s6c-sarafotoxin and Disease-Models--Animal

Endothelin ET(B) receptors mediate under normal physiological conditions vasorelaxation in coronary arteries. However, vasocontractile ET(B) receptors appear in coronary arteries of ischemic heart disease patients. Interestingly, organ culture of isolated coronary arteries also induces upregulation of vasocontractile ET(B) receptors. This study examines the early time course and mechanism behind upregulation of contractile ET(B) receptors in isolated rat coronary arteries during short-term organ culture.. Coronary artery segments were mounted in wire-myographs and incubated in physiological saline solution. Contractions were measured after exposure to the specific ET(B) receptor agonist Sarafotoxin 6c (S6c) and the endogenous agonists endothelin-1 and endothelin-3. Protein localization and levels of ET(B) and phosphorylated-extracellular-signal-regulated-kinase-1/2 (ERK1/2) were examined by immunohistochemistry.. Fresh arteries showed negligible vasoconstriction to S6c. However, incubation for only 4 and 7h increased S6c contractions two- and seven-fold, respectively. Furthermore, 7h incubation enhanced vasocontractile responses to endothelin-3 and increased ET(B) receptor density in vascular smooth muscle cells. ERK1/2 was activated rapidly after start of incubation. Moreover, incubation with either the transcriptional inhibitor actinomycin D or the mitogen-activated-protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 attenuated contractile ET(B) receptor upregulation. U0126 attenuated ET(B) receptor protein levels after 24 h of incubation.. Coronary arteries rapidly upregulate vasocontractile ET(B) receptors during organ culture via transcriptional mechanisms and MEK-ERK1/2 signalling. This model may mimic the mechanisms seen in ischemic conditions. Furthermore, these findings have important experimental implications in tissue bath experiments lasting for more than 4h.

Topics: Animals; Butadienes; Coronary Vessels; Dactinomycin; Disease Models, Animal; Endothelin-1; Endothelin-3; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocardial Ischemia; Nitriles; Organ Culture Techniques; Phosphorylation; Rats; Rats, Sprague-Dawley; Receptor, Endothelin B; Time Factors; Transcription, Genetic; Up-Regulation; Vasoconstriction; Vasoconstrictor Agents; Viper Venoms

Cerebral arteries subjected to different types of experimental stroke upregulate their expression of certain G-protein-coupled vasoconstrictor receptors, a phenomenon that worsens the ischemic brain damage. Upregulation of contractile endothelin B (ET(B)) and 5-hydroxytryptamine 1B (5-HT(1B)) receptors has been demonstrated after subarachnoid hemorrhage and global ischemic stroke, but the situation is less clear after focal ischemic stroke. Changes in smooth muscle calcium handling have been implicated in different vascular diseases but have not hitherto been investigated in cerebral arteries after stroke. Here, we evaluate changes of ET(B) and 5-HT(1B) receptors, intracellular calcium levels, and calcium channel expression in rat middle cerebral artery (MCA) after focal cerebral ischemia and in vitro organ culture, a proposed model of vasoconstrictor receptor changes after stroke. Rats were subjected to 2 h MCA occlusion followed by reperfusion for 1 or 24 h. Alternatively, MCAs from naïve rats were cultured for 1 or 24 h. ET(B) and 5-HT(1B) receptor-mediated contractions were evaluated by wire myography. Receptor and channel expressions were measured by real-time PCR and immunohistochemistry. Intracellular calcium was measured by FURA-2. Expression and contractile functions of ET(B) and 5-HT(1B) receptors were strongly upregulated and slightly downregulated, respectively, 24 h after experimental stroke or organ culture. ET(B) receptor-mediated contraction was mediated by calcium from intracellular and extracellular sources, whereas 5-HT(1B) receptor-mediated contraction was solely dependent on extracellular calcium. Organ culture and stroke increased basal intracellular calcium levels in MCA smooth muscle cells and decreased the expression of inositol triphosphate receptor and transient receptor potential canonical calcium channels, but not voltage-operated calcium channels.

