Immunoreactive Endothelin Levels Research Articles

Abstract 19347: The Mouse Mast Cell Protease 4 is Involved in the Generation of Endothelin-1 in vitro and in vivo

The serine protease chymase converts intravenously (iv)- administered big-endothelin-1 to endothelin-1 (ET-1) in the mouse in vivo. The mouse mast cell protease 4 (mMCP-4) is among eight chymase-like murine isoforms and is involved in the generation of angiotensin-II (Ang-II) from its precursor Ang-I. It remains to be determined which chymase isoform is involved in the endothelin-converting enzyme -independent production of ET-1. In the present study, the contribution of mMCP-4 was determined in the conversion of exogenous Big-ET-1 in vitro and in vivo as well as in the tissue production of endogenous ET-1. In vitro analyses were performed from cardiac left ventricle, aorta, kidneys and lungs homogenates derived from wild type (WT) and mMCP-4 KO mice. The pressor response in anaesthetized mice to Big-ET-1, but not ET-1 (1-31) or ET-1 was significantly reduced in mMCP-4 KO mice compared to their WT congeners. Furthermore, a selective chymase inhibitor, TY-51469, significantly reduced the pressor response to Big-ET-1 in WT but not mMCP-4 KO mice. Radio-telemetry analysis in free-moving, conscious mice revealed no differences in basal blood pressure between WT and mMCP-4 KO mice. TY-51469 also inhibited the in vitro hydrolysis a chymotrypsin-sensitive fluorogenic peptide in soluble fractions derived from WT but not mMCP-4 KO tissues. HPLC-MALDI-MS analysis revealed that supernatants from WT but not mMCP-4 KO mice could yield ET-1 (1-31) from Big-ET-1 in vitro, and that this conversion is blocked by TY-51469 in WT samples. The in vivo conversion of iv-administered Big-ET-1 to plasmatic ET-1 (1-31) and ET-1 was also reduced in mMCP-4 KO mice compared to their WT congeners. Finally, tissue levels of immunoreactive ET-1 were reduced by 40% in lung homogenates from mMCP-4 KO mice when compared to those from WT congeners. We conclude that mMCP-4 is the predominantly involved chymase isoform in the production of endogenous ET-1 in the mouse model.

Distinct modulation of the endothelin-1 pathway iniNOS−/−andeNOS−/−miceThis article is one of a selection of papers published in the special issue (part 2 of 2) on Forefronts in Endothelin.

We hypothesized that constitutive endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) have opposite effects on the regulation of endothelin and its receptors. We therefore sought to determine whether deletions of iNOS or eNOS genes in mice modulate pressor responses to endothelin and the expression of ETA and ETB receptors in a similar fashion. Despite unchanged baseline hemodynamic parameters, anesthetized iNOS-/- mice displayed reduced pressor responses to endothelin-1, but not to that of IRL-1620, a selective ETB agonist. Protein content of cardiac ETA receptors was reduced in iNOS-/- mice compared with wild-type mice, but that of ETB receptors was unchanged. Anesthetized eNOS-/- mice presented a hypertensive state, accompanied by an enhanced pressor response to intravenous endothelin-1, whereas the pressor response to IRL-1620 was reduced. Protein levels were also found to be increased for ETA receptors, but reduced for ETB receptors, in cardiac tissues of eNOS-/- mice. In conscious animals, both strains responded equally to the hypotensive effect of an ETA antagonist, ABT-627, whereas orally administered A-192621, an ETB antagonist, increased MAP to a greater extent in eNOS-/- than in wild-type mice. Furthermore, significant levels of immunoreactive endothelin were found in mesenteric arteries in eNOS-/- but not in iNOS-/- or wild-type congeners. Our study shows that repression of iNOS or eNOS has differential effects on endothelin-1 and its receptors. We have also shown that the heart is the main organ in which iNOS or eNOS repression induces important alterations in protein content of endothelin receptors in adult mice.

