Human Endothelin Research Articles

Effects of different preproendothelin-1 mRNA anti-sense oligodeoxynucleotides on ischemic arrhythmias in rats.

The effects of four anti-sense oligodeoxynucleotides (AS-ODNs) against rat or human preproendothelin-1 mRNA on ischemic arrhythmias in anesthetized rats were studied. AS-ODN (60 nmol/kg) or control (normal saline; sense-ODN, and scrambled-ODN, 60 nmol/kg) was injected 2 h before acute myocardial ischemia elicited by the occlusion of the left anterior descending coronary artery. Arrhythmias during 60-min ischemia were assessed, and plasma endothelin-1 was determined with an endothelin-1-specific radioimmunoassay system. The results showed that anti-senses against human preproendothelin-1 mRNA were anti-arrhythmic without significant impact on hemodynamics, whereas two against rat preproendothelin-1 mRNA and the three controls failed to be anti-arrhythmic. In human antisense groups, both the incidence of reversible ventricular fibrillation and the mortality were decreased to zero. The incidences of ventricular tachycardia and salvos were significantly decreased from almost 100% in the controls to < or =30% (p < 0.01), the arrhythmia score from an average of approximately 3.6 to 0 and 0.7, respectively (p < 0.01 versus controls), and the total ventricular ectopic beats from an average of 307-338 to < 40 (p < 0.01). The human AS-ODNs led to less plasma endothelin-1, which was associated with suppressed ischemic arrhythmias in this rat model, indicating a contributory role of endothelin-1 in ischemic arrhythmias. Conversely, considering the two- or three-base mismatches between the human AS-ODNs and rat preproendothelin-1 mRNA, and the failure of the rat AS-ODNs in suppressing arrhythmias, the possibility could not be excluded that human endothelin-1 AS-ODNs acted via an undetermined pathway other than endothelin-1.

Activated endothelin system in polyglobulia

The role of the endothelin system, the functional counterpart of NO, in the pathophysiology of polyglobulia remains still elusive. Therefore a novel erythropoietin overexpressing mouse was generated, with hematocrit levels of about 80%. Hence, we analyzed vascular contractions to ET-1 and big endothelin-1 (big ET-1), endothelin-1 (ET-1) promoter activity, ET-1 immunochemistry, endothelin-1 (ET-1)-protein tissue levels, ETA/B-receptor mRNA expression in this novel transgenic model of severe polyglobulia. For analysis of ET-1 promotor activity, EPO transgenic mice were mated with homozygous transgenic mice expressing the lacZ gene under control of the human ET-1 promoter and immunochistochemistry for gal blue was performed in lacZ transgenic animals. Notwithstanding markedly increased eNOS expression, NO-mediated endothelium-dependent relaxation and circulating and vascular tissue NO levels indicating enhanced bioavailability of NO, ET-1 tissue levels were also augmented in heart, kidney, liver and aorta (2.2±0.3 vs. 0.5±0.1 pg/mg tissue; P<0.01) of transgenic polyglobulic animals. Accordingly, immunohistochemistry demonstrated enhanced expression of ET-1 protein in the vascular wall of polyglobulic animals as compared to controls (p< 0.05), while increase of ET-1 promoter activity was confined to the perivascular tissue (P<0.05). NOS inhibition with L-NAME unmasked increased vascular reactivity to ET-1 and bigET-1 and aortic ETA/B receptor mRNA gene expression was enhanced (p<0.05 vs. controls). Administration of the NOS inhibitor L-NAME led to acute vasoconstriction of peripheral resistance vessels, hypertension and death of transgenic mice within 2 days, while wildtypes did not show increased mortality. Treatment with the ETA antagonist darusentan doubled survival time of transgenic polyglobulic mice after NO synthase inhibition (p<0.01 vs placebo). In conclusion, in this study we provide first evidence that the tissue endothelin system is activated by polyglobulia. Together with a stimulated NO system it contributes to cardiovascular regulation in pathophysiological conditions associated with increased hematocrit.

Plasma concentrations of endothelin-like immunoreactivity in healthy horses and horses with naturally acquired gastrointestinal tract disorders.

