bluuebunny/crossref_metadata_2025 huggingface dataset:
Body fatness, insulin sensitivity and muscle oxygen supply in adolescents
Authors: ARROWSMITH Fiona E. , WARD Julie , ROONEY Kieron , KRIKETOS Adamandia D. , BAUR Louise A. , THOMPSON Campbell H.
Published: August 2015

Muscle blood flow can be reduced in insulin-resistant states. The present study examined the importance of body fatness and insulin sensitivity as variables that may be associated with muscle oxygen supply. We studied 38 adolescents (22 males, 16 females; age 15.3–18.6 years; body mass index 17.7–34.7kg/m2) and used near-IR spectroscopy to measure the muscle re-oxygenation rate after ischaemic finger flexion exercise. Total body fat content was estimated by bioelectrical impedance analysis, and insulin sensitivity was assessed by homoeostasis model assessment. Regional lipid compartments were also assessed for potential associations with muscle oxygen supply. Abdominal adiposity (visceral and subcutaneous) was assessed by magnetic resonance imaging, and soleus intramyocellular lipid levels were determined by magnetic resonance spectroscopy. Total body fat content (r = 0.67, P<0.001), abdominal subcutaneous fat area (r = 0.78, P<0.001), abdominal visceral fat area (r = 0.54, P<0.001) and intramyocellular lipid levels (r = 0.68, P<0.001) were significantly related to forearm re-oxygenation half-time. After adjusting for insulin sensitivity, both total body fat content (r = 0.395, P = 0.02) and abdominal subcutaneous fat area (r = 0.543, P = 0.001) remained positively associated with relatively reduced muscle oxygen supply in adolescent subjects. After adjusting for body fat content, abdominal subcutaneous fat area (r = 0. 511, P = 0.002) was significantly associated with muscle oxygen supply. Thus muscle oxygen supply is associated with body fat content, and certain fat compartments may be more influential than others.
Effects of tumour necrosis factor-α in the human forearm: blood flow and endothelin-1 release
Authors: PATEL J.N. , JAGER A. , SCHALKWIJK C. , CORDER R. , DOUTHWAITE J.A. , YUDKIN J.S. , COPPACK S.W. , STEHOUWER C.D.A.
Published: August 2015

Increased circulating concentrations of tumour necrosis factor-α (TNF-α) are seen in several pathological conditions associated with vascular disease. TNF-α induces the synthesis of endothelin-1 (ET-1), a potent vasoconstictor, by the endothelium. However, there is profound vasodilatation in sepsis, where circulating levels of both ET-1 and TNF-α are elevated. The details of the interaction between ET-1 and TNF-α and the predominant resulting haemodynamic effect in healthy humans are unclear. The aim of the present study was to determine the effects of intra-arterial TNF-α on ET-1 spillover, vascular tone and endothelial function in the healthy human forearm. Brachial arterial and deep venous blood samples, forearm plasma flow measurements and blood flow responses to acetylcholine and sodium nitroprusside were obtained in six healthy subjects before and during a 6h infusion of TNF-α into the brachial artery. Forearm blood flow was significantly greater than baseline during exposure to TNF-α [median (lower quartile, upper quartile): baseline, 2.6 (2.1,2.8) ml·min-1·100ml-1; TNF-α, 4.6 (4.5,5.1) ml·min-1·100ml-1; P<0.05]. The rate of release of ET-1 was significantly greater than baseline after 30 and 260min of TNF-α infusion [median (lower quartile, upper quartile): baseline, 0.8 (0.6,1.1)pg·min-1·100ml-1; 30min, 2.4 (1.9,3.2)pg·min-1·100ml-1; 260min, 4.1 (3.1,4.2)pg·min-1·100ml-1; P<0.05]. The vasodilatory response to acetylcholine was diminished during TNF-α infusion, whereas the response to sodium nitroprusside remained unchanged. We thus demonstrate for the first time that local TNF-α increases ET-1 spillover from the human forearm and impairs endothelium-dependent vasodilatation. In spite of this action, TNF-α has a vasodilatory effect, resulting in an increase in forearm blood flow.
Substrate utilization in non-obese Type II diabetic patients at rest and during exercise
Authors: BORGHOUTS L.B. , WAGENMAKERS A.J.M. , GOYENS P.L.L. , KEIZER H.A.
Published: August 2015

Recently, we observed that impairments exist in skeletal muscle free fatty acid (FFA) utilization during exercise in obese subjects with Type II diabetes. The main objective of the present study was to investigate whether plasma FFA oxidation is impaired during exercise in non-obese Type II diabetic patients. Stable isotope tracers of palmitate and glucose were infused for 2h at rest and 1h of bicycle exercise at 40% peak oxygen consumption (VO2max) in volunteers with Type II diabetes and a healthy control group. At rest, plasma FFA oxidation was not significantly different between subjects with Type II diabetes and control subjects (2.13±0.51 versus 1.93±0.54μmolμkg-1μmin-1 respectively). During exercise, Type II diabetic patients and control subjects had similar rates of total fat [Type II diabetes, 9.62±1.84μmolμkg-1μmin-1; control, 12.08±4.59μmolμkg-1μmin-1; not significant (NS)] and glucose oxidation (Type II diabetes, 44.24±10.36μmolμkg-1μmin-1; control, 57.37±14.54μmolμkg-1μmin-1; NS). No aberrations were present in plasma FFA uptake [rate of disappearance (Rd); Type II diabetes, 11.78±4.82; control, 10.84±3.39; NS] and oxidation rates (Type II diabetes 8.10±1.44; control 8.00±3.12, NS) in Type II diabetic patients; triacylglycerol-derived fatty acid oxidation was 2.6-fold lower in Type II diabetic patients than in control subjects, but this difference was not statistically significant. Muscle glycogen oxidation was lower in diabetes patients than in control subjects (Type II diabetes, 25.16±13.82μmolμkg-1μmin-1; control, 42.04±10.58μmolμkg-1μmin-1; P<0.05) and plasma glucose contributed more to energy expenditure in Type II diabetes (26±3% in diabetic versus 15±2% in control, P<0.05). We conclude that plasma FFA oxidation is not impaired during exercise in non-obese Type II diabetic patients. The data confirm that Type II diabetes is a heterogeneous disease, and that the adaptation at the substrate level differs between obese and non-obese patients and may contribute to differences in the final appearance of the various phenotypes.
Contribution of endothelin-1 to renal activator protein-1 activation and macrophage infiltration in aldosterone-induced hypertension
Authors: TOSTES Rita C A , TOUYZ Rhian M , HE Gang , CHEN Xin , SCHIFFRIN Ernesto L.
Published: August 2015

