Endogenous Endothelin-1 Research Articles

Obesity and heterozygous endothelial overexpression of prepro-endothelin-1 modulate responsiveness of mouse main and segmental renal arteries to vasoconstrictor agents

AimsLevels of the endothelium-derived peptide endothelin-1 (ET-1) are elevated in obese humans, and ET-1 mediated vascular tone is increased. Renal arterial smooth muscle is highly responsive to ET-1. Whether or not endothelium-derived ET-1 affects contractions of the renal artery under normal conditions or in obesity is unknown. The present study was designed to investigate whether or not overexpression of endogenous ET-1 in the endothelium affects the responsiveness of the main and segmental renal arteries differently in obesity. Main methodsMice with tie-1 promoter-driven endothelium-restricted heterozygous overexpression of preproendothelin-1 were used (TEThet). Obesity was induced in TEThet mice and wild-type (WT) littermates by feeding a high fat diet for 30weeks; lean controls were kept on standard chow. The renal arteries were studied in wire myographs testing contractions (in the presence of l-NAME) to ET-1, serotonin, and U46619. Key findingsContractions to ET-1 were comparable between groups in main renal arteries, but augmented in segmental preparations from obese mice. Serotonin-induced responses were enhanced in obese TEThet mice renal arteries compared to lean controls. Concentration–contraction curves to U46619 were shifted significantly to the left in main renal arteries of obese animals, and the maximal response was significantly increased between lean and obese TEThet mice. SignificanceThese results indicate an augmented responsiveness of main renal arteries in obesity particularly to TP receptor activation. When combined with endothelial ET-1 overexpression this effect is even more pronounced, which may help to gain further insights into the mechanisms of hypertension in obesity.

Endogenous endothelin 1 mediates angiotensin II-induced hypertrophy in electrically paced cardiac myocytes through EGFR transactivation, reactive oxygen species and NHE-1

Emerging evidence supports a key role for endothelin-1 (ET-1) and the transactivation of the epidermal growth factor receptor (EGFR) in angiotensin II (Ang II) action. We aim to determine the potential role played by endogenous ET-1, EGFR transactivation and redox-dependent sodium hydrogen exchanger-1 (NHE-1) activation in the hypertrophic response to Ang II of cardiac myocytes. Electrically paced adult cat cardiomyocytes were placed in culture and stimulated with 1nmoll(-1) Ang II or 5nmoll(-1) ET-1. Ang II increased ~45% cell surface area (CSA) and ~37% [(3)H]-phenylalanine incorporation, effects that were blocked not only by losartan (Los) but also by BQ123 (AT1 and ETA receptor antagonists, respectively). Moreover, Ang II significantly increased ET-1 messenger RNA (mRNA) expression. ET-1 similarly increased myocyte CSA and protein synthesis, actions prevented by the reactive oxygen species scavenger MPG or the NHE-1 inhibitor cariporide (carip). ET-1 increased the phosphorylation of the redox-sensitive ERK1/2-p90(RSK) kinases, main activators of the NHE-1. This effect was prevented by MPG and the antagonist of EGFR, AG1478. Ang II, ET-1 and EGF increased myocardial superoxide production (187 ± 9%, 149 ± 8% and 163.7 ± 6% of control, respectively) and AG1478 inhibited these effects. Interestingly, Los inhibited only Ang II whilst BQ123 cancelled both Ang II and ET-1 actions, supporting the sequential and unidirectional activation of AT1, ETA and EGFR. Based on the present evidence, we propose that endogenous ET-1 mediates the hypertrophic response to Ang II by a mechanism that involves EGFR transactivation and redox-dependent activation of the ERK1/2-p90(RSK) and NHE-1 in adult cardiomyocytes.

