Specific Endothelin Receptor Research Articles

CT perfusion imaging of cerebral microcirculatory changes following subarachnoid hemorrhage in rabbits: Specific role of endothelin-1 receptor antagonist

BackgroundCerebral vasospasm may lead to delayed ischemic neurological deficits following subarachnoid hemorrhage (SAH). Endothelin (ET-1) is an important factor participating in cerebral vasospasm underlying SAH. We used a specific endothelin receptor antagonist, BQ123 to assess the specific role of endothelin-1 receptor antagonist in cerebral vasospasm in a rabbit model of SAH by examining plasma ET-1 levels and the principal CT perfusion (CTP) parameters pertinent to the hemodynamic status of microcirculation following SAH. Methods102 male New Zealand white rabbits were divided into control, SAH and SAH + BQ123 intervention group (BQ123 group). Rabbit SAH model was established by double hemorrhage injection of autologous blood into the cisterna magna; Aquilion ONE was used to collect cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT) which were used to evaluate cerebral microcirculation hemodynamics; Elisa was used to assess plasma ET-1 levels. Data were collected on days 1, 4, 7 and 14 following SAH, respectively. ResultsCompared with the control group, the CBF in the SAH group was significantly lower, while the MTT was significantly higher. The CBF decreased on the 4th day and reached the lowest on the 7th day. The MTT began to rise on the 4th day and peaked on the 7th day. While in the BQ123 intervention group, the CBF significantly increased while the MTT significantly decreased on the 1st and the 4th days, respectively. Compared with SAH group, plasma ET-1 levels in BQ123 group significantly increased on the earlier (1st and 4th days) but not later days (between the 7th and 14th days). In addition, the inflammatory infiltration of brain tissues in rabbits treated with BQ123 post-SAH was significantly reduced compared with SAH group. ConclusionCTP can quantify the therapeutic effect of BQ123 after SAH; Selective blockade of ET-1 endothelin receptor, BQ123 significantly improved microcirculatory perfusion along with a reduction in resultant vasogenic inflammatory responses. The effect of BQ123 on the cerebral microcirculation was lobe dependent.

Effect of a specific endothelin receptor A antagonist on murine lupus nephritis and IgA nephropathy.

Effect of a specific endothelin receptor A antagonist on murine lupus nephritis and IgA nephropathy.

Aspirate from human stented native coronary arteries vs. saphenous vein grafts: more endothelin but less particulate debris

Stent implantation into atherosclerotic coronary arteries releases particulate debris and soluble substances that contribute to impaired microvascular perfusion. Here we addressed the potential for microvascular obstruction in patients with stenotic native right coronary arteries (nRCA) compared with saphenous vein grafts on right coronary arteries (SVG-RCA). We enrolled symptomatic, male patients with stable angina pectoris and a flow-limiting stenosis in their nRCA or SVG-RCA (n = 18/18). Plaque volume and composition were analyzed using intravascular ultrasound before stent implantation. Coronary aspirate was retrieved during stent implantation under protection with a distal occlusion/aspiration device and divided into particulate debris and plasma. The release of catecholamines, endothelin, serotonin, thromboxane B2, and tumor necrosis factor-α was measured. The response of rat mesenteric arteries with intact (+E) and denuded (-E) endothelium to aspirate plasma (without and with selective endothelin receptor blockade) was normalized to that by potassium chloride (KClmax = 100%). Plaque volume and composition were not different between nRCA and SVG-RCA. There was less particulate debris (65 ± 8 vs. 146 ± 23 mg; P < 0.05) and more endothelin release (5.8 ± 0.8 vs. 1.3 ± 0.7 pg/ml; P < 0.05) in nRCA than in SVG-RCA, whereas the release of the other mediators was not different. Aspirate from nRCA induced stronger vasoconstriction than that from SVG-RCA [nRCA, 78 ± 6% (+E)/84 ± 5% (-E); SVG-RCA, 59 ± 6% (+E)/68 ± 3% (-E); P < 0.05 nRCA vs. SVG-RCA], which was attenuated by a nonspecific endothelin and a specific endothelin receptor A antagonist. Thus coronary aspirate from stented nRCA is characterized by less debris but more endothelin and stronger vasoconstrictor response than that from SVG-RCA.