Aldosterone-induced hypertension is associated with renal damage that may be mediated by endothelin-1 (ET-1). We evaluated whether inflammatory cell infiltration and DNA-binding activity of the transcription factors nuclear factor κB (NF-κB) and activator protein-1 (AP-1) were increased in kidneys from aldosterone-infused rats. The role of ET-1 in these processes was evaluated by treating rats with the ETA-receptor blocker, BMS 182874. Rats were infused with aldosterone (0.75µg/h) via a mini-osmotic pump and were given 1% NaCl in the drinking water in the absence and presence of BMS 182874 or of the aldosterone receptor blocker, spironolactone. Renal sections were used to assess inflammatory cell infiltration, which was identified immunocytochemically using monoclonal antibodies against macrophages (ED1+). Electrophoretic mobility shift assays evaluated the DNA-binding activity of NF-κB and AP-1 in renal tissue. Systolic blood pressure (BP) was increased in the aldosterone-infused group compared with controls (123±6 versus 110±10mmHg, P<0.05). BMS 182874 and spironolactone significantly decreased BP (P<0.05). Macrophage infiltration was increased in the kidneys of aldosterone-infused rats compared with controls. Renal binding activity (arbitrary units) of AP-1, in contrast with that of NF-κB, increased in aldosterone-infused rats compared with control rats (AP-1, 4.2±0.3 versus 1.0±0.1, P<0.05; NF-κB, 1.6±0.5 versus 1.2±0.5). BMS 182874 and spironolactone decreased macrophage infiltration (by 70% and 50% respectively) and AP-1 binding activity (1.0±0.3 and 0.8±0.3 respectively). In conclusion, kidneys from aldosterone-infused rats exhibited macrophage infiltration and increased AP-1 DNA-binding activity. These processes were attenuated by BMS 182874. Our findings suggest that renal damage in aldosterone-dependent hypertension is associated with inflammatory processes that are mediated in part via ET-1.
Blood pressure of endothelin-3 null (-/-) knockout mice and endothelin A receptor null (-/-) knockout mice under anaesthesia
Authors: KUWAKI Tomoyuki , ISHII Toru , JU Kihwan , YANAGISAWA Masashi , FUKUDA Yasuichiro
Published: August 2015

Blood pressure (BP) and heart rate (HR) in endothelin-3 (ET-3) null (-/-) knockout mice and ETA receptor (-/-) mice were measured using the servo null pressure measuring technique under halothane anaesthesia. In infant ET-3 (-/-) mice (2–3 weeks old), mean BP and HR were 55±2mmHg and 436±30beats/min respectively. These values were not different from those in age-matched wild-type mice (53±3mmHg and 430±18beats/min respectively). Baroreflex sensitivity, which was calculated as the slope of the relationship between systolic BP and RR interval on an ECG, was also similar in ET-3 (-/-) mice (0.84±0.20ms/mmHg) and wild-type mice (1.07±0.38ms/mmHg). ETA receptor (-/-) mice were obtained by caesarean section on the expected day of delivery and tracheotomized, so that they would live for more than 24h. Mean BP and HR in ETA receptor (-/-) mice were 15±1mmHg and 333±6beats/min respectively. These values were not different from those in age-matched, similarly treated wild-type mice (16±3mmHg and 308±10beats/min respectively). Baroreflex sensitivity in the newborn ETA receptor (-/-) mice (0.45±0.15ms/mmHg) and wild-type mice (0.31±0.06ms/mmHg) were very low compared with the values in infant wild-type mice, but not different between the mutant mice and their littermates. Moreover, HR in awake ETA receptor (-/-) mice (396±13beats/min) was not different from that in wild-type mice (409±13beats/min). These results show that the ETA receptor and ET-3 are not involved in cardiovascular regulation, at least during the very early life of the mice. A possible involvement of the ETA receptor in BP regulation, if any, seems to occur at later times and/or in some pathological settings.
Role of endothelin during experimental Trypanosoma cruzi infection in rats
Authors: CAMARGOS Elizabeth R.S. , MACHADO Conceição R.S. , TEIXEIRA Antonio L. , ROCHA Lamara L.V. , FERREIRA Anderson J. , ALMEIDA Alvair P. , BARTON Matthias , TEIXEIRA Mauro M.
Published: August 2015

Chagas' disease is caused by the intracellular protozoan Trypanosoma cruzi. Here we have investigated the role of endothelin-1 in T. cruzi acute infection in rats, using the orally active ETA receptor antagonist BSF-461314. Treatment with BSF-461314 markedly increased parasitaemia, but animals managed to control the infection by day 15. Histopathological analysis of heart tissue at the end of the acute phase showed greater numbers of parasite nests in BSF-461314-treated animals. The perfusion of isolated rat hearts from infected animals with bradykinin failed to induce an increase, and actually reduced, coronary blood flow. Pretreatment with BSF-461314 prevented changes in coronary flow induced by T. cruzi infection. Together these results demonstrate that endothelin-1, through ETA receptor activation, contributes to the protective immune response against acute T. cruzi infection. Moreover, these data suggest that endothelin-1 is a mediator of impaired endothelium-dependent vasomotion in the coronary microcirculation associated with acute T. cruzi infection.
HMG-CoA reductase inhibition and PPAR-alpha activation both inhibit cyclosporin A induced endothelin-1 secretion in cultured endothelial cells
Authors: KANDOUSSI Abdelmejid , MARTIN François , HAZZAN Marc , NOËL Christian , FRUCHART Jean-Charles , STAELS Bart , DURIEZ Patrick
Published: August 2015

