Doses Of ET-1 Research Articles

Sustained, delayed, and small increments in glomerular permeability to macromolecules during systemic ET-1 infusion mediated via the ETA receptor.

Emerging evidence indicates that endogenous production of endothelin (ET)-1, a 21-amino acid peptide vasoconstrictor, plays an important role in proteinuric kidney disease. Previous studies in rats have shown that chronic administration of ET-1 leads to increased glomerular albumin leakage. The underlying mechanisms are, however, currently not known. Here, we used size-exclusion chromatography to measure glomerular sieving coefficients for neutral FITC-Ficoll (molecular Stokes-Einstein radius: 15-80 Å, molecular weight: 70 kDa/400 kDa) in anesthetized male Sprague-Dawley rats (n = 12) at baseline and at 5, 15, 30, and 60 min after intravenous administration of ET-1. In separate experiments, ET-1 was given together with the selective ET type A (ETA) or ET type B (ETB) receptor antagonists JKC-301 and BQ-788, respectively. At both 15 and 30 min postadministration, the glomerular sieving coefficient for macromolecular Ficoll (70 Å) was significantly increased to 4.4 × 10-5 ± 0.7 × 10-5 (P = 0.024) and 4.5 × 10-5 ± 0.8 × 10-5 (P = 0.007), respectively, compared with baseline (2.2 × 10-5 ± 0.4 ×10-5). Decreased urine production after ET-1 prevented the use of higher doses of ET-1. Data analysis using the two-pore model indicated changes in large-pore permeability after ET-1, with no changes in the small-pore pathway. Administration of ETA blocker abrogated the permeability changes induced by ET-1 at 30 min, whereas blockade of ETB receptors was ineffective. Mean arterial pressure was only significantly increased at 60 min, being 123 ± 4 mmHg compared with 111 ± 2 mmHg at baseline (P = 0.02). We conclude that ET-1 evoked small, delayed, and sustained increases in glomerular permeability, mediated via the ETA receptor.

Dose-Dependent Effects of Endothelin-1 on Retinal and Optic Nerve Morphology in Sprague Dawley Rats

ET1 induced retinal and optic nerve injury is widely used as an experimental model to study unexplained pathophysiology and therapeutic targets involved in several ophthalmic diseases such as glaucoma and diabetic retinopathy. Although a dose-dependent effect of ET1 on retinal vascular tone and retinal blood flow has been studied, the dose-dependent effects of ET1 on retinal and optic nerve morphology remain unexplored. Current study, therefore investigated the dose-dependent effects of ET1 on retinal and optic nerve morphology. Sprague Dawley rats were injected with ET1 in the dose range of 0.025–2.5 nM, intravitreally. Seven days post-injection, eyes were enucleated and retinal sections were examined for morphological changes using H&E staining and extent of retinal cell apoptosis was determined using TUNEL staining. Optic nerve morphology was studied using toluidine blue staining. It was observed that ET1 induces significant changes in retinal and optic nerve morphology at 0.25 and 2.5 nM doses but not at 0.025 nM. The outcomes of in vivo studies using different doses of ET1, therefore, may not be comparable. Furthermore, while designing experimental studies using ET1 induced retinal ischemia, it is important to carefully make the dose selection based on the study objectives.

Linagliptin reduces effects of ET-1 and TLR2-mediated cerebrovascular hyperreactivity in diabetes