Regulation of Endothelin-1-Mediated Reactive Oxygen Species Production by Protein Disulfide Isomerase in Endothelial Cells: Role in Sickle Cell Disease

AbstractAbstract 1073Endothelial cell activation and increased levels of endothelin-1 (ET-1) have been proposed as important contributors to vaso-occlusion in the pathophysiology of Sickle Cell Disease (SCD). We have reported a role for ET-1 receptor antagonists in improving hematological complications of two transgenic mouse models of SCD in vivo (Rivera A., 2008, Amer J Physiol). Activation of endothelial cells leads to, among other factors, increased levels of reactive oxygen species (ROS) and protein disulfide isomerase (PDI). PDI mediates redox modifications, catalyzes disulfide interchange reactions in the plasma membrane and is up-regulated under hypoxic conditions as commonly observed in SCD. We tested the hypothesis that ET-1 would regulate PDI activity in endothelial cells. We now report on the detection of PDI and ET-1 receptor B by western blot analyses in membranes from the human endothelial cell line, EA.hy926 (EA). We studied the effects of ET-1 on cellular ROS production by fluorimetry of DCFDA-AM loaded EA cells and observed that 10−7 M ET-1 for 60 mins led to significant increases in cellular ROS production (1720±78 vs 1447±86 Relative Fluorescent Units (RFU), P<0.001, n=6) that were significantly blocked by ET-1 receptor B antagonist, 10−6 M BQ-788 (1720±78 vs 1426±55 RFU, P<0.0007, n=3). We also tested the effects of the specific ET-1 receptor B agonist, 10−7 M BQ-3020 on cellular ROS production and observed similar results (1748±194 vs 1447±86 RFU, P<0.02, n=3). We then studied the effect of a well-described PDI inhibitor, monoclonal antibodies to PDI (RL90), on ET-1 stimulated ROS production and showed that RL90 likewise blocked ET-1 stimulated ROS production in EA cells (P<0.001, n=3), thus suggesting a role for PDI in ET-1-stimulated ROS production. To further explore the role of PDI on ROS production, we employed a molecular approach and show that siRNA against PDI likewise led to reduced ET-1-stimulated ROS production (P<0.0001, n=2) that was associated with significantly reduced PDI mRNA levels in siRNA transfected cells, but not when cells were transfected with scrambled siRNA as determined by quantitative RT-PCR with ABI TaqMan detection probes and GAPDH and β2 microglobulin as endogenous controls. We then optimized conditions to measure PDI activity using fluorescently labeled GSSG conversion to GSH in supernatants of ET-1-stimulated cells. We observed that incubation of EA cells for 60 mins with 10−7 M ET-1 was associated with increased extracellular PDI activity (232±183 to 664±94 (RFU), n=3, P<0.02) that was blunted by PDI siRNA knockdown (1731±147 to 757±141 RFU, n=2) when compared to scrambled siRNA transfected cells. Consistent with these data we observed increases in PDI protein levels in supernatants of EA cells stimulated with ET-1 by ELISA. Moreover, incubation of EA cells for 12 hr with 10−8 M ET-1 led to increases in cell-associated PDI levels by western blot analyses. We then tested the in vivo effects of ET receptor antagonist on plasma PDI activity in the BERK mouse model of SCD. We blocked ET-1 receptors in vivo by treatment with BQ-788 and BQ-123 (360mg/Kg/Day) for 14 days and observed a reduction of plasma PDI activity when compared to vehicle treatment (67.7±3 to 34.3±6, RFU, P<0.03, n=3). We also characterized the effects of ET-1 on PDI expression in EA cells, using quantitative RT-PCR with ABI TaqMan probes and GAPDH and β-actin as endogenous controls and observed that stimulation of EA cells with 10−8 M ET-1 for 4 hr was associated with increased PDI mRNA expression levels that were 1.89 fold greater than vehicle treated cells (n=6, P<0.04). Thus, our results provide evidence to suggest that endothelin-1 receptor blockade leads to improved measures of ROS via regulation of PDI and thus implicate PDI as a novel regulator of Sickle Cell Disease pathophysiology. Disclosures:No relevant conflicts of interest to declare.