The use of cyclosporin A (CsA) in solid organ transplantation has been shown to be associated with the development of hypertension and nephrotoxicity. Several mechanisms, including endothelin (ET)-1-mediated systemic vasoconstriction, are considered to be responsible for CsA-induced hypertension. This study shows that: (i) incubation of CsA (1µM) with bovine aortic endothelial cells leads to increased ET secretion by+40%; (ii) the use of compactin, the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor and fibric acid, the peroxisome-proliferator-activated receptor (PPAR)-alpha activator, inhibit the CsA-induced ET secretion to the level below the basal ET secretion, by -32% and -26% respectively; (iii) both inhibitions were reversed by the addition of mevalonate, suggesting communication between the HMG-CoA reductase product and PPAR-alpha pathway. The present findings may be of significant clinical relevance, since statins and fibrates beyond their hypolipidaemic action may represent a potential therapeutic tool in the treatment or prophylaxis of CsA-associated side effects. Furthermore, we suggest that the mevalonate metabolism would interfere with PPAR-alpha activity.
Effects of the ECE/NEP inhibitor CGS 34225 on the big ET-1-induced pressor response and plasma atrial natriuretic peptide concentration in conscious rats
Authors: TRAPANI Angelo J. , BEIL Michael E. , BRUSEO Charles W. , FINK Cynthia A. , HOYER Denton , SAVAGE Paula , JENG Arco Y.
Published: August 2015

CGS 34226 is a thiol-containing, potent dual inhibitor of endothelin converting enzyme-1 (ECE-1) and neutral endopeptidase 24.11 (NEP) with IC50 values of 11 and 5nM respectively. The purpose of the present study was to characterize the inhibitory effects of CGS 34225, an orally active prodrug of CGS 34226, on ECE-1 and NEP in vivo. The effects on ECE-1 and NEP were assessed by determining the inhibition of big endothelin-1 (big ET-1)-induced increases in mean arterial pressure (MAP) and increases in plasma atrial natriuretic peptide (ANP) concentrations respectively, in conscious rats. Thirty and 120min after the administration of vehicle, big ET-1 (0.3nmol/kg, intravenously; i.v.) produced pressor responses of approximately 800mmHg·min (area under the curve for change in MAP×time). Treatment with CGS 34225 at 1mgEq/kg, per os (p.o.), decreased the pressor effect of big ET-1 by 39 and 53% at 30 and 120min respectively (P<0.05, both times). Increasing the dose of CGS 34255 to 30mgEq/kg, p.o., resulted in greater inhibition, 84 and 92% (P<0.05) at 30 and 120min respectively. Furthermore, at this higher dose, the inhibitory effect on ECE-1 was long-lasting, averaging 86, 75 and 30% (P<0.05, all times) at 4, 8 and 24h respectively. In rats treated with vehicle, the infusion of ANP at 450ng/kg per min i.v. resulted in plasma ANP concentrations of 3.9–4.8ng/ml that remained relatively constant for 4h. Treatment with CGS 34225 at 10mgEq/kg, p.o., increased the ANP level to 7.7±1.0 and 10.6±1.8ng/ml at 1 and 4h after dosing (P<0.05, both times). These data demonstrate that CGS 34225 is a potent, orally active and long-acting inhibitor of ECE-1 and NEP in vivo. It is anticipated that compounds with this dual function may be useful in the treatment of cardiovascular diseases where the ET system plays a pathogenic role and the potentiation of ANP elicits therapeutic benefits.
Pharmacology of endothelin receptor antagonists ABT-627, ABT-546, A-182086 and A-192621: in vitro studies
Authors: WU-WONG J. Ruth , DIXON Douglas B. , CHIOU William J. , SORENSEN Brian K. , LIU Gang , JAE H.-S. , TASKER Andrew , GELDERN Thomas W. Von , WINN Martin , OPGENORTH Terry J.
Published: August 2015

Endothelins (ETs), 21-amino-acid peptides involved in the pathogenesis of various diseases, bind to ETA and ETB receptors to initiate their effects. Based on the same core structure, we have developed four small-molecule ET receptor antagonists, ABT-627, ABT-546, A-182086 and A-192621, which exhibit difference in selectivity for ETA and ETB receptors. In this report, we compare the potency and selectivity of these four antagonists in inhibiting 125I-labelled ET-1 binding to cloned human ETA and ETB receptors, and in blocking ET-1-induced functional responses (arachidonic acid release and phosphatidylinositol hydrolysis).
Endothelin-1 (1-31) induces a thiorphan-sensitive release of eicosanoids via ETB receptors in the guinea pig perfused lung
Authors: PLANTE Mirco , HONORÉ Jean-Claude , NEUGEBAUER Witold , D'ORLÉANS-JUSTE Pedro
Published: August 2015

Maturation of big endothelin-1 (big ET-1) commonly produces the 21 amino acid vasoactive ET-1, which binds two ET receptors (ETA and ETB) to produce its effects. In the guinea pig, the systemic administration of ET-1 produces a bronchoconstrictor response that is mediated indirectly via the release of thromboxane A2 through ETB receptor activation. A new potent metabolite of big ET-1, ET-1 (1–31), has been reported to act as an ETA receptor selective agonist. In this study we investigated the effects of ET-1 (1–31), compared with ET-1, on the release of eicosanoids in the isolated and perfused guinea pig lung. We also clarified the implication of ET receptors in these effects using selective ETA or ETB receptor antagonists, BQ-123 and BQ-788 respectively. Finally, using the neutral endopeptidase 24.11 (NEP 24.11) inhibitor, thiorphan, we determined the involvement of this enzyme on ET-1 (1–31) effects. Infusion of ET-1 (1–31) (50nM) stimulates a marked release of thromboxane A2 and prostacyclin equivalent to that observed with a ten times lower concentration of ET-1 (5nM). BQ-788 (5nM and 10nM), but not BQ-123 (1µM), decreases the release of thromboxane A2 and prostacyclin triggered by both agonists. Interestingly, thiorphan (25µM) abolishes the eicosanoid-releasing properties of big ET-1 (100nM) and ET-1 (1–31). This study demonstrates that ET-1 (1–31) is less potent than ET-1 in stimulating the release of eicosanoids through ETB receptor activation in the guinea pig perfused lung. Moreover, the inhibitory properties of thiorphan indicate the possible existence of a bioactive metabolite of ET-1 (1–31). We therefore suggest that NEP 24.11 in the pulmonary vasculature, is implicated in the cleavage of ET-1 (1–31) to produce ET-1 which will further act on both ET receptors.
Cyclic stretch and endothelin-1 mediated activation of chloride channels in cultured neonatal rat ventricular myocytes
Authors: DE JONGE Henriëtte W. , DEKKERS Dick H.W. , TILLY Ben C. , LAMERS Jos M.J.
Published: August 2015

