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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↗
PubMed1990

Astrocytes are target cells for endothelins and sarafotoxin.

Endothelin-1, endothelin-3, and the snake venom toxin sarafotoxin S6b stimulate the hydrolysis of phosphatidylinositol by phospholipase C with similar potencies in primary cultures of astrocytes prepared from rat brain cortex. In indo 1-loaded cells, endothelin-1, endothelin-2, endothelin-3, and sarafotoxin induce the rapid mobilization of intracellular Ca2+ stores and promote a more slowly developing influx of Ca2+. These responses were insensitive to pertussis toxin and to inhibitors of cyclooxygenase and lipoxygenase. Similar actions of endothelins and sarafotoxin were observed using astrocytes from the cerebellum and glioma cells from the C6 and NN cell lines. The endothelin receptor of astrocytes differs from the receptor previously characterized in endothelial cells from brain microvessels in that it has a high affinity for endothelin-3. Thus, brain endothelin-1 and endothelin-3 have different target cells in the brain and may have different functions.

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↗
PubMed1990

Effects of indomethacin on the regional haemodynamic responses to low doses of endothelins and sarafotoxin.

1. Regional haemodynamic responses to i.v. bolus injections of low doses (4 pmol and 40 pmol) of endothelin-1, -2, -3 and sarafotoxin-S6b were assessed in conscious, Long Evans rats in the absence and presence of indomethacin. 2. Both doses of endothelin-3 and sarafotoxin-S6b caused early renal vasodilatations that were not affected by indomethacin. Endothelin-1 caused an initial renal vasodilatation only in the presence of indomethacin, indicating that this peptide produced concurrent release of cyclo-oxygenase products that caused renal vasoconstriction. Neither dose of endothelin-2 produced an increase in renal conductance. 3. The 4 pmol dose of all four peptides caused mesenteric vasoconstrictions only. With the 40 pmol dose of the peptides, none caused early mesenteric vasoconstriction except in the presence of indomethacin. Thus, in this vascular bed the primary vasoconstrictor effects of the peptides (seen with the 4 pmol dose) were offset, following the 40 pmol dose, by release of vasodilator cyclo-oxygenase products. Indomethacin alone caused significant vasoconstriction only in the mesenteric vascular bed, indicating that in this region of the circulation, vasodilator prostanoids might be involved also in the tonic control of vascular conductance. 4. All four peptides at both doses caused early hindquarters vasodilatation. However, only the initial hypotensive and hindquarters vasodilator effects of the 40 pmol dose of sarafotoxin-S6b were attenuated by indomethacin. Under these conditions the hindquarters vasodilator effects of sarafotoxin-S6b were similar to those of the other peptides, indicating that the more marked effects of sarafotoxin-S6b in the absence of indomethacin were contributed to by vasodilator cyclo-oxygenase products in the hindquarters.

Animals↗
PubMed1990

Binding of [125I]-endothelin-1 to rat cerebellar homogenates and its interactions with some analogues.

1. [125I]-endothelin-1, over the concentration range 6 pM-10 nM, bound to a single site in homogenates of rat cerebellum with high affinity (Kd = 2.8 +/- 0.6 x 10(-10) M). The site was present in a concentration of 321 +/- 58 fmol mg-1 protein. 2. The rates of association and dissociation of [125I]-endothelin-1 with the binding site were slow (at 25 degrees C, k+1 = 8.0 +/- 1.3 x 10(5) M-1s-1; k-1 = 2.6 x 10(-4)s-1) and, on addition of a maximally displacing concentration of endothelin-1 (100 nM), 94.0 +/- 8.4% of the [125I]-endothelin-1 was still bound after 14 h. 3. [125I]-endothelin-1 binding was inhibited by a number of naturally occurring or genetically encoded members of the endothelin/sarafotoxin family of peptides. The order of potency was endothelin-3 = sarafotoxin S6b greater than endothelin-2 = endothelin-1 much greater than porcine proendothelin1-39. 4. Binding was also inhibited by analogues in which either one or both of the cystine disulphide bridges had been replaced by substitution with 2 or 4 alanine residues. The tetra-alanyl substituted analogue, [Ala1,3,11,15]endothelin-1, was equipotent with endothelin-1 at inhibiting the binding of [125I]-endothelin-1. [Ala3,11]endothelin-1 and [Ala1,15]endothelin-1, analogues which each contained one of the disulphide bridges from the parent peptide, were respectively 3 and 14 times less potent than the parent peptide. An analogue in which the Glu10 residue had been anisylated was 25 fold less potent than endothelin-1. 5. It is concluded that the structural requirements for binding to the cerebellar sites for [1251]_ endothelin-1 do not require the presence of the disulphide bridges characteristic of the endothelin/ sarafotoxin family. Rather, the binding may be more sensitive to the presence of bulky side chain substituents, at least in the smaller intramolecular loop.