Hepatic Transaminase Abnormalities in Pulmonary Arterial Hypertension: A Report From the REVEAL Registry

PURPOSE: The Registry to EValuate Early And Long-term Pulmonary Arterial Hypertension (PAH) Disease Management (REVEAL), a 55-center observational, US study, examines current clinical practice in patients with PAH. We assessed aspartate transaminase/alanine transaminase (AST/ALT) elevation frequencies in patients by PAH-specific medication class, and by specific endothelin receptor antagonist (ERA) due to ERAs’ association with AST/ALT abnormalities.

Collecting duct-specific endothelin B receptor knockout increases ENaC activity

Collecting duct (CD)-derived endothelin-1 (ET-1) acting via endothelin B (ETB) receptors promotes Na(+) excretion. Compromise of ET-1 signaling or ETB receptors in the CD cause sodium retention and increase blood pressure. Activity of the epithelial Na(+) channel (ENaC) is limiting for Na(+) reabsorption in the CD. To test for ETB receptor regulation of ENaC, we combined patch-clamp electrophysiology with CD-specific knockout (KO) of endothelin receptors. We also tested how ET-1 signaling via specific endothelin receptors influences ENaC activity under differing dietary Na(+) regimens. ET-1 significantly decreased ENaC open probability in CD isolated from wild-type (WT) and CD ETA KO mice but not CD ETB KO and CD ETA/B KO mice. ENaC activity in WT and CD ETA but not CD ETB and CD ETA/B KO mice was inversely related to dietary Na(+) intake. ENaC activity in CD ETB and CD ETA/B KO mice tended to be elevated under all dietary Na(+) regimens compared with WT and CD ETA KO mice, reaching significance with high (2%) Na(+) feeding. These results show that the bulk of ET-1 inhibition of ENaC activity is mediated by the ETB receptor. In addition, they could explain the Na(+) retention and elevated blood pressure observed in CD ET-1 KO, CD ETB KO, and CD ETA/B KO mice consistent with ENaC regulation by ET-1 via ETB receptors contributing to the antihypertensive and natriuretic effects of the local endothelin system in the mammalian CD.

Retracted Article: Pericyte-mediated vasoconstriction underlies TBI-induced hypoperfusion

Endothelin-1 is a 21-amino acid peptide that together with specific receptors, A (ETrA) and B (ETrB) is induced following traumatic brain injury (TBI) and has been closely linked to regulation of cerebral vasospasm, oxidative stress, and hypoperfusion. Specific endothelin receptor antagonists have been shown to ameliorate early evidence of neuronal cell injury, activation of microglial cells, and hypoperfusion following TBI. The exact mechanism involved in TBI-induced hypoperfusion is still unclear; however, it is thought that endothelin-1 engagement of ETrA is primarily responsible for changes in blood flow. In this study we question the role of the microvascular pericyte in endothelin-1-mediated pathophysiology in TBI. Pericyte expression of endothelin-1, ETrA, and ETrB was examined in primary culture and in sham and impacted rat brain. Adult male rats were also given intracerebroventricular injections of ETrA (BQ-123) before being subjected to TBI using a closed head acceleration impact model. Primary pericytes express both endothelin-1 and its receptors ETrA and ETrB. Following TBI, the number of alpha-smooth muscle actin (SMA) positive pericytes located in microvessels is significantly increased by 4 hours post-traumatic impact. Increases in pericyte expression of alpha-SMA correlated with evidence of a reduction in both arteriolar and capillary diameter. Capillary endothelin-1, ETrA, and ETrB transcript and protein was also increased. Increased endothelin-1 expression was seen by 2-4 hours post-impact. Upregulation of receptors was observed by 4-8 hours and maximum by 24 hours. ETrA antagonists decreased the number of alpha-SMA(+) pericytes as well as changes in microvascular diameter. These results suggest that decreased vasoconstriction following TBI may be due to an endothelin-1-induced pericyte-mediated regulation of microvessel blood flow following TBI. Furthermore, results suggest that ETrA antagonists ameliorate trauma induced hypoperfusion, in part, by inhibiting endothelin-1-mediated upregulation of alpha-SMA in pericytes.