To date various types of Cl- currents have been recorded in cardiac myocytes from different regions of the heart and from different species. Most of these are silent under basal conditions, but are rapidly activated under the influence of various agonists or physical stress that, in the long term, also lead to development of hypertrophy. Previously, we identified three different Cl- channel activities in neonatal rat cardiomyocytes: (i) Ca2+ regulated, (ii) cAMP regulated (cystic fibrosis transmembrane conductance regulator Cl- channels) and (iii) osmoregulated Cl- channels. In this study, we examined comparatively the effects of cyclic stretch and endothelin-1 (ET-1) on Cl- channel activity in primary cultures of neonatal rat ventricular myocytes using an 125I-efflux assay. About 4min after the start of the 125I-efflux (mean basal rate amounts 6.3% of total 125I incorporated/min), the addition of 10nM ET-1 or the application of cyclic stretch rapidly and transiently increased 125I-efflux by 3.8%/min and 0.8%/min respectively above the basal rate. The stretch induced 125I-efflux rate could be blocked by 100µM Gd3+ but it had no effect on the ET-1 response. After 24h stimulation by ET-1 or cyclic stretch the myocytes responded by hypertrophy which is detected by increases of 3H-leucine incorporation into protein and protein/DNA ratio. In conclusion, cyclic stretch as well as ET rapidly and transiently activate Cl- channels in rat neonatal cardiomyocytes. The results suggest that the activation of distinct types of Cl- channels (co)transduce the stretch- and agonist-induced hypertrophic responses in these myocytes.
Potentiation by endothelin-1 and vasopressin of sympathetic vasoconstriction in male and female rats
Authors: FERNÁNDEZ Nuria , SANZ Elena , MONGE Luis , MARTÍNEZ María Angeles , DIÉGUEZ Godofredo , GARCÍA-VILLALÓN Angel Luis
Published: August 2015

The aim of this study was to analyse the modulatory role of endothelin-1 and vasopressin in sympathetic vasoconstriction, and whether there are gender differences in this modulatory role. Electrical field stimulation (4Hz, 0.2ms pulse duration at supramaximal voltage of 70V, for 1s), applied to isolated segments from rat tail arteries, produced a contraction that was lower in the vascular segments from female than in those from male rats. This arterial contraction was potentiated in a concentration-dependent way by endothelin-1 (10-10–3×10-9mol/l) and vasopressin (10-12–10-10mol/l), and the increment produced by endothelin-1 was similar in the arteries of both, whereas the increment induced by vasopressin was higher in the arteries from male than in those from female rats. These results suggest that endothelin-1 and vasopressin potentiate the vasoconstriction to sympathetic stimulation, and this potentiating effect of vasopressin may be higher, whereas that of endothelin-1 may be similar, in males than in females.
Endothelin-1 mRNA and protein in vascular wall cells is increased by reactive oxygen species
Authors: KAEHLER Jan , SILL Bjoern , KOESTER Ralf , MITTMANN Clemens , ORZECHOWSKI Hans-Dieter , MUENZEL Thomas , MEINERTZ Thomas
Published: August 2015

A dysregulated metabolism of oxygen-derived free radicals, nitric oxide and endothelin-1(ET-1) in conditions such as hypercholesterolaemia or hypertension may promote the development of atherosclerosis. We therefore subjected cultured human umbilical vein endothelial cells and coronary artery smooth muscle cells to oxidative stress induced by xanthine oxidase or hydrogen peroxide and observed alterations in ET-1 metabolism. Incubation with oxygen-derived free radicals increased preproET-1 promoter activity, ET-1 mRNA synthesis and big ET-1 concentrations in both cell types. This interaction of oxidative stress and ET-1 expression may be relevant in atherogenic conditions such as hypercholesterolaemia and hypertension since our data indicate that oxidative stress further aggravates the injurious effects attributed to ET-1.
Pericardial concentrations of adenosine, inosine and hypoxanthine in an experimental canine model of spastic ischaemia
Authors: KÉKESI Violetta , ZIMA Endre , BARÁT Erzsébet , HUSZÁR Éva , NAGY Andrea , LOSONCZI László , MERKELY Béla , HORKAY Ferenc , Juhász-Nagy Alexander
Published: August 2015

It has been shown that the adenosine concentration in the pericardial fluid of the normal heart is higher by one order of magnitude than that of the venous plasma. A further increase in the pericardial adenosine concentration was also demonstrated in myocardial ischaemia or hypoxia. It was proposed that pericardial nucleoside levels may represent the interstitial concentrations of the adenine nucleosides. An experimental model was designed to determine the intrapericardial concentrations of adenosine, inosine and hypoxanthine during coronary spasm provoked by intracoronary administration of endothelin-1 (ET-1; 0.08±0.02nmol/g of myocardial tissue). In the in situ dog heart (n = 10), adenosine, inosine and hypoxanthine concentrations were determined by HPLC in fluid samples collected from the closed pericardial sac before and after ET-1 administration, and from the systemic arterial blood. Systemic blood pressure, heart rate and standard ECG were registered continuously. We found that the nucleoside concentrations in the infusate samples increased significantly during coronary spasm [adenosine, 1.49±0.44 compared with 0.37±0.07µM (P<0.05); inosine, 27.43±11.51 compared with 0.47±0.11µM (P<0.05); hypoxanthine, 21.17±6.49 compared with 4.91±1.24µM (P<0.05)], while a significant decrease in blood pressure and an elevation in ECG ST segments were observed. The levels of the purine metabolites did not change in the systemic blood. The data indicate that changes in adenine nucleoside levels measured in pericardial infusate samples reflect activation of coronary metabolic adaptation in this model of spastic ischaemia, and that pericardial nucleoside levels may characterize alterations in interstitial adenine nucleoside concentrations.
Up-regulation of endothelin-converting-enzyme mRNA expression following cardioplegic arrest
Authors: GOODWIN Andrew T. , KHAN Mahboob A. , CHESTER Adrian H. , AMRANI Mohamed , YACOUB Magdi H.
Published: August 2015