Contractions to endogenous and exogenous endothelin-1 in segmental renal arteries of the mouse: Up-regulation in obesity

Poster Session 1: Oncology, Renal Physiology and Disease, Neurology, Infectious Diseases, New Topics of Endothelin Biology: P-26

Abstract 85: Low-grade Inflammation Participates on the Enhanced Vasoconstriction to the Endogenous Endothelin-1 in Small Vessels From Obese Patients

Objective: Obesity is characterized by vascular low-grade inflammation, with an augmented endogenous endothelin(ET)-1-mediated vasoconstriction (VC) and a blunted tonic nitric oxid(NO)-mediated relaxation (VD). We evaluated whether TNF-α, localized in the vascular wall and in perivascular adipocytes (PVA), contributes to the VC induced by endogenous ET-1 and whether this action is mediated by an effect on tonic NO release in small resistance arteries from obese patients (Ob) and controls (Ctrl). Method: Sample of visceral fat were obtained in 14 Ob (BMI:42.1±3.7) and 14 Ctrl (BMI:25.4±3.6), matched for metabolic profile and blood pressure values, undergoing laparoscopic surgical procedure. Small arteries were investigated on a pressurized micromyograph. Endogenous ET-1 was assessed by vascular response to BQ-123. TNF-α and NO were assessed by Infliximab (IFX) and L-NAME, respectively. Gene and protein expression of TNF-α, ET-1 and ETA receptors were determined by RT-PCR (gene/reference) and IHC (AU) on arterial wall and in PVA. Results: In Ctrl, L-NAME-induced VC (15.5±0.6%) was not affected by IFX (15.1±0.4%). In contrast, Ob showed a blunted VC to L-NAME (6.0±0.7%; P<0.01 vs Ctrl) which was potentiated (P<0.01) by IFX (12.5±0.8%). In Ob, the VD to BQ-123 (47.0±1.5%) was attenuated (P<0.01) by IFX (29.1±2.4%) and not affected by L-NAME (43,3±0.6%). During IFX co-infusion, L-NAME further reduced the VD to BQ-123 (19.4±3.0%; P<0.01 vs BQ-123+IFX). In Ctrl, VD to BQ-123 was blunted (26.3±1.3%; P<0.01 vs Ob), not affected by IFX (24.1±0.6%) and significantly reduced by L-NAME (12.3±1.1%). Ob showed a significant overexpression of TNF-α respect to Ctrl, either at the level of arterial wall (24.9±19.6 vs 2.8±2.5 AU, P<0.001) or in PVA (2.9±1.8 vs 1.2±0.7 gene/reference, P<0.005). These results were paralleled by a higher arterial expression of ET-1 (45.8±10.3 vs 24.3±15.0 AU, P<0.01) and ETA receptors (69.4±6.0 vs 9.6±2.8 AU, P<0.001) in Ob vs Ctrl. Conclusion: Small vessels of Ob show an enhanced ETA-mediated VC and a blunted NO-mediated VD. An excess of vascular and perivascular TNF-α, coupled with an increased expression of ET-1 and ETA in the vasculature of Ob, contributes to the enhanced ET-1-mediated VC tone partly by an impairment of tonic NO release.

Endothelin-1 Release during the Early Phase of Reperfusion Is a Mediator of Myocardial Reperfusion Injury

Purpose: In acute myocardial infarction, left ventricular (LV) unloading reduces endothelin-1 (ET-1) release. We tested that endogenous ET-1 released during acute myocardial infarction might mediate ischemia/reperfusion (I/R) injury by stimulating increased intracellular calcium concentration, [Ca²⁺]_i, and apoptosis. Methods: Rabbits were subjected to 1 h of coronary artery occlusion followed by 3 h of reperfusion. Unloading was initiated 15 min prior to reperfusion and was maintained during reperfusion. The control group was subjected to reperfusion. Animals were treated with ET-1 receptor antagonist BQ123. In parallel, isolated rabbit cardiomyocytes subjected to simulated I/R with or without ET-1 or BQ123, intracellular Ca²⁺ and cell death were assessed with flow cytometry. Results: LV unloading prior to reperfusion reduced myocardial ET-1 release at 2 h of reperfusion. Infarct size was reduced in unloaded and BQ123 groups versus controls. LV unloading and BQ123 treatment reduced the percentage of apoptotic cells associated with increases in Bcl-2 protein levels in ischemic regions. BQ123 reduced both ET-1-induced [Ca²⁺]_i increase and cell death for myocytes subjected to stimulated I/R. Conclusion: We propose that components of reperfusion injury involve ET-1 release which stimulates calcium overload and apoptosis. Intravenous ET-1 receptor blockade prior to reperfusion may be a protective adjunct to reperfusion therapy in acute myocardial infarction patients.

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.

