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PubMed1991

Localization and characterization of endothelin receptor binding sites in the rat brain visualized by in vitro autoradiography.

Endothelin binding sites in rat brain were mapped by quantitative in vitro autoradiography employing [125I]endothelin-1 as radioligand. [125I]Endothelin-1 bound with high affinity and specificity to rat cerebellar sections and was potently displaced by unlabelled endothelins (endothelin-1 greater than endothelin-2 = endothelin-3) and sarafotoxin 6B. The highest densities of endothelin binding sites were found in the cerebellum (especially Purkinje cell layer), choroid plexus and median eminence. High densities were found in the supraoptic and paraventricular hypothalamic nuclei, anterior hypothalamic area, ventromedial hypothalamic nucleus, mammillary nuclei and glomerular layer of olfactory bulb. Moderate densities were found in many thalamic nuclei, the pretectal region, interpeduncular nucleus, suprachiasmatic nucleus, raphe nuclei, tegmental nuclei, olfactory ventricle, red nucleus, subthalamic nucleus, central gray, reticular nuclei, vestibular nuclei, oculomotor and trochlear nuclei, hypoglossal nucleus, motor trigeminal nucleus, nucleus of the trapezoid body and lateral cerebellar nucleus. Low but detectable densities of endothelin binding sites were found in medial geniculate nucleus, fields of Ammon's horn, caudate-putamen, globus pallidus, entopeduncular nucleus, substantia nigra, anterior commissure, internal capsule, anterior pituitary, median preoptic nucleus, septohypothalamic nucleus, superior colliculus and area postrema. These patterns were completely abolished by 1 microM unlabelled endothelin-1, -2 and -3 and sarafotoxin S6B. Brain endothelin binding sites show high affinity for endothelin-1, -2 and -3 and sarafotoxin 6B with highest affinity for endothelin-1. Endothelin binding sites show a non-vascular pattern of distribution in the brain, suggesting that the peptide may have widespread functions as a modulator of neuronal function.

Animals↗
PubMed1992

Localization of immunoreactive endothelin and proendothelin in the human lung.

The endothelins are a family of three 21-amino-acid peptides: endothelin-1, endothelin-2 and endothelin-3. They are powerfully vasoactive, causing both contraction and relaxation of blood vessels. They are also active in the lung causing long lasting bronchoconstriction. Antibodies were raised in rabbits against the C-terminal heptapeptide of endothelin-1 (endothelin-1(15-21)) and to portions of the C-terminus of the human proendothelin-1(31-38), proendothelin-2(31-37) and proendothelin-3(31-41 amide) and tested by enzyme-linked immunosorbent assay to determine their titre and cross-reactivity. We used these antibodies to determine the localization of mature endothelin in the adult human lung and to determine the distribution of each of the three proendothelins. Mature endothelin immunoreactivity was present in airway epithelia and submucosal glands throughout the lung. In the airway epithelia immunoreactive proendothelin-1 and proendothelin-3 were detected, while immunoreactivity of all three isoforms was present in submucosal glands. Quantitative in vitro receptor autoradiography was used to locate specific endothelin binding sites. The rank order for density of endothelin binding site occurrence was: lung parenchyma greater than airway smooth muscle greater than airway epithelia. If immunoreactive endothelin is released onto these sites in vivo, endothelin may act as a paracrine mediator in the human lung.

Adult↗
PubMed1992

Monoclonal antibodies to human endothelin-1: characterization and utilization in radioimmunoassay and immunocytochemistry.

A host of monoclonal antibodies directed against human endothelin-1 (ET-1) has been developed and characterized. The antibodies reacted with ET-1 specifically and with high affinity, as determined by competition analysis and sucrose density gradients. The antibodies did not cross-react with neuropeptide YY, beta-endorphin, calcitonin gene-related peptide, secretin or somatostatin. The antibodies cross-reacted with big endothelin (B-ET), endothelin-2 (ET-2), vasointestinal constrictor peptide (VIC), and endothelin-3 (ET-3) albeit with varying affinity but did not cross-react with sarafotoxin (SRTX-6b). None of the antibodies reacted with the C-terminal hexapeptide (HXPT) of ET-1, indicating that the epitopes are not located within this region of ET-1. The monoclonal antibodies exhibited binding activity in dilutions ranging from 1:1000, to 1:10(6). The isotypes of the monoclonal antibodies were determined by competition binding assay. Six of the monoclonal antibodies were of the IgG gamma 1, two were IgM and one of the IgG gamma 2a subclass. The antibodies detected immunoreactive ETs by radioimmunoassay and in immunocytochemical localization, suggesting the potential use of these antibodies as tools to determine the concentration of ETs in biological fluids and in immunocytochemical localization of ETs in specific cell types in various tissues.