AimsThe anti-hyperglycemic agent linagliptin, a dipeptidyl peptidase-4 inhibitor, has been shown to reduce inflammation and improve endothelial cell function. In this study, we hypothesized that DPP-IV inhibition with linagliptin would improve impaired cerebral blood flow in diabetic rats through improved insulin-induced cerebrovascular relaxation and reversal of pathological cerebrovascular remodeling that subsequently leads to improvement of cognitive function. Main methodsMale type-2 diabetic Goto-Kakizaki (GK) and nondiabetic Wistar rats were treated with linagliptin, and ET-1 plasma levels and dose response curves to ET-1 (0.1–100nM) in basilar arteries were assessed. The impact of TLR2 antagonism on ET-1 mediated basilar contraction and endothelium-dependent relaxation to acetylcholine (ACh, 1nM–1M) in diabetic GK rats was examined with antibody directed against the TLR2 receptor (Santa Cruz, 5μg/mL). The expression of TLR2 in middle cerebral arteries (MCAs) from treated rats and in brain microvascular endothelial cells (BMVEC) treated with 100nM linagliptin was assessed. Key findingsLinagliptin lowered plasma ET-1 levels in diabetes, and reduced ET-1-induced vascular contraction. TLR2 antagonism in diabetic basilar arteries reduced ET-1-mediated cerebrovascular dysfunction and improved endothelium-dependent vasorelaxation. Linagliptin treatment in the BMVEC was able to reduce TLR2 expression in cells from both diabetic and nondiabetic rats. ConclusionsThese results suggest that inhibition of DPPIV using linagliptin improves the ET-1-mediated cerebrovascular dysfunction observed in diabetes through a reduction in ET-1 plasma levels and reduced cerebrovascular hyperreactivity. This effect is potentially a result of linagliptin causing a decrease in endothelial TLR2 expression and a subsequent increase in NO bioavailability.

High salt intake increases endothelin B receptor function in the renal medulla of rats

AimsEndothelin (ET)-1 promotes natriuresis via the endothelin B receptor (ETB) within the renal medulla. In male rats, direct interstitial infusion of ET-1 into the renal medulla has no effect on renal sodium and water excretion but is associated with endothelin A receptor (ETA)-dependent reductions in medullary blood flow. Loss of ETB function leads to salt-sensitive hypertension. We hypothesized that HS intake would increase the natriuretic and diuretic response to renal medullary infusion of ET peptides. Main methodsMale Sprague–Dawley (SD) rats were fed a normal (NS) or high (HS) salt diet for 7days. Rats were anesthetized and a catheter implanted in the renal medulla for interstitial infusion along with a ureteral catheter for urine collection. Medullary infusion of a low dose of ETB receptor agonist, sarafotoxin 6c (S6c; 0.15μg/kg/h), or ET-1 (0.45μg/kg/h) was used to determine changes in sodium excretion (UNaV). Key findingsIn HS fed rats, intramedullary infusion of a low dose of S6c induced a significant increase in UNaV, roughly 2-fold over baseline, compared to no response to this low dose in NS fed rats. In HS fed rats, intramedullary infusion of ET-1 induced a significantly greater increase in UNaV compared to NS fed rats, although this increase was not different from the HS time control studies. SignificanceWe conclude that high salt intake enhances the diuretic and natriuretic effects of ETB receptor activation in vivo consistent with a role for the ETB receptor in maintaining fluid-electrolyte homeostasis.

ET-1 increases reactive oxygen species following hypoxia and high-salt diet in the mouse glomerulus.

This study was designed to determine whether ET-1 derived from endothelial cells contributes to oxidative stress in the glomerulus of mice subjected to a high-salt diet and/or hypoxia. C57BL6/J control mice or vascular endothelial cell ET-1 knockout (VEET KO) mice were subjected to 3-h exposure to hypoxia (8% O₂) and/or 2 weeks of high-salt diet (4% NaCl) prior to metabolic cage assessment of renal function and isolation of glomeruli for the determination of reactive oxygen species (ROS). In control mice, hypoxia significantly increased urinary protein excretion during the initial 24 h, but only in animals on a high-salt diet. Hypoxia increased glomerular ET-1 mRNA expression in control, but not in vascular endothelial cell ET-1 knockout (VEET KO) mice. Under normoxic conditions, mice on a high-salt diet had approx. 150% higher glomerular ET-1 mRNA expression compared with a normal-salt diet (P < 0.05). High-salt diet administration significantly increased glomerular ROS production in flox control, but not in glomeruli isolated from VEET KO mice. In C57BL6/J mice, the ETA receptor-selective antagonist, ABT-627, significantly attenuated the increase in glomerular ROS production produced by high-salt diet. In addition, chronic infusion of C57BL6/J mice with a subpressor dose of ET-1 (osmotic pumps) significantly increased the levels of glomerular ROS that were prevented by ETA antagonist treatment. These data suggest that both hypoxia and a high-salt diet increase glomerular ROS production via endothelial-derived ET-1-ETA receptor activation and provide a potential mechanism for ET-1-induced nephropathy.

