ET-1 Concentrations Research Articles

Protective effect of endothelin type A receptor antagonist on brain edema and injury after transient middle cerebral artery occlusion in rats.

Recent evidence strongly suggests that endothelins (ETs) play an important role in the regulation of blood-brain barrier (BBB) functions. The aim of the present study was to evaluate the role of ETs on edema formation and BBB permeability change after cerebral ischemia/reperfusion. We examined the brain tissue ET-1 content and evaluated the time and dose response of the therapeutic effects of the specific ET type A receptor (ET(A)) antagonist, S-0139, on brain edema formation, development of infarction, and disruption of BBB after 1 hour of middle cerebral artery occlusion (MCAO) in rats. After 1-hour MCAO and reperfusion, the brain ET-1 content did not change during the first 3 hours, increased at 6 hours, and rose almost continuously over 48 hours in the ischemic region as well as in the ischemic rim. Rats infused with S-0139 (0.03 to 1.0 mg/kg per hour) during reperfusion showed dose-dependent and significant attenuation of the increase in brain water content 24 hours after reperfusion. When the infusion of S-0139 was begun after 10 minutes and 1 hour of reperfusion, the brain edema formation and infarct size were significantly attenuated. Furthermore, posttreatment with S-0139 significantly attenuated the increased Evans blue dye-quantified albumin extravasation and improved the mortality of animals after cerebral ischemia/reperfusion. Our data demonstrate that infusion with S-0139, an ET(A) antagonist, results in significant reduction of brain injury and plasma extravasation after transient MCAO. Thus, ETs may contribute to cerebral ischemia/reperfusion injury at least partly by increasing the BBB permeability via ET(A)s.

Endogenous endothelins mediate increased acidification in remnant kidneys.

Because endothelins (ET) mediate increased renal acidification induced by dietary acid and animals with reduced renal mass exhibit increased urinary ET-1 excretion, the hypothesis that ET mediate increased renal acidification in remnant kidneys was tested. Four weeks before the study, rats underwent a 5/6 nephrectomy (Nx) and a microdialysis apparatus was inserted into the remnant left kidney and the left kidney of sham-treated control animals, for measurements of renal ET-1 contents. Nx animals exhibited greater ET-1 addition to the renal dialysate than did control animals (681 +/- 91 versus 290 +/- 39 fmol/g kidney wt per min, P < 0.002) and greater urinary ET-1 excretion (346 +/- 79 versus 125 +/- 24 fmol/d, P < 0.02). Urinary net acid excretion rates were similar for Nx and control animals (732 +/- 106 versus 1005 +/- 293 microEq/d, P = 0.4), but Nx animals exhibited greater in situ HCO(3)(-) reabsorption in proximal (972.3 +/- 77 versus 482.6 +/- 42.4 pmol/min, P < 0.001) and distal (62.7 +/- 6.7 versus 24.3 +/- 2.5 pmol/min, P < 0.001) tubules. Orally administered bosentan, an ET(A/B) receptor antagonist, decreased urinary net acid excretion in Nx animals (to 394 +/- 99 microEq/d, P < 0.04 versus without bosentan); the decrease was mediated by decreased HCO(3)(-) reabsorption in both the proximal and distal tubules. Furthermore, bosentan decreased blood base excess in Nx animals (0.1 +/- 0.3 to -0.12 +/- 0.03 microM/ml blood, P < 0.002), consistent with acid retention. The data demonstrate that endogenous ET mediate increased urinary acid excretion in the remnant kidneys of Nx animals.

The intrarenal endothelin system and hypertension.

The kidney is both a source of endothelin (ET) generation and an important target organ of this peptide. The highest concentrations of ET-1 in the body exist in the renal medulla, where it mediates natriuretic and diuretic effects through the ET(B) receptor subtype. It is proposed that aberrations in the renal ET system may lead to sodium and water retention and subsequently to the development of hypertension.

Endothelin-1 and nitric oxide synthase in short rebound reaction to short exposure to inhaled nitric oxide.