Sex Hormones and Vascular Protection in Pulmonary Arterial Hypertension

Pulmonary Arterial Hypertension (PAH) is a serious disease and a major public health problem with approximately 1000 new patients diagnosed every year in the United States.1, 2 PAH is a progressive disease involving impaired pulmonary vascular structure and function and is ultimately lethal due to right ventricular failure. Recent insights into the pathogenesis of PAH have lead to more promising therapeutic approaches and improved outcomes; however, the mortality rates associated with PAH remain unacceptably high.1, 3, 4

Up-Regulation of Endothelin Type A Receptor in Human and Rat Radiation Proctitis: Preclinical Therapeutic Approach With Endothelin Receptor Blockade

Rectum radiation damage and fibrosis are often associated with radiation therapy of pelvic tumors. The endothelin (ET) system has been implicated in several fibrotic diseases but never studied in the context of gastrointestinal radiation damage. This study assessed modifications in ET type 1 (ET-1), ET type A receptor (ET(A)), and ET type B receptor (ET(B)) localization and/or expression in irradiated human rectal tissue and in a rat model of delayed colorectal injury. We also evaluated the therapeutic potential of long-term ET receptor blockade. Routine histological studies of sections of healthy and radiation-injured human rectum tissue were done; the sections were also immunostained for ET(A) and ET(B) receptors. The rat model involved the delivery of 27 Gy in a single dose to the colons and rectums of the animals. The ET-1/ET(A)/ET(B) expression and ET(A)/ET(B) localization were studied at 10 weeks postexposure. The abilities of bosentan and atrasentan to protect against delayed rectal injury were also investigated. The immunolocalization of ET(A) and ET(B) in healthy human rectums was similar to that in rat rectums. However, strong ET(A) immunostaining was seen in the presence of human radiation proctitis, and increased ET(A) mRNA levels were seen in the rat following colorectal irradiation. Immunostaining for ET(A) was also strongly positive in rats in areas of radiation-induced mucosal ulceration, atypia, and fibroproliferation. However, neither bosentan nor atrasentan prevented radiation damage to the rectum when given long term. The only effect seen for atrasentan was an increased number of sclerotic vessel sections in injured tissues. As the result of the overexpression of ET(A), radiation exposure deregulates the endothelin system through an "ET(A) profile" in the human and rodent rectum. However, therapeutic interventions involving mixed or specific ET(A) receptor blockade do not prevent radiation damage. Further studies are necessary to identify the precise roles of ET in the gastrointestinal response to radiation exposure.

The science of endothelin‐1 and endothelin receptor antagonists in the management of pulmonary arterial hypertension: current understanding and future studies

Pathological vascular remodelling is a key contributor to the symptomatology of pulmonary arterial hypertension (PAH), and reversing this process may offer the best hope for improving this debilitating condition. The vascular remodelling process is believed to be due to endothelial cell dysfunction and to involve altered production of endothelial cell-derived vasoactive mediators. The observation that circulating plasma levels of the vasoactive peptide endothelin (ET)-1 are raised in patients with PAH, and that ET-1 production is increased in the pulmonary tissue of affected individuals, makes it a particularly interesting target for a therapeutic intervention in PAH. Clinical trials with ET receptor antagonists (ETRAs) show that they provide symptomatic benefit in patients with PAH, thereby proving the clinical relevance of the ET system as a therapeutic target. In this paper, we review the role of ET-1 together with the available data on the roles of the specific ET receptors and ETRAs in PAH. In particular, we discuss the possible role of ET receptor selectivity in the vascular remodelling process in PAH and whether selective ET(A) or nonselective ET(A)/ET(B) blockade offers the greatest potential to improve symptoms and alter the clinical course of the disease.