To compare plasma endothelin (ET)- like immunoreactivity between healthy horses and those with naturally acquired gastrointestinal tract disorders. 29 healthy horses and 142 horses with gastrointestinal tract disorders. Blood samples were collected from healthy horses and from horses with gastrointestinal tract disorders prior to treatment. Magnitude and duration of abnormal clinical signs were recorded, and clinical variables were assessed via thorough physical examinations. Plasma concentrations of ET-like immunoreactivity were measured by use of a radioimmunoassay for human endothelin-1, and CBC and plasma biochemical analyses were performed. Plasma ET-like immunoreactivity concentration was significantly increased in horses with gastrointestinal tract disorders, compared with healthy horses. Median plasma concentration of ET-like immunoreactivity was 1.80 pg/ml (range, 1.09 to 3.2 pg/ml) in healthy horses. Plasma ET-like immunoreactivity was greatest in horses with strangulating large-intestinal obstruction (median, 10.02 pg/ml; range, 3.8 to 22.62 pg/ml), peritonitis (9.19 pg/ml; 789 to 25.83 pg/ml), and enterocolitis (8.89 pg/mI; 6.30 to 18.36 pg/ml). Concentration of ET-like immunoreactivity was significantly associated with survival, PCV, and duration of signs of pain. However, correlations for associations with PCV and duration of pain were low. Horses with gastrointestinal tract disorders have increased plasma concentrations of ET-like immunoreactivity, compared with healthy horses. The greatest values were detected in horses with large-intestinal strangulating obstructions, peritonitis, and enterocolitis. This suggests a potential involvement of ET in the pathogenesis of certain gastrointestinal tract disorders in horses.

Transfer of Antisense Oligodeoxynucleotides Against Endothelin Receptors A and B Into Human Coronary Smooth Muscle Cells and Endothelial Cells by Apolipoprotein E Peptide. An in Vitro Study.

Antisense oligodeoxynucleotides (AS-ODNs) are a new generation of therapeutic agents for gene therapy. To develop a new approach in regulating the expression of endothelin (ET) receptor, N,N-dipalmitylglycyl-apolipoprotein E (129-169) peptide (dpGapoE), an efficient gene delivery system, was used to transfect phosphorothioated AS-ODNs against nucleotides of human ET type A (ETA) receptors in human coronary smooth muscle cells (HCSMCs) and type B (ETB) receptors in human coronary endothelial cells (HCECs). After transfection, translocation to the nuclei and concentration in nuclear structures were observed in approximately 40% of HCSMCs and 60% of HCECs, respectively, at 48 h by fluorescence microscopy. Both the cellular ETA mRNA concentration in HCSMCs and ETB mRNA concentration in HCECs significantly declined. This approach may enable gene regulation in vivo and could be used to regulate vascular tone and constriction through ET receptors.

Endothelin receptors in cultured and native human radial artery smooth muscle.

In human vascular smooth muscle cells endothelin-1, acting at both endothelin A and endothelin B receptors, has been demonstrated to be both a potent vasoconstrictor and mitogen. Our aim was to study the functional expression of endothelin receptors in human radial artery smooth muscle using both native tissue and cultured cells (RASMCs). Radial artery smooth muscle cells were cultured from arterial explants and loaded with the calcium fluorescent dye fura-2. Cells responded to endothelin-1 and a variety of other vasoconstrictors with rises in cytoplasmic calcium ([Ca2+]c). Arterial rings responded to endothelin-1 with an increase in tension. The response of both cells and arterial rings to endothelin-1 was characterized using the selective endothelin A receptor antagonist BQ123 and the endothelin B receptor antagonist BQ788. The RASMCs were found to express [Ca2+]c responses consistent with the expression of only the endothelin A receptor. Endothelin-1-mediated vasoconstriction in radial artery rings was unaffected by BQ788 but was completely blocked by BQ123. Using the selective radioligands [125I]-PD151242 and [125I]-BQ3020 and a combination of in vitro receptor autoradiography and isolated cell preparations, endothelin A receptors were confirmed to be present on RASMCs and on arterial sections, whereas endothelin B binding was barely detectable on native smooth muscle and on RASMCs.

Human Endothelin Subtype A Receptor Enhancement during Tissue Culture via de Novo Transcription

Human Endothelin Subtype A Receptor Enhancement during Tissue Culture via de Novo Transcription

Human endothelin subtype A receptor enhancement during tissue culture via de novo transcription.