Amino Acid Sequence↗
PubMed1992

Endothelin modulation of tissue plasminogen activator release from human vascular endothelial cells in culture.

To clarify a possible involvement of the vasoconstrictive peptide endothelin in the regulation of endothelial cell-mediated fibrinolytic system, confluent cultures of vascular endothelial cells from human umbilical vein were incubated in serum-free medium in the presence of endothelin-1 at 100 nM and below, and tissue plasminogen activator antigen (t-PA:Ag) in the medium was determined by enzyme immunoassay. Endothelin-1 at 1 nM and above significantly decreased the release of t-PA:Ag from the endothelial cells after a 24 h incubation. The t-PA:Ag release was also decreased by either endothelin-2 or endothelin-3 at 10 nM. The activity of lactate dehydrogenase in the medium was not changed by endothelin-1 at 100 nM and below, suggesting that the peptide did not cause nonspecific cell damage. The decrease in the t-PA:Ag release induced by endothelin-1 occurred in the presence or absence of 8-bromo cyclic AMP, which is an active congener of cyclic AMP; 3-isobutyl-1-methylxanthine, which is an inhibitor of phosphodiesterase; and forskolin, which is a stimulator of adenylate cyclase. These results strongly indicated that cyclic AMP which is known to down-regulate t-PA:Ag release was not involved in the endothelin-1 effect. However, endothelin-1 failed to decrease the t-PA:Ag release in the presence of either calcium ionophore A23187 or EGTA; the ionophore itself markedly decreased the release. The cytosolic calcium accumulation was significantly increased by endothelin-1. These results suggest that endothelin-1 decreases the release of t-PA:Ag from human endothelial cells through an excess accumulation of intracellular, especially cytosolic which would be mediated by an extracellular, calcium-dependent mechanism.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Anatomical localization and pharmacological activity of mature endothelins and their precursors in human vascular tissue.

OBJECTIVES: We developed four selective rabbit antisera in order to compare the distribution of immunoreactive mature endothelins and their precursors, proendothelin-1, proendothelin-2 and proendothelin-3, in the endothelium from human vascular tissue. Our second aim was to use in vitro pharmacological assays to test the vasoconstrictor actions of the mature endothelin and proendothelin peptides. METHODS: The antisera were shown to be selective by enzyme-linked immunosorbent assays. With these antisera, we detected immunoreactivity in serial cryostat sections from saphenous and mesenteric veins, and mesenteric and internal mammary arteries, using a peroxidase-antiperoxidase technique. In pharmacological experiments, segments of human coronary and mesenteric arteries were exposed to cumulative (0.06-60 nmol/l) concentrations of the endothelins and their precursors. RESULTS: Antisera directed against mature endothelin stained the cytoplasm of endothelial cells in all vessels tested. Immunoreactive proendothelin-1 and proendothelin-2 were also detected, but not proendothelin-3. Endothelin-1 and endothelin-2 were strongly vasoactive, with similar molar potencies, and caused a dose-related increase in contractile force in human coronary arteries (0.06-60 nmol/l). However, proendothelin-1 and proendothelin-2 were 100-fold and 1000-fold less vasoactive than their respective mature peptides. No contractile effect was seen with proendothelin-3 or endothelin-3 at the concentrations tested in human coronary arteries, and similar results were obtained with human mesenteric arteries. CONCLUSIONS: These results suggest that proendothelin-1 and proendothelin-2 must be converted to their corresponding mature peptides to produce vasoconstrictor activity in human vessels. Immunoreactive mature endothelin is widely distributed in human vascular endothelial cells and, if released, may produce endothelin-mediated vasoconstriction.