Characterization and localization of the endothelin receptors in human end-stage heart failure: ET A/ET B receptor system—Role in the failing human heart

Endothelin (ET) is a potent vasoconstrictor peptide implicated in numerous human diseases, including ischemic cardiomyopathy (ICM). ET binds to receptors ET A and ET B. Specific ET receptors were characterized in left atria of patients with end-stage heart failure due to ischemic cardiomyopathy (ICM) ( n = 9) and healthy controls ( n = 9). Saturation assays revealed a K d and B max of 28 ± 8 pM and 87 ± 22 fmol mg −1 of protein, respectively, from healthy atria and 50 ± 9 pM and 162 ± 19 fmol mg −1 of protein, respectively, from diseased atria ( p < 0.05). For competition studies, we obtained a percentage of ET A receptors using BQ123, 55% ± 5 and 66% ± 4 in healthy and diseased atria, respectively ( p < 0.05). The percentage of ET B using BQ3020 was 55% ± 14 in healthy and 59% ± 10 in diseased atria. Microautoradiography studies showed a greater number of ET receptors, predominately ET A, existed in the myocardial layer of diseased atria compared with healthy atria. The percentage of occupied area was greater in the endocardium than the epicardium in diseased atria. These results showed an increase in the ET A/ET B receptor system in the failing human heart compared with the non-failing human heart.

Functional and Molecular Characterization of the Endothelin System in Retinal Arterioles

Activation of the endothelin (ET) system has been implicated in the pathogenesis of retinal ischemic disease. Although ET-1, the predominant endogenous isoform of ET, has been shown to cause constriction of retinal vessels, the expression and functional significance of its synthesis and the involved specific ET receptors in retinal arterioles remain unknown. The authors examined the roles of ET(A) and ET(B) receptors and of endothelin-converting enzyme (ECE)-1 in ET-1-induced vasomotor responses of single retinal arterioles. To exclude systemic confounding effects, porcine retinal arterioles were isolated for vasoreactivity and molecular studies. Isolated and pressurized retinal arterioles developed basal tone and constricted in a manner dependent on concentration to ET-1. ET-1 precursor big ET-1 elicited time-dependent vasoconstriction over 20 minutes, which was blocked by the ECE-1 inhibitor phosphoramidon. ET(A) receptor antagonist BQ123 inhibited most (approximately 90%) of vasoconstrictions to ET-1 and big ET-1. ET(B) receptor agonist sarafotoxin also elicited concentration-dependent constriction of retinal arterioles but with significantly less potency than ET-1. ET(B) receptor antagonist BQ788 abolished vasoconstriction to sarafotoxin but only slightly reduced responses to ET-1 and big ET-1. Protein and mRNA expressions of ET(A), ET(B), and ECE-1 were detected in retinal arterioles. Immunohistochemistry revealed ET(A) and ET(B) receptors predominantly in smooth muscle and ECE-1 predominantly in endothelium and smooth muscle. ET-1 elicits constriction of retinal arterioles predominantly through the activation of smooth muscle ET(A) receptors. Endogenous production of ET-1 from vascular ECE-1 is sufficient to evoke ET(A) receptor-dependent constriction in retinal arterioles.