The role of endothelin pathway in modulation of airway reactivity to methacholine in C57Bl/6 and BALB/c mice

Endothelin peptides have been shown to increase cholinergic neurotransmission in the airway. Genetic differences in airway responsiveness to methacholine where reported in mice. The present study compared the airway reactivity to methacholine in C57Bl/6 and BALB/c mice, the involvement of endothelin on this reactivity and endothelin levels in lung homogenates. Whole airway reactivity was analyzed by means of an isolated lung preparation where lungs were perfused through the trachea with warm gassed Krebs solution at 5 ml/min, and changes in perfusion pressure triggered by methacholine at increasing bolus doses (0.1–100 µg) were recorded. We found that the maximal airway response to methacholine was much greater in C57Bl/6 than in BALB/c (Emax 34 ± 2 vs 12 ± 1 cmH 2O, respectively). Bosentan (mixed endothelin A/B receptor antagonist; 10 mg/kg, i.p., 30 min before sacrifice) reduced lung responsiveness to methacholine in C57Bl/6 (58% at EC50 level) but had no effect in BALB/c mouse strain. This effect seems to be mediated by the endothelin ET A receptor since it was significantly reduced by the selective endothelin ET A receptor antagonist, BQ 123. Immunoreactive endothelin levels were higher in C57Bl/6 than in BALB/c lungs (43 ± 5 vs 19 ± 5 pg/g of tissue). In conclusion, airway reactivity to methacholine and lung endothelins content varies markedly between C57Bl/6 and BALB/c strains. Endothelins upregulate lung responsiveness to methacholine only in C57Bl/6, an effect achieved through the endothelin ET A receptor.

RETINAL DETACHMENT AND PROLIFERATIVE VITREORETINOPATHY

To analyze if endothelin-1 (ET-1) may have an effect on ophthalmic artery (OA) blood flow velocities and intraocular pressure (IOP) in retinal detachment (RD). Using radioimmunoassay, immunoreactive (IR) ET-1 levels were tested in both plasma and subretinal fluid (SRF) specimens from patients with RD, while only plasma specimens from normal (healthy) subjects were tested. OA Doppler sonography parameters and IOP were measured in eyes with RD, with and without proliferative vitreoretinopathy (PVR), their respective healthy fellow eyes, and normal eyes. RD eyes had lower OA peak systolic velocity (PSV) and end diastolic velocity (EDV), higher resistivity index (RI), lower IOP, and higher plasma IR ET-1 levels than normal eyes (P < 0.0001). Eyes with PVR had lower OA PSV and EDV, higher RI, lower IOP, higher plasma IR ET-1 levels, and higher SRF IR ET-1 than eyes without PVR (P < 0.0001). A statistically significant linear correlation was found among OA parameters, IOP, and SRF IR ET-1 measurements. Decreased OA blood flow velocities may explain lower IOP found in RD patients, and ET-1 levels may be responsible for both measurements.

Errata

Acta PaediatricaVolume 96, Issue 11 p. 1717-1717 Free Access Errata This article corrects the following: Bed-sharing in the first four months of life: a risk factor for sudden infant death Jan H Ruys, Guus A De Jonge, Ronald Brand, Adèle C Engelberts, Ben A Semmekrot, Volume 96Issue 10Acta Pædiatrica pages: 1399-1403 First Published online: August 20, 2007 First published: 12 October 2007 https://doi.org/10.1111/j.1651-2227.2007.00555.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat In (1, 2), the following errors were published: Nikolaidou P should have read Nicolaidou P. In (3) the commentary by Bernt Alm was cross-referenced to the incorrect article. It should have read: Invited commentary for Ruys et al. Bed-sharing in the first four months of life: a risk factor for sudden infant death, pp. 1399–1403. We apologize for these errors. Reference 1 Siahanidou T, Nikolaidou P, Doudounakis S, Georgouli E, Kanavakis M, Papadimitriou A, Karpathios Th. Plasma immunoreactive endothelin levels in children with cystic fibrosis. Acta Paediatrica 2000; 89: 915– 20. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 2 Anatoliotaki M, Tsilimigaki A, Tsekoura Th, Schinaki A, Stefanaki S and Nikolaidou P. Congenital rickets due to maternal vitamin D deficiency in a sunny island of Greece. Acta Paediatrica 2003; 92: 389– 91. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 3 Alm B. To co-sleep or not to sleep? Acta Paediatrica 2007; 96: 1385– 1386. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Volume96, Issue11November 2007Pages 1717-1717 ReferencesRelatedInformation

