Immunoreactive Endothelin Research Articles

Thiazolidinediones Suppress Endothelin-1 Secretion from Bovine Vascular Endothelial Cells: A New Possible Role of PPARγ on Vascular Endothelial Function

We examined the effect of troglitazone on immunoreactive endothelin-1 (ET-1) secretion from cultured bovine vascular endothelial cells (bVECs). Insulin (10−9–10−7M) stimulated ET-1 secretion in a dose-dependent fashion without any kinetic change. Troglitazone (1–20 μM) dose-dependently inhibited both spontaneous and insulin-stimulated ET-1 secretion. This inhibitory effect of troglitazone was associated with reduced ET-1 mRNA levels. Addition of indomethacin (100 μM) orNw-nitro-l-arginine methyl ester (1 mM) and downregulation of protein kinase C by prolonged pretreatment of the cells with a phorbol ester, 12-O-tetradecanoylphorbol 13-acetate, did not affect the inhibitory effect of troglitazone at concentrations up to 10 μM. Troglitazone did not change the intracellular Ca2+concentration stimulated by angiotensin II (10 μM). Other PPARγ ligands, pioglitazone (1–10 μM) and 15-deoxy-delta 12, 14-prostaglandin J2(1–10 μM), but not a PPARα ligand, bezafibrate (1–10 μM), dose-dependently suppressed spontaneous ET-1 secretion from bVECs. These results, taken together, suggest that troglitazone inhibits ET-1 mRNA expression and secretion in bVECs possibly through activation of PPARγ. This inhibition may contribute to the hypotensive effect of troglitazone in insulin-resistant subjects.

Renal and vascular effects of chronic nitric oxide synthase inhibition: involvement of endothelin 1 and angiotensin II

Endothelin 1 (ET-1) is a potent vasoconstrictor implicated in the control of blood pressure and renal function. Its effects can be modulated by nitric oxide (NO), which inhibits ET-1 production and action. Recently, we reported that ET-1 production can also be modulated by angiotensin II (AngII) in vivo. To investigate the interactions between NO, ET-1, and AngII in hypertension and renal dysfunction, we assessed immunoreactive ET-1 (ir-ET-1) concentration in plasma and urine as well as in vascular and renal tissues of rats with chronic inhibition of NO synthesis, in the presence and the absence of the AngII type 1 receptor antagonist losartan. Normal (protocols A and B) and uninephrectomized rats (protocol C) received the L-arginine analog NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthesis, 0.05% (protocol A) or 0.1% (protocols B and C), with or without losartan (20 mg·kg-1·day-1). After 6 weeks, systolic blood pressure was significantly increased in L-NAME rats compared with the controls (p < 0.01), while serum creatinine and urea, creatinine clearance, and proteinuria were similar to control values. However, ir-ET-1 concentration in plasma and in the thoracic aorta was augmented in animals receiving 0.1% L-NAME (p < 0.01), while it was unchanged in the mesenteric arterial bed, preglomerular arteries, and glomeruli. In contrast, ir-ET-1 concentration was decreased in the renal papilla (p < 0.05) as well as in the urine of L-NAME rats (p < 0.01). Treatment with losartan significantly attenuated the rise in systolic blood pressure induced by L-NAME (p < 0.01). Losartan also normalized the increased ir-ET-1 concentration in plasma and in the thoracic aorta, but had no effect on tissues with normal or reduced ir-ET-1 levels. These results indicate that chronic inhibition of NO synthase with L-NAME induces hypertension without renal dysfunction. Increased ET-1 production in some blood vessels and elevated circulating ET-1 concentration may contribute to the maintenance of high blood pressure. The reduction of systolic blood pressure by losartan supports a role for AngII in the pathogenesis of this form of hypertension, which may be due, at least in part, to the modulation of ET-1 production.Key words: endothelin, nitric oxide, L-NAME, angiotensin II, losartan, hypertension, blood vessel, glomeruli.

Renal and vascular effects of chronic nitric oxide synthase inhibition: involvement of endothelin 1 and angiotensin II.