Endothelin (ET) has, since its discovery, increasingly been considered a key player in the pathophysiological processes of cerebral vasospasm in the course of subarachnoid hemorrhage, although it remains unclear how ET is involved. We present data that indicate an inherent capacity of human cerebral arteries to change their sensitivity to ET. Human cerebral arteries were obtained from patients undergoing intracranial tumor surgery. The vessels were divided into segments and subjected to organ culture for 48 hours. The vessels were then examined by using in vitro pharmacological methods and molecular biological techniques. After organ culture of the cerebral arteries, both the sensitivity to and potency of ET were enhanced (maximal response, 152 +/- 9%; -log (50% effective concentration), 10.3 +/- 0.3), in comparison with data for fresh cerebral arteries. Contractions were inhibited by both FR139317 (a specific ET(A) receptor antagonist) and bosentan (a mixed ET(A) and ET(B) receptor antagonist), in a manner indicating the sole presence of contractile ET(A) receptors. An inconsistent dilative response to the selective ET(B) receptor agonist sarafotoxin 6c was observed; the response was preserved in some segments and abolished in others, and potentiation of the precontraction was observed in yet other segments. No isolated contractile response to sarafotoxin 6c was observed, however. In reverse transcription-polymerase chain reaction assays, both ET(A) and ET(B) receptor messenger ribonucleic acid was detected. These results demonstrate that human cerebral arteries are capable of enhancing the function of ET(A) receptors.

Distinct expression of endothelin receptor subtypes A and B in luteinized human granulosa cells.

Endothelins (ET) are potent vasoconstrictive peptides originally isolated from vascular endothelial cells. Their biological effects are mediated through two different receptors, the endothelin-1 (ET-1)-selective endothelin receptor subtype ETA and the non-selective receptor subtype ETB. ET-1 protein has been found in human ovarian follicular fluid and ET-1 mRNA expression has been demonstrated in ovarian tissue. These findings indicate that the endothelin-system participates in the modulation of ovarian function, probably acting in an autocrine/paracrine manner. In the current study we used freshly aspirated, luteinized human granulosa cells (hGC) representing an in vitro model of the early corpus luteum. By means of RT-PCR and immunocytochemistry we investigated whether luteinized human granulosa cells express ETA and ETB receptors. Specific amplification products of ETA transcripts were detected in all samples investigated. In contrast, only after using a three-fold amount of ETB reverse transcripts we were able to demonstrate specific, but weak amplification products. In addition, immunocytochemical staining for ETA but not for ETB was found in granulosa cell preparations. The present study provides clear evidence that human granulosa cells predominantly express ETA receptor subtype mRNA and protein hinting to its possible role in follicle maturation and corpus luteum formation.

Vezf1/DB1 Is an Endothelial Cell-specific Transcription Factor That Regulates Expression of the Endothelin-1 Promoter

Coordinated gene regulation within the vascular endothelium is required for normal cardiovascular patterning during development and for vascular homeostasis during adulthood, yet little is known about the mechanisms that regulate endothelial transcriptional events. Vascular endothelial zinc finger 1 (Vezf1)/DB1 is a recently identified zinc finger-containing protein that is expressed specifically within endothelial cells during development. In this report, we demonstrate that Vezf1/DB1 is a nuclear localizing protein that potently and specifically activates transcription mediated by the human endothelin-1 promoter, in a Tax-independent manner, in transient transfection assays. Using a combination of deletion mutagenesis and electrophoretic mobility shift assays, a novel Vezf1/DB1-responsive element was localized to a 6-base pair (bp) motif, ACCCCC, located 47 bp upstream of the endothelin-1 transcription start site. Recombinant Vezf1/DB1 also bound to this sequence, and a 2-bp mutation in this element abolished Vezf1/DB1 responsiveness by the endothelin-1 promoter. Vezf1/DB1 could be identified with a specific antibody in nuclear complexes from endothelial cells that bound to this element. Regulation of endothelin-1 promoter activity by Vezf1/DB1 provides a mechanism for endothelin-1 expression in the vascular endothelium during development and to maintain vascular tone; Vezf1/DB1 itself is a candidate transcription factor for modifying endothelial cell phenotypes in order to appropriately assemble and maintain the cardiovascular system.