Coronary Vessels↗
PubMed1991

Differential pharmacological profile of endothelin-1 and its precursor, big endothelin.

In pentobarbital-anesthetized rats endothelin-1 (ET-1), endothelin-2 (ET-2), endothelin-3 (ET-3), and mouse ET-2 (mET-2), in contrast to human big ET-1 (h-big ET), administered as i.v. bolus injections (0.25 nmol/kg i.v.) produced rapidly appearing and short-lasting blood pressure decreases. This effect was markedly inhibited (80-100%) after an 8-min i.v. infusion (0.1 nmol/kg/min over 10 min) of any of the ET studied, but not by h-big ET, the precursor of ET-1. Similarly, in pithed rats given a 10 min i.v. infusion of an equipressor dose (0.1 nmol/kg/min) of ET-1 or h-big ET, the hypotensive effects of ET-1 were entirely blocked only in the group of animals pretreated with ET-1. In pithed rats, ET-1 (0.25 nmol/kg i.v.) and h-big ET (0.5 nmol/kg i.v.) produced equivalent maximal pressor responses and the same pattern of increase in systemic, hindquarter, and renal vascular resistance. However, ET-1 was three times more potent than h-big ET as a vasoconstrictor of the mesenteric bed. Also the pressor response to h-big ET, but not ET-1 (0.25 nmol/kg i.v.), was markedly inhibited by the metalloprotease inhibitor phosphoramidon (5 mg/kg i.v.). These results indicate that the hypotensive effects of ET isopeptides have a common mechanism because they elicit cross tachyphylaxis, although h-big ET did not inhibit the decrease in blood pressure produced by ET-1. A possible explanation for this finding is that h-big ET has intrinsic pressor activity but does not have affinity for receptors mediating the vasodepressor effects of ET isoforms. Alternatively, h-big ET is converted into ET-1 too slowly to yield biophase concentrations of ET-1 necessary for lowering blood pressure and developing tachyphylaxis to ET-isoform-induced hypotension. Finally, if the pressor effects of h-big ET are mediated by ET-1 formation, phosphoramidon can be considered as an inhibitor of the endothelin-converting enzyme.

Animals↗
PubMed1991

Localization of [125I]endothelin binding sites in the region of the carotid bifurcation and brainstem of the cat: possible baro- and chemoreceptor involvement.

We have demonstrated by autoradiography, displaceable binding for [125I]endothelin-1 ([125I]ET-1), [125I]endothelin-2 ([125I]ET-2), and [125I]endothelin-3 ([125I]ET-3) in the cat carotid bifurcation as well as in the nucleus of the tractus solitarius, where baroreceptor and chemoreceptor afferents from the carotid body and sinus terminate. There was also significant binding in the nodose and superior cervical ganglia. Barosensory and chemosensory discharge was recorded from filaments of the carotid sinus nerve in cats anesthetized with pentobarbitone. Intra-carotid injection of ET-1 or ET-3 (4-402 pmoles) caused transient dose-related depression of baroreceptor discharge without any immediate effects on systemic blood pressure (BP) or heart rate; there was a delayed biphasic effect on BP. ET-1 had little effect on chemosensory discharge during the first 15 s post-injection, but there was a delayed (45-90 s) dose-related increase in discharge. The effects of all three ETs were qualitatively similar, and ET enhanced chemoexcitation evoked by either acetylcholine or sodium cyanide. Our results show that (a) ET binding sites are located in the baroreceptor and chemoreceptor afferent pathways and (b) ETs can influence afferent activity of baroreceptors and chemoreceptors. Further studies are needed to determine the significance of these findings, particularly with regard to reflex control of the cardiovascular system.

Animals↗
PubMed1991

Immunoreactive endothelin in human plasma, urine, milk, and saliva.