Topics: Animals; Calcium; Cerebral Arteries; Disease Models, Animal; Dose-Response Relationship, Drug; Intracellular Fluid; Male; Muscle, Smooth, Vascular; Organ Culture Techniques; Rats; Rats, Wistar; Receptor, Endothelin B; Receptor, Serotonin, 5-HT1B; Stroke; Vasoconstriction; Viper Venoms

Diabetes increases the risk of stroke and contributes to poor clinical outcomes in this patient population. Myogenic tone of the cerebral vasculature, including basilar arteries, plays a key role in controlling cerebral blood flow. Increased myogenic tone is ameliorated with ET receptor antagonism in Type 1 diabetes. However, the role of endothelin-1 (ET-1) and its receptors in cerebrovascular dysfunction in Type 2 diabetes, a common comorbidity in stroke patients, remains poorly elucidated. Therefore, we hypothesized that 1) cerebrovascular dysfunction occurs in the Goto-Kakizaki (GK) model of Type 2 diabetes, and 2) pharmacological antagonism of ETA receptors ameliorates, while ETB receptor blockade augments vascular dysfunction. GK or control rats were treated with antagonists to either ETA (atrasentan, 5 mg.kg(-1).day(-1)) or ETB (A-192621, 15 or 30 mg.kg(-1).day(-1)) receptors for 4 wk and vascular function of basilar arteries was assessed using a wire myograph. GK rats exhibited increased sensitivity to ET-1. ET(A) receptor antagonism caused a rightward shift, indicating decreased sensitivity in diabetes, while it increased sensitivity to ET-1 in control rats. Endothelium-dependent relaxation was impaired in diabetes. ETA receptor blockade restored relaxation to control values in the GK animals with no significant effect in Wistar rats and ETB blockade with 30 mg.kg(-1).day(-1) A-192621 caused paradoxical constriction in diabetes. These studies demonstrate that cerebrovascular dysfunction occurs and may contribute to altered regulation of myogenic tone and cerebral blood flow in diabetes. While ETA receptors mediate vascular dysfunction, ETB receptors display differential effects. These results underscore the importance of ETA/ETB receptor balance and interactions in cerebrovascular dysfunction in diabetes.

Topics: Acetylcholine; Animals; Atrasentan; Basilar Artery; Diabetes Mellitus, Type 2; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Male; Pyrrolidines; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents; Viper Venoms

Vascular dysfunction, which presents either as an increased response to vasoconstrictors or an impaired relaxation to dilator agents, results in worsened cardiovascular outcomes in diabetes. We have established that the mesenteric circulation in Type 2 diabetes is hyperreactive to the potent vasoconstrictor endothelin-1 (ET-1) and displays increased nitric oxide-dependent vasodilation. The current study examined the individual and/or the relative roles of the ET receptors governing vascular function in the Goto-Kakizaki rat, a mildly hyperglycemic, normotensive, and nonobese model of Type 2 diabetes. Diabetic and control rats received an antagonist to either the ET type A (ETA; atrasentan; 5 mg x kg(-1) x day(-1)) or type B (ET(B); A-192621; 15 or 30 mg x kg(-1) x day(-1)) receptors for 4 wk. Third-order mesenteric arteries were isolated, and vascular function was assessed with a wire myograph. Maximum response to ET-1 was increased in diabetes and attenuated by ETA antagonism. ETB blockade with 15 mg/kg A-192621 augmented vasoconstriction in controls, whereas it had no further effect on ET-1 hyperreactivity in diabetes. The higher dose of A-192621 showed an ETA-like effect and decreased vasoconstriction in diabetes. Maximum relaxation to acetylcholine (ACh) was similar across groups and treatments. ETB antagonism at either dose had no effect on vasorelaxation in control rats, whereas in diabetes the dose-response curve to ACh was shifted to the right, indicating a decreased relaxation at 15 mg/kg A-192621. These results suggest that ETA receptor blockade attenuates vascular dysfunction and that ETB receptor antagonism exhibits differential effects depending on the dose of the antagonists and the disease state.

Topics: Acetylcholine; Animals; Atrasentan; Cardiovascular Agents; Diabetes Mellitus, Type 2; Diabetic Angiopathies; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Male; Mesenteric Arteries; Microcirculation; Myography; Peptides, Cyclic; Pyrrolidines; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B; Up-Regulation; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents; Viper Venoms

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disease that causes kidney failure and accounts for 10% of all patients who are on renal replacement therapy. However, the marked phenotypic variation between patients who carry the same PKD1 or PKD2 mutation suggests that nonallelic factors may have a greater influence on the cystic phenotype. Endothelin-1 (ET-1) transgenic mice have been reported to develop profound renal cystic disease and interstitial fibrosis without hypertension. The hypothesis that ET-1 acts as a modifying factor for cystic disease progression was tested in an orthologous mouse model of ADPKD (Pkd2(WS25/-)). Four experimental groups (n = 8 to 11) were treated from 5 to 16 wk of age with the highly selective orally active receptor antagonists ABT-627 (ETA) and A-192621 (ETB) singly or in combination. Unexpected, ETB blockade led to accelerated cystic kidney disease. Of significance, this was associated with a reduction in urine volume and sodium excretion and increases in urine osmolarity and renal cAMP and ET-1 concentrations. The deleterious effect of chronic ETB blockade was neutralized by simultaneous ETA blockade. ETA blockade alone resulted in a significant increase in tubular cell proliferation but did not alter the cystic phenotype. It is concluded that the balance between ETA and ETB signaling is critical for maintaining tubular structure and function in the cystic kidney. These results implicate ET, acting via vasopressin-dependent and independent pathways, as a major modifying factor for cystic disease progression in human ADPKD.