Taming the “sleeping giant”: the role of endothelin-1 in the regulation of skeletal muscle blood flow and arterial blood pressure during exercise

The cardiovascular response to exercise is governed by a combination of vasodilating and vasoconstricting influences that optimize exercising muscle perfusion while protecting mean arterial pressure (MAP). The degree to which endogenous endothelin (ET)-1, the body's most potent vasoconstrictor, participates in this response is unknown. Thus, in eight young (24 ± 2 yr), healthy volunteers, we examined leg blood flow, MAP, tissue oxygenation, heart rate, leg arterial-venous O(2) difference, leg O(2) consumption, pH, and net ET-1 and lactate release at rest and during knee extensor exercise (0, 5, 10, 15, 20, and 30 W) before and after an intra-arterial infusion of BQ-123 [ET subtype A (ET(A)) receptor antagonist]. At rest, BQ-123 did not evoke a change in leg blood flow or MAP. During exercise, net ET-1 release across the exercising leg increased approximately threefold. BQ-123 increased leg blood flow by ~20% across all work rates (changes of 113 ± 76, 176 ± 83, 304 ± 108, 364 ± 130, 502 ± 117, and 570 ± 178 ml/min at 0, 5, 10, 15, 20, and 30 W, respectively) and attenuated the exercise-induced increase in MAP by ~6%. The increase in leg blood flow was accompanied by a ~9% increase in leg O(2) consumption with an unchanged arterial-venous O(2) difference and deoxyhemoglobin, suggesting a decline in intramuscular efficiency after ET(A) receptor blockade. Together, these findings identify a significant role of the ET-1 pathway in the cardiovascular response to exercise, implicating vasoconstriction via the ET(A) receptor as an important mechanism for both the restraint of blood flow in the exercising limb and maintenance of MAP in healthy, young adults.

Role of endothelin-1 in renal regulation of acid-base equilibrium in acidotic humans

Endothelin-1 inhibits collecting duct sodium reabsorption and stimulates proximal and distal tubule acidification in experimental animals both directly and indirectly via increased mineralocorticoid activity. Diet-induced acid loads have been shown to increase renal endothelin-1 activity, and it is hypothesized that increased dietary acid-induced endothelin-1 activity may be a causative progression factor in human renal insufficiency and that this might be reversed by provision of dietary alkali. We sought to clarify, in normal human volunteers, the role of endothelin-1 in renal acidification and to determine whether the effect is dependent on dietary sodium chloride. Acid-base equilibrium was studied in seven normal human volunteers with experimentally induced metabolic acidosis [NH(4)Cl 2.1 mmol·kg body weight (BW)(-1)·day(-1)] with and without inhibition of endogenous endothelin-1 activity by the endothelin A/B-receptor antagonist bosentan (125 BID p.o./day) both during dietary NaCl restriction (20 mmol/day) and NaCl repletion (2 mmol NaCl·kg BW(-1)·day(-1)). During NaCl restriction, but not in the NaCl replete state, bosentan significantly increased renal net acid excretion in association with stimulation of ammoniagenesis resulting in a significantly increased plasma bicarbonate concentration (19.0 ± 0.8 to 20.1 ± 0.9 mmol/l) despite a decrease in mineralocorticoid activity and an increase in endogenous acid production. In pre-existing human metabolic acidosis, endothelin-1 activity worsens acidosis by decreasing the set-point for renal regulation of plasma bicarbonate concentration, but only when dietary NaCl provision is restricted.

The inhibitory effect of miRNA-1 on ET-1 gene expression

There is a close correlation between endothelin-1 (ET-1) and microRNA-1 (miRNA-1) expression in the heart, but whether ET-1 expression is regulated by miRNA-1 warrants further research. Our results revealed multiple clues suggesting that miRNA-1 may participate in inhibiting ET-1 gene expression. The inhibitory effect of miRNA-1 on recombinant luciferase reporter gene was mediated by the target sequence at the 127th nucleotide on ET-1 3′UTR. We further confirmed that miRNA-1 could inhibit endogenous ET-1 gene expression at the post-transcriptional level. Our study provides a new perspective on the regulatory mechanism of ET-1 gene.