Molecular Regulation of the Endothelin-1 Gene by Hypoxia: CONTRIBUTIONS OF HYPOXIA-INDUCIBLE FACTOR-1, ACTIVATOR PROTEIN-1, GATA-2, AND p300/CBP

Endothelin-1 (ET-1) is a peptide hormone with potent vasoconstrictor properties which is synthesized and secreted predominantly by vascular endothelial cells. Its production is regulated by numerous stimuli including ischemia and hypoxia, and the enhanced levels that occur during myocardial ischemia may contribute to the progression of heart failure. We reported previously a preliminary characterization of a hypoxia-inducible factor-1 (HIF-1) binding site in the human ET-1 promoter which contributed to the activation of ET-1 expression in endothelial cells. We report here that the HIF-1 binding site alone is not sufficient for the response to hypoxia but requires an additional 50 base pairs of flanking sequence that includes binding sites for the factors activator protein-1 (AP-1), GATA-2, and CAAT-binding factor (NF-1). Mutation of any one of these sites or the HIF-1 site eliminated induction by hypoxia. Mutations of the AP-1 and GATA-2 sites, but not the HIF-1 site, were complemented by overexpressing AP-1, GATA-2, HIF-1alpha, or the activator protein p300/CBP, restoring the response to hypoxia. Binding studies in vitro confirmed physical associations among GATA-2, AP-1, and HIF-1 factors. Overexpression or depletion of p300/CBP modulated the level of ET-1 promoter expression as well as the endogenous ET-1 transcript but did not change the fold induction by hypoxia in either case. Regulation of the ET-1 promoter by hypoxia in non-endothelial cells required overexpression of GATA-2 and HIF-1alpha. The results support essential roles for AP-1, GATA-2, and NF-1 in stabilizing the binding of HIF-1 and promoting recruitment of p300/CBP to the ET-1 hypoxia response complex.

Functional Characterization of Three Mutations of the Endothelin B Receptor Gene in Patients With Hirschsprung’s Disease: Evidence for Selective Loss of Gi Coupling

Hirschsprung's disease (HSCR) is one the most common congenital intestinal disease. It leads to aganglionic megacolon in the early childhood. Several susceptibility genes have been identified : RET protooncogene and its ligand, glial cell derived neutrophic factor (GDNF), Sox 10, Endothelin-3 (EDN3) and its receptor B (EDNRB). EDNRB mutations are found in 5% of familial or sporadic HSCR. Only few EDNRB mutations found in HSCR have been explored and some of them seem to be non fonctional variants. The properties of three mutant human endothelin B receptor (hETB) (G57S, R319W and P383L) in isolated HSCR were analyzed. Stable recombinant cells expressing the three mutants and the wild-type (WT) were established. The hETB receptors were characterized for 125I ET-1 binding, ET-1 induced signaling: calcium transient, AP-1 transcriptional factor activation and cAMP accumulation. Immunofluorescence experiments showed normal cellular distributions of the mutant G57S, R319W and WT hETB receptors. In contrast, the P383L hETB mutant receptor was concentrated near the nucleus and essentially no ET-1 binding was detected. The two other mutants (G57S and R319W) bound ET-1 normally, induced calcium transients and activated the AP-1 pathway in the same way as wild type, but did not inhibit adenylate cyclase. The G57S hETB mutant even stimulated cAMP accumulation which was blocked by pertussis toxin. The absence of the P383L mutant receptor from the membrane clearly indicates that this mutation could be involved in HSCR. The G57S and R319W mutant receptors, despite their normal coupling to Gaq, have a defect in the Galphai signaling pathway and the G57S mutation couples to Galphas. These observations allow us to hypothesize that cAMP signaling might be involved in the differenciation of neural cells in the bowel.