Topics: Animals; Crosses, Genetic; Disease Models, Animal; Disease Progression; Endothelin B Receptor Antagonists; Endothelin-1; Fibrosis; Humans; Mice; Polycystic Kidney, Autosomal Dominant; Vasoconstrictor Agents; Viper Venoms

Endothelial ET(B) receptor activation by exogenously administered sarafotoxin 6c(a snake venom peptide with a sequence homology to ET-1 prior to ischemia activates release of nitric oxide(NO) and previous studies have shown that NO facilitates mitochondrial K(ATP) activation in cardiac cells and cardioprotection.. The aim of this investigation was to test whether the administration of sarafotoxin 6c(a selective ET(B) receptor agonist) has cardioprotective and antiarrhythmic effects against ischemia and reperfusion injury in a well-standardized model of reperfusion arrhythmias in anesthetized adult male rabbits (n = 53) subjected to 30 min occlusion of the left coronary artery followed by 120 min of reperfusion.. Pretreatment with sarafotoxin 6c (0.24 nmol/kg, i.v.) prior to the period of coronary occlusion offers significant infarct size reduction (19.1 +/- 2.0% versus 39.7 +/- 3.7% in the saline control group; P < 0.01) and antiarrhythmic effects. Sarafotoxin 6c treatment significantly attenuated the incidence of life-threatening arrhythmias like sustained VT (13 versus 100% in the saline control group; P < 0.005) and other arrhythmias (25 versus 100% in the saline control group; P < 0.005), and increased the number of surviving animals without arrhythmias. Pretreatment with 5-HD but not HMR 1883 abolished the beneficial effects of sarafotoxin 6c on reperfusion induced arrhythmias and cardioprotection suggesting that benefits have been achieved via the selective activation of cardiomyocyte mitochondrial K(ATP) channels. Sarafotoxin 6c evoked NO release and selective activation of mitoK(ATP) channels in cardiomyocytes contributes to cardioprotection and antiarrhythmic activity during ischemia-reperfusion in the anesthetized rabbit.. We conclude that the selective activation of ET(B) receptors by sarafotoxin 6c prior to coronary occlusion contributes to cardioprotective and antiarrhythmic properties.

Topics: Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Cardiotonic Agents; Disease Models, Animal; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Nitric Oxide; Potassium Channels; Rabbits; Sarcolemma; Viper Venoms

Endothelin (ET) exerts powerful pressor actions primarily through activation of the ET(A) receptor subtype. The ET(B) receptor (ET(B)R) subtype, on the other hand, is generally thought to initiate physiological actions that decrease arterial pressure. Such actions include clearing ET from the bloodstream, initiating endothelium-mediated vasodilation, and facilitating renal sodium and water excretion. The effect of long-term activation of the ET(B)R on arterial pressure, however, never has been directly tested. In this study we evaluated cardiovascular responses to chronic (5-day) activation of ET(B)R in male rats using continuous intravenous infusion of the selective agonist sarafotoxin 6c. Surprisingly, we found that sarafotoxin 6c caused a sustained increase in arterial pressure that rapidly reversed on termination of infusion. The hypertension was associated with increased renal excretion of sodium and water and decreased plasma volume. Alterations in daily sodium intake did not affect the magnitude of the hypertension. Hemodynamic studies revealed a decreased cardiac output and increased total peripheral resistance during sarafotoxin 6c infusion. Infusion of sarafotoxin 6c caused a small increase in plasma ET levels. Nevertheless, the hypertension was not affected by coadministration of a selective ET(A) receptor antagonist (atrasentan) but was completely prevented by treatment with a combined ET(A) receptor and ET(B)R antagonist (A186280). These experiments reveal for the first time that chronic activation of ET(B)R in rats causes sustained hypertension.