Using a highly sensitive radioimmunoassay, elevated plasma immunoreactive endothelin (ir-ET) levels were found in patients with diabetes mellitus (1.88 +/- 0.12 pmol/L, mean +/- SEM, n = 100), patients undergoing maintenance hemodialysis (4.28 +/- 0.76 pmol/L, n = 14), patients with acute myocardial infarction (3.43 +/- 1.03 pmol/L, n = 6), and patients with subarachnoid hemorrhage (4.92 +/- 0.64 pmol/L, n = 14) (normal controls: 0.54 +/- 0.05 pmol/L, n = 19). ir-ET was also present in urine (2.1 +/- 0.3 pmol/L, n = 12), breast milk (6.8 +/- 1.6 pmol/L, n = 16), and saliva (2.0 +/- 0.2 pmol/L, n = 15) obtained from healthy subjects. Chromatography studies verify the identity of endothelin. Fast protein liquid chromatography (FPLC) showed one peak in the normal plasma extract, three peaks in the plasma extracts from diabetic patients and patients undergoing maintenance hemodialysis, three peaks in the urine extract, four peaks in the milk extract, and five peaks in the saliva extract. When the materials eluting in the void volume on FPLC of urine and saliva extracts were loaded onto a Sephadex G-25 column, the ir-ET was eluted in a higher molecular weight region. Incubation of endothelin-1, endothelin-2, and endothelin-3 in urine for 5 h showed that the total amount of ir-ET decreased to less than 30% of the initial levels, suggesting that endothelins are very unstable in urine.

Adult↗
PubMed1991

Sandwich-enzyme immunoassays for endothelin family peptides.

Sandwich-enzyme immunoassays (sandwich-EIAs) for endothelin-1 (ET-1), endothelin-3 (ET-3), big ET-1, big endothelin-2 (big ET-2), and big endothelin-3 (big ET-3) have been established using two antibodies directed against the N-terminal and C-terminal portions of each ET and big ET. These sandwich-EIAs are sensitive enough to detect 0.1-0.4 pg/well of each ET or big ET with cross-reactivity of less than 1% with other ETs and big ETs (except in the case of the EIA for ET-1, which detects immunoreactivity of ET-2 as well as ET-1). Plasma levels of immunoreactive (ir-) ET-1, big ET-1, and ET-3 in normal humans were approximately 2.0, 5.1, and 0.9 pg/ml, respectively, when corrected for recovery efficiency. In reverse-phase high-performance liquid chromatography (HPLC) analysis, immunoreactivities detected by these sandwich-EIAs were eluted at the positions of authentic ET-1, big ET-1, and ET-3, indicating that plasma levels of ET-1, big ET-1, and ET-3 were precisely measured by our methods. Levels of ir-big ET-2 and ir-big ET-3 were also detected in normal human plasma; however, further studies are needed to determine their precise values. The establishment of reliable sandwich-EIAs and the measurement of specific ETs and big ETs in normal human plasma will contribute to clarifying the biological and clinical significance of ET family peptides.

Adult↗
PubMed1991

Autoradiographic localization of endothelin-1 binding sites in porcine skin.

Autoradiographic techniques and 125I-labeled endothelin-1 were used to study the distribution of endothelin-1 binding sites in porcine skin. Specific endothelin-1 binding sites were localized to blood vessels (capillaries, deep cutaneous vascular plexus, arteries, and arterioles), the deep dermal and connective tissue sheath of hair follicles, sebaceous and sweat glands, and arrector pili muscle. Specific binding was inhibited by endothelin-2 and endothelin-3 as well as endothelin-1. Non-specific binding was found in the epidermis and the medulla of hair follicles. No binding was found in connective tissue or fat. These vascular binding sites may represent endothelin receptors, in keeping with the known cutaneous vasoconstrictor actions of the peptide. If all binding sites are receptors, the results suggest that endothelin could also regulate the function of sweat glands and may have trophic effects in the skin.

Animals↗
PubMed1991

Identification of multiple phosphoinositide-linked receptors on human SK-N-MC neuroepithelioma cells.