Endothelin-1 (ET-1) is believed to play an important role in cardiac ischaemia/reperfusion injury. ET-1 is synthesized from preproET-1 by the action of ET-converting enzyme (ECE). It is unclear to what extent the ET system is activated following prolonged ischaemia. In this study we used a model mimicking the conditions of the donor heart during transplantation. Isolated rat hearts perfused with Krebs–Henseleit buffer were subjected to 30min of normothermic perfusion, then 4h of cardioplegic arrest at 4°C with St Thomas' Hospital solution, followed by reperfusion for 2h. Hearts were freeze-clamped at different time points during the protocol. Using quantitative reverse transcription–PCR, relative levels of ET-1 and ECE mRNA expression were measured and compared with a housekeeping gene (ribosomal protein L32). During reperfusion there was a consistent decrease in coronary flow to approx. 85–90% of pre-ischaemic flow. There was no significant alteration in preproET-1 mRNA expression during 2h of reperfusion. However, ECE mRNA expression was increased by 77.5% at 1h and by 74.6% at 2h following ischaemia compared with pre-ischaemic values (P<0.05). Thus we conclude that ECE mRNA expression is increased following prolonged hypothermic cardioplegic arrest. Elevations in the expression of this enzyme may help to explain the role of the ET system in the pathogenesis of ischaemia/reperfusion injury following cardiac surgery and transplantation.
Increased expression of endothelin-converting enzyme-1 in failing human myocardium
Authors: MORAWIETZ Henning , GOETTSCH Winfried , SZIBOR Marten , BARTON Matthias , SHAW Sidney , HAKIM Kavous , ZERKOWSKI Hans-Reinhard , HOLTZ Juergen
Published: August 2015

Endothelin-1 (ET-1) is considered to be involved in the development and progression of heart failure. Therefore, we analysed the expression of endothelin-converting enzyme-1 (ECE-1), endothelin receptors A (ETA) and B (ETB) mRNAs by standard-calibrated, competitive reverse transcriptase-PCR using an internal-deleted in vitro-transcribed cRNA standard. ET-1 peptide levels were measured using isoform-specific rabbit antibodies against synthetic ET-1. mRNA and protein expression was determined in the right atrial myocardium of New York Heart Association class I patients and class IV patients undergoing aorto-coronary bypass surgery. ECE-1 mRNA was upregulated in failing atrial myocardium. Furthermore, ET-1 peptide levels were increased in failing atrial myocardium. Atrial ETA mRNA expression was not changed, while ETB mRNA was downregulated in the failing atrial myocardium. Our results support an upregulation of ET-1 synthesis by induction of ECE-1 in failing atrial myocardium. Pharmacological inhibition of augmented ECE-1 expression might provide a new therapeutic perspective in the treatment of heart failure.
Effects of endothelin receptors ETA and ETB blockade on renal haemodynamics in normal rats and in rats with experimental congestive heart failure
Authors: ABASSI Zaid , FRANCIS Bahaa , WESSALE Jerry , OVCHARENKO Elena , WINAVER Joseph , HOFFMAN Aaron
Published: August 2015

The present study examined the effects of two highly selective endothelin-1 (ET-1) receptor antagonists, ABT-627 (ETA blocker) and A-192621 (ETB blocker), on the systemic and renal haemodynamic effects of ET-1 in normal rats and in rats with experimental congestive heart failure (CHF) produced by aortocaval fistula. Intravenous injection of ET-1 (1.0nmol·kg-1 of body weight) to anaesthetized normal rats produced sustained decreases in renal blood flow (RBF) (assessed by ultrasonic flowmetry) and glomerular filtration rate (GFR), and significant increases in renal vascular resistance (RVR) and mean arterial pressure (MAP). Pretreatment with ABT-627 (1mg·h-1·kg-1 of body weight) abolished the pressor response to ET-1 without affecting the depressor phase, and significantly impaired the renal vasoconstriction. The systemic and renal vasoconstrictive effects of ET-1 in normal rats were significantly augmented by pretreatment with 3.0mg·h-1·kg-1 of A-192621. Baseline RBF and GFR in rats with CHF were reduced significantly compared with control rats, whereas RVR was elevated. The hypertensive effect of ET-1 was attenuated in rats with CHF. In the presence of ETA blockade, the pressor response to ET-1 was completely abolished in CHF rats. Furthermore, pretreatment with ABT-627 enhanced the recovery from ET-1- dependent vasoconstriction and remarkably reversed the ET-1-induced hypofiltration. Blockade of ETB receptors in rats with CHF further exposed the exaggerated ET-1-induced renal vasoconstriction. Our data demonstrate that experimental CHF is associated with altered responsiveness to ETA- and ETB-mediated systemic and renal effects of ET-1. Furthermore, in CHF, as in control rats, the ETB-mediated vasodilatory response may serve as an important compensatory counterbalance to the adverse ETA-mediated effects.
Role of cardiac myocyte-derived endothelin-1 in chagasic cardiomyopathy: molecular genetic evidence
Authors: HUANG Huan , YANAGISAWA Masashi , KISANUKI Yaz Y. , JELICKS Linda A. , CHANDRA Madhulika , FACTOR Stephen M. , WITTNER Murray , WEISS Louis M. , PESTELL Richard G. , SHTUTIN Vitaliy , SHIRANI Jamshid , TANOWITZ Herbert B.
Published: August 2015