Topics: Animals; Atrasentan; Blood Pressure; Blood Volume; Cardiac Output; Disease Models, Animal; Diuresis; Drug Administration Schedule; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Heart Rate; Hypertension; Male; Natriuresis; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptor, Endothelin B; Stroke Volume; Sulfonamides; Vascular Resistance; Viper Venoms

The aims of this study were to determine if endothelin-1 (ET-1) under normal and ischaemic conditions exhibits a direct arrhythmogenic effect that is independent of its ability to cause coronary vasoconstriction, and to determine the contribution of the ET(A) and ET(B) receptor subtype. ET(A/B) (with ET-1) and ET(A) (ET-1 in the presence of BQ-788) receptor activation resulted in a significant reduction in both epi- and endocardial monophasic action potential duration (MAPD(90)). ET(A) receptor activation reduced both epi- and endocardial effective refractory period (ERP). This MAPD(90) and ERP shortening were associated with a reduction in coronary flow, myocardial contractility and induction of ventricular fibrillation (VF) during ERP measurement. The ET(B) agonist sarafotoxin (S6c) had no marked, or concentration-dependent, effect on MAPD(90), ERP, myocardial contractility or induction of arrhythmias. Neither ET-1 nor S6c, given prior to coronary artery occlusion, significantly changed the ischaemia-induced dispersion of MAPD(90), ERP or the % incidence of VF. In conclusion, neither ET(A) nor ET(B) receptor stimulation has a direct arrhythmogenic effect in isolated rabbit hearts under normal or ischaemic conditions. The ET-1-induced arrhythmogenic effect observed in nonischaemic hearts is likely to be the result of the associated coronary vasoconstriction caused by ET(A) receptor stimulation resulting in myocardial ischaemia.

Topics: Action Potentials; Animals; Arrhythmias, Cardiac; Blood Pressure; Coronary Circulation; Disease Models, Animal; Endocardium; Endothelin B Receptor Antagonists; Endothelin-1; In Vitro Techniques; Male; Myocardial Contraction; Myocardial Ischemia; Oligopeptides; Pericardium; Piperidines; Rabbits; Receptor, Endothelin A; Receptor, Endothelin B; Ventricular Fibrillation; Viper Venoms

The aims of this study were to determine if the ETB receptor agonist, sarafotoxin 6c (S6c) reduces myocardial infarct size following myocardial ischemia and reperfusion and to investigate whether any changes in mRNA for endothelin receptors in the injured myocardium were modified by S6c pretreatment. Hypnorm/Hypnovel anesthetized rats were subjected to occlusion of the left main coronary artery for 30 minutes, followed by 120 minutes reperfusion. Animals were administered a bolus dose of S6c (0.24 nmol kg-1 i.v., n = 10) or saline (n = 15) 5 minutes prior to occlusion. At the end of reperfusion, hearts were stained with Evan's Blue dye to delineate area at risk. A 1.5- to 2.0-mm thick slice was cut transmurally 1 mm below the site of ligation for assessment of infarct size by triphenyltetrazolium chloride. A further transmural slice (2.5-3-mm thick) was cut for assessment of receptor mRNA levels by RTPCR. Administration of S6c caused a transient fall in mean arterial blood pressure (MABP) prior to occlusion and attenuated the fall in MABP induced by coronary occlusion. S6c significantly reduced infarct size (13 +/- 4% of area of slice at risk) compared with control hearts (35 +/- 5%; P < 0.05). In control hearts, there was a marked reduction in mRNA content for both ETA (50% reduction) and ETB (70% reduction) receptors in the ischemic zone, compared with non-ischemic tissue. In hearts pre-treated with S6c there was a reduction in ETA, but not ETB receptor mRNA in the ischemic zone. This study has shown that S6c reduces myocardial infarct size and results in preservation of ETB receptor mRNA in ischemic/reperfused tissue.

Topics: Animals; Blood Pressure; Coronary Disease; Disease Models, Animal; Drug Administration Schedule; Evans Blue; Glyceraldehyde-3-Phosphate Dehydrogenases; Injections, Intravenous; Ligation; Male; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Receptor, Endothelin B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrazolium Salts; Time Factors; Ventricular Fibrillation; Viper Venoms