On withdrawal of inhalation of nitric oxide (INO) administered after lung injury, pulmonary artery pressure (PAP) and arterial oxygen tension (Pa(O(2))) may deteriorate more than before INO (rebound response). In this study, we investigated the possible roles of endothelin (ET)-1 and nitric oxide (NO) synthase (NOS) activity in the short rebound reaction to short-term inhalation of NO. Twenty-six anesthetized mechanically ventilated piglets were given endotoxin infusion. Twelve animals then received INO (30 parts per million) for two 30-min periods. Nine controls were not given NO. Measurements were made of blood gases and hemodynamic parameters, lung tissue ET-1 expression and NOS activity, and plasma ET-1 concentration. INO decreased PAP and increased Pa(O(2)), but INO withdrawal caused a short rebound reaction with an increase in PAP. Lung tissue expression and plasma concentration of ET-1 increased during INO, and plasma ET-1 increased further after its withdrawal. Activity of constitutive NOS decreased during INO, whereas that of inducible NOS was unchanged. Upregulation of ET-1 and downregulation of NOS activity may have influenced the short rebound reaction to short-term INO.

Endothelin receptor mediating contraction of isolated bovine coronary artery.

We studied endothelin (ET) receptors and their subtypes on isolated bovine coronary arteries. Endothelin receptors that mediated contraction of isolated bovine coronary artery were characterized by the use of antagonists and agonists. Contractions induced by the nonselective agonist ET-1 (10-10-10-7 M) were not affected by the removal of the endothelium (pEC50: 8.52, maximal contraction: 105% of that induced by 60 mM KCl). BQ-123 (3 x 10-7 M) antagonized contractions of endothelium-denuded coronary rings induced by low concentrations of ET-1 (10-10 or 10-9 M), but potentiated the contractions induced by higher concentrations of ET-1 (3 x 10-8 and 10-7 M). BQ-788 (10-6 M) potentiated contractions induced by ET-1 (3 x 10-10 and 10-7 M). In the presence of BQ-788 (10-6 M), BQ-123 (3 x 10-8-3 x 10-6 M) concentration - dependently inhibited contractions induced by ET-1 (3 x 10-10 and 10-7 M) (pA2: 6.61). Sarafotoxin S6b (10-9-3 x 10-7 M) evoked contractions in the denuded coronary artery (pEC50: 8.49, maximal contraction: 139% of 60 mM KCl). The BQ-123 caused a concentration-dependent rightward shift of contractions induced by sarafotoxin S6b (pA2: 7.89). The present study indicates that ET-1 and sarafotoxin S6b contract the isolated bovine coronary artery by stimulating ETA receptors on smooth muscle cells, and that ETB receptors might suppress the ET-1-induced contractions.

Endothelin-1, endothelin-1 receptors and cardiac natriuretic peptides in failing human heart

Endothelin (ET)-1 is a potent vasoconstrictor peptide produced in the myocardium that can exert important effects on cardiac myocyte growth and phenotype; cardiac natriuretic peptides (ANP and BNP) are known to act as physiological antagonists of ET-1. In this study a comparative determination of ET-1 receptors and of the local productions of ET-1 and of ANP and BNP was made in different sites of failing and nonfailing hearts. Tissue from right and left atrium, right and left ventricle and interventricular septum from seven adult heart transplant recipients with end-stage idiopathic dilated cardiomyopathy (functional class III and IV, with ejection fraction < 35%) and from four post-mortem subjects without cardiac complications was analyzed. In failing hearts we observed a tendency to increase of density of binding sites, most evident in left ventricle (62.6 ± 22.6 fmol/mg protein vs. 29.0 ± 3.3, mean ± SEM, p = ns). A prevalence of ET-A subclass, observed in all samples, resulted more pronounced in failing hearts where this increase, found in all the cardiac regions, was more evident in left ventricle (p = 0.0007 vs nonfailing hearts). The local concentrations of ET-1, ANP and BNP resulted significantly increased in failing hearts with respect to controls in all sides of the heart. In failing hearts we have observed a tendency to increase in endothelin receptor density mainly due to a significant upregulation of ET-A subtype and a parallel increase of the tissue levels of ANP, BNP and ET-1 indicating an activation of these systems in heart failure.

Effect of endothelin antagonism on the production of cytokines in eosinophilic airway inflammation.