The biochemical and pharmacological characteristics of receptor-stimulated phosphoinositide (PPI) hydrolysis in human SK-N-MC neuroepithelioma cells have been examined. Of 11 ligands tested, the addition of four, i.e., norepinephrine, oxotremorine-M, endothelin-1, and ATP, each resulted in an increased release (three- to eightfold) of inositol phosphates from [3H]inositol-prelabeled cells. Agonist-stimulated PPI turnover was sustained for at least 30 min and required the addition of Ca2+ for full effect. An increased release of inositol phosphates could also be elicited by the addition of the Ca2+ ionophore, ionomycin. All four agonists enhanced the release of radiolabeled inositol mono- and bisphosphates, inositol 1,3,4-trisphosphate, and inositol tetrakisphosphate. Increases in inositol 1,4,5-trisphosphate were smaller and only consistently observed in the presence of norepinephrine or oxotremorine-M. Norepinephrine-stimulated PPI turnover was potently inhibited by prazosin, WB-4101, and 5-methylurapidil (Ki less than 2.5 nM), but was relatively insensitive to chlorethylclonidine pretreatment. This pharmacological profile is consistent with the involvement of an alpha 1A-receptor subtype. The presence of an M1 muscarinic cholinergic receptor is also indicated, because pirenzepine blocked oxotremorine-M-stimulated inositol phosphate release (Ki = 35 nM) with a 30-fold greater potency than the M2-selective antagonist, AF-DX 116. Of the three endothelins tested, only the addition of endothelin-1 and endothelin-2 promoted PPI hydrolysis, whereas endothelin-3 was essentially inactive. A P2 nucleotide receptor of broad agonist specificity is also present on these cells and activates PPI turnover in the absence of a generalized increase in plasma membrane permeability. These results indicate that SK-N-MC cells express at least four PPI-linked receptors. Because the functional coupling of three of these receptors, i.e., alpha 1A-adrenergic, endothelin, and P2 nucleotide, has not been extensively characterized previously in neural tissues, the SK-N-MC cell line may provide a useful model system for studies of these receptors and their regulation.

Adrenergic Agonists↗
PubMed1991

The mechanism of action of endothelin in human lung.

1 The peptides endothelin-1 (ET-1) and endothelin-2 (ET-2) elicited potent and sustained contractions of human isolated bronchus and pulmonary artery. 2 ET-1 is one of the most potent contractile agonists investigated in these tissues with an EC50 value of 18.3 nM (95% confidence interval: 12.9, 25.9 nM: n = 26) in bronchus and 3.2 nM (95% confidence interval: 0.4, 23.9 nM; n = 5) in the arterial preparation. 3 ET-1 is 2.5 times more potent than ET-2 in both the airway and vascular tissues, and both forms of the peptide have geometric mean EC50 values 5 times greater than in the isolated bronchial tissue than in the pulmonary artery. 4 Neither pretreatment with the voltage-dependent calcium (VDC) channel antagonist verapamil (10 microM) nor with indomethacin (25 microM) significantly altered the response curve to ET-1 in human isolated bronchus. Removal of calcium from the Krebs-Henseleit solution did not affect ET-1-induced responses. 5 Specific binding on the smooth muscle of human airway and pulmonary arterial tissue to both ET-1 and ET-2 was detected in autoradiographic studies. There appeared to be no difference between the peptides in the location nor the density of binding sites. 6 We conclude that contraction of human bronchial tissue by ET-1 is not dependent upon influence of extracellular calcium nor release of prostaglandins or thromboxane A2. It is likely that the action of ET-1 in this tissue is due to binding of this peptide to specific receptors located on the smooth muscle.

Binding Sites↗
PubMed1991

Cross tachyphylaxis to endothelin isopeptide-induced hypotension: a phenomenon not seen with proendothelin.