Activation of AMPK inhibits pulmonary arterial smooth muscle cells proliferation

ABSTRACTThe aims of the present study were to examine the effect of AMPK activation on pulmonary arterial smooth muscle cells (PASMCs) proliferation and to address its potential mechanisms. ET-1 dose and time-dependently induced PASMCs proliferation, and this effect was suppressed by a selective AMPK activator metformin. The results of the study further indicated that the proliferation of PASMCs stimulated by ET-1 was associated with the increase of Skp2 and decrease of p27, and metformin reversed ET-1-induced Skp2 elevation and raised p27 protein level. Our study suggests that activation of AMPK suppresses PASMCs proliferation and has potential value in negatively modulating pulmonary vascular remodeling and therefore could prevent or treat the development of pulmonary arterial hypertension (PAH).

Vasoactive effects of endothelin‐1 in the mesentery and cremaster muscle of leptin deficient mice (674.11)

Leptin has been shown to up‐regulate endothelin‐1 production. We investigated the responses to endothelin‐1 (ET) in leptin deficient diabetic mice (db/db) in comparison to C57BL/6J (C57) genetic background mice in the absence or presence of endogenous NO (LNNA), and with blockade of the ETB receptor (PD142893, BQ788). The cremaster muscle of anesthetized (50 mg/kg Nembutal or 4% isoflurane) mice (n=33) was prepared for intravital microscopy. Arteriole(A)/venule(V) pairs were chosen for study. Baseline diameters in the cremaster (A/V, 30/35 um) were ~20‐30% larger than in db/dbs; in the mesentery (60/120 um) they were similar for the 2 strains. Vasodilatory tone (adenosine, 10‐4M) was not significantly different between strains, and was lower in mesentery vs. cremaster. Vasoconstrictor tone (Phenylephrine, 10‐4M) was decreased in db/db compared to C57 in the cremaster, but not mesentery. ET1 dose dependent constriction in the C57 or db/db cremaster (logM EC50: C57 ‐11.9±29.6 A, ‐11.7±2.8 V; db/db ‐11.7±4.4 A, ‐10.0 V) was suppressed by LNNA, and enhanced with ETB antagonism to a greater extent in the A vs. V. In the mesentery, baseline constriction to ET1 was diminished to a greater extent in the db/db vs C57 and was suppressed by LNNA and enhanced with ETB antagonism. Thus, the leptin deficient mouse exhibits a tissue specific loss of ET1 mediated constriction. Yet, the potential role for ETB mediated vasodilation appears similar for C57 and db/db in both tissuesGrant Funding Source: NIH DK68401, The UWI

Impaired Endothelin Calcium Signaling Coupled to Endothelin Type B Receptors in Penile Arteries from Insulin-Resistant Obese Zucker Rats

