ET-1 Release Research Articles

Gene expression and gene therapy in experimental duodenal ulceration

Gastroduodenal ulceration is still poorly understood and changes in gene expression may provide new mechanistic insights. Previously, we demonstrated that angiogenic growth factors are potent ulcer healing agents, and the synthesis of bFGF, PDGF and VEGF is enhanced early in duodenal ulcer healing. The initial molecular event in duodenal ulceration seems to be the organ-specific early release of ET-1 in the pre-ulcerogenic stages after the administration of duodenal ulcerogen cysteamine in rats. We also briefly review here data from literature indicating a central role of ET-1 in gastroduodenal ulceration. After studying the involvement of immediate early genes (e.g. egr-1, Sp1) in ulcer development, we now investigated expression of other genes in the duodenal mucosa in the early stages of chemically induced duodenal ulceration in rats. Following a brief review of principles of gene expression and gene therapy, we review our preliminary gene expression studies, involving monitoring about 1200 genes which revealed about 160 signals and prominent changes in about 30 genes in the early stages of experimental duodenal ulceration. Cysteamine enhanced ET-B receptor gene expression in 30 min, while transcription factors (MAX, STAT 3) showed increased expression in 12 h. We recently also initiated gene therapy studies to enhance the local synthesis of PDGF and VEGF to accelerate duodenal ulcer healing, using a single dose of naked DNA (ND) or adenoviral (AV) vectors of VEGF and PDGF in rats with cysteamine-induced duodenal ulcers. Gene therapy with ND or AV of VEGF or PDGF significantly accelerated chronic duodenal ulcer healing, and increased levels of VEGF and PDGF were detected by Western blotting and ELISA in duodenal mucosa after both VEGF and PDGF gene therapy. Thus, gene expression studies provide new insights into the molecular mechanisms of duodenal ulceration and VEGF or PDGF gene therapy seems to be a new option to achieve a rapid ulcer healing.

Ischaemia/reperfusion contributes to colonic injury following experimental aortic surgery.

ischaemia of the colon is an important complication of abdominal aortic aneurysm (AAA) repair. The aim of this animal study was to investigate the effect of sequential ischaemia and reperfusion on sigmoid mucosal pO2 and its association with local ET-1 release. twelve pigs underwent colonic ischaemia followed by complete reperfusion. Six other animals were sham controls. A Clark-type microcatheter was used for continuous mucosal pO2 measurements. Serial systemic and inferior mesenteric vein blood samples were obtained for determination of ET-1 concentration. Neutrophil extravasation was assessed by tissue myeloperoxidase (MPO) activity. arterial occlusion was associated with a gradual decrease of mucosal pO2 and local release of ET-1. After restoration of blood flow, mucosal pO2 returned to near baseline values, whereas ET-1 reached its maximum concentration during the reperfusion period. MPO activity was significantly increased. colonic ischaemia and reperfusion causes neutrophil extravasation and local ET-1.

Strategies to modulate the deleterious effects of endothelin in hepatic ischemia-reperfusion.

This study evaluates whether bosentan (endothelin [ET] receptor antagonist) or preconditioning (mechanism that inhibits the postischemic ET release) could reduce the microvascular disorders and the injurious effects of tumor necrosis factor (TNF) associated with hepatic ischemia-reperfusion (I/R). Hepatic I/R was induced in rats and the effects of bosentan or preconditioning on the deleterious effects of ET in hepatic I/R were evaluated. Transaminase and TNF levels in plasma; edema, vascular permeability, lactate, ET, and TNF levels in liver; and edema and myeloperoxidase activity levels in lung were measured after hepatic reperfusion. The administration of bosentan or the induction of preconditioning previous to I/R attenuated the increase in vascular permeability, edema and lactate levels observed in liver after I/R. However, the addition of ET before preconditioning abolished its benefits. Preconditioning prevented both the increase in hepatic TNF and its release from the liver into the systemic circulation. This resulted in an attenuation of liver and lung damage. Addition of ET or TNF to the preconditioned group abolished the benefits of preconditioning, whereas the previous inhibition of TNF release with GdCl3 in the preconditioned group pretreated with ET did not modify the effects of preconditioning. The inhibition of ET with bosentan prevented the increase of both hepatic and plasma TNF, thus attenuating the liver and lung injury, whereas TNF addition abolished the benefits of bosentan. These findings suggest that both bosentan and preconditioning, by inhibition of ET could attenuate the microvascular disorders and the deleterious effect of TNF on the liver and lung elicited by hepatic I/R.