Endothelin (ET)-1 has been launched as an important mediator in bronchial asthma, which is an eosinophilic airway inflammation. However, the interplay between ET-1 and other proinflammatory mediators during the development of airway inflammation has not been elucidated. We wanted to study 1) whether the production of ET-1 precedes the production of other proinflammatory mediators and 2) whether ET-1 stimulates the production of these mediators within the airways. These hypotheses were studied during the development of an eosinophilic airway inflammation in rats. The increase in ET-1 mRNA level in lung tissue preceded the increase in mRNA levels of tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-8. Treatment of the animals with the ET receptor antagonist bosentan resulted in a substantial decrease in the concentrations of tumor necrosis factor-alpha, IL-4, IL-1beta, interferon-gamma, and ET-1 in bronchoalveolar lavage fluid. In conclusion, the synthesis of ET-1 as measured by increased mRNA level precedes the synthesis of other proinflammatory cytokines of importance for the development of an eosinophilic airway inflammation, and ET antagonism inhibits the production of these mediators within the airways. Whether treatment with ET antagonists will prove beneficial for patients with eosinophilic airway inflammations like bronchial asthma is not yet known.

Expressions of endothelin-1, fibronectin, and interleukin-1alpha of human umbilical vein endothelial cells under prolonged culture.

We examined human umbilical vein endothelial cells (HUVECs) under prolonged culture by electron microscopy and by light and electron immunocytochemistry including double immunolabeling. Based on the cell area of HUVECs through multiple passages, we divided the cells into first, second, and third stages, which exhibited distinct morphological and immunocytochemical characteristics. During the first stage, HUVECs were polygonal in shape and had already formed the monolayer confluence. During the second stage, they were characterized by an increased number of Weibel-Palade (WP) bodies, which were actively segregated from Golgi cisterns. Endothelin (ET)-1 and von Willebrand factor, an endothelial cell marker, were occasionally colocalized in WP bodies. The increase in WP bodies correlated with high ET-1 concentration in the cultured medium, suggesting that these inclusions are involved in storage and release of ET-1 in a manner indicating a regulatory pathway. During the third stage, fibronectin and interleukin (IL)-1alpha were expressed in HUVECs as well as in multinucleated giant cells, which originated from HUVECs, but WP bodies decreased in number in association with a decrease in ET-1 immunoreactivity and concentration. The foregoing changes in immunoreactivities for ET-1, fibronectin, and IL-1alpha affecting HUVECs under prolonged culture may reflect a senescent process of these cells.

Amplification effect and mechanism of action of ET-1 in U-46619-induced vasoconstriction in pig skin.

The aim of this study was to investigate if a low concentration of endothelin-1 (ET-1; 8 x 10(-10) M) may amplify the skin vasoconstrictor effect of other vasoactive substances in the pathogenesis of skin vasospasm. Pig skin flaps (6 x 16 cm) were perfused with Krebs buffer equilibrated with 95% O(2) and 5% CO(2) at 37 degrees C and pH 7.4. Skin perfusion pressure measured by a pressure transducer and skin perfusion assessed by the dermofluorometry technique were used for assessment of skin vasoconstriction. We observed that ET-1 (8 x 10(-10) M) significantly amplified the concentration-dependent (10(-7)-10(-5) M) skin vasoconstrictor effect of norepinephrine. More importantly, we observed for the first time that this low concentration of ET-1 also amplified the concentration-dependent (10(-8)-10(-6) M) skin vasoconstrictor effect of the thromboxane A(2) mimetic U-46619, and this amplification effect of ET-1 was completely blocked by the protein kinase C (PKC) inhibitor chelerythrine (5 x 10(-6) M). Conversely, the PKC activator phorbol 12,13-dibutyrate (10(-7) M) amplified the vasoconstrictor effect of U-46619. Furthermore, the sensitivity of the skin vasculature to the vasoconstrictor effect of extracellular Ca(2+) in U-46619-induced skin vasoconstriction was significantly enhanced in the presence of 8 x 10(-10) M ET-1. Finally, the cyclooxygenase inhibitor indomethacin (5 x 10(-6) M) did not affect the amplification effect of ET-1 on U-46619-induced skin vasoconstriction. We conclude that a low concentration of ET-1 can amplify the skin vasoconstrictor effect of U-46619 independent of endogenous cyclooxygenase products, and the mechanism may involve activation of PKC and increase in sensitivity of the contractile apparatus to Ca(2+) in smooth muscle cells.

In vitro pharmacologic effect of two endothelin-1 antagonists on equine colonic arteries and veins.