Erectile dysfunction is considered as an early sign of subclinical vascular disease and endothelial dysfunction and a highly prevalent condition in diabetic patients. The current study assessed whether impaired vascular effects of endothelin (ET)-1 may contribute to the vascular dysfunction of penile arteries from a rat model of insulin resistance. The effect of ETA and ETB receptor antagonists was assessed on the intracellular Ca(2+) [Ca(2+) ]i and contractile responses to ET-1 in penile arteries from obese Zucker rats (OZR) and lean Zucker rats (LZR), and ET receptor expression in the arterial wall was assessed by immunohistochemistry. Changes in ET-1 [Ca(2+) ]i and vasoconstriction and ET receptor expression were evaluated in penile arteries from insulin-resistant rats. ET-1-induced vasoconstriction was associated with a higher increase in smooth muscle [Ca(2+) ]i in penile arteries from OZR compared with LZR. Removal of the endothelium inhibited and enhanced contractions to the lowest and highest doses of ET-1, respectively, mainly in OZR. The selective ETA receptor antagonist BQ-123 inhibited ET-1 vasoconstriction and [Ca(2+) ]i response in both LZR and OZR. The ETB receptor antagonist BQ-788 had little effect in healthy arteries but markedly inhibited ET-1-induced increases in [Ca(2+) ]i and vasoconstriction in arteries from OZR. ETA receptors were located on the smooth muscle and endothelium of penile arteries, whereas ETB receptors were found on the arterial endothelium in LZR and OZR, and also on the smooth muscle in OZR, immunostaining for both receptors being higher in OZR. Penile arteries from OZR exhibit an impaired ET-1 Ca(2+) signaling along with changes in the ET receptor profile. Thus, whereas ET-1 contraction and the associated [Ca(2+) ]i increase are mediated by smooth muscle ETA receptors in healthy arteries, ETB receptors contribute to contraction and are coupled to the augmented ET-1 [Ca(2+) ]i response under conditions of insulin resistance.

Endothelin-1 and -3 induce choleresis in the rat through ETB receptors coupled to nitric oxide and vagovagal reflexes

We have reported previously that centrally applied ET (endothelin)-1 and ET-3 induce either choleresis or cholestasis depending on the dose. In the present study, we sought to establish the role of these endothelins in the short-term peripheral regulation of bile secretion in the rat. Intravenously infused endothelins induced significant choleresis in a dose-dependent fashion, ET-1 being more potent than ET-3. Endothelins (with the exception of a higher dose of ET-1) did not affect BP (blood pressure), portal venous pressure or portal blood flow. ET-1 and ET-3 augmented the biliary excretion of bile salts, glutathione and electrolytes, suggesting enhanced bile acid-dependent and -independent bile flows. ET-induced choleresis was mediated by ET(B) receptors coupled to NO and inhibited by truncal vagotomy, atropine administration and capsaicin perivagal application, supporting the participation of vagovagal reflexes. RT (reverse transcription)-PCR and Western blot analysis revealed ETA and ET(B) receptor expression in the vagus nerve. Endothelins, through ET(B) receptors, augmented the hepatocyte plasma membrane expression of Ntcp (Na⁺/taurocholate co-transporting polypeptide; Slc10a1), Bsep (bile-salt export pump; Abcb11), Mrp2 (multidrug resistance protein-2; Abcc2) and Aqp8 (aquaporin 8). Endothelins also increased the mRNAs of these transporters. ET-1 and ET-3 induced choleresis mediated by ET(B) receptors coupled to NO release and vagovagal reflexes without involving haemodynamic changes. Endothelin-induced choleresis seems to be caused by increased plasma membrane translocation and transcriptional expression of key bile transporters. These findings indicate that endothelins are able to elicit haemodynamic-independent biological effects in the liver and suggest that these peptides may play a beneficial role in pathophysiological situations where bile secretion is impaired.

Increased Neurogenesis Contributes to the Promoted Behavioral Recovery by Constraint‐Induced Movement Therapy after Stroke in Adult Rats

Increased Neurogenesis Contributes to the Promoted Behavioral Recovery by Constraint‐Induced Movement Therapy after Stroke in Adult Rats

Abstract WP50: Defining the Hyper-acute Infarct Core in Porcine Model of Cerebral Ischemia: A Multi-modality Imaging Study