Endothelin-1 Levels in Severe Burn Injuries

Most investigators have reported high levels of endothelin (ET)-1 in patients with thermal injury. We attempted to examine the hypothesis that ET-1 levels increase in patients with severe burn injury. Plasma from 28 adult subjects, 14 of whom had thermal injuries with a median (range) percentage of total burn surface area of 22% (20%-76%), was assessed for ET-1 and tumor necrosis factor (TNF) alpha. Samples from closely age-matched patients were obtained on admission (day 1) and 24 hours postinjury (day 2). Samples were obtained before blood transfusion or surgical treatment occurred. Enzyme immunoassay techniques suitable for the measurements of the cytokines were used. Median (range) of TNF-alpha was higher in patients (day 1, 10.0 ng/L [1.2-35.0 ng/L]; day 2, 12.0 ng/L [0.4-39.0 ng/L]) than controls (0. 8 ng/L [0.3-3.2 ng/L]) (P<.005) while ET-1 levels remained significantly unchanged in patients (mean [SD], day 1, 183.0 [42.2] ng/L; day 2, 204.7 [41.7] ng/L) compared with controls (170.0 [59.8] ng/L) (P>.05). We observed no significantly raised levels of ET-1 in patients with thermal injury within 24 hours after burn injury. We found no significant correlation between the plasma levels of TNF-alpha and ET-1. Endothelin-1 levels did not seem to reflect severity of illness. The actual evaluation of ET-1 release in patients with thermal injury could enhance the pathophysiological study of human thermal injury.

Periovulatory changes in the local release of vasoactive peptides, prostaglandin f(2alpha), and steroid hormones from bovine mature follicles in vivo.

We previously proposed that an endothelin-angiotensin-atrial natriuretic peptide system may contribute to inducing ovulation of mature bovine follicles by modulating follicular secretion of steroids and prostaglandins (PGs). Thus, this study aimed to determine the real-time changes in the local release of angiotensin II (Ang II), endothelin (ET), atrial natriuretic peptide (ANP), PGF(2alpha), and steroid hormones from bovine mature follicles during the periovulatory period in vivo. Seven cows were treated for superovulation using FSH and PGF(2alpha) injections. Two dialysis capillary membranes per follicle were surgically implanted into the theca layer of mature follicles and connected to a microdialysis system (MDS). Fractions of the perfusate were collected from Day -1 (Day 0 = LH surge) to Day 3. Five out of seven treated cows were normally ovulated, and the newly formed corpora lutea were observed at the end of the experiment. In these five ovulated cows, the release of estradiol, androstenedione, and progesterone in the theca layer increased (P < 0.05) synchronously with the LH surge. Acute increases in PGF(2alpha) and Ang II concentrations in the ovarian venous plasma (OVP) were observed at 24-48 h after the peak of the LH surge, when multiple ovulations were expected to occur. The follicular Ang II release was low during the pre-LH surge period and rose (P < 0.05) at the beginning of the increase in the LH surge. On the other hand, ET-1 release dropped (P < 0.05) when plasma LH started to increase. However, no clear changes in ANP concentration in the MDS perfusate and plasma were observed. The above local changes in Ang II, PGF(2alpha), as well as steroid hormones were not observed in cows (n = 2) that did not show an LH surge and ovulation. The present results demonstrate for the first time the local release of Ang II, ET-1, and ANP from the bovine mature follicle in real-time in vivo and show that Ang II and PGF(2alpha) concentrations in the OVP acutely increase around the time of ovulation. The overall results support the concept of a local functional ET-Ang-ANP system in the bovine mature follicle that may be involved in the ovulatory process.

Thrombin induces endothelin expression in arterial smooth muscle cells.

