunohistochemistry, using antibodies against fibroblasts (AS02), endothelial cells (CD31) or nerve fibres (NF200) and in-vitro receptor microautoradiography, using ETAR-specific and ETBR-specific radioligands. ETARs were upregulated in all cell types, apart from nerve, in cancer compared with normal colon (1:1.59 normal to cancer). Specifically, ETAR binding was highest in cancer-associated blood vessels and fibroblasts and to a lesser extent in epithelial cancer cells. In contrast, ETBRs were the predominant receptors in normal colon (1:0.59 normal to cancer) and were markedly down-regulated in cancer-associated blood vessels, fibroblasts and to a lesser extent in epithelial cells. Nerve colocalization was demonstrated, but remained unchanged for all tissues. The shift in ET receptor binding observed in epithelial cancer cells and cancer-associated fibroblasts and endothelial cells may favour ET-1 signals contributing to colorectal cancer growth and neovascularization via ETAR. This may provide the basis for therapeutic use of specific ETAR antagonists as adjuvant treatment of colorectal cancer.

Engineered Sarafotoxins as Tissue Inhibitor of Metalloproteinases-like Matrix Metalloproteinase Inhibitors

The sarafotoxins and endothelins are approximately 25-residue peptides that spontaneously fold into a defined tertiary structure with specific pairing of four cysteines into two disulfide bonds. Their structures show an interesting topological similarity to the core of the metalloproteinase interaction sites of the tissue inhibitors of metalloproteinases. Previous work indicates that sarafotoxins and endothelins can be engineered to eliminate or greatly reduce their vasopressive action and that their structural framework can withstand multiple sequence changes. When sarafotoxin 6b, which possesses modest matrix metalloproteinase inhibitory activity, was C-terminally truncated to remove its toxic vasopressive activity, the metalloproteinase inhibitory activity was essentially abolished. However, further changes, based on the sequences of peptides selected from libraries of sarafotoxin variants or suggested by analogy with tissue inhibitors of metalloproteinases, progressively enhanced the matrix metalloproteinase inhibitory activity. Peptide variants with multiple substitutions folded correctly and formed native disulfide bonds. Improvements in matrix metalloproteinase affinity have generated a peptide with micromolar K(i) values for matrix metalloproteinase-1 and -9 that are selective inhibitors of different metalloproteinases. Characterization of its solution structure indicates a close similarity to sarafotoxin but with a more extended C-terminal helix. The effects of N-acetylation and other changes, as well as docking studies, support the hypothesis that the engineered sarafotoxins bind to matrix metalloproteinases in a manner analogous to the tissue inhibitors of metalloproteinases.

Antagonism of endothelin action normalizes altered levels of VEGF and its signaling in the brain of stroke-prone spontaneously hypertensive rat

Stroke-prone spontaneously hypertensive rats (SHRSP) often suffer from spontaneous stroke, in part, due to abnormalities in the cerebrovasculature. Here, we investigate the profile of key angiogenic factors and their basic signaling molecules in the brain of SHRSP during the age-dependent stages of hypertension. The profile of VEGF and its receptor, Flk-1, was dependent on age and stage of hypertension (i.e., down regulated at pre-hypertensive and malignant hypertensive stages, but up regulated at typical hypertensive stage), while that of its downstream components, pAkt and eNOS, were down regulated in a time-dependent manner in the frontal cortex of SHRSP compared to age-matched genetic control, normotensive WKY rats. On the other hand, the expression of endothelin-1 and its type A receptor (endothelin ETA receptor) were up regulated, depending on age and stage of hypertension. In contrast, levels of endothelin type B receptor were down regulated. The regional cerebral blood flow decreased during the development of malignant hypertension. Thus, subsequent experiments were designed to investigate whether endothelin-1 receptor antagonism, using endothelin-A/-B dual receptor antagonist SB209670, could normalize the molecular profile of these factors in SHRSP brain. Interestingly, blockage of endothelin-1 receptor restored to normal, levels of cerebral endothelin-1, endothelin ETA receptor and endothelin ETB receptor; VEGF and Flk-1; endothelial nitric oxide synthase (eNOS) and pAkt, in SHRSP, compared to age-matched WKY. Endothelin receptor blocker might be important to prevent the progression in the defect in VEGF and its angiogenic signaling cascade in the pathogenesis of hypertension-induced vascular remodeling in frontal cortex of SHRSP rats.

Endothelin receptors as drug targets in chronic cardiovascular diseases: the rationale for dual antagonism

AbstractEndothelin has been described as the most potent vasoconstrictor known. Recent research shows that it is also a growth factor, a promoter of fibrosis and inflammation, and a key initiator of endothelial dysfunction. Endothelin becomes, therefore, a candidate in the pathogenesis of many chronic cardiovascular and fibrotic diseases, and inhibition of endothelin function is a potential therapeutic approach for those diseases. Endothelin receptor antagonists are now established therapy in the treatment of pulmonary arterial hypertension, and present promising perspectives in diabetic nephropathy, peripheral arterial disease, hypertension, heart failure, and pulmonary diseases. Endothelin binds to two endothelin receptors, ETA and ETB, and their respective roles are actively debated. A major challenge lies in the understanding of what is the preferred profile for new drugs: selective ETA antagonism or dual ETA and ETB receptor antagonism. A number of cardiovascular diseases are characterized by an up‐regulation of smooth muscle cell ETB receptors mediating vasoconstriction and proliferation, and a down‐regulation of endothelial ETB receptors, as compared to the physiological state, creating a situation where both ETA and ETB receptors contribute to vasoconstriction and remodeling. When these two receptor subtypes co‐exist, a cross‐talk between them, probably a result of receptor heterodimerization, suggests that dual ETA/ETB receptor antagonism may be necessary to obtain maximal efficacy. Drug Dev. Res. 67:825–834, 2006. © 2007 Wiley‐Liss, Inc.