Animals↗
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↗
PubMed1991

Endothelin activation of an inwardly rectifying K+ current in atrial cells.

Various tissues including heart express specific binding sites for endothelin. Endothelins have been reported to increase the force of contraction of cardiac muscle, presumably via specific receptors. Specific binding of endothelin to atrial tissue is particularly high. In spontaneously contracting rat atrial cells used in this study, all three isoforms of endothelin (endothelin-1, endothelin-2, and endothelin-3) decreased the rate of beating and caused an increase in inwardly rectifying K+ current in voltage-clamped whole cells. Endothelin-3 was the most potent isoform, and its effects on beating rate and K+ current were present at a concentration as low as 100 pM (Kd, approximately 1 nM). the atrial cells did not have the hyperpolarization-activated current (the pacemaker current), If. In excised inside-out patches, all three isoforms of endothelin activated a population of K+ channels with kinetic properties identical to those of acetylcholine (muscarinic)-activated K+ channels, and this was GTP dependent. Endothelin failed to decrease the beating rate or to elicit the K+ current in pertussis toxin-treated cells. These results indicate that endothelin has a potent negatively chronotropic effect by activation of the inwardly rectifying, muscarinic K+ channel and therefore could be an important regulator of heart function.

Acetylcholine↗
PubMed1990

Endothelin family in human plasma and cerebrospinal fluid.

To clarify whether endothelin may be present in human cerebrospinal fluid (CSF) and, if it exists, to compare its molecular forms with those of endothelin in human plasma, we analyzed pooled human CSF and plasma by high performance liquid chromatography with specific enzyme immunoassays for each endothelin peptide. Of the four human endothelin peptides hitherto identified, big endothelin-1 was the major molecular form of endothelin present in human CSF. In addition, there was a small but significant amount of endothelin-1 and endothelin-3 in CSF, while endothelin-2 was not detectable. Similarly, big endothelin-1, endothelin-1, and endothelin-3 were identified in human plasma. Although big endothelin-1 was the most abundant, a substantial amount of endothelin-1 and endothelin-3 was found in plasma with a resultant lower molar ratio of big endothelin-1 to endothelin-1 than in CSF. In all CSF samples from 17 patients requiring diagnostic lumbar puncture or lumbar anesthesia, endothelin was detectable, with a preponderance of big endothelin-1 relative to endothelin-1 and endothelin-3. The mean concentrations of endothelin-1 and endothelin-3 in simultaneously collected plasma were significantly (P less than 0.01) higher than those in CSF, while there was no significant difference between the mean big endothelin-1 concentration in plasma and that in CSF. There was no significant correlation among concentrations of big endothelin-1, endothelin-1, and endothelin-3 in each paired sample. These results indicate that endothelin is present in human CSF that is differently processed from endothelin in vascular endothelial cells and suggest a possible role for endothelin as a modulator of neuronal functions.

Adult↗
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↗
PubMed1991

Scintillation proximity assay: competitive binding studies with [125I]endothelin-1 in human placenta and porcine lung.

Scintillation proximity assay (SPA) technology has been used to investigate the competitive binding of [125I]endothelin-1 (ET-1) by ET-1, endothelin-2 (ET-2), endothelin-3 (ET-3), sarafotoxin 6b (SFTX-b), and big endothelin (big ET) to endothelin receptors in human placenta and porcine lung. Specific binding of [125I]ET-1 to high-affinity receptors was detected in membranes coupled to wheat germ agglutinin (WGA)-coated beads impregnated with scintillant (fluomicrospheres). The binding characteristics of ET-1 were similar in both of the tissues studied. In contrast, the receptor-binding properties of ET-2, ET-3, and SFTX-b were different in human placental and porcine lung membranes. This suggests that different endothelin receptor populations exist in these tissues. In addition, the binding characteristics of ET-3 and SFTX-b differed markedly to those of ET-1 and ET-2 in both tissues. This may be due to the four amino acid residue substitution in the N-terminal loop of both ET-3 and SFTX-b. We have also demonstrated that the ET-1 precursor, big ET, competitively inhibits [125I]ET-1 binding in these tissues, but is 100-fold less potent than ET-1.

Animals↗