To evaluate the effectiveness of 2 potential endothelin (ET)-1 antagonists in blocking the contractile responses of equine colonic vessels to increasing concentrations of ET-1. Mesenteric vessels from 6 clinically healthy horses. Colonic vessels (arterial and venous rings) were placed in organ baths with oxygenated Tyrode solution at 37 C. Each was attached to a force transducer interfaced with a polygraph, and 2 g of tension was applied and equilibrated for 45 minutes. Then, B-1 (PD 142893) and B-2 (PD 145065) ET-1 antagonists were tested. One ring from each vessel type was used as a control for determining concentration-response relationships of ET-1 (10(-10) to 10(-6)M). Three rings of each vessel type were incubated with 3 concentrations of each antagonist (10(-7), 10(-6), and 10(-5) M) for 30 minutes before ET induced contractions were determined. The maximum contractile response and pA2 values were determined. Vessels contracted in a concentration-dependent manner to ET-1. Arteries responded slowly but reached greater contractions. Veins responded immediately with sustained contractions. Both antagonists inhibited contractions in a concentration-dependent manner with significant differences at 10(-6) and 10(-5)M for arteries and 10(-5) M for veins. Complete blockade of contractions was observed with B-2 (10(-5)M). The pA2 values for B-1 were 8.26 and 6.82 for arteries and veins, respectively, whereas they were 8.25 and 7.21 for B-2. Both antagonists effectively blocked ET-1-induced contractions of equine colonic vessels. Because B-2 is water soluble and caused complete blockade at 10(-5) M, it appears to be the preferred antagonist.

Inhaled nitric oxide-induced rebound pulmonary hypertension: role for endothelin-1.

Clinically significant increases in pulmonary vascular resistance have been noted on acute withdrawal of inhaled nitric oxide (NO). Endothelin (ET)-1 is a vasoactive peptide produced by the vascular endothelium that may participate in the pathophysiology of pulmonary hypertension. The objectives of this study were to determine the effects of inhaled NO on endogenous ET-1 production in vivo in the intact lamb and to determine the potential role of ET-1 in the rebound pulmonary hypertension associated with the withdrawal of inhaled NO. Seven 1-mo-old vehicle-treated control lambs and six PD-156707 (an ET(A) receptor antagonist)-treated lambs were mechanically ventilated. Inhaled NO (40 parts per million) was administered for 24 h and then acutely withdrawn. After 24 h of inhaled NO, plasma ET-1 levels increased by 119.5 +/- 42.2% (P < 0.05). Western blot analysis revealed that protein levels of preproET-1, endothelin-converting enzyme-1alpha, and ET(A) and ET(B) receptors were unchanged. On acute withdrawal of NO, pulmonary vascular resistance (PVR) increased by 77.8% (P < 0.05) in control lambs but was unchanged (-5.5%) in PD-156707-treated lambs. Inhaled NO increased plasma ET-1 concentrations but not gene expression in the intact lamb, and ET(A) receptor blockade prevented the increase in PVR after NO withdrawal. These data suggest a role for ET-1 in the rebound pulmonary hypertension noted on acute withdrawal of inhaled NO.

Associations of plasma endothelin concentration with carotid atherosclerosis and asymptomatic cerebrovascular lesions in patients with essential hypertension.

We studied the association of endothelin (ET)-1 with carotid atherosclerosis and asymptomatic cerebrovascular lesions in patients with essential hypertension. Neurologically normal patients with essential hypertension (n=293; 138 male, 155 female; mean age, 65 years) and age-matched control subjects (n=242) were studied with B-mode ultrasonography of the common and internal carotid arteries and magnetic resonance imaging of the brain. Plasma ET-1 was measured by enzyme immunoassay. Hypertensive patients were divided into groups with carotid plaques and low ET-1 concentrations (< 0.75 pg/ml; PL group); carotid plaques and mid-range ET-1 (0.75 to 1.55 pg/ml; PM group); carotid plaques and high ET-1 (> or = 1.55 pg/ml; PH group); no plaques and low ET-1 (NPL); no plaques and mid-range ET-1 (NPM); and no plaques and high ET-1 (NPH). Overall, ET-1 concentrations were significantly higher in patients than in control subjects. Carotid plaque prevalence was significantly related to ET-1 in hypertensive patients. ET-1 showed a significant positive relationship with the number of asymptomatic lacunar infarcts of the brain in hypertensive patients with carotid plaques (rho=0.48, p<0.001). No significant relationship was seen between ET-1 and periventricular hyperintensity scores in patients with plaques. ET-1 did not show a relationship to either brain lesion type in patients without carotid plaques. Thus, ET-1 may foster asymptomatic lacunar cerebral infarcts by promoting carotid atherosclerosis in patients with essential hypertension.

