Papel de los factores natriuréticos (atrial y urodilatina) y antagonistas de la endotelina / Role of natriurético factors (atrial and urodilatin) and endothelin antagonists

El Péptido Natriurético Atrial (PNA) ha sido estudiado en el tratamiento del fracaso renal agudo (FRA). Aunque algunos trabajos ofrecieron resultados alentadores dos estudios randomizados, multicéntricos, con un gran número de enfermos no obtuvieron diferencias frente a placebo. Últimamente dos artículos, con pequeño tamaño muestral han objetivado buenos resultados, pero dados los antecedentes sería recomendable la realización de estudios a mayor escala antes de poder extenderlo en el tratamiento del FRA. (Recomendación B.) La urodilatina también mostró resultados iniciales positivos, pero en un estudio con un mayor número de pacientes tampoco demostró superioridad respecto al placebo (Recomendación A.) La endotelina es una molécula vasoactiva muy importante a nivel renal. En fase experimental se han estudiado las acciones de varios fármacos que bloquean los receptores de la endotelina A o mixtos A/B. Hasta el momento no ha habido estudios en humanos que pudieran ser susceptibles de análisis. (Recomendación A.)

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Central endothelin-B receptor stimulation does not affect morphine analgesia in rats.

Several neurotransmitter mechanisms have been proposed to play a role in the actions of morphine. We reported that centrally administered endothelin A (ETA) receptor antagonists potentiate morphine analgesia in rats. It has also been reported that ETB agonist, IRL1620, has antinociceptive action mediated through opiate receptors in the periphery. The present study was conducted to determine if central ETB receptors are involved in analgesic actions of morphine. The effect of intracerebroventricular (i.c.v.) administration of ETB receptor agonist, IRL1620, on morphine-induced analgesia and hyperthermia was determined in the rat. Morphine (4 mg/kg, s.c.) produced a significant increase (84%) in tail-flick latency compared to the control group and the analgesic response lasted for 4 h. IRL1620 (30 microg, i.c.v.) did not produce any increase (16%) in tail-flick latency over the 5-hour observation period in vehicle-treated rats. Pretreatment with IRL1620 (3, 10, and 30 microg, i.c.v.) did not have any significant effect on the intensity and duration of morphine (4 mg/kg, s.c.)-induced analgesia. Morphine (4 mg/kg, s.c.) administration produced an increase in body temperature compared to the control group. In vehicle-pretreated rats, IRL1620 (30 microg, i.c.v.) did not produce any change in body temperature. The morphine-induced hyperthermic effect was not altered in IRL1620-pretreated rats. These studies demonstrate that IRL1620, a specific ETB receptor agonist, did not affect the morphine-induced analgesic and hyperthermic effect in rats. It can be concluded that central ETB receptors are not involved in modulation of pharmacological actions of morphine.

Submucosal microinfusion of endothelin and adrenaline mobilizes ECL-cell histamine in rat stomach, and causes mucosal damage: a microdialysis study.

Rat stomach ECL cells release histamine in response to gastrin. Submucosal microinfusion of endothelin or adrenaline, known to cause vasoconstriction and gastric lesions, mobilized striking amounts of histamine. While the histamine response to gastrin is sustainable for hours, that to endothelin and adrenaline was characteristically short-lasting (1-2 h). The aims of this study were to identify the cellular source of histamine mobilized by endothelin and adrenaline, and examine the differences between the histamine-mobilizing effects of gastrin, and of endothelin and adrenaline. Endothelin, adrenaline or gastrin were administered by submucosal microinfusion. Gastric histamine mobilization was monitored by microdialysis. Local pretreatment with the H1-receptor antagonist mepyramine and the H2-receptor antagonist ranitidine did not prevent endothelin- or adrenaline-induced mucosal damage. Submucosal microinfusion of histamine did not cause damage. Acid blockade by ranitidine or omeprazole prevented the damage, suggesting that acid back diffusion contributes. Gastrin raised histidine decarboxylase (HDC) activity close to the probe, without affecting the histamine concentration. Endothelin and adrenaline lowered histamine by 50-70%, without activating HDC. Histamine mobilization declined upon repeated administration. Endothelin reduced the number of histamine-immunoreactive ECL cells locally, and reduced the number of secretory vesicles. Thus, unlike gastrin, endothelin (and adrenaline) is capable of exhausting ECL-cell histamine. Microinfusion of alpha-fluoromethylhistidine (known to deplete ECL cells but not mast cells of histamine) reduced the histamine-mobilizing effect of endothelin by 80%, while 1-week pretreatment with omeprazole enhanced it, supporting the involvement of ECL cells. Somatostatin or the prostanoid misoprostol inhibited gastrin-, but not endothelin-stimulated histamine release, suggesting that endothelin and gastrin mobilize histamine via different mechanisms. While gastrin effectively mobilized histamine from ECL cells in primary culture, endothelin had no effect, and adrenaline, a modest effect. Hence, the striking effects of endothelin and adrenaline on ECL cells in situ are probably indirect, possibly a consequence of ischemia.