Endothelin 1 (ET-1) is a potent vasoconstrictor implicated in the control of blood pressure and renal function. Its effects can be modulated by nitric oxide (NO), which inhibits ET-1 production and action. Recently, we reported that ET-1 production can also be modulated by angiotensin II (AngII) in vivo. To investigate the interactions between NO, ET-1, and AngII in hypertension and renal dysfunction, we assessed immunoreactive ET-1 (ir-ET-1) concentration in plasma and urine as well as in vascular and renal tissues of rats with chronic inhibition of NO synthesis, in the presence and the absence of the AngII type 1 receptor antagonist losartan. Normal (protocols A and B) and uninephrectomized rats (protocol C) received the L-arginine analog N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthesis, 0.05% (protocol A) or 0.1% (protocols B and C), with or without losartan (20 mg x kg(-1) x day(-1)). After 6 weeks, systolic blood pressure was significantly increased in L-NAME rats compared with the controls (p < 0.01), while serum creatinine and urea, creatinine clearance, and proteinuria were similar to control values. However, ir-ET-1 concentration in plasma and in the thoracic aorta was augmented in animals receiving 0.1% L-NAME (1 < 0.01), while it was unchanged in the mesenteric arterial bed, preglomerular arteries, and glomeruli. In contrast, ir-ET-1 concentration was decreased in the renal papilla (p < 0.05) as well as in the urine of L-NAME rats (p < 0.01). Treatment with losartan significantly attenuated the rise in systolic blood pressure induced by L-NAME (p < 0.01). Losartan also normalized the increased ir-ET-1 concentration in plasma and in the thoracic aorta, but had no effect on tissues with normal or reduced ir-ET-1 levels. These results indicate that chronic inhibition of NO synthase with L-NAME induces hypertension without renal dysfunction. Increased ET-1 production in some blood vessels and elevated circulating ET-1 concentration may contribute to the maintenance of high blood pressure. The reduction of systolic blood pressure by losartan supports a role for AngII in the pathogenesis of this form of hypertension, which may be due, at least in part, to the modulation of ET-1 production.

Secretory pathways in endothelin synthesis

British Journal of Pharmacology (1999) 126, 391–398; doi:10.1038/sj.bjp.0702315

Identification of amino acid residues in the C-terminal tail of big endothelin-1 involved in processing to endothelin-1.

Big endothelin-1 (big ET-1) is converted to 21-amino acid residue endothelin-1 (ET-1) via a specific cleavage at Trp21-Val22 by endothelin converting enzyme (ECE). This conversion is an essential step to produce bioactive ET-1 and represents a regulatory site in the biosynthesis of this potent vasoconstrictor. ECE-1a, a unique membrane-bound enzyme, processes big ET-1 more efficiently than other big ET isoforms, which mainly differ in the C-terminal tail (residues 22-38). In this study, each of the highly conserved residues, Val22, Pro25, Pro30, Gly32, Leu33, and Gly34 were replaced with Ala in the preproendothelin-1 (PPET-1) cDNA using site-directed mutagenesis. The mutant and wild-type cDNAs were transiently transfected into Chinese hamster ovary cells along with ECE-1a cDNAs, and concentrations of the resulting recombinant peptides, ET-1 and big ET-1, in the transfection media were then measured. The concentration of immunoreactive ET-1 in the media from Val22, Pro25, Pro30, Gly32, and Leu33 mutant PPET-1-transfected cells was 4- to 6-fold lower than that of wild type and (Gly34-->Ala)PPET-1. Moreover, with the exception of Gly34 there was a corresponding increase in the concentrations of immunoreactive big ET-1 in the media from mutants. Similar results were obtained when His27, Val28, and Ser35 of big ET-1( )were substituted with the corresponding residues in big ET-2 and big ET-3. These findings suggest that the C-terminal tail has an important role in the intracellular processing of big ET-1 by ECE-1a. Herein we also report that a recombinant big ET-1 analog we previously generated and characterized, (Ala21)big ET-1, inhibits ECE-1a activity in a dose-dependent (K i=1 microM) and competitive manner.

Subthreshold concentration of endothelin-1-enhanced, capsaicin-induced bronchoconstriction in anaesthetized guinea-pigs.