Asthma is a chronic airway disease, known to involve several inflammatory mediators. Little is known about how these mediators interact in order to produce or attenuate even basic features of the disease, like airway hyper-reactivity and remodelling. Endothelin-1 (ET-1) and IL-1beta are two mediators suggested to play important roles in the induction of airway inflammation.. To investigate the interactions between ET-1 and IL-1beta, using a novel in vitro model of asthma, focusing on airway smooth muscle contractility.. Isolated murine tracheal segments were cultured from 1 to 8 days in the absence and presence of IL-1beta. The subsequent contractile responses to sarafotoxin 6c (S6c) (selective agonist for ETB receptor) and sarafotoxin 6b (S6b) (ETA and ETB receptor agonist) were recorded by a myographs system. In all experiments, ETB receptors were desensitized before the contractile response to S6b was recorded. Thus, the response to S6b is only mediated by ETA receptors in the present study. The mRNA expressions for ET-1 and endothelin (ET) receptors were quantified by real-time PCR.. Organ culture in the presence of IL-1beta attenuated the maximal contraction induced by S6c, but not S6b. This reduction was concentration-dependent and was significant after 2, 4 and 8 days of culture. To investigate the mechanisms behind this, inhibitors for endothelin converting enzyme (ECE) phosphoramidon, c-JUN N-terminal kinase (JNK) SP600125, extracellular-signal-regulated kinase 1/2(ERK 1/2) PD98059 and p38 pathway SB203580 were used. Individually, SP600125 and PD98059, but not SB203580, could partly reverse the reduction induced by IL-1beta. An additional effect was obtained when SP600125 and PD98059 were combined. The mRNA expressions for ET-1 and ETB receptor were up- and down-regulated, respectively, by IL-1beta.. Presence of IL-1beta in the airways attenuate the contractile response mediated via ETB receptors, an effect dependent on ECE, JNK and ERK 1/2 pathways.

Topics: Animals; Aspartic Acid Endopeptidases; Asthma; Disease Models, Animal; Endothelin-1; Endothelin-Converting Enzymes; Extracellular Signal-Regulated MAP Kinases; Interleukin-1; JNK Mitogen-Activated Protein Kinases; Male; Metalloendopeptidases; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; Muscle Contraction; Muscle, Smooth; Organ Culture Techniques; p38 Mitogen-Activated Protein Kinases; Receptor, Endothelin A; Receptor, Endothelin B; RNA, Messenger; Time Factors; Trachea; Vasoconstrictor Agents; Viper Venoms

To clarify the functional role of endothelin-A/endothelin- B (ETA/ETB) receptors in congestive heart failure (CHF), we examined the effects of a non-selective endothelin receptor agonist, endothelin-1 (ET-1), and a selective ETB receptor agonist, sarafotoxin S6c. CHF was induced in dogs by rapid ventricular pacing and resulted in decreased left ventricular dp/dtmax, decreased cardiac output and increased pulmonary vascular resistance. Sarafotoxin S6c (0.3 nmol/kg) resulted in decreased left ventricular dp/dtmax (-26 +/- 2%), decreased cardiac output (-47 +/- 3%) and increased pulmonary vascular resistance (+48 +/- 10%) in dogs without CHF. The effects of sarafotoxin S6c were attenuated in dogs with CHF (-12 +/- 5% in left ventricular dp/dtmax, -19 +/- 5% in cardiac output and +7 +/- 5% in pulmonary vascular resistance). In contrast, ET-1 (0.5 nmol/kg) had no effect on left ventricular dp/dtmax in dogs without CHF and increased left ventricular dp/dtmax by 16 +/- 3% in dogs with CHF. These data indicate that reduced cardiac contractile and pulmonary vasoconstrictor responses via the ETB receptor are attenuated and that responses mediated by the ETA receptor are more prominent in the context of CHF. This suggests a functional shift of endothelin receptor subtypes in CHF.

Topics: Animals; Cardiac Output; Cardiac Pacing, Artificial; Disease Models, Animal; Dogs; Endothelin-1; Heart Failure; Lung; Male; Myocardial Contraction; Myocardium; Receptor, Endothelin A; Receptor, Endothelin B; Up-Regulation; Vascular Resistance; Vasoconstriction; Ventricular Pressure; Viper Venoms