Endothelin B Receptor Antagonist Increases Preproendothelin-1 Transcription in Bovine Aortic Endothelial Cells and In Vivo

Endothelin-1 (ET-1) receptor antagonists increase plasma immunoreactive ET-1 levels. However, their effect on preproendothelin-1 (PPE-1) mRNA levels is still controversial. Few studies have found a decrease in PPE-1 mRNA levels in endothelial cells treated with the nonselective ETA/B receptor antagonist, and others demonstrated that an ETB blockade by the selective antagonist BQ788 increases PPE-1 mRNA levels. We studied the effect of ETA and ETB selective receptor antagonists on PPE-1 transcription, both in vitro and in vivo. Endothelial cells, transiently transfected with PPE-1 luciferase plasmid, were treated with ET-1 receptor antagonists. Bosentan, a dual ETA/B receptor antagonist, and BQ788 (ETB receptor antagonist) treatment resulted in a 1.6-fold and 1.3-fold increase, respectively in luciferase activity as compared with the untreated control. In contrast, the ETA receptor antagonist, BQ123, had no effect on luciferase activity. Transgenic mice that express the luciferase gene under the control of PPE-1 promoter were treated with Bosentan. Luciferase activity, PPE-1 mRNA levels, and plasma immunoreactive ET-1 levels were increased by 1.6-fold to 2.0-fold in the Bosentan-treated group compared with the untreated, control group. ET-1 receptor blockade increased PPE-1 transcription both in vitro and in vivo. The increased transcription can be attributed to ETB receptor blockade, because BQ-788, but not BQ-123, increased PPE-1 transcription.

Endothelium‐dependent regulation of arterial blood pressure by ANP: role of endogenous vasoactive endothelial factors

Atrial natriuretic peptide (ANP) has an important physiological role in the maintenance of arterial blood pressure and volume. Its guanylyl cyclase-A (GC-A) receptor is highly expressed in vascular endothelium, but the functional relevance is controversial. To dissect the endothelium - mediated actions of ANP in vivo, we recently inactivated the GC-A gene selectively in endothelial cells by homologous loxP/Tie2-Cre-mediated recombination. Notably, mice with endothelial-restricted GC-A deletion (EC GC-A KO mice) exhibit significant arterial hypertension, hypervolemia and cardiac hypertrophy. This hypervolemia is due to a diminished permeability of the microvascular endothelium to macromolecules such as albumin (J Clin Invest 2005; 115:). Published studies suggested that ANP not only modulates endothelial permeability, but also the endothelial synthesis/release of vasoactive factors such as endothelin-1 (ET-1) and nitric oxide (NO). Therefore, in the present study we investigated whether the chronic arterial hypertension of EC GC-A KO mice is due to altered endogenous release and/or actions of ET-1 and NO. Plasma and tissue levels of immunoreactive ET-1 were not different in EC GC-A KO and corresponding control mice. Even more, blood pressure responses to exogenous or endogenous ET-1 as well as functional NO activity levels were not different between genotypes. This new genetic mouse model demonstrates that the vascular endothelium is critically involved in the hypovolemic and hypotensive effects of ANP. However, these effects are not mediated by ANP-dependent alterations in the endothelial synthesis of ET-1 or NO.

Suppression of Cytokine-induced Expression of Endothelin-1 by Dexamethasone in Human Retinal Pigment Epithelial Cells

We have recently shown that retinal pigment epithelial cells secrete endothelin-1 (ET-1). In the present study, we studied effects of cytokines and dexamethasone on expression of ET-1 in two types of cultured human retinal pigment epithelial cells, ARPE- 19 cells and D407 cells. Treatment for 24 hours with a combination of three cytokines, interferon-gamma, tumor necrosis factor-alpha and interleukin-1beta, induced expression of ET-1 in both cells. When treated with dexamethasone (10(-7) mol/L) alone, the immunoreactive endothelin level in the medium increased about three-fold in D407 cells, but not in ARPE-19 cells. Dexamethasone (10(-7) mol/L), however, suppressed the cytokine-mediated increases in ET-1 mRNA expression levels and immunoreactive endothelin levels in the medium. These findings raise the possibility that antiinflammatory effects of dexamethasone in eyes may be partly mediated or modulated by its effects on expression of ET-1.