Plasma levels of von Willebrand factor, endothelin-1, prostacyclin and thromboxane in children from families with high risk of premature coronary artery disease

The aim of the study was to assess the plasma levels of endothelial injury markers in children from families with high risk of premature coronary heart disease (CHD) without other common CHD risk factors (hyperlipidaemia, obesity, hypertension, low physical activity). The study comprised 48 children, including 24 children from high-risk families (HR), according to the NCEP (National Cholesterol Education Programme) criteria: one or two parents had clinical manifestation of cardiovascular disease before the age of 65 years (mother) or 55 years (father). The control group included 24 healthy children with no familial history of cardiovascular disease. All the children were normolipidaemic according to the NCEP and the European Atherosclerosis Society criteria for children aged 2-19 years. In the HR group, the concentration of vWf was significantly elevated in comparison to that in the control group (p<0.0001). Plasma concentrations of ET-1 and TxB

Ischaemia/reperfusion contributes to colonic injury following experimental aortic surgery.

ischaemia of the colon is an important complication of abdominal aortic aneurysm (AAA) repair. The aim of this animal study was to investigate the effect of sequential ischaemia and reperfusion on sigmoid mucosal pO2 and its association with local ET-1 release. twelve pigs underwent colonic ischaemia followed by complete reperfusion. Six other animals were sham controls. A Clark-type microcatheter was used for continuous mucosal pO2 measurements. Serial systemic and inferior mesenteric vein blood samples were obtained for determination of ET-1 concentration. Neutrophil extravasation was assessed by tissue myeloperoxidase (MPO) activity. arterial occlusion was associated with a gradual decrease of mucosal pO2 and local release of ET-1. After restoration of blood flow, mucosal pO2 returned to near baseline values, whereas ET-1 reached its maximum concentration during the reperfusion period. MPO activity was significantly increased. colonic ischaemia and reperfusion causes neutrophil extravasation and local ET-1.

Involvement of the Endothelin System in Experimental Critical Hind Limb Ischemia

Endothelin- (ET-1) is involved in the pathogenesis of several ischemic diseases. We investigated the hypotheses that ET-1 is involved in the pathogenesis of experimental critical hind limb ischemia and that ET-1 receptor antagonists have a protective effect. Critical hind limb ischemia was achieved by exclusion of the femoral artery and embolization of collateral vessels in rats. The induction of endothelin system components by ischemia was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) (mRNAs) and immunoassay (peptides) in the plasma and ischemic muscles 5 hr (H5), 5 days (D5) and 14 days (D14) after ischemia. Two groups of rats received 100 mg/kg/day of either Bosentan, a mixed ET(A/B) receptor antagonist (n = 12), or LU 135252, a selective ET(A) receptor antagonist (n = 9), and a control group without treatment (n = 12) served as control. Muscle blood flow and ischemia were monitored in the ischemic limb by laser Doppler and phosphorylase activity, respectively. The procedure induced an 80% decrease in muscle blood flow and complete suppression of phosphorylase activity without necrosis. At day 14, the tissue blood flow remained reduced by 70% and phosphorylase activity was suppressed completely. There was up-regulation of preproendothelin-1, preproET-3, endothelin converting enzyme-1, and ET(A). ET(B) receptor mRNAs in ischemic muscle at day 5 and day 14 was accompanied by an increase in muscle concentration of ET-1 at day 5, without significant changes in plasma endothelin. Treatment with Bosentan and LU 135252 increased tissue blood flow and reduced muscle ischemia at day 14. Tissue production of ET- 1 is up-regulated in experimental critical hind limb ischemia. Inhibition of the endothelin system by a mixed ET(A/B) receptor antagonist may protect, at least in part, against muscle injury.