Elevated levels of endothelin-1 (ET-1) have been reported in cerebral ischemia. A role for ET may prove more important if the vascular receptors were changed. We addressed whether there is any change in ET receptor expression in cerebral ischemia.. The right middle cerebral artery (MCA) was occluded in male Wistar rats for 2 hours with the intraluminal filament method. The basilar artery and both MCAs were removed after 46 hours of recirculation. The contractile responses to ET-1, a combined ET(A) and ET(B) receptor agonist, and sarafotoxin 6c (S6c), a selective ET(B) receptor agonist, were examined in vitro, and ET receptor mRNA was quantified by real-time polymerase chain reaction.. S6c, which had no contractile effect per se on fresh or sham-operated rat cerebral arteries, induced a marked contraction in the occluded MCA (E(max) [maximum contraction, calculated as percentage of the contractile capacity of 63.5 mmol/L K+]=68+/-68%; P<0.0001), while there was no difference in the responses to ET-1 after cerebral ischemia. Real-time polymerase chain reaction revealed a significant upregulation of both the ET(A) and ET(B) receptors (both P<0.05) in the occluded MCA compared with the nonoccluded MCA from the same rats.. Focal cerebral ischemia in rat induces increased transcription of both ET(A) and ET(B) receptors, which results in the appearance of a contractile response to the ET(B) receptor agonist S6c. These results suggest a role for ET receptors in the pathogenesis of a vascular component after cerebral ischemia.

Topics: Animals; Basilar Artery; Brain Ischemia; Disease Models, Animal; Endothelin-1; In Vitro Techniques; Infarction, Middle Cerebral Artery; Male; Middle Cerebral Artery; Peptide Elongation Factor 1; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; RNA, Messenger; Up-Regulation; Vasoconstriction; Vasoconstrictor Agents; Viper Venoms

Studies of congestive heart failure (CHF) in man and in experimental CHF have demonstrated elevated circulating levels of endothelin (ET). In order to examine whether there are concomitant ET receptor alterations, the vasomotor effects of endothelin-1 (ET-1) and sarafotoxin 6c (S6c) were examined in endothelium-intact and -denuded isolated mesenteric arteries from rats with CHF. CHF was induced by ligation of the left anterior descending coronary artery. Vasomotor responses were studied using small mesenteric arteries (approx. 250 microm in diameter, determined after normalisation). The antagonists IRL2500 and FR139317 were used in order to characterise the ET-1-induced response. In mesenteric arteries with intact endothelium, ET-1-induced contractions were more potent in CHF as compared to sham (pEC(50) 9.6+/-0.2 and 9.1+/-0.1, respectively, P<0.01). In endothelium-denuded arteries, there was no difference in potency of ET-1 between CHF and sham arteries, or in maximum contraction. In the presence of IRL2500, a selective ET(B)-receptor antagonist, ET-1 was more potent in endothelium-denuded arteries of CHF rats, while this difference was not seen in sham arteries. S6c had no consistent contractile or dilatory effect in CHF and sham rats. The results indicate that the enhanced contractile effects of ET-1 noted in CHF might be due to an attenuated endothelial function and that inhibition of smooth muscle cell ET(B) receptors increase the effects of contractile ET(A) receptors in CHF rats.

Topics: Animals; Disease Models, Animal; Endothelin Receptor Antagonists; Endothelin-1; Endothelium, Vascular; Heart Failure; Male; Mesenteric Arteries; Myocardial Contraction; Myocardial Infarction; Potassium; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Vasoconstrictor Agents; Viper Venoms

To test whether hemorrhagic shock and resuscitation (HSR) alters the vascular responsiveness of the portohepatic circulation to endothelins (ETs), we studied the macro- and microcirculatory effects of the preferential ET(A) receptor agonist ET-1 and of the selective ET(B) receptor agonist sarafotoxin 6c (S6c) after 1 h of hemorrhagic hypotension and 5 h of volume resuscitation in the isolated perfused rat liver ex vivo using portal pressure-flow relationships and epifluorescence microscopy. Although HSR did not cause major disturbances of hepatic perfusion per se, the response to ET-1 (0.5 x 10(-9) M) was enhanced, leading to greater increases in portal driving pressure, total portal resistance, and zero-flow pressures and more pronounced decreases in portal flow, sinusoidal diameters, and hepatic oxygen delivery compared with time-matched sham shock controls. In sharp contrast, the constrictive response to S6c (0.25 x 10(-9) M) remained unchanged. Thus HSR primes the portohepatic circulation for the vasoconstrictive effects of ET-1 but does not alter the effects of the ET(B) receptor agonist S6c. The enhanced sinusoidal response may contribute to the subsequent development of hepatic microcirculatory failure after secondary insults that are associated with increased generation of ET-1.