Salivary Immunoreactive Endothelin in Patients with Upper Gastrointestinal Diseases

Endothelins have been implicated in gastric mucosal damage in a variety of animal models. Furthermore, clinical reports also show elevated gastric mucosal endothelin-1 levels in patients suffering from peptic ulcer diseases. We have demonstrated, first, the presence of immunoreactive endothelin (IR-ET) in human saliva. We also show that endothelins are rather stable in human saliva. The present study was undertaken to determine whether patients with endoscopically proven upper gastrointestinal diseases have a salivary excess of IR-ET, compared with patients with a normal esophagogastroduodenoscopy. Saliva was collected from fasting subjects prior to esophagogastroduodenoscopy. The levels of IR-ET were measured by the radioimmunoassay method. The salivary concentrations of IR-ET in the studied subjects were as follows: 8.9 +/- 1.0 fmol/mL (mean +/- standard error of the mean) for patients with gastric ulcers (n = 18); 7.3 +/- 1.0 fmol/mL for patients with duodenal ulcers (n = 22); and 6.8 +/- 0.6 fmol/mL for patients with gastritis (n = 28). These values are all higher than that of normal subjects (4.4 +/- 0.5 fmol/mL, n = 20; P < 0.001, P < 0.01, and P < 0.05, respectively). No significant differences in salivary IR-ET were noted between patients with a normal esophagogastroduodenoscopy and patients with esophagitis (3.8 +/- 0.7 fmol/mL, n = 4) or gastric cancer (5.3 +/- 1.4 fmol/mL, n = 4). There were no significant differences in the salivary IR-ET levels between males and females. However, the salivary IR-ET levels in the smokers (8.0 +/- 0.6 fmol/mL, n = 38) were significantly higher (P < 0.01) than those of the non-smokers (6.0 +/- 0.4 fmol/mL, n = 58). There was no correlation of IR-ET levels with age. Our findings suggest that salivary endothelin may have a contributing role in certain gastroduodenal diseases.

Enhanced or Repressed Pressor Responses to Endothelin-1 or IRL-1620, respectively, in eNOS Knockout Mice

Nitric oxide (NO) has been shown to be a physiological antagonist of the potent pressor agent endothelin-1, a transcriptional and post-transcriptional repressor of the production of this particular peptide as well as an endogenous modulator of vascular resistance in several animal species as well as in man. The aim of the present study was to assess the hemodynamic properties of endothelin-1 in endothelial NO synthase knockout mice (eNOS (-/-) KO). Ketamine- xylazine anesthetized eNOS (-/-) KO mice show a significantly higher mean arterial pressure (+25.7 +/- 4.3 mmHg, n = 10) than their wild-type congeners (C57BL/6 background). eNOS (-/-) KO mice, when subjected to systemically administered endothelin-1 or the selective endothelin-B agonist IRL-1620 (1 nmol/kg, each), responded by markedly enhanced and reduced pressor responses (systolic, diastolic and mean arterial pressure), respectively, when compared with wild-type mice. Surprisingly, plasma levels of immunoreactive endothelin are not significantly different in endothelial nitric oxide synthase knockout mice and wild-type mice. Our results suggest that chronic repression of endothelial nitric oxide synthase affects in opposite fashion the functionality of endothelin-A and endothelin-B receptor-dependent responses. The increase in mean arterial pressure afforded in endothelial nitric oxide synthase knockout mice does not appear to involve an increase in production of endogenous endothelin in vivo.

Effect of elevated intraocular pressure on endothelin-1 in a rat model of glaucoma

The role of endothelin-1 (ET-1) a potent vasoactive peptide, in glaucoma pathogenesis is receiving increasing attention, particularly in astroglial activation in optic nerve damage. Our laboratory has also shown that ET-1 treatment causes proliferation of cultured human optic nerve head astrocytes to possibly initiate astrogliosis. ET-1 is distributed in retina, optic nerve, and ciliary epithelium, however the effects of elevated intraocular pressure (IOP) (as seen in glaucoma) on ET-1 and ET B receptors are not clearly understood. In the present study, the levels of immunoreactive ET-1 (ir-ET-1) in aqueous humour (AH) and optic nerve head (ONH) were determined in the Morrison elevated IOP model of glaucoma. Additionally in the ONH of these rats, immunohistochemical analyses of ET B receptors and glial fibrillary acidic protein (GFAP; a marker for astroglial cells and for astrogliosis) were performed. There was 2- to 2.5-fold increase in AH ir-ET-1 levels for rats subjected to elevated IOP, compared to their respective controls. In the Morrison rat model of glaucoma, elevated IOP increased optic nerve ir-ET-1 with concomitant increases in ir-ET B and ir-GFAP labelling (possibly indicative of astrogliosis and hypertrophy). As seen in brain astrocytes subjected to neurotrauma, the present findings are suggestive of ET-1’s role in astroglial activation, particularly in response to elevated IOP in glaucoma.