Trypanosoma cruzi is the aetiological agent of Chagas' disease, an important cause of chronic cardiomyopathy. We previously demonstrated a role for endothelin-1 (ET-1) in the pathogenesis of chagasic heart disease. In order to explore further the significance of ET-1 in chagasic heart disease, we infected ET-1 (flox/flox); α-MHC-Cre(+) (ET-1KO) mice, in which the ET-1 gene has been deleted from cardiac myocytes, with 104T. cruzi (Brazil strain) trypomastigotes. As controls, we used ET-1 (flox/flox);Cre(-) (FLOX) and C57BL/6×129sv (WT) mice. All mice survived and were evaluated 150–160 days post-infection. Cardiac magnetic resonance imaging revealed a significant increase in right ventricular internal diameter in all infected animals except ET-1KO mice (control WT, 1.6±0.10mm; infected WT, 2.8±0.15mm; control FLOX, 2.04±0.02mm; infected FLOX, 2.76±0.28mm). There was no significant difference in right ventricular internal diameter between infected and uninfected ET-1KO mice (control ET-1KO, 1.83±0.11mm; infected ET-1KO, 2.14±0.20mm). In another series of experiments, transthoracic echocardiography was performed on uninfected as well as infected ET-1KO mice, and uninfected and infected FLOX mice. Both infected groups had an increased left ventricular end-diastolic diameter and reduced fractional shortening. In addition, relative wall thickness was also decreased in infected animals. However, the magnitude of these changes was less in infected ET-1KO mice. These data provide further support for a role for ET-1 in the pathogenesis of chronic chagasic heart disease, and indicate that cardiac myocytes are an important source of ET-1 in this disease.
ABT-627, a potent endothelin receptor A antagonist, inhibits ovarian carcinoma growth in vitro
Authors: SALANI Debora , ROSANÒ Laura , DI CASTRO Valeriana , SPINELLA Francesca , VENUTI Aldo , PADLEY Robert J. , NICOTRA Maria Rita , NATALI Pier Giorgio , BAGNATO Anna
Published: August 2015

Endothelin-1 (ET-1) is present at high concentrations in ovarian cancer ascites and is overexpressed in primary and metastatic ovarian carcinomas. In these tumours the presence of ET-1 is associated with enhanced neovascularization and with vascular endothelial growth factor (VEGF) expression. In these tumour cells, ET-1 acts as an autocrine growth factor selectively through the receptor ETA, which is predominantly expressed in tumour cells. Furthermore, ET-1 produced by ovarian tumour cells stimulates VEGF production and VEGF-mediated angiogenic effects through ETA binding. These results demonstrate that activation of the ETA in ovarian carcinoma cells promotes cell proliferation, neovascularization and invasion, which are the principal hallmarks of malignant transformation. The present study was designed to investigate the effects of the ETA-selective antagonist ABT-627 on the ET-1-induced mitogenic effect in both primary cultures (PMOV1 and PMOV2) and cell lines (OVCA 433 and HEY) of ovarian carcinoma. All tumour cells express the components of the ET-1 system and secrete ET-1. ETA blockade by ABT-627 inhibits ET-1-induced mitogenic effects. The ETB antagonist BQ-788 is ineffective although all cell lines express both ETA and ETB mRNAs. In conclusion, our results demonstrate that ABT-627 is capable of inhibiting the proliferative activity of ET-1, suggesting that this potent ETA antagonist may provide a novel approach to the multidisciplinary treatment of ovarian carcinoma.
Endothelins may modulate invasion and proliferation of Ewing's sarcoma and neuroblastoma
Authors: BERRY Paul , BURCHILL Susan
Published: August 2015

Tumours of the Ewing's sarcoma (ES) family and neuroblastoma (NBL) were examined by reverse transcriptase-PCR for expression of mRNA for endothelin (ET) receptors ET-A and ET-B, and the ligands ET-1, ET-2 and ET-3. The effect of ET-1, ET-3, an ET-1-neutralizing antibody and ET-A receptor antagonist BQ-123 on cell proliferation was examined using an ELISA. Loss of ET-B receptor mRNA occurred in 57% of ES and 42% of NBL tumours. This appeared to be associated with the presence of metastatic disease and disease progression. ET-A receptor mRNA was expressed in all ES and 85% of NBL tumours, and in all ES and NBL cell lines examined. All ET ligands were detected in NBL cell lines, but only ET-1 and ET-2 were expressed in ES cell lines. Treatment of ES and NBL cells with ET-1 increased proliferation, but ET-3 had no effect. Incubation of ES and NBL cells with an ET-1-neutralizing antibody or BQ-123 decreased proliferation. The ET-3 ligand and ET-B receptor may be associated with migration and metastasis of ES and NBL, whereas ET-1 (acting through the ET-A receptor) may regulate their proliferation.
Enhanced ETA receptor-mediated contractile function is associated with reduced ETA receptor density in sheep bronchial explants maintained in culture
Authors: GOLDIE Roy G. , D'APRILE Angela C. , BETTS Richard J. , FERNANDES Lynette B.
Published: August 2015

We are interested in developing an airway explant culture system using sheep bronchi in which to establish respiratory viral infection and from which tissue can be used for functional, biochemical and immunohistochemical studies involving the endothelins (ETs). Freshly harvested sheep bronchial airway smooth muscle contains a homogeneous population of the ETA receptor. However, the potency of ET-1 and maximum contractile response of sheep bronchial explants to ET-1 increased with time in culture, despite these parameters remaining constant for carbachol in explants maintained for up to 48h. The possibility that this was caused by changes in ET receptor density was assessed using light microscopic quantitative autoradiography. In view of the increased responsiveness to ET-1 in cultured explants, it was surprising to demonstrate a significant decrease in total ET receptor (59±6% compared with the initial value, n = 4–5; P<0.01) and ETA receptor (51±2% compared with the initial value, n = 4–5, P<0.01) density in sheep bronchial explants after 48h. No ETB receptors were detected. Thus, the culture of sheep bronchial explants was associated with an increase in ETA receptor-mediated contractile function that was accompanied by a decrease in ETA receptor density. In addition, the structural integrity of the ciliated epithelium was preserved using this culture protocol, a feature that is critical to successful respiratory viral infection. The significant changes in ET receptor density and function in these bronchial explants must be carefully considered when assessing any effects of respiratory viral infection in this model.
Effects of a selective endothelin A receptor antagonist, ABT-627, in healthy normotensive anaesthetized rats developing acute pulmonary air embolism
Authors: AYACH Bilal , TSANG John , JENG Arco Y. , BLOUIN André , GOSSELIN Mélanie , WANG Fu-Hou , WU-WONG Jinshyun R. , WESSALE Jerry , OPGENORTH Terry J. , BATTISTINI Bruno
Published: August 2015