Topics: Animals; Disease Models, Animal; Endothelin-1; Hemodynamics; Liver; Male; Microcirculation; Oxygen Consumption; Portal System; Rats; Rats, Sprague-Dawley; Receptor, Endothelin B; Receptors, Endothelin; Resuscitation; Shock, Hemorrhagic; Vascular Resistance; Vasoconstriction; Viper Venoms

The aim of this study was to assess the role of nitric oxide (NO) and endothelin (ET)-1 in the pathophysiology of persistent pulmonary hypertension of the newborn in fetal lambs with a surgically created congenital diaphragmatic hernia (CDH). The pulmonary vascular response to various agonists and antagonists was assessed in vivo between 128 and 132 days gestation. Age-matched fetal lambs served as control animals. Control and CDH lambs had similar pulmonary vasodilator responses to acetylcholine, sodium nitroprusside, zaprinast, and dipyridamole. The ET(A)-receptor antagonist BQ-123 caused a significantly greater pulmonary vasodilatation in CDH than in control animals. The ET(B)-receptor agonist sarafotoxin 6c induced a biphasic response, with a sustained pulmonary vasoconstriction after a transient pulmonary vasodilatation that was not seen in CDH animals. We conclude that the NO signaling pathway in vivo is intact in experimental CDH. In contrast, ET(A)-receptor blockade and ET(B)-receptor stimulation significantly differed in CDH animals compared with control animals. Imbalance of ET-1-receptor activation favoring pulmonary vasoconstriction rather than altered NO-mediated pulmonary vasodilatation is likely to account for persistent pulmonary hypertension of the newborn in fetal lambs with a surgically created CDH.

Topics: Acetylcholine; Animals; Antihypertensive Agents; Cyclic GMP; Dipyridamole; Disease Models, Animal; Endothelin Receptor Antagonists; Endothelin-1; Endothelium, Vascular; Female; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; Hypertension, Pulmonary; Nitric Oxide; Nitroprusside; Peptides, Cyclic; Phosphodiesterase Inhibitors; Pregnancy; Pulmonary Circulation; Purinones; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Sheep; Vasoconstrictor Agents; Vasodilator Agents; Viper Venoms

Endothelin-1, unlike the selective endothelin ETB receptor agonist sarafotoxin S6c, causes nociception in the rat when injected intraarticularly 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 ETA receptor antagonist BQ-123 (cyclo [D-Asp-Pro-D-Val-Leu]; by approximately/= 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-gamma-methoxycarbonyl- tryptophanil-D-norleucine; 10 nmol). Prior joint challenge with carrageenan (300 microg) 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 approximately/= 80% inhibition at 10 nmol). Priming also enhanced PET responses to carrageenan itself and to LPS (1 microg) 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 (1

Topics: Animals; Antihypertensive Agents; Arthritis, Reactive; Bosentan; Carrageenan; Disease Models, Animal; Endothelin Receptor Antagonists; Endothelin-1; Excipients; Knee Joint; Lipopolysaccharides; Male; Nociceptors; Oligopeptides; Peptides, Cyclic; Piperidines; Rats; Rats, Wistar; Sulfonamides; Vasoconstrictor Agents; Viper Venoms

Endothelin (ET), a coronary vasoconstrictor, mediates its activity through the specific receptors ET-A and ET-B, which may demonstrate different activity under pathophysiological conditions. Thoracic inferior vena cava constriction (TIVCC) is an experimental model of congestive heart failure that is characterized by a decrease in cardiac output and an increase in circulating ET concentrations. The present study was designed to test the hypothesis that experimental heart failure altered coronary vascular responsiveness to ET-A- and ET-B-receptor stimulation in vivo.. ET-1 was infused at a rate of 2 ng/kg per minute into the left circumflex coronary artery in normal dogs (n = 5) and in dogs subjected to TIVCC (TIVCC dogs, n = 6). Similarly, sarafotoxin, an ET-B-receptor agonist, was infused at the same dosage in normal (n = 5) and TIVCC (n = 6) dogs. Intracoronary infusion of ET-1 significantly decreased coronary blood flow and increased coronary vascular resistance in normal dogs; this effect was significantly attenuated in TIVCC compared with normal dogs. The percent changes in coronary blood flow and coronary vascular resistance in the TIVCC compared with the normal dogs was -11 +/- 8% versus -48 +/- 7% (P < .01) and 29 +/- 10% versus 105 +/- 23% (P < .01), respectively. There was no significant effect on coronary blood flow, coronary vascular resistance, or coronary artery diameter in normal dogs that received an intracoronary infusion of sarafotoxin. In contrast, the administration of intracoronary sarafotoxin in TIVCC compared with normal dogs resulted in significant percent changes in coronary blood flow and coronary vascular resistance (-31 +/- 4% versus -7 +/- 3% [P < .001] and 53 +/- 12% versus 12 +/- 8% [P < .02], respectively).. The present study demonstrates an attenuated coronary vasoconstrictor response to ET-1 with an enhanced vasoconstrictor response to sarafotoxin and suggests an alteration in coronary ET receptor sensitivity in experimental heart failure.