Eicosanoids Inhibit the G-protein-gated Inwardly Rectifying Potassium Channel (Kir3) at the Na+/PIP2 Gating Site

We previously showed that activation of the human endothelin A receptor (HETAR) by endothelin-1 (Et-1) selectively inhibits the response to mu opioid receptor (MOR) activation of the G-protein-gated inwardly rectifying potassium channel (Kir3). The Et-1 effect resulted from PLA2 production of an eicosanoid that inhibited Kir3. In this study, we show that Kir3 inhibition by eicosanoids is channel subunit-specific, and we identify the site within the channel required for arachidonic acid sensitivity. Activation of the G-protein-coupled MOR by the selective opioid agonist D-Ala(2)Glyol, enkephalin, released Gbetagamma that activated Kir3. The response to MOR activation was significantly inhibited by Et-1 activation of HETAR in homomeric channels composed of either Kir3.2 or Kir3.4. In contrast, homomeric channels of Kir3.1 were substantially less sensitive. Domain deletion and channel chimera studies suggested that the sites within the channel required for Et-1-induced inhibition were within the region responsible for channel gating. Mutation of a single amino acid in the homomeric Kir3.1 to produce Kir3.1(F137S)(N217D) dramatically increased the channel sensitivity to arachidonic acid and Et-1 treatment. Complementary mutation of the equivalent amino acid in Kir3.4 to produce Kir3.4(S143T)(D223N) significantly reduced the sensitivity of the channel to arachidonic acid- and Et-1-induced inhibition. The critical aspartate residue required for eicosanoid sensitivity is the same residue required for Na(+) regulation of PIP(2) gating. The results suggest a model of Kir3 gating that incorporates a series of regulatory steps, including Gbetagamma, PIP(2), Na(+), and arachidonic acid binding to the channel gating domain.

Modeling and Docking the Endothelin G-Protein-Coupled Receptor

A model of the endothelin G-protein-coupled receptor (ET A) has been constructed using a segmented approach. The model was produced using a bovine rhodopsin model as a template for the seven transmembrane α-helices. The three cytoplasmic loop regions and the C-terminal region were modeled on NMR structures of corresponding segments from bovine rhodopsin. The three extracellular loops were modeled on homologous loop regions in other proteins of known structure. The N-terminal region was modeled as a three-helix domain based on its homology with a hydrolase protein. To test the model, the FTDOCK algorithm was used to predict the ligand-binding site for the crystal structure of human endothelin. The site of docking is consistent with mutational and biochemical data. The principal sites of interaction in the endothelin ligand all lie on one face of a helix that has been implicated by structure-activity relationship studies as being essential for binding. As further support for the model, attempts to dock bigET, an inactive precursor to endothelin that does not bind to the receptor, found no sites for tight binding. The model of the receptor-ligand complex produced forms a basis for rational drug design of agonists and antagonists for this G-protein-coupled receptor.

Effect of losartan on angiotensin II-mediated endothelin and prostanoid excretion in humans

Angiotensin II (Ang II) stimulates renal prostanoid and vascular endothelin-1 (ET-1) release. Most known Ang II effects are mediated by AT 1 receptors. Our aim was to determine whether AT 1 receptor activation mediates Ang II-evoked renal prostanoid and ET-1 release. Eleven healthy men were randomized in a crossover, double-blind fashion to receive 100 mg/day of losartan or matching placebo, for 8 days. Blood and urine were sampled before and after a 2-h infusion of Ang II at a rate previously determined to increase mean arterial pressure (MAP) by 25 to 30 mm Hg in each subject. After a 14-day washout, subjects received the alternate treatment. Pretreatment with losartan had little effect on baseline MAP, but increased plasma renin activity, and virtually eliminated the pressor response to Ang II infusion. Angiotensin II significantly increased prostanoid excretion after placebo; the prostanoid response to Ang II was even greater after losartan. Plasma ET-1 was not altered by Ang II infusion, with or without losartan. In contrast, urine ET-1 excretion rate decreased to 40% of baseline after Ang II but not after losartan pretreatment; losartan alone had no effect. We conclude that Ang II decreases renal ET-1 synthesis and release through the AT 1 receptor. In contrast, Angiotensin II-mediated renal prostanoid synthesis does not require activation of AT 1 receptors. These findings indicate that AT 1 receptor antagonists could provide renal protection through indirect mechanisms.

Percutaneous in vivo gene transfer to the peripheral lungs using plasmid-liposome complexes.