An increasing number of studies have been performed to address a possible role for endothelin-1 (ET-1) as a significant mediator in asthma. However, the effects of subthreshold concentrations of ET-1, which cannot elicit bronchial smooth muscle contraction itself, in asthma has yet to be determined. This study determined these effects of ET-1 on capsaicin-induced bronchoconstriction in anaesthetized guinea-pigs. Aerosolized ET-1 administered at doses of 10(-9) M and higher induced a dose-dependent increase in pulmonary resistance, but ET-1 at 10(-10) M did not have any bronchoconstrictive effect. However, this subthreshold concentration of ET-1 potentiated capsaicin-induced bronchoconstriction. In addition, the potentiation of capsaicin-induced bronchoconstriction by this subthreshold concentration of ET-1 was completely abolished by BQ788 (ET(B) receptor antagonist), but not BQ123 (ET(A) receptor antagonists). Immunoreactive substance P (SP) levels in bronchoalveolar lavage fluid after capsaicin administration were significantly higher than those after solvent administration. However, ET-1 alone did not significantly stimulate immunoreactive SP release and ET-1 (10(-10) M) did not potentiate capsaicin-induced immunoreactive SP release. In contrast, ET-1 (10(-10) M) potentiated exogenous neurokinin A- and SP-induced bronchoconstriction. These findings suggest that a subthreshold concentration of endothelin-1 does not potentiate the tachykinin release induced by capsaicin but the airway smooth muscle contraction through endothelin-B receptors.

Role of neurosympathetic pathways in the vascular response to sepsis

Purpose: The aim of this study was to determine the role of sympathetic neural activity in the hemodynamic adaptations to sepsis in pigs with an emphasis on circuit adaptations. A fall in resistance to venous return (RVR) was predicted in contrast to what was previously observed in sympathetically intact animals that had no change in RVR. Materials and Methods: We anesthetized and ventilated 13 pigs and gave 5 mg/kg of indomethacin. We measured cardiac output (CO) by thermodilution and measured pulmonary arterial (PAP), pulmonary capillary wedge (Pcw), right atria) pressure (Pra), and arterial pressure (MAP). Intermittent inflation of a 50-mL balloon in the right atrium was used to transiently arrest the circulation for the measurement of mean circulatory filling pressure (MCFP). RVR was calculated from (MCFP — Pra)/CO. Animals were divided into two groups; 6 received 10 mg/kg of the ganglionic blocker, hexamethonium and norepinephrine to maintain MAP; 7 had their spinal cords cut at C-2. After baseline measurements, all animals received 10 μg/kg/h of endotoxin for 2 hours, and hemodynamic measurements were repeated. Plasma samples were obtained for measurements of immunoreactive endothelin-1 (ET-1), which was assayed by a radioimmunoassay. Results: Hexamethonium had no significant effect on hemodynamics except for an increase in heart rate. After endotoxin, MAP and SVR fell, PAP rose, and CO and RVR did not change. Spinal section resulted in an increase in heart rate and small increase in PAP and MCFP After endotoxin, there was a further increase in heart rate, PAP, and MCFP with a marked fall in MAP and CO. RVR increased from 2.1 ± 0.46 after spinal section to 3.6 ± 54 mm · min/L ( P < .05). ET -1 in the hexamethonium group (n = 2) rose from 2.21 ± .14 to 11.5 ± 2.1 pg/ml at 2 hours, and in the spinal group (n = 7) from 2.04 ± 0.77 to 6.85 ± 3.9 pg/mL at 45 minutes. Conclusion: Spinal section resulted in a more profound fall in blood pressure and less increase in MCFP than in previously studied animals with sympathetic nervous system intact, but there was still an increase in RVR and PAP ET-1 is a possible mediator of the increase in RVR and PAP.