Suppression of cytokine-induced expression of adrenomedullin and endothelin-1 by dexamethasone in T98G human glioblastoma cells

There is accumulating evidence showing that glial cells and gliomas secrete some neuropeptides and vasoactive peptides, such as adrenomedullin and endothelin-1. We have previously shown that expression of these two peptides is induced by inflammatory cytokines in T98G human glioblastoma cells. Glucocorticoids are frequently used for the treatment of inflammatory diseases and glioblastomas. We therefore studied effects of dexamethasone on expression of adrenomedullin and endothelin-1 in T98G human glioblastoma cells. Dexamethasone dose-dependently increased adrenomedullin mRNA levels and immunoreactive-adrenomedullin levels in the medium in T98G cells, whereas it decreased immunoreactive-endothelin levels in the medium. A combination of three cytokines, interferon-γ (100 U/ml), tumor necrosis factor-α (20 ng/ml) and interleukin-1β (10 ng/ml) induced expression of adrenomedullin and endothelin-1 in T98G cells. Dexamethasone (10 −8 mol/l) suppressed increases in expression of both adrenomedullin and endothelin-1 induced by these three cytokines. Thus, dexamethasone alone increased adrenomedullin expression whereas it suppressed the cytokine-induced expression of adrenomedullin in T98G cells. These findings raised the possibility that effects of dexamethasone on brain inflammation and glioblastomas may be partly mediated or modulated by its effects on expression of adrenomedullin and endothelin-1.

Plasma immunoreactive endothelin-1 levels in hypertensive rats and human subjects

Plasma immunoreactive endothelin-1 levels in hypertensive rats and human subjects

Renal endothelin in chronic angiotensin II hypertension.

To determine the influence of chronic ANG II infusion on urinary, plasma, and renal tissue levels of immunoreactive endothelin (ET), ANG II (65 ng/min) or saline vehicle was delivered via osmotic minipump in male Sprague-Dawley rats given either a high-salt diet (10% NaCl) or normal-salt diet (0.8% NaCl). High-salt diet alone caused a slight but not statistically significant increase (7 +/- 1%) in mean arterial pressure (MAP). MAP was significantly increased in ANG II-infused rats (41 +/- 10%), and the increase in MAP was significantly greater in ANG II rats given a high-salt diet (59 +/- 1%) compared with the increase observed in rats given a high-salt diet alone or ANG II infusion and normal-salt diet. After a 2-wk treatment, urinary excretion of immunoreactive ET was significantly increased by approximately 50% in ANG II-infused animals and by over 250% in rats on high-salt diet, with or without ANG II infusion. ANG II infusion combined with high-salt diet significantly increased immunoreactive ET content in the cortex and outer medulla, but this effect was not observed in other groups. In contrast, high-salt diet, with or without ANG II infusion, significantly decreased immunoreactive ET content within the inner medulla. These data indicate that chronic elevations in ANG II levels and sodium intake differentially affect ET levels within the kidney and provide further support for the hypothesis that the hypertensive effects of ANG II may be due to interaction with the renal ET system.

Elevated immunoreactive endothelin-1 levels in newborn rabbit lungs after meconium aspiration.