Rapid functional upregulation of vasocontractile endothelin ETB receptors in rat coronary arteries

AimsEndothelin ETB receptors mediate under normal physiological conditions vasorelaxation in coronary arteries. However, vasocontractile ETB receptors appear in coronary arteries of ischemic heart disease patients. Interestingly, organ culture of isolated coronary arteries also induces upregulation of vasocontractile ETB receptors. This study examines the early time course and mechanism behind upregulation of contractile ETB receptors in isolated rat coronary arteries during short-term organ culture. Main methodsCoronary artery segments were mounted in wire-myographs and incubated in physiological saline solution. Contractions were measured after exposure to the specific ETB receptor agonist Sarafotoxin 6c (S6c) and the endogenous agonists endothelin-1 and endothelin-3. Protein localization and levels of ETB and phosphorylated-extracellular-signal-regulated-kinase-1/2 (ERK1/2) were examined by immunohistochemistry. Key findingsFresh 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 ETB 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 ETB receptor upregulation. U0126 attenuated ETB receptor protein levels after 24h of incubation. SignificanceCoronary arteries rapidly upregulate vasocontractile ETB 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.

Inhibitory Effects of the Dual Endothelin Receptor Blocker Bosentan on Thrombin-Activated Lung Fibroblasts Isolated from Scleroderma Patients

Background: Endothelin-1 and thrombin are elevated during lung injury and repair, as seen in scleroderma and other interstitial lung diseases, and each plays important roles in remodeling epithelium, blood vessels and connective tissue. Both factors promote lung myofibroblast differentiation, the hallmark of pulmonary fibrosis. This study was undertaken to investigate whether bosentan, the dual, specific and competitive inhibitor of endothelin, interferes with thrombin signaling in scleroderma lung fibroblasts. Methods: Endothelin-1 secretion was measured by enzyme-linked immunosorbent assay. Lung fibroblast proliferation was studied by DNA Synthesis and Quick Cell Proliferation assays. Expression of α-smooth muscle actin (α-SMA) was analyzed on Western blots. Contractile activity of lung fibroblasts was measured by a collagen gel contraction assay. Lung fibroblast migration was studied by wound-healing “scratch” assay. Results: We show that thrombin significantly induces endothelin-1 expression in human lung fibroblasts. Bosentan significantly decreases α-SMA and collagen gel contraction of human lung fibroblasts stimulated by thrombin, suggesting that thrombin-induced differentiation of fibroblasts to the myofibroblast phenotype is at least in part regulated by endothelin-1. Bosentan decreases thrombin-induced migration of normal and scleroderma lung fibroblasts and inhibits innately increased migration of scleroderma lung fibroblasts suggesting that endogenous endothelin may be in part responsible for enhanced migration of lung fibroblasts in pulmonary fibrosis. We also report that bosentan inhibits thrombin-induced thymidine incorporation and decreases lung fibroblast proliferation. Conclusions: Fibrogenic effects of thrombin in scleroderma lung fibroblasts are mediated in part by endothelin-1. The dual endothelin receptor blocker bosentan restrains profibrotic effects of endogenous and thrombin-induced endothelin-1 in scleroderma lung fibroblasts.

Up-regulation of endothelial endothelin-1 expression prior to vasogenic edema formation in the rat piriform cortex following status epilepticus

Endothelin-1 (ET-1) is one of potential factors to induce vasogenic edema formation, since exogenous ET-1 treatment decreases aquaporin 4 (AQP4) expression and increases chemokines induction. To identify the role of endogenous ET-1 in vasogenic edema formation, we examined the correlation between endogenous ET-1 expression and vasogenic edema formation in the pirifom cortex following status epilepticus (SE). In the present study, SMI-71 (a brain–blood barrier marker) immunoreactivity was significantly reduced in blood vessels at 1 day after SE when vasogenic edema and neuronal damage were observed. ET-1 expression was up-regulated in endothelial cells prior to reduction in SMI-71 immunoreactivity. Furthermore, ET-1 expressing endothelial cells showed the absence of SMI-71 immunoreactivity. Increase in ET-1 expression was followed by reduced AQP4 immunoreactivity prior to vasogenic edema formation. Only a few microglia showed monocyte chemotactic protein-1 (a chemokine induced by ET-1) outside vasogenic edema lesion. Taken together, our findings suggest that endothelial ET-1 expression may contribute to SE-induced vasogenic edema formation via brain–blood barrier disruption at AQP4/MCP-1 independent manners.