Acute pulmonary air embolism (APAE) injures the vascular endothelium in the lung and results in pulmonary hypertension (PH). Endothelins (ETs), a family of potent vasoactive peptides, are known to be associated with PH of various aetiologies. We evaluated the effects of ABT-627, a selective ETA receptor (ETA-R) antagonist in a rat model of APAE over 3h. APAE rats developed a higher right ventricular systolic pressure (RVSP), lower mean arterial blood pressure (MABP), and had lower PaO2. At 3h, arterial plasma levels of ET-1 were increased. ABT-627-treated controls showed no effects. However, ABT-627 significantly lowered RVSP during APAE, abolished the short recovery phase (within 10–25min) of MABP without affecting the subsequent lowering of MABP, and improved oxygen saturation in APAE rats. These results show that ETA-R subtype is involved in the pathogenesis of APAE since a blockade of this receptor subtype attenuated the cardiopulmonary deterioration and improved blood gas exchanges in rats with this disease.
Role of endothelin receptors in the hypertensive state of kinin B2 knockout mice subjected to a high-salt diet
Authors: BROCHU Isabelle , LABONTÉ Julie , BKAILY Ghassan , D'ORLÉANS-JUSTE Pedro
Published: August 2015

Mice with disruption of the kinin B2 receptor (B2KO mice) are sensitive to salt-rich diets, which causes hypertension. The aim of the study was to assess the role of endothelin-1 (ET-1) and angiotensin-II in hypertensive B2KO mice on a salt-rich diet. We also wanted to verify if there is an upregulation of the mRNA expression of the precursors or receptors for these hormones. Two groups of B2KO mice (20–25g) were investigated. The first group received an 8% NaCl diet with 1% NaCl in drinking water (HS) and the second was fed with normal food with tap water (NS). The antagonists tested were the ETA receptor antagonist BQ-123 (1 and 5mg/kg), the ETB receptor antagonist BQ-788 (0.25 and 1mg/kg), the angiotensin receptor type 1 antagonist losartan (10mg/kg) and the angiotensin-converting enzyme inhibitor captopril (3mg/kg). These were injected intraperitoneally 30min prior to blood pressure measurement by the tail-cuff method. We also studied the level of expression of preproET-1, ET-1 receptors, angiotensinogen and angiotensin receptors by RNA extraction from the heart and kidneys of these mice followed by reverse transcriptase (RT)-PCR. B2KO mice (HS) were hypertensive after 8 weeks compared with B2KO mice on normal diet (HS, 93.4±1.5mmHg, n = 7; NS, 61.4±2.7mmHg, n = 7). In the HS group, the mean arterial blood pressure was significantly reduced by BQ-123 (5mg/kg) to 61.9±1.8mmHg (n = 7), by BQ-788 (1mg/kg) to 58.8±2.6mmHg (n = 6), by losartan (10mg/kg) to 73.2±1.7mmHg (n = 8) and by captopril (3mg/kg) to 86.0±2.3mmHg (n = 8). The expression studied by RT-PCR did not show any difference (either in precursors or receptors expression) between hypertensive and normal mice. The four antagonists used seemed to reverse the hypertension. These results suggest that ET-1 and angiotensin-II are probably involved in the mechanism that leads to hypertension since the effect of these hormones is probably not compensated by kinins in B2KO mice. Further studies are necessary to understand the implication of the cross-talk between these hormones in the hypertensive state.
Interaction of the endothelin system and calcineurin inhibitors after kidney transplantation
Authors: SLOWINSKI Torsten , SUBKOWSKI Thomas , DIEHR Petra , BACHERT Daniela , FRITSCHE Lutz , NEUMAYER Hans.-H. , HOCHER Berthold
Published: August 2015

Plasma endothelin (ET)-1 concentrations have been shown to be elevated in patients receiving calcineurin-inhibitors (CI). We investigated urinary and plasma ET-1 (uET-1, pET-1), BigET-1 (uBigET-1, pBigET-1) concentrations, and plasma soluble endothelin-converting enzyme (ECE) concentrations in 381 adult caucasian kidney allograft recipients with graft survival of more than 2 years from the outpatients department of our clinic. Blood and urine probes were always drawn immediately before morning dosage of immunosuppressants. Mean of urinary protein excretion (meanProt) and mean of serum creatinine (meanCrea) were calculated from all available measurements in the most recent year. uET-1 and uBigET-1 were adjusted for urinary protein excretion by calculating uET-1/meanProt and uBigET-1/meanProt. Patients (n = 310) were on a cyclosporine A or FK506 (CI-group) based immunosuppression protocol, and 71 patients were immunosuppressed without use of CI (nonCI group). Time since transplantation was similar in both groups (mean±S.D.; CI-group: 7.55±2.50; nonCI-group: 7.74±3.06 years, P = 0.240) as well as meanCrea (mean±S.D.; CI-group: 1.97±1.34; nonCI-group: 1.77±1.29mg/dl, P = 0.326). pET-1 was significantly higher in the CI-group, compared with nonCI (mean±S.D.; 0.87±1.4 versus 0.56±0.76fmol/ml, P = 0.011). pBigET-1 was similar (mean±S.D.; 0.85±1.41 versus 0.70±1.21fmol/ml, P = 0.33). ECE concentrations were higher in the CI group (mean±S.D.; 14.30±18.02 versus 9.23±7.42ng/ml, P = 0.001). uET-1/meanProt and uBigET-1/meanProt concentration were similar in the CI-group compared with the nonCI-group (mean±S.D.; uET-1/meanProt: 15±24 versus 21±40pmol/g, P = 0.139; uBigET-1/meanProt: 34±55 versus 19±23pmol/g, P = 0.248). pET-1 elevation in patients receiving CI might be more likely to be due to elevated conversion of pBig-ET-1 by more ECE, and not to higher concentrations of pBigET-1.
Endothelin A-receptor blockade in experimental diabetes improves glucose balance and gastrointestinal function
Authors: BALSIGER Bruno , RICKENBACHER Andreas , BODEN Penelope Jane , BIECKER Erwin , TSUI Janice , DASHWOOD Michael , REICHEN Jürg , SHAW Sidney George
Published: August 2015