OBJECTIVE: Our prime objective was to study the production of big endothelin-1 (Big ET-1) and its conversion to ET-1 in the lungs of newborn rabbits exposed to meconium. Our second objective was to study the effect of captopril on endothelin expression. DESIGN: Prospective, comparative study. SETTING: Research laboratory of the Michael Reese Hospital and the University of Illinois, Chicago. SUBJECTS: Two-wk-old rabbit pups. INTERVENTIONS: Rabbit pups were instilled with meconium or saline into the lungs. Another group, pretreated with captopril, was also instilled with either meconium or saline. MEASUREMENTS AND MAIN RESULTS: After meconium or saline instillation, lung lavage was performed. Big ET-1 and ET-1 were measured in lung lavage fluid by using a commercially available enzyme-linked immunosorbent assay kits in all groups. Also, lungs were studied by histochemistry analysis for a morphologic evaluation of meconium-induced damage. In the lavage fluid of saline-instilled pups, ET-1 remained low and no increase in Big ET-1 levels was observed. In meconium-instilled animals, bioactive ET-1 levels were significantly higher, with a peak at 8 hrs after instillation. The conversion ratio of Big ET-1 to ET-1 in the meconium group increased from 2.19 at the initial period to 7.19 at 8 hrs after meconium instillation. CONCLUSIONS: Our conclusion is that aspiration of meconium causes lung injury in the newborn and that this injury is associated with a significant increase in ET peptide production in the lungs. We also showed that ET production is inhibited by pretreatment of rabbits with captopril before meconium-induced injury. ET-1 and its conversion from ET-1 in response to meconium may play important roles in increasing pulmonary vascular resistance and lung cell death, even in the absence of hypoxia. In general, we conclude, that ET-1 levels are significantly elevated in meconium-instilled rabbits compared with saline-instilled ones, and both can be significantly inhibited by pretreatment with captopril. Whether ET-1 contributes directly to the pathophysiology of or is simply a marker of meconium aspiration syndrome remains speculative.

Elevated immunoreactive endothelin-1 levels in newborn rabbit lungs after meconium aspiration

Elevated immunoreactive endothelin-1 levels in newborn rabbit lungs after meconium aspiration

Increased plasma endothelin levels in angina patients with rapid coronary artery disease progression.

To investigate the association between plasma endothelin levels and rapid coronary artery disease progression, as assessed by quantitative angiography. Changes in diameter were assessed in 224 coronary stenoses of 92 consecutive patients (62 men) with chronic stable angina pectoris who were on a waiting list for routine coronary angioplasty and underwent coronary angiography on two occasions: the first (diagnostic) angiogram was carried out at study entry and the second 5.5+/-3.0 months later, immediately prior to coronary angioplasty. A digital quantitative angiographic analysis system was used to assess differences in stenosis diameter between the first and second angiogram. Plasma immunoreactive endothelin levels were estimated by radioimmunoassay at study entry. Rapid coronary artery disease progression occurred in 29 (31.5%) patients according to pre-established criteria: 12 (41%) had a > or =10% diameter reduction of at least one pre-existing stenosis > or =50%, 10 (34%) had a > or =30% diameter reduction of a pre-existing stenosis <50%, 5 (17%) patients developed a new stenosis and 2 (7%) had progression of a lesion to total occlusion by the second angiogram. Baseline demographic, clinical and angiographic data were similar in patients with and without stenosis progression. Plasma endothelin levels were significantly higher in patients with rapid disease progression than in those without (5.7+/-2.0 pg. ml(-1)vs 3.9+/-1.6 pg. ml(-1), P<0.001). Multiple logistic regression analysis revealed that endothelin was an independent predictor of disease progression (P=0.001). Moreover, endothelin levels above 4.26 pg. ml(-1)(the median of the total endothelin concentrations) were associated with a sixfold increase in the risk of developing rapid stenosis progression. Plasma endothelin is raised in patients with coronary artery disease progression and may be a marker of risk of rapid stenosis progression. Endothelin may also play a pathogenic role in this process.