Thrombin has been shown to stimulate endothelin release by endothelial cells, but the ability of thrombin to induce endothelin in nonendothelial cells is less well-known. Incubation of rat aortic smooth muscle cells with thrombin resulted in a stimulation of preproendothelin-1 (preproET-1) mRNA expression. This induction of preproET-1 mRNA expression by thrombin was accompanied by the release of immunoreactive peptide ET-1 into the extracellular medium. The synthetic thrombin receptor activator peptide (TRAP) confirmed ligand-specific receptor action to induce preproET-1 mRNA. Nuclear run-on analysis revealed that the transcriptional rate of preproET-1 mRNA increases twofold after 1 h of incubation with thrombin. In cells treated with thrombin, the half-life of preproET-1 mRNA was identical to that in untreated control cells. These results demonstrated that thrombin regulates endothelin synthesis at a transcriptional level but does not influence mRNA stability. Inhibition of protein kinase C (PKC) with selective inhibitors (chelerythrine and bisindolylmaleimide I) before thrombin stimulation failed to significantly inhibit preproET-1 gene expression. Inhibition of mitogen-activated protein (MAP) kinase kinase and protein tyrosine kinase decreased preproET-1 mRNA expression in thrombin-stimulated smooth muscle cells. Furthermore, addition of an activator of peroxisome proliferator-activated receptors-alpha (PPARalpha), fenofibrate, prevented the preproET-1 gene induction in response to thrombin. These results demonstrated that thrombin-induced endothelin gene transcription involved MAP kinase kinase rather than the PKC cascade in smooth muscle cells, which was repressed by PPARalpha stimulation.

Regulation of ET: pulmonary release of ET contributes to increased plasma ET levels and vasoconstriction in CHF.

Endothelin (ET) contributes to the increased systemic vascular resistance and elevated cardiac filling pressures seen in congestive heart failure (CHF). We investigated to what extent ET-mediated vasoconstriction in CHF occurs through an endocrine action of elevated plasma ET or by an autocrine/paracrine mechanism related to induction of vascular ET gene expression. Three weeks of pacing (225 beats/min) induced a marked release of ET-1 from the pulmonary circulation with a sixfold elevation of arterial plasma ET in CHF pigs compared with sham-operated pigs. Arterial plasma ET was the strongest and only independent predictor of systemic vascular resistance. In contrast, vascular preproET-1 and ET-receptor mRNA expression were unaltered or decreased in CHF pigs and did not correlate with indexes of vascular tone. However, myocardial preproET-1 mRNA expression increased twofold in CHF pigs. PreproET-2 and preproET-3 mRNAs were not detectable in cardiovascular tissues. In conclusion, plasma ET was markedly increased because of an augmented release from the pulmonary circulation during CHF, and arterial plasma ET correlated with systemic vascular resistance. The absence of ET induction in the peripheral vasculature suggests that ET increases vascular tone during CHF by an endocrine, not an autocrine/paracrine, mechanism.

Angiotensin II formation and endothelin clearance in ARDS patients in supine and prone positions.

In patients with acute respiratory distress syndrome (ARDS), the prone position may enhance oxygenation by changing ventilation/perfusion ratio. In this study, we investigated whether the prone position affects the net balance between pulmonary endothelin (ET-1) and angiotensin II (Ang II) production and clearance, two metabolic functions of lung endothelial cells. Anaesthesiological intensive care unit of a university hospital. Ten ARDS patients (Murray score > 2.5) were studied in both the supine position (SP) and the prone position (PP). MEASUREMENTS AND DESIGN: Blood samples were taken simultaneously from the patient in SP for assessment of mixed venous and arterial ET-1 and Ang II concentrations, and plasma renin concentration (PRC). This was repeated after 60 min in SP, immediately after turning the patient into PP, and 60 min thereafter. Net arterial/mixed venous ET-1 clearances and net Ang II formations were calculated. arterial oxygen tension increased from SP to PP by an average of 60 mmHg, about 20%. Arterial ET-1 concentrations of ARDS patients were 1.57 +/- 1.1 pg/ml (mean +/- SD) and within the range of healthy persons. Net ET-1 clearances were negative in SP, indicating pulmonary release of ET-1, and did not change in PP. Arterial Ang II concentrations (73 +/- 56 pg/ml) as well as PRC (126 +/- 85 pg/ml) were markedly elevated. Net transpulmonary Ang II formation did not change. Acute changes of oxygenation in ARDS patients by positioning do not induce any short-term effects on pulmonary ET-1 net clearance or Ang II net formation.

Endothelin antagonists and renal protection.