Transactivation of epidermal growth factor receptor by enhanced levels of endogenous angiotensin II contributes to the overexpression of Giα proteins in vascular smooth muscle cells from SHR

We earlier showed that the increased expression of Gi proteins exhibited by vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) was attributed to the enhanced levels of endogenous endothelin. Since the levels of angiotensin II (Ang II) are also enhanced in VSMC from SHR, the present study was undertaken to examine the role of enhanced levels of endogenous Ang II in the overexpression of Giα proteins in VSMC from SHR and to further explore the underlying mechanisms responsible for this increase. The enhanced expression of Giα-2 and Giα-3 proteins in VSMC from SHR compared to WKY was attenuated by the captopril, losartan and AG1478, inhibitors of angiotensin converting enzyme, AT 1 receptor and epidermal growth factor receptor (EGFR) respectively as well as by the siRNAs of AT1, cSrc and EGFR. The enhanced inhibition of forskolin-stimulated adenylyl cyclase activity by low concentrations of GTPγS (receptor-independent functions) and of inhibitory responses of hormones on adenylyl cyclase activity (receptor-dependent functions) in VSMC from SHR was also attenuated by losartan. Furthermore, the enhanced phosphorylation of EGFR in VSMC from SHR was also restored to control levels by captopril, losartan, PP2, a c-Src inhibitor and N-acetyl-L-cysteine (NAC), superoxide anion (O 2 −) scavenger, whereas enhanced ERK1/2 phosphorylation was attenuated by captopril and losartan. Furthermore, NAC also restored the enhanced phosphorylation of c-Src in SHR to control levels. These results suggest that the enhanced levels of endogenous Ang II in VSMC from SHR, transactivate EGFR, which through MAP kinase signaling, enhance the expression of Giα proteins and associated adenylyl cyclase signaling.

Prostanoids suppress the coronary vasoconstrictor influence of endothelin after myocardial infarction

Myocardial infarction (MI) is associated with endothelial dysfunction resulting in an imbalance in endothelium-derived vasodilators and vasoconstrictors. We have previously shown that despite increased endothelin (ET) plasma levels, the coronary vasoconstrictor effect of endogenous ET is abolished after MI. In normal swine, nitric oxide (NO) and prostanoids modulate the vasoconstrictor effect of ET. In light of the interaction among NO, prostanoids, and ET combined with endothelial dysfunction present after MI, we investigated this interaction in control of coronary vasomotor tone in the remote noninfarcted myocardium after MI. Studies were performed in chronically instrumented swine (18 normal swine; 13 swine with MI) at rest and during treadmill exercise. Furthermore, endothelial nitric oxide synthase (eNOS) and cyclooxygenase protein levels were measured in the anterior (noninfarcted) wall of six normal and six swine with MI. eNOS inhibition with N(ω)-nitro-L-arginine (L-NNA) and cyclooxygenase inhibition with indomethacin each resulted in coronary vasoconstriction at rest and during exercise, as evidenced by a decrease in coronary venous oxygen levels. The effect of l-NNA was slightly decreased in swine with MI, although eNOS expression was not altered. Conversely, in accordance with the unaltered expression of cyclooxygenase-1 after MI, the effect of indomethacin was similar in normal and MI swine. L-NNA enhanced the vasodilator effect of the ET(A/B) receptor blocker tezosentan but exclusively during exercise in both normal and MI swine. Interestingly, this effect of L-NNA was blunted in MI compared with normal swine. In contrast, whereas indomethacin increased the vasodilator effect of tezosentan only during exercise in normal swine, indomethacin unmasked a coronary vasodilator effect of tezosentan in MI swine both at rest and during exercise. In conclusion, the present study shows that endothelial control of the coronary vasculature is altered in post-MI remodeled myocardium. Thus the overall vasodilator influences of NO as well as its inhibition of the vasoconstrictor influence of ET on the coronary resistance vessels were reduced after MI. In contrast, while the overall prostanoid vasodilator influence was maintained, its inhibition of ET vasoconstrictor influences was enhanced in post-MI remote myocardium.