Endothelin-1 Enhances Eicosanoids-Induced Coronary Smooth Muscle Contractility and [Ca 2+ ] I by Activating a Protein Kinase C-Sensitive Ca 2+ Entry Pathway

P151 Endothelin-1 (ET-1) has been implicated in the pathogenesis of coronary vasospasm by enhancing the coronary vasoconstrictor response to vasoactive eicosanoids; however, the cellular mechanisms involved are unclear. We investigated whether physiological concentrations of ET-1 enhance prostaglandin F 2α (PGF 2α )-induced coronary smooth muscle contraction by increasing [Ca 2+ ] i and the Ca 2+ mobilization mechanisms of coronary vasoconstriction. Single smooth muscle cells were freshly isolated from the left anterior descending coronary artery of castrated male pigs and labeled with the Ca 2+ indicator fura-2 or with antibodies to specific protein kinase C (PKC) isoforms. In Hank’s solution (1 mM Ca 2+ ), ET-1 (30 pM) caused a small increase in cell contraction (9%) and [Ca 2+ ] i (106±5 nM). PGF 2α (10 -7 M) increased cell contraction to 11% and [Ca 2+ ] i to 121±7 nM. In cells pretreated with ET-1 (30 pM) for 30 min, PGF 2α (10 -7 M) significantly increased cell contraction to 32% and [Ca 2+ ] i to 217±8 nM. Membrane depolarization by 24 mM KCl, which stimulates Ca 2+ entry from the extracellular space, caused 12% cell contraction and increased [Ca 2+ ] i to 152±6 nM. In cells pretreated with ET-1, KCl significantly increased cell contraction to 34% and [Ca 2+ ] i to 306±8 nM. In Ca 2+ -free Hank’s, caffeine (10 mM), which stimulates Ca 2+ release from intracellular stores, caused cell contraction (9%) and a transient increase in [Ca 2+ ] i (417±14 nM). ET-1 did not significantly affect the caffeine contraction or [Ca 2+ ] i . ET-1 caused significant translocation of α-PKC from the cytosol to the cell membrane, suggesting activation of the isoform. The PKC inhibitor Gö6976 (10 -6 M) completely inhibited the ET-1 induced α-PKC translocation and the enhancement of PGF 2α − and KCl-induced cell contraction and [Ca 2+ ] i . Thus, ET-1 enhances PGF 2α -induced coronary smooth muscle contractility and [Ca 2+ ] i by enhancing Ca 2+ entry from the extracellular space, but not Ca 2+ release from intracellular stores. The results suggest that the enhancement of PGF 2α -induced Ca 2+ entry by ET-1 involves activation and translocation of α-PKC isoform.

Endothelin-1-induced PMN infiltration and mucosal dysfunction in the rat small intestine.

The objectives of this study were to characterize the effects of endothelin (ET)-1 on intestinal mucosal parameters and to assess the contribution of polymorphonuclear leukocytes (PMNs), intercellular adhesion molecule-1 (ICAM-1), and a platelet-activating factor (PAF) to the mucosal dysfunction induced by ET-1. Different concentrations of ET-1 (100, 200, and 400 pmol/kg) were infused into the superior mesenteric artery for 10 min, and tissue samples were obtained 30 min after terminating the infusion. ET-1 administration significantly elevated tissue myeloperoxidase activity, plasma carbonyl content, and tissue chemiluminescence intensity, indicating that ET-1 produces PMN infiltration and oxidant stress. Blood-to-lumen clearance of (51)Cr-EDTA significantly increased after ET-1 infusion (400 pmol/kg). Monoclonal antibodies against ICAM-1 (1A29, 2 mg/kg), antineutrophil serum, and PAF antagonist (WEB-2086, 10 mg/kg) attenuated the mucosal barrier dysfunction induced by ET-1. Overall, our data indicate that ET-1 causes PMN accumulation, oxidant stress, and mucosal dysfunction in the rat small intestine and that ET-1-induced mucosal dysfunction involves a mechanism that includes a role for PMNs, ICAM-1, and PAF.

Endothelin-1 Changes Polymorphonuclear Leukocytes' Deformability and CD11b Expression and Promotes Their Retention in the Lung