Proteinuric nephropathies either of diabetic or nondiabetic origin tend to develop renal structural damage associated with progressive renal function decline over time. In proteinuric glomerular disease excessive protein reabsorption by proximal tubular epithelial cells modulates tubular cell function to the extent that cell growth and their phenotypic expression of growth factors and inflammatory chemokines and cytokines is upregulated. Recent evidence is available that renal tubular cells synthesize endothelins (ETs), a family of peptides with potent vasoconstrictor and proliferation properties, and that overloading these cells with proteins induces a dose-dependent increase in the synthesis and release of ET-1. This peptide, accumulating in the renal interstitium, eventually participates in activation of the sequence of events that leads to interstitial inflammation and ultimately renal scarring. Increased renal synthesis of ET-1 occurs in vivo as documented in several animal models of proteinuric progressive nephropathies, in which enhanced renal ET-1 gene expression as well as the excretion of the peptide in the urine correlated with the urinary protein excretion rate. Similarly, in patients with chronic renal disease an association has been found between increased urinary excretion of ET-1 and renal damage. A strong argument in favor of ET-1 as a mediator of renal injury derives from preclinical studies with selective and non-selective ET receptor antagonists that have became available in the past few years. They block the effect of ET to their specific receptors called ET(A) and ET(B), and have been reported to have a renoprotective effect in several animal models of progressive renal disease, including in rats with remnant kidney or experimental diabetes as well as mice with lupus nephritis. The peptide nature of some of these compounds, which are currently appearing in the literature, may however hamper their future use in humans. Administration of nonpeptide ET receptor antagonists to humans would hopefully overcome this problem and possibly provide a new therapeutic approach for patients with renal disease who do not adequately respond to the currently available therapy with angiotensin-converting enzyme inhibitors or angiotensin II receptor antagonists.

Effects of quercetin on the release of endothelin, prostacyclin and tissue plasminogen activator from human endothelial cells in culture

Quercetin and related flavonoids are naturally occurring polyphenolic compounds with multiple pharmacological activities. Using cultured human umbilical vein endothelial cells, we investigated the effects of quercetin on endothelin (ET-1) and tissue plasminogen activator (t-PA) release induced by thrombin. We observed that when endothelial cells pretreated with 5 or 50 μM of quercetin were incubated for 4 and 24 h with thrombin, ET-1 concentration-dependently decreased ( n=6, P<0.01, at 4 h IC 50=1.54 μM, at 24 h IC 50=2.78 μM). Under the same experimental conditions, quercetin significantly increased t-PA ( n=6, P<0.01, at 4 h EC 50=0.71 μM and at 24 hrs EC 50=0.74 μM). In the same preparation, we evaluated prostacyclin (PGI 2) release, induced by thrombin activated platelets, as determined by a 6-Keto-PGF 1α radioimmunoassay. Following the treatment of cultured endothelial cells with activated platelets, the concentration of 6-Keto-PGF 1α was significantly increased ( P<0.01). Quercetin (1, 5, and 20 μM) inhibited PGI 2, in a concentration-dependent manner ( n=6, P<0.05). Our data indicate that quercetin modulates the release of ET-1, t-PA, and PGI 2 from vascular endothelial cells.

Cardiac endothelin and big endothelin in right-heart hypertrophy due to monocrotaline-induced pulmonary hypertension in rat.

Recent observations suggest the existence of a myocardial endothelin (ET) system and its possible involvement in left-ventricular myocardial hypertrophy and failure. However, nothing is known about the role of myocardial ET in right-ventricular hypertrophy. Rats (80-100 g) were given an intraperitoneal injection of saline (controls) or monocrotaline (50 mg/kg) resulting in pulmonary hypertension-induced myocardial hypertrophy (n = 11 in both groups). After 10 weeks, the animals were sacrificed and hearts perfused in vitro to determine levels of big ET-1 and ET-1 in coronary effluent, interstitial fluid and ventricular tissue homogenates; plasma levels were also determined. In monocrotaline-treated animals, weights of right ventricles were 1.5 and of right atria 1.8-fold higher than in controls (p < 0.05), indicating substantial right-ventricular hypertrophy as also evident from greatly increased myocardial production of atrial natriuretic peptide. Left-ventricular weights were not different. Release of big ET-1 in coronary effluent, and of ET-1 in coronary effluent and interstitial transudate were similar in control and hypertrophic hearts (p > 0.05). Disruption of endothelium with collagenase reduced release of both peptides close to zero, indicating endothelial (not myocardial) origin of the peptides. Levels of big ET-1 and ET-1 were similar in left ventricles of both experimental groups, but lower in right ventricles of hypertrophic than control hearts (p < 0.05), reflecting increased tissue mass rather than reduced peptide production. On the other hand, plasma levels of both peptides and of ANP were twofold and levels of angiotensin II 1.3-fold higher in rats with right-heart hypertrophy than in controls (p < 0.05 in each case). These data do not support a role for local cardiac ET-1 and/or big ET-1 in right-ventricular hypertrophy, but point to blood-borne endothelins as possible mediators.