Background: Defining the hyper-acute infarct core can inform thrombolysis treatment. Clinical outcome and hemorrhagic transformation are correlated with infarct volume at admission. CT perfusion (CTP)-derived parameters and MR-diffusion weighted imaging (DWI) are currently used to delineate infarct volume; however, reversibility of such defects has been shown for both modalities. Methods: Six domestic pigs (mean weight 34.7kg) had a CTP scan prior to intracranial injection of endothelin-1 (ET-1; 0.01mL/kg) into the left striatum. Subsequent CTP scans at 30min, 1hr, 1.5hrs were done to monitor stroke progression. A second dose of ET-1 (0.01mL/kg) was injected at 2hrs from the first injection. Twenty minutes after the second ET-1 injection, 18F-FDG was injected. The animal was moved to a 3T MRI scanner where MR-DWI was performed. The animal was then moved back to the CT scanner for a final CTP/PET acquisition. The brain was quickly removed, cut into 10mm slices and stained with tetrazolium-chloride (TTC), the gold standard for defining final infarct volume. Using custom software, the infarct volume defined by low intensity TTC stain, low CBF (&lt; 9.3ml•min-1•100g-1), low CBV (&lt; 1.07ml•100g-1), DWI hyper-intensity and low F-18 FDG uptake were determined. A linear regression model was used to correlate the infarct volume measured by each imaging modality to that by the histological gold standard and the coefficients of correlation (R2) compared. Results: R2 values for CBF, CBV, DWI and FDG versus TTC-histology were 0.83, 0.69, 0.95 and 0.61, respectively. For the CBF and DWI parameters the slope of the fitted line was greater than 1, while the slope of the fitted line for the CBV and CBFxCBV parameters was less than 1. Mean normalized (relative to the histologically defined infarct) values were 1.38, 0.82, 0.99, and 0.67 for CBF, CBV, DWI and FDG, respectively. Conclusion: In this small sample study, the CTP-CBF and the imaging gold standard DWI both overestimated the TTC-infarct in 100% and 50% of cases, while CTP-CBV and FDG-PET both underestimated the final infarct volume in 100% of animals.

ET‐1‐induced vascular contraction is mediated by RhoA/Rho‐kinase pathway activation via O‐GlcNAcylation

O‐GlcNAc‐proteins and ET‐1‐production are increased in DOCA‐salt rats. We hypothesized that ET‐1 augments O‐GlcNAc levels and this modification contributes to increased vascular contractile responses via activation of RhoA/Rho‐kinase pathway. Incubation of smooth muscle cells with ET‐1 (0.1μM) increased O‐GlcNAc levels and decreased OGA, but not OGT expression, key enzymes in the O‐GlcNAc process. Overnight treatment of rat aortas (no endothelium) with ET‐1 increased contractile response to phenylephrine (PE). However, the effects of ET‐1 were not observed when vessels were previously transfected with an anti‐OGT antibody (chariot OGT) or after incubation with an OGT inhibitor (ST045849 100μM). Aortas from rats chronically infused with low doses of ET‐1 exhibited increased O‐GlcNAc‐proteins and displayed changes in reactivity similar to those induced by PugNAc, an inhibitor of OGA. Augmented aortic contractions to PE, after treatment with PugNAc or ET‐1 were abolished by the Rho kinase inhibitor Y‐27632. ET‐1 or PugNAc increased expression of RhoA and phosphorylated forms of MYPT‐1, a Rho kinase target. The effects of ET‐1 on RhoA and MYPT‐1 expression and activation were not observed after incubation with atrasentan or ST045849. Our data show that ET‐1 augments O‐GlcNAc levels and we speculate that this modification contributes to the vascular changes mediated by RhoA/Rho‐kinase pathway activation.