Endothelin release by rabbit proximal tubule cells: Modulatory effects of cyclosporine A, tacrolimus, HGF and EGF

Previous studies have suggested that endothelins, a family of 21 amino acid peptides with potent vasoconstrictive and mitogenic properties, are involved in the pathogenesis of acute and chronic renal failure. In addition, endothelin seems to play an important role in mediating the nephrotoxic side effects of cyclosporine A (CsA) and tacrolimus. The present study investigated the production of endothelin-1 (ET-1) and endothelin-3 (ET-3) by bipolar differentiated rabbit proximal tubule cells (PT-1 cells), and the modulatory effect of CsA, tacrolimus, hepatocyte growth factor (HGF) and epidermal growth factor (EGF) on ET-1 and ET-3 release. ET-1 and ET-3 mRNA was detected by RT-PCR, immunoreactive endothelin was localized to PT-1 cells by immunofluorescence staining with antibodies against ET-1 and ET-3. ET-1 and ET-3 release into the culture medium was determined by specific radioimmunoassays after solid phase extraction. PT-1 cells exhibited a time-dependent increase of ET-1 release up to an incubation period of 36 hours, whereas ET-3 release already reached a steady state level after four hours. PT-1 cells, cultured on filter membranes, released a significantly higher amount of immunoreactive ET-1 into the basolateral compartment than into the apical compartment. ET-3 release did not differ significantly between the basolateral and the apical compartment. Supplementation of the cell culture medium with 10% fetal calf serum induced a marked increase of the basolateral and apical ET-1 release, whereas ET-3 release was only slightly increased. CsA and tacrolimus (0.5 to 5000 microgram/liter) induced a significant, dose-dependent increase of ET-1 and ET-3 release by PT-1 cells with a maximum stimulation at a CsA concentration of 500 microgram/liter (P < 0.001) and a tacrolimus concentration of 50 microgram/liter (P < 0.001). HGF and EGF (10-10 to 10-8 mol/liter) exerted a significant (P < 0.001) dose-dependent inhibitory effect on ET-1 release, whereas ET-3 release was not significantly reduced. Coincubation of PT-1 cells with CsA or tacrolimus and HGF or EGF also resulted in a marked reduction of ET-1 release. The present data suggest that ET-1 and ET-3 release by cultured rabbit proximal tubule cells are regulated differently, and that the stimulatory effect of CsA and tacrolimus on ET-1 release is antagonized by HGF and EGF.

Thrombin Receptor Activation Inhibits Monocyte Spreading by Induction of ETB Receptor-Coupled Nitric Oxide Release

AbstractThe effect of thrombin receptor activation on monocyte conformation was evaluated using the human monocyte cell line, THP-1, and the thrombin mimetic peptide, Trap-14. Treatment of THP-1 cells with Trap-14 induced rapid rounding of ameboid cells adherent to fibronectin-coated slides, whereas cell rounding was abrogated in the presence of the nitric oxide synthase inhibitor, NG-nitro-l-arginine or the endothelin B receptor antagonist, BQ-788. Endothelin-1 (ET-1) levels in the culture supernatant increased markedly within minutes of Trap-14 exposure with a concomitant loss in cellular ET-1 immunoreactivity. Importantly, loss of ET-1 immunoreactivity was blocked by pretreatment with the vesicle translocation inhibitor, nocodazole. Trap-14 potently induced the release of NO from THP-1 cells, whereas NO release was ablated by preincubation with BQ-788. These data demonstrate that thrombin receptor activation may inhibit cellular spreading as a result of autocrine ET-1 release and subsequent endothelin B receptor-dependent NO production, and suggest that initial exposure of inflammatory cells to thrombin may limit cellular activation and recruitment.

Endothelin-dependent tone limits acetylcholine-induced dilation of resistance coronary vessels after blockade of NO formation in conscious dogs.