1. In anaesthetized rats, an i.v. injection of endothelin-1 (0.25 nmol kg-1) evoked a rapidly appearing (maximal effect within 15 s) and short lasting (3 min) fall in blood pressure with tachyphylaxis occurring so that it was reduced by 50% by the last of 4 injections given 10 min apart. This property was also shared by endothelin-2, endothelin-3 and vasoactive intestinal contractor (VIC). 2. Cross tachyphylaxis between the isopeptides occurred. However, under the same experimental conditions the hypotensive effects of acetylcholine, adenosine, atrial natriuretic peptide (ANP) and substance P were reproducible and not modified in animals in which endothelin-1 no longer lowered blood pressure. Thus, the mechanism of the hypotensive action of endothelin peptides is different from that of acetylcholine, adenosine, ANP, and substance P. 3. In pithed rats, endothelin-1 (0.25 nmol kg-1) and its precursor human proendothelin (h-proendothelin) (0.5 nmol kg-1) induced pressor responses of a similar magnitude, which for h-proendothelin (up to 5.0 nmol kg-1) were not preceded by a hypotensive phase. The pressor effects of endothelin-1, like those of vasopressin, were reproducible upon repeated i.v. injections. 4. Rats given a 10 min infusion (0.1 nmol kg-1 min-1) of endothelin-1 showed no hypotensive response to an i.v. bolus injection of endothelin-1, whereas animals pretreated with an equipressor infusion of h-proendothelin did not develop tachyphylaxis to endothelin-1. 5. In pitched rats, endothelin-1, at a dose inducing the same maximal increase in blood pressure as h-proendothelin, was approximately 3 fold more potent as a mesenteric vasoconstrictor than h-proendothelin. These results suggest that if h-proendothelin is processed to endothelin-1, this transformation is not uniform throughout the vascular system. 6. The pressor response of h-proendothelin in pithed rats was dose-dependently inhibited by phosphoramidon (2.5-5.0mgkg '). However, this compound did not antagonize the effects of endothelin-1(0.25 nmol kg- ) or those of h-proendothelin (0.5 nmol kg- ) once developed. 7. Although some of these results may suggest that h-proendothelin does not undergo in vivo conversion to endothelin-1, the results obtained with phosphoramidon suggest that h-proendothelin is converted into endothelin-1. Therefore, the amount of endothelin-1 so produced can elicit pressor responses or regional vasoconstriction, but is insufficient to lower blood pressure and to inhibit endothelin-1-induced hypotension. 8. The mechanism of the tachyphylaxis does not appear to be depletion of endothelium-derived relaxing factor, since agents coupled to the latter endogenous vasorelaxant substance do not exhibit crosstachyphylaxis with endothelin-1. It is suggested that upon repeated or sustained exposure to endothelin-1, the endothelin-1 receptors mediating hypotension decrease in number and/or undergo conformational changes making them refractory to activation. Alternatively, the depletion of a blood-borne agent responsible for the hypotension could be involved.

Anesthesia↗
PubMed1992

Relative potency of endothelin analogues on changes in gastric vascular resistance.

The purpose of this study was to examine the effects of several endothelin analogues on the gastric microcirculation. We measured changes in vascular resistance of an ex vivo gastric segment of chloralose-anesthetized dogs. Changes in luminal pressure were used to quantitate contractile force. Graded doses of endothelin-2 (ET-2), endothelin-3 (ET-3), [Ala3,11]endothelin (Ala-ET), and the COOH-terminal hexapeptide (ET-C) were infused intra-arterially to the stomach. ET-2 and Ala-ET produced dose-related sustained increases in vascular resistance (10(-10) to 10(-8) M), with Ala-ET less potent than ET-2. In contrast, ET-3 was a weak vasoconstrictor while ET-C had no significant effects. There were no detectable changes in filtration with any of these analogues. However, the force of gastric contractions was significantly attenuated by both ET-2 and Ala-ET in a dose-dependent manner. We found that endothelin analogues produced sustained vasoconstriction of the gastric segment with a potency order of ET-2 greater than Ala-ET-3 much greater than ET-C. Our results suggest that endothelin analogues are potent vasoconstrictors but have little effect on filtration across the gastric vasculature.

Animals↗
PubMed1992

Properties and localization of endothelin-1-specific receptors in rat testicles.

The characterization and localization of rat testicular endothelin receptors were studied. Receptor binding assay with radiolabeled members of the endothelin family revealed that endothelin-1-specific receptors were present in rat testes; a maximal binding capacity of 250 +/- 62 fmol/mg protein and a dissociation constant value of 0.35 +/- 0.06 nM were calculated from the Scatchard plot. The affinities for endothelin analogues were endothelin-1 = endothelin-2 much greater than endothelin-3 much greater big endothelin-1 for the membrane-bound receptors. Receptors of endothelin-1 were localized in peritubular myoid cells and interstitial cells of rat testis by 125I-labeled endothelin-1 autoradiography; the receptors were undetectable in spermatogenic cells. The presence of endothelin-1 receptors in the rat testis raises the possibility that one or more of the endothelins may play a physiological role in the regulation of testicular function.