Topics: Animals; Blood Pressure; Cardiac Output; Disease Models, Animal; Dogs; Endothelins; Heart Failure; Receptor, Endothelin B; Receptors, Endothelin; Vasoconstriction; Vasoconstrictor Agents; Viper Venoms

(+/-)-SB 209670, a potent nonpeptide endothelin (ET) receptor antagonist, was used to investigate the potential role of ET in cerebral vasospasm associated with subarachnoid hemorrhage.. The effects of (+/-)-SB 209670 were evaluated in isolated segments of canine posterior cerebral arteries in vitro, vascular smooth muscle cells in culture, and in the canine two-hemorrhage model of delayed cerebral vasospasm in vivo.. In the canine basilar and anterior spinal arteries, (+/-)-SB 209670 caused a dose-related inhibition of contractile responses mediated by ET (KB = 4.6 nmol/L and apparent KB = 2.7 nmol/L, respectively). The effects of (+/-)-SB 209670 were mediated by inhibition of ETA receptors since the ETB selective agonist sarafotoxin 6c did not contract these posterior cerebral vessels. (+/-)-SB 209670 also produced a concentration-dependent inhibition (IC50 = 1 nmol/L) of the mitogenic response induced by ET-1 in vascular smooth muscle cell culture. In the canine model of delayed cerebral vasospasm, animals received intracisternal vehicle (saline) or (+/-)-SB 209670 (360 +/- 10 micrograms/d) via osmotic minipump for 7 days. On day 7, the cross-sectional areas in the (+/-)-SB 209670 group were significantly greater than those in the vehicle group in both the basilar artery (68% versus 27%) and anterior spinal artery (78% versus 38%). No differences in blood pressure or heart rate were noted in the two groups, and the vasospasm in the vehicle group did not differ from that of historic controls in this model.. The results suggest that ET plays a significant role in the development of delayed cerebral vasospasm via an interaction with ETA receptors. Furthermore, ETA receptor antagonists may represent a novel therapeutic approach to the treatment of subarachnoid hemorrhage.

Topics: Animals; Arteries; Basilar Artery; Cells, Cultured; Cerebral Arteries; Cerebrovascular Circulation; Disease Models, Animal; Dogs; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelins; Indans; Ischemic Attack, Transient; Male; Mitogens; Muscle, Smooth, Vascular; Spinal Cord; Subarachnoid Hemorrhage; Vasoconstriction; Vasoconstrictor Agents; Viper Venoms

The two endothelin (ET) receptor subtypes (ETA and ETB) have been characterized in rat kidney from normal rats and rats with acute renal failure induced by hypertonic glycerol administration. In control rats, the total number of ET receptors in kidney cortex and medulla was 155 and 386 fmol/mg of protein, respectively. The ratio of ETA to ETB receptors was 54:46 in renal cortex and 35:65 in renal medulla. Treatment of rats with 10 ml/kg glycerol (50%, w/v) intramuscularly resulted in severe renal dysfunction; the serum urea concentration increased from 0.46 to 2.65 g/liter and the creatinine clearance decreased from 1.06 to 0.30 ml/min. Ligand binding studies showed that glycerol-induced acute renal failure was associated with a marked up-regulation of ETA and ETB receptor subtypes in both cortex and medulla. In glycerol-treated rats, the total ET receptor density in kidney cortex and medulla was increased to 294 and 1172 fmol/mg of protein, with ETA/ETB ratios of 52:48 and 31:69, respectively. The upregulatory effect of glycerol treatment was significantly more pronounced in renal medulla than renal cortex and affected ETB receptors preferentially, compared with ETA receptors. Subsequently, ETA and ETB receptor mRNA levels were markedly increased by glycerol administration in both kidney cortex and medulla, as assessed by polymerase chain reaction coupled to reverse transcription. These results suggest that up-regulation of renal ET receptors, particularly ETB receptors in kidney medulla, may account for or contribute to renal function impairment induced by glycerol, and they support a pathophysiological role for ET in acute renal failure.

Topics: Acute Kidney Injury; Animals; Base Sequence; Binding, Competitive; Disease Models, Animal; Endothelin Receptor Antagonists; Glycerol; Kidney Cortex; Kidney Medulla; Male; Molecular Sequence Data; Peptides, Cyclic; Polymerase Chain Reaction; Rats; Rats, Wistar; Receptors, Endothelin; Up-Regulation; Vasoconstrictor Agents; Viper Venoms