Nitric oxide (NO) impairs endothelin (ET) formation and/or action in isolated vessels. We hypothesized that ET may magnify the consequences of NO formation blockade on receptor-operated dilation of resistance coronary vessels in conscious dogs. In conscious instrumented dogs, graded intracoronary (IC) doses of acetylcholine (ACh) were delivered before IC administration of Nomega-nitro-L-arginine methyl ester (L-NAME), after L-NAME, and after L-NAME plus IC bosentan, an ETA/ETB receptor blocker. Before L-NAME, ACh (100 ng. kg-1. min-1) increased coronary blood flow (CBF) by 43+/-4% from 47+/-6 mL. min-1. After L-NAME, ACh failed to increase CBF (-3+/-2% from 50+/-7 mL. min-1). CBF responses to ACh were partially restored (+10+/-2% from 50+/-7 mL. min-1, P<0.01) after the addition of bosentan. Bosentan alone (without L-NAME) did not alter CBF responses to ACh. Blockade of ETA (Ro 61-1790) but not ETB (Ro 46-8443) receptors partially restored CBF responses to ACh after L-NAME. Myocardial immunoreactive ET levels in the perfusion territories of the circumflex and left anterior descending coronary arteries did not differ. ETA-dependent tone magnified the inhibitory effects of blockade of NO formation on receptor-operated dilation to ACh in resistance coronary vessels. Presumably, stimulated NO release has an inhibitory action on endogenous ET production and/or action at the level of resistance coronary vessels.

The effect of regulation of high blood pressure on plasma endothelin-1 levels in blacks with hypertension

Plasma concentrations of immunoreactive endothelin-1 (irET-1) are significantly elevated in blacks with hypertension. In the present study, we investigated the effect of the regulation of high blood pressure on plasma irET-1 levels in black hypertensive individuals. After the initial blood samples were collected from 20 black patients with uncontrolled high blood pressure (Day 1), an intensive antihypertensive treatment was initiated, and the blood pressure and plasma irET-1 levels were monitored on days 2, 8, and 22. When the high blood pressure was brought under control with commonly used antihypertensive medications, plasma irET-1 concentrations dropped dramatically, suggesting that ET-1 concentrations rise as a consequence of high blood pressure in this study group.

Expression of endothelin(A) receptors in human gliomas and meningiomas, with high affinity for the selective antagonist PD156707.

Endothelin (ET) immunoreactivity, ET production, and specific ET receptors have been identified in the brain. Changes in ET concentration or receptor expression have been implicated in the pathophysiological changes in vasospasm after subarachnoid hemorrhage and in cerebral neoplasia. In this study, we have characterized the ET(A) and ET(B) receptor subtypes present in human normal cerebral cortex (NCC) and two common central nervous system tumors, i.e., meningioma (MNG) and glioblastoma multiforme (GBM). A knowledge of the ET receptor subtypes present may provide a novel therapeutic target for newly developed ET antagonists. Saturation, competition, and autoradiographic studies were performed with the subtype-specific radioligands 125I-PD151242 and 125I-BQ3020, to characterize the ET(A) and ET(B) receptors present in NCC, MNG, and GBM. NCC expresses high-affinity ET(A) receptors on pial and intraparenchymal vessels and high-affinity ET(B) receptors on glia and neurons. MNGs express mainly (85%) high-affinity ET(A) receptors in a diffuse pattern, whereas GBMs express high-affinity ET(A) receptors on the neovasculature and ET(B) receptors on the nonvascular elements. The ET antagonist PD156707 (Kd = 0.059 nmol/L) showed a higher affinity for the ET(A) receptor subtype than did bosentan (Kd = 1.1 nmol/L). ET(A) receptors are expressed in high concentrations in MNGs and in the vasculature of NCC and GBMs. The ET(A)-selective antagonist PD156707 may be of potential therapeutic value in vascular and neoplastic diseases of the central nervous system.

Differential plasma endothelin levels in subgroups of patients with angina and angiographically normal coronary arteries