Increased renal glomerular endothelin-1 release in gentamicin-induced nephrotoxicity.

Gentamicin-induced acute renal failure is characterized by a decrease in renal plasma flow and creatinine clearance. Endothelins (ET) are potent renal vasoconstrictors. The aim of this work is to assess the role of ET-1 in gentamicin-induced renal failure. Renal glomerular release of ET-1 was measured in rats with gentamicin-induced nephrotoxicity (100 mg/kg/day, s.c. for 2, 4 or 6 days). Glomeruli were isolated and incubated for 24 h in RPMI-1640. Glomerular supernatant and plasma concentration of ET-1 were measured by RIA. Renal failure was assessed by insulin, para-aminohippuric and creatinine clearance and histological studies. Gentamicin induced a dose number-dependent increase in plasma creatinine and a decrease in creatinine clearance. This was accompanied by a marked decrease in inulin and para-aminohippuric acid clearance, as well as by a marked tubular necrosis, without alterations in glomerular structures. Plasma ET-1 concentration and glomerular ET-1 release were also increased in gentamicin-treated rats. When 10-5 M gentamicin was added to control glomeruli, ET-1 production was not modified (36.4 +/- 2.2 vs. 35.2 +/- 1.7 pg/ml/24 h). All these results suggest that elevated ET-1 plasma levels and increased glomerular release of ET-1 could mediate, at least in part, the decrease in glomerular filtration rate observed in gentamicin-induced ARF.

Cardiac endothelin release into the coronary sinus in myocardial ischemia and coronary endothelial injury

Endothelin has both vasoconstrictor and mitogenic properties and might, therefore, play a role in the pathogenesis of acute coronary syndromes and coronary atherosclerosis. The aim of the study was to characterize the mechanisms and kinetics of cardiac endothelin-1 (ET-1) release following a local endothelial injury during PTCA (group A) and after sustained myocardial ischemia (group B). Additionally, the precision of agreement between measurements in coronary sinus and peripheral venous samples should be analyzed. In group A, elective PTCA was performed in 20 patients with stable angina pectoris and a > 70% type A stenosis. Simultaneous determinations of ET-1 from coronary venous and peripheral venous blood were done before balloon inflation and during the several hours following the last dilatation procedure. A coronary sinus study with high rate atrial pacing was performed in 20 group B patients with coronary multivessel disease. ET-1 was determined from coronary sinus and peripheral venous blood samples prior to stimulation and during several hours after cessation of pacing. Control groups were provided for both groups. The control group consisted of 10 patients with coronary angiography without PTCA for group A and 10 patients with angiographic normal coronary arteries for group B.PTCA induced an instantaneous increase of coronary sinus ET levels from 4.1 +/- 1.1 pg/ml to 13.7 +/- 2.3 pg/ml (peripheral venous 7.9 +/- 2.5 pg/ml), which was more pronounced if the target vessel was the left anterior descending artery. This peak was followed by a gradual decrease of ET-1 to the limit of normal within 6 hours. The concentrations of ET, furthermore, remained higher in the coronary sinus compared with the peripheral vein indicating a persisting cardiac release of ET. In group B, incremental atrial pacing resulted in myocardial ischemia, and a significant increase in ET-1 from 4.6 +/- 0.6 pg/ml to 13.1 +/- 2.8 pg/ml was detected in the coronary sinus samples. A persistent cardiac release of ET-1, as reflected by sustained elevated coronary sinus concentrations, was observed for up to one hour after cessation of pacing. The analysis of measurement agreement between coronary venous and peripheral venous samples revealed considerable variations of the differences between the two sampling sites indicating wide limits of agreement. Despite a significant positive correlation, our date reflecting a remarkable lack of agreement. 1) An enhanced release of ET-1 following PTCA is mainly due to the localized endothelial injury, and the ET-1 levels remain elevated for up to hours after the mechanical stimulus. 2) A short-lasting myocardial ischemia is associated with a significant ET-1 increase. 3) For refined evaluations of release kinetics of cardiac ET-1, blood sampling from the coronary sinus seems to be essential.