Differential effects of endothelins on histological and ultrastructural changes and trypsinogen activation in the secretagogue-induced acute pancreatitis in rats

The role of endothelins in acute pancreatitis remains obscure. To assess the effects of endothelins (ETs) in early (4 h) caerulein-induced acute pancreatitis (AP) in rats, ET-1, ET-2 and ET-3 (0.5 or 1.0 nmol/kg) were applied twice with i.p. caerulein (2×40 μg/kg) at 1 h interval. Histological and ultrastructural examinations of pancreases and the assay of trypsinogen activation in whole homogenate were performed. All ETs, especially ET-1 at the higher dose, decreased inflammatory cell infiltration despite an increase in the edema score. The vacuolization and necrosis of acinar cells were slightly increased after the lower dose of ET-1 and ET-2. Ultrastructural changes were generally improved after the higher dose of ETs. Trypsinogen activation increased from 4.8±1.3% in control to 18.4±3.8% in AP ( p<0.01). It was attenuated to 6.4±1.3% ( p<0.01) by the higher dose of ET-1 and to 8.8±1.5% ( p<0.05) by the lower dose of ET-3. In summary, ETs, especially ET-1 at the higher dose, were found to have some beneficial effects on morphological changes and trypsinogen activation in the pancreas in early caerulein-induced AP.

Augmented vascular reactivity induced by ET‐1 is associated with increased O‐GlcNAcylation

O-GlcNAcylation, the modification of proteins with O-linked beta-N-acetylglucosamine (O-GlcNAc), modulates many functions including vascular reactivity. We hypothesized that ET-1-induced changes in vascular responses are associated with increased levels of O-GlcNAc-proteins. Incubation of rat aortas with ET-1 (0.1 ?M) produced a time-dependent increase in vascular O-GlcNAc levels (at 24h, arbitrary units = 2.2±0.1 vs. 1.0±0.05 control; n=4) and decreased expression of OGT and OGA, key enzymes in the O-GlcNAcylation process. Overnight incubation with ET-1 increased phenylephrine (PE) reactivity [Emax (mN) = 19±5 vs. 11±2 control; n=4]. The effects of ET-1 we not observed when vessels were previously transfected with antibodies against-OGT or after incubation with atrasentan (ETA antagonist, 1μM, Fig.1). Aorta and resistance arteries from rats chronically treated with low doses of ET-1 (2pmol/min/Kg, for 14 day) exhibit increased O-GlcNAc-proteins (Fig.1) and displayed changes in reactivity similar to those induced by PugNAc, an inhibitor of OGA. PugNAc treatment increased O-GlcNAc levels and enhanced PE responses. Our data show that ET-1 augments O-GlcNAc levels and that this modification contributes to the vascular changes induced by this peptide. We speculate that reactive oxygen species may provide a mechanism for the augmented vascular O-GlcNAc content induced by ET-1. Figure 1Open in figure viewerPowerPoint

Selective enhanced constriction and attenuated dilation in leptin deficient mice.

We tested responses to endothelin‐1 (ET), interleukin‐6 (IL6) and IL8 in leptin deficient diabetic mice (db/db or ob/ob), in comparison to C57 genetic background mice in the absence or presence of inhibitors. The cremaster muscle of anesthetized (50 mg/kg Nembutal) mice (n=63) was prepared for intravital microscopy. Arteriole(A)/venule(V) pairs were chosen for study; baseline diameters A 26±8 ?m, V 50±13 ?m were not different between strains. Micropipette applied adenosine (10‐4M) was attenuated in A and V from db/db or nitroarginine (LNNA) pre‐treated C57 vs. control C57. Phenylephrine (10‐4M) constriction was unchanged in A, but attenuated in V from db/db, ob/ob or LNNA vs. C57. ET1 dose dependent constriction (C57 logM EC50 ‐10±5 A; ‐5±4 V) was significantly attenuated in either db/db or ob/ob mice, yet enhanced with LNNA pretreatment in C57. Responses to IL6 or IL8 were biphasic and dose dependent in A from C57, while V showed only constriction. Responses to IL6 were blocked by PD142893. IL6 caused only constriction in A and V of ob/ob or db/db. IL8 caused biphasic responses in some diabetics. Leptin deficient diabetes thus shows attenuated constriction to ET1, selective absence of dilation to IL6 and inconsistent absence of dilation to IL8. NIH DK68401