Effects of the endothelin receptor antagonist Bosentan on ischaemia/reperfusion injury in rat skeletal muscle

We examined the role of endothelin in ischaemia/reperfusion injury in skeletal muscle, using the endothelin receptor antagonist Bosentan. In the rat hindlimb tourniquet ischaemia model, one hindlimb was rendered ischaemic for 2 h at 36 °C, then blood flow was re-established for either 24 h to assess muscle survival or 1.5 h for a study of capillary perfusion. In the first set of rats, the gastrocnemius muscle was removed from the postischaemic limb and assessed for viability histochemically using the nitro blue tetrazolium stain. Tissue water content (a measure of oedema) and myeloperoxidase activity (a measure of neutrophil accumulation) were also assessed in the ischaemic muscle, the contralateral non-ischaemic muscle and the lungs. In the second set of rats, the hind limb was infused with India ink after 2-h ischaemia and 1.5-h reperfusion and the muscle was harvested, fixed and cleared. In control rats, muscle viability was 17±2% (S.E.M.). In rats treated with Bosentan (10 mg/kg, i.p.) 30 min before release of the tourniquet, muscle viability (48±7%) was significantly increased compared to the control group ( P<0.01). Bosentan treatment had no significant effect on tissue water content or myeloperoxidase activity in the ischaemic muscle, the contralateral non-ischaemic muscle or the lung. Immunoreactive endothelin levels in serum increased to a peak at 90 min of reperfusion and returned to control levels by 24-h reperfusion. India ink studies demonstrated a significantly increased functional capillary density in postischaemic Bosentan-treated muscles compared with postischaemic control muscles ( P<0.05). These results suggest that endothelin plays an important role in the necrosis which results from a period of ischaemia and reperfusion in skeletal muscle, by mediating a decrease in postischaemic microvascular perfusion.

High plasma immunoreactive endothelin levels in patients with Chagas’ cardiomyopathy

Chagas’disease is a major cause of morbidity and death in South America,1 where a great proportion of seropositive patients with chronic infection by Trypanosoma cruzi (T. cruzi) develop dilated cardiomyopathy, whose pathophysiology remains elusive. Because T. cruzi is seldom isolated from blood or tissue of chronically infected patients, several pathophysiologic mechanisms other than direct myocardial aggression by the parasite have been proposed.2,3 One of these focuses on the frequent coronary microvascular alterations found in patients with Chagas’ cardiomyopathy, which may lead to myocardial damage and dysfunction through different mechanisms, including myocardial ischemia. This “microvascular hypothesis” has received strong support from experimental4,5 and clinical observations.6,7 Plasma endothelin-1, a powerful vasoconstrictor peptide, has been found to be elevated in a number of cardiac conditions associated with endothelial injury and dysfunction.8,9 Wittner et al10 recently reported that human endothelial cells infected with T. cruzi release endothelin-1, suggesting a role for endothelins in the genesis of microvascular dysfunction as observed in patients with Chagas’cardiomyopathy. In the present study, we sought to assess the relation between plasma endothelin-1 levels and Chagas’cardiomyopathy in a group of patients with positive serology for chronic T. cruzi infection.

Plasma immunoreactive endothelin levels in children with cystic fibrosis.

Plasma immunoreactive endothelin levels were determined in 31 children and adolescents with cystic fibrosis and it was examined whether these levels correlated with the severity of the disease. The study comprised 16 cystic fibrosis patients (mean (SD) age 13.0 (4.9) y) with impaired lung function (Group A), 15 cystic fibrosis patients (11.2 (5.5) y) with unimpaired lung function (Group B) and 28 healthy controls (10.6 (4.3) y) (Group C). The selection and classification of patients into groups was based on criteria including the grade of finger-clubbing, the Brasfield chest radiograph score and spirometric and arterial blood gas values. In all subjects, plasma immunoreactive endothelin, atrial natriuretic peptide, renin, serum aldosterone levels and serum and urine electrolytes were measured. Plasma endothelin levels were significantly higher in Group A (range 2.5-8.4 pg/ml, median 3.2 pg/ml) than those in Group B (1.3-3.8 pg/ml, median 2.0 pg/ml, p < 0.001) and Group C (1.5-3.5 pg/ml, median 2.5 pg/ml, p < 0.001), whereas they did not differ between groups B and C. They correlated positively with the severity of finger-clubbing, heart rate, arterial blood PCO2, plasma atrial natriuretic peptide levels and serum aldosterone levels and negatively with the arterial blood PO2, forced vital capacity (FVC), forced expiratory volume in 1 sec (FEV1) and the Brasfield chest radiograph score. In multivariate regression analysis PO2 was the only independent factor found to significantly affect plasma endothelin levels. In conclusion, plasma immunoreactive endothelin levels are increased in cystic fibrosis patients with impaired pulmonary function and are related to the severity of the disease.