Lipids and the endothelium.

The normal endothelium is characterised by the production of a number of molecules which affect the contractile state of adjacent myocytes and the behavior of formed elements within the blood stream, and by the absence of cell surface adhesion molecules. In addition, endothelial cells are important modulators of coagulation and fibrinolysis. Whilst effects of lipids have been documented on many of these endothelial processes, there is particularly strong evidence for effects on the vasodilatation mediated by endothelium derived nitric oxide and on the interaction between leukocytes and the endothelial surface. Both LDL cholesterol and triglyceride rich lipoproteins impair endothelium dependent vasodilatation. The effects of LDL cholesterol are primarily evident for lipoprotein particles that have been oxidised with evidence for effects of specific constituents of oxidised LDL, such as lysophosphatidylcholine (LPC). LDL effects have been demonstrated at numerous sites of the nitric oxide signaling pathway including receptor-G protein coupling, nitric oxide synthase and NO bioactivity, with evidence for enhanced superoxide formation and the consequent production of the less potent dilator peroxynitrite. The effects of lipids on endothelium dependent vasodilatation can be reversed not only by reducing the level of elevated lipids levels but also by provision of the NOS substrate, L-arginine and by the provision of antioxidants, although the mechanism for these effects are not fully elucidated. The adhesion of leukocytes to the endothelial surface is stimulated by low density and triglyceride rich lipoproteins. As with endothelium dependent vasodilatation, the effects of LDL cholesterol are primarily evident for low-density lipoprotein particles that have been oxidised, and many of the effects of oxidised LDL can be mimicked by LPC. HDL can overcome pro-adhesive effects of oxidised LDL. The effects of LDL on leukocyte adhesion are secondary to the expression of adhesion molecules on the luminal surfaces of endothelial cells. In addition to the likely deleterious effects of lipids on endothelium-mediated vasodilatation and leukocyte-endothelial cell interaction, lipids have been shown to affect a number of other endothelial processes and function. Thus, oxidised LDL affects endothelial ET1 and PGI2 release. Although effects have been shown on endothelial cell growth and apoptosis and on endothelial processes related to thrombosis and fibrinolysis, these effects have been less extensively studied than endothelial dependent vasodilatation and leukocyte-endothelial cell interaction. Many of the effects of elevated or modified low density and TG rich lipoproteins on endothelial cells and endothelial cell processes could be expected to contribute to the development of atherosclerosis and therefore, to the association between lipids and atherosclerotic, particularly coronary, vascular disease. However, the extent to which "endothelial dysfunction" accounts for the known relationships between serum lipid concentrations and CHD is yet to be established.

Suppression of endothelin system in Vero cells latently infected with influenza virus B/Lee/40.

In Vero cells latently infected with influenza virus B/Lee/40 (L/V cells), the endothelin (ET) system was examined as a possible mediator in pathogenesis of latent viral infection by radioimmunoassay (RIA) and quantitative reverse transcription polymerase reaction (RT-PCR). During viral latency of more than two months, ET secretion, preproendothelin-1 (PPET-1) mRNA, and endothelin receptor subtype A (ETAR) mRNA within the cells remained suppressed. Our data indicate that not only the release of ET-1 was downregulated at the transcriptional level but also ETAR mRNA was downregulated rather than upregulated compensatively in L/V cells.

Effects of endothelin-1 on vasoactivity and its synthesis, storage, and acting sites in the rat superior mesenteric vasculature: an ultrastructural and immunocytochemical study.

Vasoactivity after treatment with endothelin (ET)-1 and immunoreactivity for ET-1 and its receptors were investigated in the rat superior mesenteric vasculature (SMV). By measurements of corrosion cast images of the SMVs, it was seen that ET-1 induces remarkable vasocontraction of the distal arterial branches, consisting of small arteries and arterioles, and localized vasoconstriction throughout the venous branches which possess localized medial thickenings. Immunoreactivity for ET-1 was preferentially seen along the endothelia of the proximal arterial branches. Cisterns of the rough endoplasmic reticulum and Weibel-Palade (WP) bodies of the endothelial cells were immunoreacted. Immunoreactivity for the ETa receptor was preferentially seen on the media of the distal arterial branches. These findings indicate that endothelial cells of the proximal arterial branches synthesize ET-1 and store it in the WP bodies. Because WP bodies are involved in the release of ET-1, this suggests that this endogenous ET-1, which is released from the proximal arterial branches, may be involved in the regulation of blood flow through the distal arterial branches by mediation of the ETa receptor. In addition, it seems likely that ET-1-induced vasoconstriction of the venous branches may act to impel the portal blood flow.