Endothelin and Aged Blood Vessels

Age is one of the most important risk factors for cardiovascular disease. For example, a 30-year-old male smoker with untreated hypertension, diabetes, dyslipidemia, and a strong family history of premature coronary heart disease (CHD) would have a 10-year coronary heart disease event rate of only 16% (“intermediate risk”) by Framingham criteria; however, given exactly the same risk profile for a 60-year-old, the risk climbs to >50%. Aging, particularly sedentary aging, leads not only to the development of atherosclerosis but also to ventricular and arterial stiffening.1 This reduces functional capacity and contributes to chronic diseases of the elderly, such as systolic hypertension or heart failure with a preserved ejection fraction. In the search for the underlying mechanism for this effect of sedentary aging, one common feature that has been identified universally in older men is an increased baseline vascular tone,2 which has both hemodynamic and metabolic consequences.3 Although this age-related increase in vascular tone is mediated in part by a chronically elevated sympathetic α-adrenergic vasoconstriction,2 endothelial function also plays a critical role in vascular stability and the regulation of vasomotor function. The endothelium regulates vascular tone through the release of dilator and constrictor substances. Endothelin (ET)-1 is the most potent endothelium-derived constricting factor and influences peripheral vascular tone by interacting with ETA and ETB receptors on smooth muscle cells and the endothelium.4 Several animal studies have examined the profound effects of vascular aging on the ET-1 pathway and support a central role for ET-1 to explain the age-related increase in vascular tone.5–7 In the present issue of Hypertension , Van Guilder et al8 present new and important information regarding the role of aging and physical fitness on the vasomotor effects of ET-1. Using a cross-sectional design in a small but carefully studied …

POTENTIAL FOR ENDOTHELIN‐1‐MEDIATED IMPAIRMENT OF CONTRACTILE ACTIVITY IN HYPERTENSION

1. The present study examined the potential for reduced exercise capacity observed in hypertensive patients as a result of elevated levels of endothelin (ET)-1. We have previously reported that ET-1 exerts low-dose stimulatory or high-dose inhibitory effects on the metabolism of the rat isolated perfused hindlimb from its vasoconstrictor activity. 2. Herein, we determined whether there are similar effects on tension development by the rat isolated constant-flow hindlimb during ET-1-mediated vasoconstriction. 3. The dose-dependent vasoconstrictor effects of ET-1 on metabolism in contracting muscle were the same as those observed previously in resting muscle. Highest concentrations of ET-1 gave rise to a transient stimulation followed by a marked inhibition of tension development, consistent with a decrease in aerobic capacity of the muscle. The vasoconstriction due to the higher doses of ET-1 was not dilated by electrical stimulation. 4. In conclusion, the biphasic nature of the actions of ET-1 suggests that although lower concentrations of ET-1 do not affect exercise capacity, higher concentrations that may occur in hypertension are inhibitory to metabolism and aerobic capacity of muscle. The inhibitory effects of ET-1 appear to result from enhanced functional shunting.

Effect of endothelin-1 on erythropoietin production in a rat model under normoxia and functional carbon monoxide-induced hypoxia

It has been hypothesized that autacoids, such as endothelin-1 (ET), may modulate erythropoietin (Epo) secretion. Therefore, we studied the effect of ET-1 infusion and of a nonselective ET(A/B) receptor antagonist on Epo secretion under carbon monoxide (CO) exposure. Anesthetized rats were supplied with room temperature air containing increasing concentrations of CO by an aerating cap. A CO-Epo dose-response curve over the range of 0.02-0.14 vol% CO was conducted. Subpressor doses of ET-1 (3 pmol/min/kg BW) and the ET(A/B) receptor antagonist LU302872 (LU; 30 mg/kg) were applied to anaesthetized rats under normoxia (controls CON, ET, LU) and following hypoxia (CO exposure; H-CON, H-ET, H-LU). Mean arterial blood pressure (MAP), glomerular filtration rate (GFR, inulin clearance), Epo and ET-1 serum concentrations (ELISA) and renal Epo mRNA (Light Cycler) were determined. The EC50 value for CO was 0.1 vol% with a 70-fold increase in Epo serum concentrations. CO exposure increased Epo serum and Epo mRNA concentrations in the expected range in all groups. None of the treatments with ET or LU influenced the effect of hypoxia on Epo serum concentrations and renal Epo mRNA content. Under hypoxia, administration of ET-1 as well as LU prevented the hypoxia-induced decrease in MAP (p<0.05). Under hypoxia, GFR was reduced by 50% except for H-LU with values comparable to normoxia. Taken together, the influence of hypoxia exceeds by far the effect of ET-1 on Epo production, irrespective of the presence or absence of exogenous ET-1. Thus, ET-1 does not appear to be a major modulator of Epo production.