Background Raised plasma endothelin concentrations have previously been reported in patients with cardiac syndrome X, but it is not known whether these levels vary between clinically distinct subgroups in this heterogeneous condition. Methods and Results We compared plasma immunoreactive endothelin levels in 54 patients with angina pectoris and normal coronary angiograms and 21 healthy control subjects. The patient group was divided into 4 clinically distinct subgroups: 7 with left bundle branch block (group A); 7 with previous myocardial infarction (group B); 24 with positive exercise electrocardiography (group C); and 16 with negative exercise electrocardiography (group D). The plasma endothelin concentration was significantly higher in patients compared with control subjects (3.7 [2.9 to 4.3] vs 2.96 [2.4 to 3.4] pg/mL, respectively, median [interquartile range]; P = 0.002). Endothelin concentrations were most significantly elevated in group A and group B (4.5 [3.6 to 5.2] pg/mL; P = 0.005 and 4.1 [3.9 to 4.5] pg/mL; P = 0.001, respectively). Plasma endothelin concentrations were also significantly elevated in group C (3.7 [2.8 to 4.1] pg/mL; P = 0.02) but not in group D (3.0 [2.5 to 3.8] pg/mL; P = 0.3). Conclusions Plasma endothelin concentration is elevated in patients with angina pectoris and angiographically normal coronary arteries, particularly those with left bundle branch block or previous myocardial infarction. (Am Heart J 1998;136:412-7.)

Increased response to big endothelin-1 in atherosclerotic human coronary artery: functional evidence for up-regulation of endothelin-converting enzyme activity in disease.

Overproduction of the potent vasoconstrictor peptide endothelin-1 (ET-1) is implicated in the pathogenesis of coronary artery disease. In endothelium-denuded human coronary arteries the response to big ET-1 was significantly enhanced in atherosclerotic arteries (coronary artery disease, CAD; n=7) with an EC50 value of 96 nM (57- 161 nM, 95% C.I.) compared to 274 nM (205-365 nM) in non-diseased arteries (dilated cardiomyopathy, DCM; n=10) (Mann-Whitney U-test, P<0.05). Higher levels of immunoreactive endothelin (ET) could be detected by radioimmunoassay in bathing medium taken from CAD arteries than from DCM arteries (2.8+/-0.5 nM, n=5 vs 1.1+/-0.2 nM, n=7) (Student's two-tailed t-test, P<0.05). There were no differences in responses of arteries from either group to ET-1 (EC50 10 nM, CAD vs 14 nM, DCM). The enhanced response of atherosclerotic human coronary arteries to big ET-1 appears to be due to up-regulation of endothelin-converting enzyme (ECE) activity rather than to an augmented response of the arteries to ET-1. This non-endothelial ECE may therefore be an important therapeutic target in coronary artery disease.

Endothelin-1 has haemodynamic effects at pathophysiological concentrations in patients with left ventricular dysfunction.

Plasma levels of immunoreactive endothelin-1 (ET-1) are raised in chronic heart failure. Whether plasma ET-1 contributes to the haemodynamic derangement found in chronic heart failure is not known. We investigated the effects of exogenous ET-1 on the pulmonary and systemic vasculature in patients with left ventricular systolic dysfunction (LVD), with or without overt heart failure. ET-1 was infused at 1, 5 and 15 pmol/min into a distal pulmonary artery of ten patients with LVD to achieve plasma concentrations of ET-1 similar to those found in patients with heart failure and pulmonary hypertension. Haemodynamics were measured using a pulmonary thermodilution catheter and an arterial line. Intravascular Doppler and local pulmonary angiography were used to assess local pulmonary blood flow in the first four patients. Systemic haemodynamic changes occurred with ET-1 infusion: mean arterial pressure (100 +/- 3 [standard error of the mean]) to 107 +/- 3 mmHg; p < 0.01) and systemic vascular resistance (1699 +/- 118 to 2033 +/- 135 dynes s/cm5; p < 0.001) rose, while the cardiac index fell from 2.43 +/- 0.17 to 2.20 +/- 0.16 l/min/m2 (p < 0.002). Mean pulmonary artery pressure (21 +/- 2 mmHg) and pulmonary vascular resistance (151 +/- 14 to 147 +/- 14 dynes s/cm5) did not change however. Exogenous ET-1, when infused to achieve plasma concentrations similar to those in severe heart failure and pulmonary hypertension, causes systemic but not pulmonary vasoconstriction.