Effects of equine oestrogens on markers of vasoactive function in human coronary artery endothelial cells

A large proportion of the beneficial effects that oestrogens demonstrate on the vasculature are believed to be mediated via direct effects on the vascular wall. In this study we compared a number of oestrogenic compounds isolated from pregnant mare’s urine including 17β-oestradiol and oestrone, in terms of their abilities to inhibit stimulated endothelin-1 release from normal human coronary artery endothelial cells (CAEC). We also examined their ability to stimulate expression of constitutive endothelial nitric oxide synthase (eNOS) and explored their effects on cellular angiotensin converting enzyme (ACE). All the oestrogens tested were able to inhibit serum-stimulated ET-1 release. Oestrone and 17α-dihydroequilenin failed to significantly affect cellular eNOS levels. 17β-oestradiol and oestrone significantly increased cellular ACE levels while 17β,Δ 8,9-dehydroestradiol decreased cellular ACE. We discuss these observations in terms of their potential clinical relevance and use as a means of screening novel oestrogen-like compounds.

Endothelin-A and -B antagonists protect myocardial and endothelial function after ischemia/reperfusion in a rat heart transplantation model.

Previous studies suggested that endothelin-1 (ET-1) may play a pathophysiological role in myocardial ischemia/reperfusion injury. This study was designed to investigate the effects of the selective ET-A receptor antagonist BQ123 and the selective ET-B receptor antagonist BQ788 on myocardial and endothelial function after reversible deep hypothermic ischemia in a heterotopic rat heart transplantation model. Isogenic intraabdominal heterotopic transplantation was performed in Lewis rats. After 1 h of cold ischemic preservation reperfusion was started either after application of placebo (control), BQ123 (3 mumol/kg/min). BQ788 (3 mumol/kg/min), ET-1 (8 pmol/kg/min) or simultaneous infusion of BQ123 or BQ788 and ET-1, respectively (n = 12 each). An implanted balloon was used to obtain pressure-volume relations of the transplanted heart. Myocardial blood flow (MBF) was assessed by the hydrogen-clearance method. Measurements were taken after 1 and 24 h of reperfusion. Endothelium-dependent vasodilation to acetylcholine (ACH) and endothelium-independent vasodilation to sodium nitroprusside were also determined. Both BQ123 and BQ788 significantly improved myocardial and endothelial functional recovery during early reperfusion, whereas ET-1 significantly impaired myocardial and endothelial function. Simultaneous infusion of ET-1 diminished the effects of BQ123 and BQ788. Although myocardial function and baseline MBF were similar in all groups after 24 h of reperfusion, endothelium dependent vasodilation to ACH was still significantly higher in the BQ123 and BQ788 groups and lower in the ET-1 groups (p < 0.05). These results suggest that endogenous ET release is involved in the pathogenesis of reperfusion injury after heart transplantation. ET-A and ET-B receptor antagonists may be useful to reduce ischemia/reperfusion injury.

Endothelin blockers and renal protection: a new strategy to prevent end-organ damage in cardiovascular disease?