Secondary complications of diabetes mellitus often involve gastrointestinal dysfunction. In the experimental Goto Kakizaki rat, a model of Type II diabetes, hyperglycaemia and reduced glucose clearance is associated with elevated plasma endothelin (ET)-1 levels and selective decreases in nitric oxide synthase in circular muscle, longitudinal muscle and neuronal elements of the gastrointestinal tract. Functionally, this is accompanied by decreased nitrergic relaxatory responses of jejunal longitudinal muscle to tetrodotoxin-sensitive electrical field stimulation. Long-term treatment with a selective ET A-type receptor antagonist, markedly reduced hyperglycaemia and restored plasma glucose clearance rates towards normal. This was associated with a restoration of NG-nitro-L-arginine methyl ester-sensitive relaxatory responses of jejunal longitudinal muscle to electrical field stimulation. The results indicate that beneficial effects of ETA receptor blockade on gastrointestinal function may result from an improvement in insulin sensitivity with concomitant reduction of the severity of hyperglycaemia. ETA receptor blockade may represent a new therapeutic principle for improving glucose tolerance in Type II diabetes and could be beneficial in alleviating or preventing hyperglycaemia-related secondary complications in this condition.
Role of transforming growth factor β in conjunctival scarring
Authors: CORDEIRO M. Francesca
Published: August 2015

Glaucoma is the major cause of irreversible blindness throughout the world. Of all of the treatments that are available at present, the most effective appears to be surgery; however, excessive conjunctival scarring can lead to surgical failure. In the last decade, the introduction of the anti-metabolites mitomycin-C and 5-fluorouracil as anti-scarring treatments have greatly improved the results of glaucoma surgery, but these agents are associated with complications that can potentially result in blindness. A possible target for a more physiological approach to anti-scarring is transforming growth factor β. This review examines the role of transforming growth factor β in conjunctival scarring and discusses promising new ways of modifying its activity.
Mechanisms for the role of tetrahydrobiopterin in endothelial function and vascular disease
Authors: Schmidt Tim S. , Alp Nicholas J.
Published: August 2015

NO produced by eNOS (endothelial nitric oxide synthase) is a key mediator of vascular homoeostasis. NO bioavailability is reduced early in vascular disease states, such as hypercholesterolaemia, diabetes and hypertension, and throughout the progression of atherosclerosis. This is a result of both reduced NO synthesis and increased NO consumption by reactive oxygen species. eNOS enzymatic activity appears to be determined by the availability of its cofactor BH4 (tetrahydrobiopterin). When BH4 levels are adequate, eNOS produces NO; when BH4 levels are limiting, eNOS becomes enzymatically uncoupled and generates superoxide, contributing to vascular oxidative stress and endothelial dysfunction. BH4 bioavailability is determined by a balance of enzymatic de novo synthesis and recycling, versus oxidative degradation in dysfunctional endothelium. Augmenting vascular BH4 levels by pharmacological supplementation, by enhancing the rate of de novo biosynthesis or by measures to reduce BH4 oxidation have been shown in experimental studies to enhance NO bioavailability. Thus BH4 represents a potential therapeutic target for preserving eNOS function in vascular disease.
Cardiac peroxisome-proliferator-activated receptor expression in hypertension co-existing with diabetes
Authors: Lee Ting-I , Kao Yu-Hsun , Chen Yao-Chang , Pan Nan-Hung , Lin Yung-Kuo , Chen Yi-Jen
Published: August 2015

Hypertension and DM (diabetes mellitus) are common chronic disorders that often co-exist. DM and PPAR (peroxisome-proliferator-activated receptor)-γ agonists may directly impair heart function. However, the effects of DM and PPAR-γ agonists on hypertensive myocardium are not known. Hence the aim of the present study was to investigate whether DM and a PPAR-γ agonist [RGZ (rosiglitazone)] modulated the effects of hypertension on myocardial expression of PPAR isoforms. Cardiac PPAR isoforms, TNF (tumour necrosis factor)-α and IL (interleukin)-6 were evaluated by real-time PCR and Western blotting in SHRs (spontaneously hypertensive rats), diabetic SHRs, diabetic SHRs treated with RGZ (5 mg/kg of body weight) and control WKY (Wistar–Kyoto) rats. Cardiac NADPH oxidase activity was quantified using a SOD (superoxide dismutase)-sensitive cytochrome c reduction assay. When compared with hearts from control WKY rats, hearts from SHRs had decreased PPAR-α and PPAR-δ mRNA and protein levels (39 and 44% respectively for PPAR-α, and 37 and 42% respectively for PPAR-δ), but had increased PPAR-γ mRNA and protein levels (1.9- and 1.4-fold respectively). The hypertension-induced changes in mRNA and protein of cardiac PPAR isoforms were enhanced in diabetic SHRs, which were attenuated in diabetic SHRs treated with RGZ. Cardiac TNF-α and IL-6 protein levels and NADPH oxidase activities were increased in SHRs and were increased further in diabetic SHRs. RGZ treatment decreased TNF-α and IL-6 protein levels and NADPH oxidase activities in hearts from diabetic SHRs. In conclusion, these findings suggest that DM and the PPAR-γ agonist modulated the hypertensive effects on cardiac PPAR isoform expression.