Effects of losartan and captopril on endothelin-1 production in blood vessels and glomeruli of rats with reduced renal mass

Recently, we have reported that endothelin-1 (ET-1) production is increased in blood vessels and glomeruli of rats with chronic renal failure. This study was design to investigate the role of angiotensin II (Ang II) in endogenous ET-1 production in rats with reduced renal mass. One week after subtotal (5/6) nephrectomy, uremic rats were divided into three groups, and received either no treatment, the Ang II subtype 1 receptor (AT 1) antagonist losartan (10 mg/kg/day), or the angiotensin-converting enzyme inhibitor (ACE-I) captopril (30 mg/kg/day) for 6 weeks. Sham-operated rats were used as controls and received no treatment. The levels of immunoreactive ET-1 (ir-ET-1) in plasma and urine, as well as in vascular and renal tissues, were determined by radioimmunoassay (RIA) after extraction. In uremic rats, losartan and captopril completely prevented the increase in systolic blood pressure. At week 6, plasma ir-ET-1 was similar in the different groups of uremic rats and in the controls. However, ir-ET-1 concentration in the mesenteric arterial bed, the thoracic aorta, preglomerular arteries, and glomeruli, as well as urinary ir-ET-1 excretion were significantly greater in uremic-untreated rats compared to controls ( P < .01). Treatment of uremic rats with losartan or captopril reduced ir-ET-1 concentration in the thoracic aorta and preglomerular arteries ( P < .05), but ir-ET-1 concentration in the mesenteric arterial bed was unchanged. Although both drugs completely prevented the increase in proteinuria, losartan but not captopril significantly reduced ir-ET-1 concentration in glomeruli ( P < .05) and normalized urinary ir-ET-1 excretion. This indicates that increased ET-1 production in blood vessels and glomeruli of uremic rats is modulated, at least in part, by Ang II through the AT 1 receptor. The beneficial effects of the AT 1 antagonist losartan could be attributable to the attenuation of Ang II-induced ET-1 production in this rat remnant kidney model of chronic renal failure, whereas those of the ACE-I captopril are not related to changes in ET-1 production in glomeruli.

Dietary L-arginine normalizes endothelin-induced vascular contractions in cholesterol-fed rabbits.

The endothelium regulates vascular function by releasing the vasodilator autacoid nitric oxide (NO) and the vasoconstrictor peptide endothelin-1 (ET-1). Impaired activity of NO as well as excessive activity of ET-1 have been demonstrated in hypercholesterolemia and atherosclerosis. Because dietary L-arginine can restore NO function and improve abnormal endothelium-dependent relaxation in hypercholesterolemic rabbits, we examined the effects of dietary supplementation with L-arginine in cholesterol-fed rabbits on endothelium-dependent vascular relaxation and ET-1-induced vascular contraction, as well as the systemic synthesis of ET-1. Rabbits were initially fed a diet enriched with 1% cholesterol for 4 weeks, followed by 0.5% cholesterol alone or supplemented with 2% L-arginine in drinking water during the next 12 weeks. Cholesterol feeding impaired endothelium-dependent relaxation of rabbit aortic rings ex vivo and increased urinary immunoreactive ET-1 excretion, along with decreased urinary nitrate excretion, an index of NO production. L-Arginine partially restored endothelium-dependent relaxation in parallel to increased urinary nitrate excretion and decreased urinary immunoreactive ET-1 excretion. Selective inhibition of ET-A receptors with BQ123 partially restored endothelium-dependent relaxation in hypercholesterolemic rabbits but had no effect on arterial rings from rabbits supplemented with L-arginine or from control animals. The contractile vascular response of aortic rings to exogenous ET-1 was increased in rabbits fed a high-cholesterol diet; this enhanced contractility to ET-1 was completely reversed by L-arginine. These data suggest that L-arginine restores endothelial function and normalizes the synthesis and vasoconstrictor response to ET-1 in hypercholesterolemia.

Role of endogenous endothelin in the development of graft arteriosclerosis in rat cardiac allografts: antiproliferative effects of bosentan, a nonselective endothelin receptor antagonist.