Time for primary review 27 days. The endothelin (ET) family comprises three 21-amino acid peptides: ET-1, ET-2 and ET-3. Endothelin-1 has been identified as the major cardiovascular isopeptide. Release of the active peptide ET-1 requires cleavage of a Trp21–Val22 bond in the carboxyterminal of the precursor molecule, big ET-1 [1]. This reaction is catalyzed by a membrane bound metalloprotease, endothelin converting enzyme (ECE-1) [1, 2]. An intracellularly located ECE (ECE-2) has been identified as well [3]. Endothelin release is increased transcriptionally when the endothelium is exposed to vasoconstrictor peptides, inflammatory cytokines and physical factors (e.g. angiotensin II, thrombin, TGF-β). Although the human kidney contains mRNA for all three isoforms of endothelin (ET), ET-1 appears to be the only peptide expressed at the protein level [4]. Expression of ET-1, its precursor, big ET-1, and the ECE in the non-diseased kidney is largely confined to the glomerular and vascular endothelium [4, 5]. However, when proteinuria is present, there is tubular expression of ET-1 as well [6]. ET-1 released by the renal vasculature or nephron segments mainly acts on cells in the immediate vicinity in an autocrine/paracrine fashion. ET-1 mediates its biological effects by interacting with two receptors, the ETA and ETB receptors, which have been cloned and well characterized. The ETA receptor is preferentially expressed in vascular smooth muscle cells and mediates the potent constrictor actions of ET-1 [7]. The ETB receptor is primarily expressed in endothelial cells and binds all three ET isoforms. When this receptor is activated, nitric oxide and prostacyclin are released, possibly as a feedback loop to the constrictor actions of ET-1. This is exemplified by recent observations where both administration of a selective ETB antagonist in animals as well as specific knock-out … * Corresponding author. Tel: +31-30-250-7329; Fax: +31-30-254-3492; E-mail: t.rabelink@digd.azu.nl

Articular nociception induced by endothelin-1, carrageenan and LPS in naive and previously inflamed knee-joints in the rat: inhibition by endothelin receptor antagonists

Endothelin-1, unlike the selective endothelin ET B receptor agonist sarafotoxin S6c, causes nociception in the rat when injected intra-articularly into the naive knee-joint. By using selective antagonists, the present study further characterizes the receptors underlying the articular nociceptive actions of endothelin-1, as well as the possible contribution of endogenous endothelins towards nociception induced by carrageenan or E. coli lipopolysaccharide (LPS) in this tissue. Nociception was evaluated by placing the animal for 1 min each hour on a revolving (3 rpm) cylinder and measuring the increase in time the hindpaw of the limb affected by the intra-articular (i.a.) injection of the nociceptive agent, failed to touch its metallic surface (i.e. paw elevation time, PET). In naive joints, endothelin-1 (120 pmol) increased the area under the PET curve (AUC 0–6 h, in arbitrary units) from 61±3 (control) to 156±12. This nociceptive effect was reduced by prior intravenous (i.v.) injection of the mixed ET A/ET B receptor antagonist bosentan (by 54 and 73% with 10 and 30 mg/kg) or i.a. administration of the selective ET A receptor antagonist BQ-123 (cyclo [D-Asp-Pro-D-Val-Leu]; by ≅45% with 10 or 30 nmol), but was unaffected by the selective ET B receptor antagonist BQ-788 (N- cis-2,6-dimethyl-piperidinocarbonyl- l- γ-methyl-leucyl- d-1-methoxycarbonyl-tryptophanil-D-norleucine; 10 nmol). Prior joint challenge with carrageenan (300 μg) 72 h beforehand (i.e. priming) rendered the joint more sensitive to nociception induced by either endothelin-1 or sarafotoxin S6c (15, 30 and 60 pmol). Responses elicited by endothelin (30 pmol) in the primed joint were sensitive to inhibition by either BQ-123 or BQ-788 (each causing ≅80% inhibition at 10 nmol). Priming also enhanced PET responses to carrageenan itself and to LPS (1 μg) markedly and persistently, increasing the area under the curve (AUC 0–12 h, in arbitrary units) from 241±19 to 409±50 and from 312±40 to 466±25, respectively ( P<0.05), without changing that measured following vehicle injection (from 121±3 to 117±4). Bosentan (up to 30 mg/kg, i.v.) failed to modify nociception caused by carrageenan or LPS in naive joints, by carrageenan in the primed joint, or control PET responses. LPS-induced nociception in the primed joint, however, was inhibited by 52 to 56% by bosentan (3 or 10 mg/kg) or 59% by local injection of the selective endothelin ET B receptor antagonist BQ-788 (10 nmol, i.a.), but was unaffected by the selective endothelin ET A receptor antagonist BQ-123. Thus, nociception induced by endothelin-1 in the naive joint is mediated largely via endothelin ET A receptors, whereas both ET A and ET B receptors contribute to its action in the carrageenan-primed joint. Furthermore, LPS-induced nociception in the primed joint is mediated to a large extent via endothelin release and activation of ET B receptors within the joint itself. These findings may be relevant to the etiology of pain underlying chronic arthritic disease in humans.