Tezosentan Reduces the Renal Injury Induced by Abdominal Aortic Ischemia-Reperfusion in Rats

Renal injury induced by aortic ischemia-reperfusion (IR) is an important factor in the development of postoperative acute renal failure following abdominal aortic surgery. Endothelin (ET) is involved in the development of renal injury induced by aortic IR and tezosentan (R0 61-0612) is a specific ET receptor antagonist. The aim of this study was to examine the effect of tezosentan on renal injury induced by abdominal aortic IR in rats. Twenty-four Wistar-Albino rats were randomized into three groups (eight per group). Control group underwent laparotomy and dissection of the infrarenal abdominal aorta (IAA) without occlusion. The aortic IR group underwent laparotomy and clamping of the IAA for 120 min followed by 120 min of reperfusion. Aortic IR + tezosentan group underwent same aortic IR periods, and received a bolus intravenous injection of 10 mg/kg tezosentan before ischemia plus continuous intravenous infusion of 1 mg/kg/h tezosentan during 120 min ischemia and 120 min reperfusion. At the end of the experiment, blood and kidney tissue specimens were obtained for biochemical analysis. Histological evaluation of the rat kidney tissues was also done. Biochemical analysis showed that aortic IR significantly increased (P < 0.05 versus control) while tezosentan significantly decreased (P < 0.05 versus aortic IR) the tissue levels of malondialdehyde, superoxide dismutase, catalase and myeloperoxidase. Histological analyses showed that aortic IR significantly increased (P < 0.05 versus control) while tezosentan significantly decreased (P < 0.05 versus aortic IR) focal glomerular necrosis, dilatation of Bowman's capsule, degeneration of tubular epithelium, necrosis in tubular epithelium and tubular dilatation in the renal tissue samples. The results of this study indicate that tezosentan reduces renal injury induced by aortic IR in rats. We think that tezosentan exerted this beneficial effect via reducing oxidative stress and lipid peroxidation, inhibition of leukocyte infiltration into renal tissue and acting cytoprotective on renal tubular cells after aortic IR.

Endothelin is a downstream mediator of profibrotic responses to transforming growth factor β in human lung fibroblasts

Fibrosis is excessive scarring caused by the accumulation and contraction of extracellular matrix proteins and is a common end pathway in many chronic diseases, including scleroderma (systemic sclerosis [SSc]). Indeed, pulmonary fibrosis is a major cause of death in SSc. Transforming growth factor beta (TGFbeta) induces endothelin 1 (ET-1) in human lung fibroblasts by a Smad-independent, JNK-dependent mechanism. The goal of this study was to assess whether ET-1 is a downstream mediator of the profibrotic effects of TGFbeta in lung fibroblasts. We used a specific endothelin receptor antagonist to determine whether ET-1 is a downstream mediator of TGFbeta responses in lung fibroblasts, using microarray technology, real-time polymerase chain reaction, and Western blot analyses. The ability of TGFbeta to induce the expression of a cohort of profibrotic genes, including type I collagen, fibronectin, and CCN2, and to contract a collagen gel matrix, depends on ET-1. ET-1 contributes to the ability of TGFbeta to promote a profibrotic phenotype in human lung fibroblasts, consistent with the notion that endothelin receptor antagonism may be beneficial in controlling fibrogenic responses in lung fibroblasts.

Arginine Diet for Sickle Transgenic Mice Reduces Endothelin-1-Stimulated Gardos Channel Activity.