The purpose of this study was to determine whether endothelin-1 (ET-1) contributes to the development of graft arteriosclerosis and whether the orally active nonpeptide endothelin receptor antagonist bosentan, which blocks both ETA and ETB receptors, can protect against this pathologic damage. Recipient male Lewis rats were divided into three groups; group 1 received heterotopic heart transplantations from Lewis donors and groups 2 and 3 received transplantations from Brown-Norway donors; group 3 recipients also received bosentan orally at the dose of 20 mg/kg per day for 120 days. All recipients were given cyclosporine and were euthanized at examination 120 days after transplantation. Plasma ET-1 levels were significantly higher in group 2 than in group 1 (6.99+/-0.91 and 4.15+/-.83 pg/mL, respectively). Strong ET-1 immunoreactivity was seen in both the thickened neointima and the media of the coronary arteries in group 2 but not in group 1. The mean ratio of the coronary luminal area to the total vascular area in group 2 (19.0+/-11.7%) was significantly lower than that in group 1 (34.2+/-9.9%) and was significantly increased in group 3 (33.2+/-9.2%). These results show that local upregulation of ET-1, mainly in the thickened neointima and the media of the coronary arteries, may play an important role in the pathogenesis of graft arteriosclerosis by stimulating ETA receptors, ETB receptors, or both. Orally active bosentan might be a useful agent for the clinical prevention of graft arteriosclerosis.

Effects of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, atorvastatin and simvastatin, on the expression of endothelin-1 and endothelial nitric oxide synthase in vascular endothelial cells.

Endothelial dysfunction associated with atherosclerosis has been attributed to alterations in the L-arginine-nitric oxide (NO)-cGMP pathway or to an excess of endothelin-1 (ET-1). The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to ameliorate endothelial function. However, the physiological basis of this observation is largely unknown. We investigated the effects of Atorvastatin and Simvastatin on the pre-proET-1 mRNA expression and ET-1 synthesis and on the endothelial NO synthase (eNOS) transcript and protein levels in bovine aortic endothelial cells. These agents inhibited pre-proET-1 mRNA expression in a concentration- and time-dependent fashion (60-70% maximum inhibition) and reduced immunoreactive ET-1 levels (25-50%). This inhibitory effect was maintained in the presence of oxidized LDL (1-50 microg/ml). No significant modification of pre-proET-1 mRNA half-life was observed. In addition, mevalonate, but not cholesterol, reversed the statin-mediated decrease of pre-proET-1 mRNA levels. eNOS mRNA expression was reduced by oxidized LDL in a dose-dependent fashion (up to 57% inhibition), whereas native LDL had no effect. Statins were able to prevent the inhibitory action exerted by oxidized LDL on eNOS mRNA and protein levels. Hence, these drugs might influence vascular tone by modulating the expression of endothelial vasoactive factors.

Regulation of endothelin synthesis in hepatic endothelial cells.

Endothelin (ET) stimulates vasoconstriction and glucose production and mediator synthesis in the liver. Only hepatic endothelial cells express ET-1 mRNA, and during endotoxemia in the intact rat, a ninefold increase in hepatic ET-1 mRNA occurs within 3 h of lipopolysaccharide (LPS) infusion [A. T. Eakes, K. M. Howard, J. E.Miller, and M. S. Olson. Am. J. Physiol. 272 (Gastrointest. Liver Physiol. 35): G605-G611, 1997]. The present study defines the mechanism by which hepatic ET production is enhanced during endotoxin exposure. Culture media conditioned by exposure to endotoxin-treated Kupffer cells stimulated a twofold increase in immunoreactive ET-1 (irET-1) secretion by liver endothelial cells. Transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), LPS, and platelet-activating factor (PAF) were tested for their ability to stimulate cultured liver endothelial cells to secrete irET-1. Although TNF-alpha, LPS, and PAF had no significant effect on ET-1 synthesis, TGF-beta increased ET-1 mRNA expression and irET-1 secretion. In coculture experiments, treating Kupffer cells with endotoxin caused a doubling of the ET-1 mRNA level in the liver endothelial cells.This increase in ET-1 mRNA was attenuated by a TGF-beta-neutralizing antibody. Hence, a paracrine signaling mechanism operates between Kupffer cells that release TGF-beta on endotoxin challenge and hepatic endothelial cells in which TGF-beta stimulates ET-1 mRNA expression and ET-1 secretion; this intercellular signaling relationship is an important component in the hepatic responses to endotoxin exposure.