Effects of Antihypertensive Treatment on Endothelial Function

Essential hypertension is characterized by endothelial dysfunction due to reduced availability of nitric oxide (NO) secondary to increased generation of oxygen-free radicals. Some antihypertensive drugs may improve or restore endothelial function independently of their blood pressure lowering effect. The newer generation of β-blockers, such as nebivolol and carvedilol, which provide antioxidant activity, can improve endothelial function in patients with hypertension. Dihydropyridine and non-dihydropyridine calcium antagonists reverse impaired endothelium-dependent vasodilatation in different vascular districts, through a mechanism related to an antioxidant effect. However, conflicting results are found in the brachial artery. Angiotensin-converting enzyme (ACE) inhibitors improve endothelial function in subcutaneous, epicardial, brachial, and renal circulation, but they are ineffective in potentiating the impaired response to acetylcholine in the forearm of hypertensive patients. Angiotensin II receptor antagonists can restore endothelium-dependent vasodilatation to acetylcholine in subcutaneous microcirculation but not in that of the forearm muscle. They also improve basal NO release and decrease the vasoconstrictor effect of endogenous endothelin-1. Large-scale clinical trials are required to definitively demonstrate that treatment of endothelial dysfunction can improve the prognosis of patients with essential hypertension.

Effect of chronic central endothelin-1 on hemodynamics and plasma vasopressin in conscious rats

<title/>Objectives: These studies were designed to test whether chronic central administration of endothelin-1 induces changes in systemic hemodynamics and plasma vasopressin similar to those observed with acute microinjections of endothelin-1.Methods: Sprague Dawley rats underwent sham denervation or sinoaortic denervation. Three days later, baseline mean arterial blood pressure, heart rate, and vasopressin were assessed in conscious rats. Then, a cannula was stereotaxically inserted into the lateral ventricle and attached to an osmotic minipump that delivered one of the following: (i) artificial cerebrospinal fluid; (ii) endothelin-1, 10 pmol/hour; (iii) BQ-123, 400 pmol/hour; or (iv) endothelin-1+BQ-123. Mean arterial blood pressure and heart rate were monitored daily and blood was obtained for plasma vasopressin on days 3 and 9. On day 10, the rats were euthanized, the hypothalami were removed, and vasopressin messenger ribonucleic acid content was assessed.Results: The pressor effect of intracerebroventricular endothelin-1 was similar in intact and sinoaortic denervation rats and was prevented by endothelin receptor A antagonism with BQ-123. Administration of BQ-123 alone resulted in a depressor and bradycardia in sinoaortically denervated rats. Chronic endothelin-1 administration did not change plasma vasopressin but resulted in a significant decrease in hypothalamic vasopressin messenger ribonucleic acid levels, which was reversed by endothelin receptor A inhibition.Discussion: Although the pressor effect of chronic central endothelin-1 is similar to that reported with acute endothelin-1, plasma vasopressin levels do not increase, at least in part, due to downregulation of hypothalamic vasopressin gene expression. Sinoaortic denervation increases endogenous central endothelin receptor A tone. Furthermore, these observations confirm that the pressor effect of central endothelin-1 is not mediated by plasma vasopressin.

Interactions Between Sympathetic Nervous System and Endogenous Endothelin in Patients With Essential Hypertension

Experimental evidence indicates that endothelin 1 stimulates the sympathetic nervous system by activation of the subtype A receptor. The aim of the present study was to assess whether this mechanism is active in humans and to investigate its potential role in the pathogenesis of essential hypertension. In 15 hypertensive patients and 12 normotensive subjects, blood pressure, heart rate, and muscle sympathetic nerve activity were evaluated during intravenous 20-minute infusion of BQ123 (0.1 mg/kg per hour), an endothelin A receptor antagonist, and sodium nitroprusside (SNP; 0.4 μg/kg per minute). In hypertensive patients, blood pressure was reduced similarly by BQ123 and SNP. In contrast, the increase in muscle sympathetic nerve activity induced by BQ123 (from 52.0 ± 4.9 to 56.8 ± 5.5 bursts per 100 heartbeats; P<0.05 versus baseline) was significantly lower (P<0.05) than that induced by SNP (from 50.6 ± 4.9 to 61.1 ± 5.1 bursts per 100 heartbeats; P<0.05 versus baseline). In normotensive subjects, SNP reduced blood pressure and increased muscle sympathetic activity, whereas BQ123 was ineffective. Thus, in a subgroup (n = 9) of normotensive subjects, we administered BQ123 at a higher dose (0.2 mg/kg per hour), representing an equidepressor dose of SNP, inducing a blunted increase in sympathetic activity (from 44.1 ± 2.4 to 50.1 ± 6.4 bursts per 100 heartbeats; P<0.05 versus baseline). Finally, administration of a different vasodilator (papaverine, 0.5 mg/kg per hour) exerted results superimposable to SNP. Endogenous endothelin 1 appears to have a sympathoexcitatory effect both in normotensive and hypertensive subjects through endothelin A receptors, contributing to basal sympathetic vasomotor tone. Moreover, essential hypertension shows an increased susceptibility to the sympathoexcitatory effect of endogenous endothelin 1.