Endothelin is a dose-dependent trophic factor and a mitogen in small arteries in vivo

Endothelin (ET) modulates cellular processes relevant to vascular remodeling, but there is still some debate as to the potential of ET to be a trophic factor or a mitogen. Moreover, the signaling of ET in vivo to produce these effects is largely unknown. 3H-leucine and 3H-thymidine incorporation in rat small mesenteric arteries was studied with several doses of ET-1 (0.1-10 pmol/kg/min) administered for 26 h in vivo. The EC50 for protein synthesis was four times lower than that of DNA synthesis, with maximal effects around 1 and 3 pmol/kg/min, respectively. At 5 pmol/kg/min, ET enhanced CDK2 activity by reducing the binding of its inhibitor p27(Kip1). In contrast, the binding was enhanced at 0.5 pmol/kg/min. The reduced binding observed at 5 pmol/kg/min could not be explained by changes of p27(Kip1) or CDK2 content. Phosphorylation of p27(Kip1) on serine 10 was significantly reduced at 5 pmol/kg/min ET. Although the phosphoinositide 3-kinase pathway was activated, it did not contribute to the protein or DNA synthesis responses. Administration of 1 or 5 pmol/kg/min ET-1 for 28 days increased the thickness and cross-sectional area of the small mesenteric artery due to hypertrophy and hyperplasia, respectively, thus confirming the results obtained in acute conditions. ET modulates p27(Kip1) binding to CDK2, producing hypertrophy at low and hyperplasia at higher concentrations. Taken together, these results suggest that ET can act both as a trophic factor and as a mitogen in an in vivo environment, depending on its local concentration.

In vitro evaluation of the contractile response to endothelin-1 of the circular and longitudinal myometrial layers of the uterine horn of nongravid mares

To characterize the in vitro response of circular and longitudinal myometrial layers of the uterine horn (CMLH and LMLH, respectively) of horses to endothelin (ET)-1 by use of specific ETA (BQ-123) and ETB (IRL-1038) receptor antagonists. Uteruses from 10 nongravid mares in anestrus. Muscle strips from the CMLH and LMLH were suspended in tissue baths and connected to force-displacement transducers interfaced with a polygraph. Strips were incubated for 45-minute intervals with no antagonist (control specimens), and 3 concentrations (10(-9), 10(-7), and 10(-5)M) of BQ-123, IRL-1038, or BQ-123 and IRL-1038 before concentration-response curves to ET-1 were generated. Contractile response to cumulative concentrations of ET-1 (10(-9) to 10(-6)M) was quantified by measuring change in the area under the curve (AUC) for the 3-minute period after each ET-1 dose. ET-1 caused concentration-dependent contraction of the CMLH and LMLH specimens. Application of BQ-123 decreased AUC values for both layers. Application of IRL-1038 increased the AUC value for LMLH specimens but did not affect the CMLH value. The combination of BQ-123 and IRL-1038 decreased the AUC value for LMLH tissue and increased that for CMLH tissue. ET-1 causes contraction of the CMLH and LMLH in nongravid horses. In both layers, ETA receptors mediate contraction but the role of ETB receptors remains unclear. In the LMLH, ETA receptors have a dominant role; the presence of another receptor or receptor subtype within this layer is suggested. These findings support a physiologic role for ET-1 in uterine contractility.