Arginine (ARG) supplementation (5% arginine in chow) restores plasma ARG levels in S+S-Antilles mice to that found in C57BL mice and is associated with a reduction in Ca++ activated K+ channel (Gardos channel) activity, MCHC and high density red cells (Romero, Blood 99(4):2002). However the mechanisms for these effects are not entirely clear. Endothelin-1 (ET) is a potent vasoconstrictor that has been shown to activate Gardos channel activity and reduce MCHC. We hypothesized that reduced ET could account for the decrease in Gardos channel activity. We report here that arginine diet reduced ET levels in mouse plasma from 137 ± 10 to 89 ± 17 pmol/ml using a modified HPLC technique originally proposed by Kumarathasan (Anal Biochem 299:37, 2001). We measured Gardos channel activity in ex vivo red cells as the influx of 86Rb in the presence or absence 10μM clotrimazole. Consistent with our previous report, we observed that ARG supplementation was associated with a decrease in Gardos Channel activity. In addition, incubation of red cells with 500 nmol/L of ET for 15 min at 37°C stimulated an increase in Gardos channel activity in cells from both ARG supplemented vs non-supplemented mice that was sensitive to 1 mmol/L of the specific ET receptor B antagonist, BQ788 (9.4 ± 2.5 vs 14.6 ± 3.8 mmol/L cell x h, respectively). However, the maximal response of the Gardos channel activity to ET was significantly blunted in ARG supplemented vs non-supplemented mice (15.7 ± 2.8 vs 22.1 ± 2.2 mmol/L cell x h, p<0.03, n=5). We also performed QT-RT PCR using Taqman assays on aortas from these mice and observed that mRNA expression of both ET and ET receptor A levels were significantly decreased in ARG supplemented vs non-supplemented mice (p<0.05, n=5) while ET receptor B levels were not significantly affected (n=5). Thus, our results suggest that reduced ET is in part responsible for the blunted Gardos channel activity in arginine supplemented mice and raises the possibility that ET metabolism is modulated by arginine supplementation. We conclude that arginine supplementation of sickle cell patients may have multiple benefits including reduced polymer formation, reduced vasoconstriction, and reduced inflammatory response.

Endothelin-2 induces oviductal contraction via endothelin receptor subtype A in rats

Proper function of the oviduct is critical to reproductive success with regulated contraction and relaxation facilitating transportation of the germ cells to the site of fertilization. Endothelin-2 (EDN2) is a potent vasoconstrictor produced by granulosa cells of the preovulatory follicle at the time of ovulation; however, whether this gonadotropin surge-induced peptide played a role in facilitating germ cell transportation by inducing oviductal contraction was unknown. The objectives of these experiments were (1) to determine whether the endothelin receptor system was present in the oviduct, (2) to test the hypothesis that EDN2 induces oviductal contraction via a specific endothelin receptor subtype, (3) to determine, as a possible alternate source of the ligand, whether mRNA for EDN2 was expressed in cumulus-oocyte complexes (COCs) within the oviduct, and (4) to determine whether EDN2 could overcome prostaglandin E(2) (PGE(2))-induced oviductal relaxation. Microarray and real-time PCR analysis indicated that mRNA for both the endothelin receptor subtypes (ET(A) and ET(B)) was present in the oviduct, whereas immunohistochemical examination revealed that ET(A) protein was the dominant isoform, present in the luminal epithelial cells of the oviduct. Real-time PCR analysis demonstrated that mRNA for EDN2 was expressed in COCs after ovulation. Isometric tension analysis indicated that EDN2 was a potent oviductal constrictor and that the contractile effect of EDN2 was mediated by the ET(A) and not the ET(B) receptor subtype. The oviductal contraction induced by EDN2 also reversed oviductal relaxation induced by PGE(2). In summary, ET(A) receptor-specific EDN2-induced contraction as a facilitator of oviductal function suggests a novel pathway involved in germ cell transport and hence mammalian fertility.

Inflammatory Murine Skin Responses to UV-B Light Are Partially Dependent on Endothelin-1 and Mast Cells

Endothelin (ET-1) has been shown to crucially contribute to UV-induced skin responses such as tanning. To test whether ET-1 is also involved in early cutaneous reactions to UV, we assessed ET-1 skin levels in UV-irradiated mice. In correlation with the levels of UV-induced skin inflammation, ET-1 concentrations increased substantially and continually. Moreover, blocking of ET-1 receptors (ETA) resulted in significantly decreased cutaneous inflammation following UV irradiation. When we assessed skin responses to ET-1 injections, we observed prominent mast cell degranulation and mast cell-dependent inflammation. Since mast cells also critically contributed to UV-induced inflammation, we determined the ET-1-dependent inflammatory response to UV in the absence and presence of these cells. Interestingly, ETA blockade did not decrease UV-induced inflammation in mast cell-deficient mice, unless these mice had been adoptively transferred with mast cells before irradiation. This indicates that skin inflammation due to UV irradiation is caused in part by ET-1 acting on skin mast cells.