Different Impact of Endogenous Endothelin-1 and Angiotensin II Inhibition Within the Corpus Luteum During Prostaglandin F2alpha-Induced Luteolysis in the Cow.

Different Impact of Endogenous Endothelin-1 and Angiotensin II Inhibition Within the Corpus Luteum During Prostaglandin F2alpha-Induced Luteolysis in the Cow.

Enhanced levels of endogenous endothelin-1 contribute to the over expression of Giα protein in vascular smooth muscle cells from SHR: Role of growth factor receptor activation

We earlier showed that vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit increased expression of Gi proteins. Since the levels of endothelin-1 (ET-1) are enhanced in VSMC from SHR, we undertook the present study to examine the implication of endogenous ET-1 and the underlying mechanisms in the enhanced expression of Giα proteins in VSMC from SHR. The enhanced expression of Giα-2 and Giα-3 proteins in VSMC from SHR was inhibited by ETA and ETB receptor antagonists, BQ123 and BQ788 respectively. In addition, these antagonists also attenuated the enhanced inhibition of forskolin-stimulated adenylyl cyclase activity by low concentrations of GTPγS and by inhibitory hormones in VSMC from SHR compared to WKY. Furthermore, AG1295, AG1024 and PP2, inhibitors of platelet derived growth factor receptor (PDGFR), insulin-like growth factor 1 receptor (IGF-1R) and c-Src respectively, inhibited the enhanced expression of Giα protein and the enhanced phosphorylation of PDGFR and IGF-1R in VSMC from SHR to WKY levels. In addition, NAD(P)H oxidase inhibitor DPI and N-acetylcysteine (NAC), a scavenger of superoxide anion (O2−) also inhibited the enhanced phosphorylation of PDGFR and IGF-1R and c-Src in VSMC from SHR to control levels. Furthermore, the augmented phosphorylation of ERK1/2 in VSMC from SHR was attenuated by BQ123 and BQ788, growth factor receptors inhibitors and PP2. These results suggest that the enhanced levels of endogenous ET-1 in VSMC from SHR increase oxidative stress, which through c-Src-mediated activation of growth factor receptors and associated MAP kinase signaling, contribute to the enhanced expression of Giα proteins.

Long-Term Administration of Endothelin Receptor Antagonist Improves Coronary Endothelial Function in Patients With Early Atherosclerosis

Endothelin (ET-1) is one of the most potent vasoconstrictors and plays a seminal role in the pathogenesis of atherosclerosis. The present study was designed to test the hypothesis that long-term treatment with an endothelin-A (ET(A)) receptor antagonist improves coronary endothelial function in patients with early coronary atherosclerosis. Forty-seven patients with multiple cardiovascular risk factors, nonobstructive coronary artery disease, and coronary endothelial dysfunction were randomized in a double-blind manner to either the ET(A) receptor antagonist atrasentan (10 mg) or placebo for 6 months. Coronary endothelium-dependent vasodilation was examined by infusing acetylcholine (10(-6) to 10(-4) mol/L) in the left anterior descending coronary artery. N(G)-monomethyl-l-arginine was administered to a subgroup of patients. Endothelium-independent coronary flow reserve was examined by use of intracoronary adenosine and nitroglycerin. Baseline characteristics and incidence of adverse effects were similar between the 2 groups. There was a significant improvement in percent change of coronary blood flow in response to acetylcholine at 6 months from baseline in the atrasentan group compared with the placebo group (39.67%, 95% confidence interval 23.23% to 68.21%, versus -2.22%, 95% confidence interval -27.37% to 15.28%; P<0.001). No significant difference in the percent change of coronary artery diameter or change in coronary flow reserve was demonstrated. Coronary blood flow, coronary artery diameter, and the effect of N(G)-monomethyl-l-arginine were similar between the groups at baseline and at 6 months. This study demonstrates that 6-month treatment with atrasentan improves coronary microvascular endothelial function and supports the role of the endogenous endothelin system in the regulation of endothelial function in early atherosclerosis in humans. Clinical Trial Registration Information- URL: http://www.clinicaltrials.gov. Unique identifier: NCT00271492.