The purpose of this study was to investigate a new method of in vivo gene transfer to the lung parenchyma by the percutaneous approach. The plasmid that contains the gene for firefly luciferase driven by a cytomegalovirus (CMV) promoter (pCMVL) in combination with cationic lipids was percutaneously injected into the lung parenchyma. Luciferase activities were localized to the lobes of the lung where the plasmids with cationic lipids were injected. Percutaneous injection of the plasmid containing the human endothelin-1 (hET-1) gene driven by a CMV promoter (pRc/CMVhET-1) in combination with cationic lipids into the lungs caused pulmonary fibrosis localized to the injection site in the peripheral lungs. We concluded that percutaneous in vivo gene transfer to the lungs is a unique and important approach to introduce exogenous gene expression in the limited area of the lung parenchyma. This method of gene transfer will be applicable for human gene therapy for targeted areas of peripheral lung and will also be useful to assess the function of the proteins expressed by a gene in the local area of the lungs.

Effect of a novel bifunctional endothelin receptor antagonist, IRL 3630A, on guinea pig respiratory mechanics

This study characterized the in vitro pharmacological properties of a newly developed endothelin receptor antagonist, N-butanesulfonyl-[ N-(3,5-dimethylbenzoyl)- N-methyl-3-[4-(5-isoxazolyl)-phenyl]-( d)-alanyl]-( l)-valineamide sodium salt (IRL 3630A), and its in vivo effects on respiratory mechanics were determined. IRL 3630A showed highly balanced affinities to human endothelin ET A and ET B receptors, giving apparent K i values of 1.5 and 1.2 nM, respectively. This compound also potently antagonized the endothelin-1-induced intracellular Ca 2+ increases in both embryonic bovine tracheal (EBTr) cells expressing endothelin ET A receptors and human Girardi heart (hGH) cells expressing endothelin ET B receptors. In guinea pig isolated tracheas having both endothelin ET A and ET B receptors, IRL 3630A greatly inhibited endothelin-1-induced contraction (p A 2=7.1), which was partially or scarcely suppressed by the endothelin ET A receptor antagonist cyclo[-( d)-Trp-( d)-Asp-( l)-Pro-( d)-Val-( l)-Leu-] (BQ-123) or the endothelin ET B receptor antagonist N-(3,5-dimethylbenzoyl)- N-methyl-3-(4-phenyl)-( d)-phenylalanyl-( l)-tryptophan (IRL 2500), respectively. Bolus i.v. injections of IRL 3630A administered into anaesthetized guinea pigs at 10 and 30 μg/kg inhibited endothelin-1 (1.3 μg/kg)-induced changes in respiratory resistance and compliance in a dose dependent manner, whereas both sodium 2-benzo[1,3]dioxol-5-yl-4-(4-methoxy-phenyl)-4-oxo-3-(3,4,5-trimethoxy-benzyl)-but-2-enoate (an endothelin ET A receptor antagonist: PD 156707) and IRL 2500 at doses of up to 30 μg/kg did not affect endothelin-1-induced changes in respiratory mechanics, reflecting the in vitro results. IRL 3630A is thus an effective bifunctional endothelin receptor antagonist, and will be useful in clarifying the role of endothelin in pulmonary diseases such as bronchial asthma.

AGE-RELATED CHANGES IN CONTRACTILE RESPONSES OF RABBIT LOWER URINARY TRACT TO ENDOTHELIN

AGE-RELATED CHANGES IN CONTRACTILE RESPONSES OF RABBIT LOWER URINARY TRACT TO ENDOTHELIN

Expression and Co-Localization of C-Type Natriuretic Peptide and Endothelin in Human Kidney

Abstract C-type natriuretic peptide (CNP) is an endothelium-drived vasorelaxing factor which is present in human plasma, urine and renal epithelial cells. Endothelin (ET) is a potent renal and systemic vasoconstrictor and sodium regulating peptide. To date, the localization of C-type natriuretic peptide and endothelin gene expression in human kidney remains poorly defined. We determined the co-localization of C-type natriuretic peptide and endothelin mRNA in normal human kidney using in situ hybridization (ISH) with synthesized human C-type natriuretic peptide or human endothelin oligonucleotide probes together with immunohistochemical staining (IHCS) with human CNP-22 and ET-1 antibodies with no crossreactivity. The sections treated with nonimmune rabbit serum (NRS) demonstrated no immunoperoxidase activity. Human renal tissue (n=7) was obtained from biopsy of normal cadaveric donor kidneys and normal autopsy specimens. In situ hybridization (Figure 1) demonstrated that C-type natriuretic peptide and endothelin mRNA presence in human kidney which was localized to proximal and distal tubes with similar intensities.