Animals↗
PubMed1992

Endothelin-1 stimulates chloride secretion across canine tracheal epithelium.

Although much attention has been paid to the effect of endothelin on the bronchopulmonary system, there are few reports concerning the effect of endothelin on Cl- secretion across airway epithelium. We examined the effects of endothelin-1, -2 and -3 on bioelectric parameters of canine tracheal epithelium, a tissue in which Cl- is actively secreted and Na+ is absorbed. Potential difference (PD) and short-circuit current (SCC) were measured using an Ussing chamber with 0.5 cm2 of exposed area, and conductance (G) was calculated by dividing SCC by PD. Luminal endothelin-1 produced transient increases in PD and SCC, returning to baseline values within 5 min after stimulation in a dose-dependent fashion and reached mean responses of 123 and 126% of baseline PD and SCC, respectively, at 10(-6) M of endothelin-1, whereas submucosal endothelin-1 did not alter PD and SCC. G remained unchanged when stimulated by endothelin-1. Endothelin-2 and -3 did not produce any significant alterations in PD and SCC. Endothelin-1 evoked increases in PD and SCC, which were not altered by pretreatment with luminal amiloride (10(-4) M), or by treatment with submucosal propranolol (10(-5) M). Ionic substitution of Cl- with nontransported anions, iodide and gluconate, inhibited endothelin-1-induced increases in PD and SCC. Pretreatment with 10(-5) M indomethacin partially inhibited Endothelin-1-evoked increases in PD and SCC. These findings indicate that endothelin-1 stimulates Cl- secretion partially through the generation of cyclooxygenase products of arachidonic acid.

Amiloride↗
PubMed1992

Functional expression of human myometrial endothelin receptors in Xenopus laevis oocytes.

We demonstrate the existence of functional endothelin receptors in human uterine myometrium using the Xenopus oocyte expression system. Fifty nanograms of poly(A)+RNA from myometrium was injected into Xenopus laevis oocytes and incubated for 70-80 h. The membrane potential of the oocyte was clamped at -60 mV and membrane current was measured during and after endothelin stimulation. Endothelin-1 elicited a large inward membrane current in the oocytes injected with poly(A)+RNA; endothelin-2 elicited a small current; while endothelin-3 did not induce any membrane current. These results indicate the existence of messenger RNA encoding functional endothelin-1 receptors in human uterine myometrium.

Animals↗
PubMed1992

Radioimmunoassay of endothelin in human plasma.

A specific and sensitive radioimmunoassay (RIA) for determination of endothelin-1 (ET-1) in human plasma has been developed. Antibodies were raised in rabbits using synthetic ET-1 conjugated to thyroglobulin as immunogen. The antibodies obtained were used at a final dilution of 1:300,000 yielding maximum binding of 61.7 +/- 3.0% (mean +/- 1 SD, n = 20) of 125I-ET-1. The ID50 (inhibitory dose 50%) was 4.5 +/- 0.6 fmol/100 microliters (mean +/- 1 SD, n = 20). The sensitivity of the RIA was 0.33 fmol/100 microliters standard solution. No cross reactivity was observed with endothelin-3, big-endothelin-1, atrial natriuretic factor, angiotensin I or angiotensin II. The cross-reactivity with endothelin-2 was 100%. Endothelin was extracted from acidified plasma with Sep-pak C18 cartridges and recovery of ET-1 added to normal plasma was 70.9 +/- 10.3% (mean +/- 1 SD, n = 12). The concentration of ET-1 in plasma from normal subjects was 1.5 +/- 0.4 pmol/l (mean +/- 1 SD, n = 11) ranging from 1.0 to 2.2 pmol/1. Extracts of normal human plasma subjected to high performance liquid chromatography on a reverse phase C18 column showed one peak of immunoreactivity co-eluting with the standard for ET-1. From these data it is concluded that the immunoreactive material measured in normal plasma with the present RIA is identical to ET-1.

Animals↗