Endothelin (ET)1 influences polymorphonuclear leukocyte (PMN)- endothelial cell interactions. The aim of this study was to examine the effect of ET-1 on factors that influence PMN-endothelial interaction and retention in the lung both in vitro and in vivo. In vitro, high concentration of ET-1 (> or = 10(-8) M) rapidly increased PMN F-actin content (10(-7) M: 58 +/- 6% increase, P<0.01), whereas lower concentration of ET-1 (< or = 10(-9) M) caused a small but consistent decrease in F-actin content (10(-10) M: 6.9+/-1.5% decrease, P< 0.01). Preincubation of PMNs with the nitric oxide donor sodium nitroprusside (SNP) inhibited the F-actin content increase by 10(-7) M of ET-1 (P<0.01), and enhanced the F-actin content decrease by 10(-10) M of ET-1 (P<0.01). Preincubation of PMNs with Nomega-nitro-L-arginine methylester prevented the F-actin content decrease by 10(-10) M of ET-1. ET-1 (10(-7) M) reduced the deformability of PMNs (P<0.01), which was inhibited by preincubation of PMNs with SNP (P<0.05). ET-1 (10(-9) to 10(-7) M) increased CD11b expression of PMNs (P<0.01), which was inhibited by preincubation of PMNs with SNP. In vivo studies showed that the retention of PMNs treated with ET-1 increased from 45+/-8 to 70+/-5% compared with naive PMNs during their first pass through the lung (P<0.05). We conclude that ET-1 changes the F-actin content, the deformability, and the CD11b expression of PMNs in a dose-dependent fashion and that this leads to increased PMN sequestration in pulmonary microvessels.

Comparative effects of pioglitazone, glibenclamide, and voglibose on urinary endothelin-1 and albumin excretion in diabetes patients

Urinary endothelin (ET)-1 excretion is present in non-insulin dependent diabetes (NIDDM) patients with microalbuminuria, and an increase in circulating ET-1 precedes the microalbuminuric phase of renal injury related to diabetes. The aim of the present study was to determine whether various drugs alter urinary ET-1 levels and urinary albumin excretion (UAE) in NIDDM patients with microalbuminuria. Forty-five NIDDM patients with microalbuminuria were randomly assigned to three groups: those treated with pioglitazone at 30 mg/day ( n=15), those treated with glibenclamide at 5 mg/day ( n=15), and those treated with voglibose at 0.6 mg/day ( n=15). Patients received these drugs for 3 months. UAE, urinary ET-1, and plasma ET-1 levels were measured in these patients before and after treatment. Before treatment, UAE, urinary ET-1, and plasma ET-1 levels differed little among the three groups. UAE in the 45 NIDDM patients (156.2±42.8 μg/min) was greater than that in 30 healthy controls (8.2±2.6 μg/min) ( P<.001). Urinary ET-1 levels in the NIDDM patients (8.7±1.3 ng/g urinary creatinine (UC)) were significantly higher than that in the controls (2.4±0.2 ng/g UC) ( P<.01). Plasma ET-1 levels, however, in the NIDDM patients (1.3±0.4 pg/ml) did not differ significantly from the levels in healthy controls (1.0±0.6 pg/ml). Pioglitazone but no glibenclamide or voglibose reduced UAE from 142.8±42.2 to 48.4±18.2 μg/min ( P<.01) and urinary ET-1 levels from 8.6±1.3 to 3.4±0.5 ng/g UC ( P<.01). These data suggest pioglitazone to be effective in reducing UAE and urinary ET-1 concentrations in NIDDM patients with microalbuminuria.

Lack of nuclear apoptosis in cardiomyocytes and increased endothelin-1 levels in a rat heart model of myocardial stunning.

Reperfusion injury may affect the cardiac NO and endothelin production. We investigated whether 20 min of total ischemia followed by 40 min of reperfusion can induce apoptosis in a Langendorff model of retrogradely perfused rat hearts (37 degrees C; paced at 300/'), and we attempted to correlate these findings with measured tissue NO and ET-1 levels. An apoptosis detection system was utilized which catalytically incorporates fluorescein-12-dUTP at the 3'-OH DNA ends using the principle of the TUNEL assay, with direct visualization of the labeled DNA. ET-1 was measured by radioimmunoassay and NO3/NO2 by ion pairing HPLC on C18 reverse phase columns. None of the postischemic (n = 6) nor of the control perfused (90 min, n = 6) hearts showed signs of apoptosis, while those exposed to longer ischemia (40 min) and reperfusion (2 h) confirmed the presence of apoptotic cells. Myocardial ET-1 concentrations were 4.8 +/- 1.0 versus 8.3 +/- 2.5 pg/100 mg (control vs. ischemic hearts, respectively; mean +/- SD; p < 0.05). Myocardial NO contents showed no differences. These data suggest that the time window of apoptosis with detectable DNA fragmentation exceeds 20 min of global total ischemia and 40 min of reperfusion, a model frequently used for inducing myocardial stunning. While NO was not increased in postischemic hearts, increased ET-1 levels indirectly argue for a role of ET-1 as inducer of apoptosis, but only at a later stage of reperfusion.