Regulation of the endothelin system by shear stress in human endothelial cells.

In this study, the effect of shear stress on the expression of genes of the human endothelin-1 system was examined. Primary cultures of human umbilical vein endothelial cells (HUVEC) were exposed to laminar shear stress of 1, 15 or 30 dyn cm-2 (i.e. 0.1, 1.5 or 3 N m-2) (venous and two different arterial levels of shear stress) in a cone-and-plate viscometer. Laminar shear stress transiently upregulates preproendothelin-1 (ppET-1) mRNA, reaching its maximum after 30 min (approx 1.7-fold increase). In contrast, long-term application of shear stress (24 h) causes downregulation of ppET-1 mRNA in a dose-dependent manner. Arterial levels of shear stress result in downregulation of endothelin-converting enzyme-1 isoform ECE-1a (predominating in HUVEC) to 36.2 +/- 8.5 %, and isoform ECE-1b mRNA to 72.3 +/- 1.9 % of static control level. The endothelin-1 (ET-1) release is downregulated by laminar shear stress in a dose-dependent manner. This downregulation of ppET-1 mRNA and ET-1 release is not affected by inhibition of protein kinase C (PKC), or tyrosine kinase. Inhibition of endothelial NO synthase (L-NAME, 500 microm) prevents downregulation of ppET-1 mRNA by shear stress. In contrast, increasing degrees of long-term shear stress upregulate endothelin receptor type B (ETB) mRNA by a NO- and PKC-, but not tyrosine kinase-dependent mechanism. In conclusion, our data suggest the downregulation of human endothelin synthesis, and an upregulation of the ETB receptor by long-term arterial laminar shear stress. These effects might contribute to the vasoprotective and anti-arteriosclerotic potential of arterial laminar shear stress.

Enhanced endothelin ET B receptor down-regulation in human tumor cells

The characteristics of specific binding of human [ 125I]Tyr 13-endothelin-(1–21), [ 125I]-Tyr 13-Suc-[Glu 9,Ala 11,15]-endothelin-(8–21), ([ 125I]IRL-1620) and endothelin ET A receptor antagonist [ 125I]Tyr 3-( N-[(hexahydro-1 H-azepin-1-yl)carbonyl]- l-Leu]-1Me)- d-Trp ([ 125I]PD151242) (number of sites and their affinity) and proliferation responses to exogenous endothelin receptor agonists (endothelin-1 and the endothelin ET B receptor-selective, truncated N-acetyl-[Ala 11,15]-endothelin-(6–21) analogue BQ3020) were determined in cultured human fibroblasts and in tumorigenic HeLa cells. The cells were pre-incubated with equimolar concentrations of human endothelin-1 or its truncated analogue BQ3020. After pre-incubation (2 h), both peptides induced down-regulation of surface-membrane endothelin-1 receptors. This process was specific for endothelin ET B receptors and was much more intensive in tumorigenic cells. BQ3020, acting mostly through its C-terminus, induced nearly maximal endothelin ET B receptor down-regulation in HeLa cells. Staurosporine, a wide spectrum protein kinase inhibitor, significantly reduced, and N-[ N-[ N-[2,6-dimethyl-1piperidinyl)carbonyl]-4-Me- l-Leu]-1-(methoxycarbonyl)- d-tryptophanyl]- d-norleucine (BQ788), an endothelin ET B receptor antagonist, attenuated the down-regulation of endothelin receptors induced by endothelin receptor agonists. The down-regulation of endothelin ET B receptors was prevented by pre-incubation of the cells with the lysosomal enzyme blocker chloroquine. The endothelin-1-induced cell proliferation was attenuated by pre-incubation of the cells with the non-selective endothelin receptor antagonist Ac- d-10,11-dihydro-5 H-dibenzo[ a, d] cycloheptene-glycine-3,3- d-diphenyl-Ala-Leu-Asp-Ile-Ile-Trp (PD142893) and it was only partially reduced by the endothelin ET A receptor-selective endothelin antagonist PD151242.