Effects of Specific Endothelin-1 Receptor Antagonists on Proliferation and Fibronectin Production of Glomerular Mesangial Cells Stimulated with Angiotensin II

Angiotensin II (Ang-II) is a potent vasoactive hormone, which plays an important role in the pathogenesis of glomerulosclerosis. Ang-II activates many cytokine systems in the kidney. Recent studies indicate that Ang-II is closely related to the activation of the endothelin-1 (ET-1) system. The present study was designed to measure the [H3]-thymidine uptake and fibronectin production of cultured rat mesangial cells stimulated with Ang-II, and to evaluate the effects of specific ET-1 receptor antagonists, BQ123 (type A receptor antagonist) and IRL1038 (type B receptor antagonist) on the cells. ET-1 was measured by radioimmunoassay and fibronectin by Western blot analysis. The results were as follows: (1) Ang-II enhanced ET-1 production, [H3]-thymidine uptake, number of cells, and fibronectin production of mesangial cells; (2) all the baseline [H3]-thymidine uptake, number of cells, and fibronectin production of mesangial cells can be partly suppressed by BQ123, but not by IRL1038; (3) the increment of Ang-II-enhanced number of cells can be partly suppressed by BQ123, but not by IRL1038; and (4) the increment of Ang-II-enhanced fibronectin production can be partly suppressed by both BQ123 and IRL1038. Our results indicate that Ang-II is an active stimulant for the proliferation and fibronectin production of mesangial cells, and the effect is partly suppressed mainly by ET-1 type A receptor antagonists.

Effects of ETA Receptor Antagonism on Proinflammatory Gene Expression and Microcirculation Following Hepatic Ischemia/Reperfusion

The objective of this study was to investigate the effect of a specific endothelin(A) receptor antagonist (ET(A)-RA) on mRNA expression of genes encoding vasoactive mediators and proinflammatory cytokines and on the microhemodynamics (assessed by measurement of laser Doppler flow and tissue blood gases) following complete vascular exclusion of the porcine liver. Sixteen adult German landrace pigs were subjected to 120 min of warm hepatic ischemia by total vascular exclusion. To avoid portal congestion, a passive porto-femoro/jugular bypass was implanted. The animals were divided into 2 groups: the control group received saline solution and the therapy group was given the selective ET(A)-RA BSF 208075. Hepatic microcirculation was evaluated by p(O(2)) and p(CO(2)) measurement with the Paratrend sensor and by laser Doppler flow measurement. Liver tissue samples were collected 1 h after reperfusion and quantitative mRNA expression for prepro-ET-1, pro-IL-1beta, pro-IL-6, pro-TNF-alpha, eNOS was analyzed using the TaqMan system. Additionally, immunohistochemical analysis using a semiquantitative score for ET-1 was performed. Postischemic liver damage was monitored by measurement of liver enzymes and assessed by histological analysis using a semiquantitative scoring classification. Partial oxygen pressure in the hepatic tissue and laser Doppler flow were significantly improved in the therapy group. One hour after reperfusion, quantitative RT-PCR revealed significantly lower expression of prepro-ET-1, eNOS, pro-TNF-alpha, and pro-IL-6 in the therapy group compared to controls. Immunohistochemical analysis demonstrated significantly reduced ET-1 immunostaining after therapy. Furthermore, blockade of ET(A) receptors prevents tissue damage. Treatment with the selective ET(A)-RA BSF 208075 has protective effects on microcirculation after 120 min liver ischemia and reperfusion. The authors were able to show that ET(A)-RA not only affects the expression of vasoactive genes, but also decreases gene expression of proinflammatory cytokines such as TNF-alpha and IL-6.

Abstracts

Abstracts