Both endothelin-A and endothelin-B receptors are present on adult rat cardiac ventricular myocytes.

Endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors have been demonstrated in intact heart and cardiac membranes. ET(A) receptors have been demonstrated on adult ventricular myocytes. The aim of the present study was to determine the presence of ET(B) and the relative contribution of this receptor subtype to total endothelin-1 (ET-1) binding on adult ventricular myocytes. Saturation binding experiments indicated that ET-1 bound to a single population of receptors (Kd = 0.52 +/- 0.13 nM, n = 4) with an apparent maximum binding (Bmax) of 2.10 +/- 0.25 sites (x 10(5))/cell (n = 4). Competition experiments using 40 pM [125I]ET-1 and nonradioactive ET-1 revealed a Ki of 660 +/- 71 pM (n = 10) and a Hill coefficient (nH) of 0.99 +/- 0.10 (n = 10). A selective ET(A) antagonist, BQ610, displaced 80% of the bound [125I]ET-1. No displacement was observed by concentrations of an ET(B)-selective antagonist, BQ788, up to 1.0 microM. However, in the presence of 1.0 microM BQ610, BQ788 inhibited the remaining [125I]ET-1 binding. Similarly, in the presence of 1.0 microM BQ788, BQ610 inhibited the remaining specific [125I]ET-1 binding. Binding of an ET(B1)-selective agonist, [125I]IRL-1620, confirmed the presence of ET(B). ET(B) bound to ET-1 irreversibly, whereas binding to ET(A) demonstrated both reversible and irreversible components, and BQ610 and BQ788 bound reversibly. Reducing the incubation temperature to 0 degrees C did not alter the irreversible component of ET-1 binding. Hence, both ET(A) and ET(B) receptors are present on intact adult rat ventricular myocytes, and the ratio of ET(A):ET(B) binding sites is 4:1. Both receptor subtypes bind to ET-1 by a two-step association involving the formation of a tight receptor-ligand complex; however, the kinetics of ET-1 binding to ET(A) versus ET(B) differ.

A novel bioactive 31-amino acid ET-1 peptide stimulates eosinophil recruitment and increases the levels of eotaxin and IL-5.

OBJECTIVE AND DESIGN: Investigation of the role of a novel inflammatory mediator 31-amino acid endothelin-1 [ET-1 (1-31)], a major ET derivative in granulocytes, in eosinophil recruitment after its subcutaneous administration to mice. METHODS: Various ET-1 derivatives (100 pmol), with or without ET receptor antagonists (200 pmol), were administered subcutaneously to mice, and then the eosinophil migration into and chemokine levels in the injected loci were analyzed. RESULTS: ET-1 (1-31) and a 21-amino acid endothelin-1 (ET-1), but not big ET-1, induced eosinophil migration into the injected loci with a peak after administration for 12 h, and increased the levels of eotaxin and interleukin-5 with peaks at 6 and 24 h, respectively. These effects of ET-1(1-31) and ET-1 were significantly inhibited by an ETA receptor antagonist, BQ-123, but not by an ETB receptor antagonist, BQ-788. CONCLUSION: Novel bioactive ET-1 (1-31) induces local eosinophil migration, and increases in eotaxin and interleukin-5 through an ETA or ETA-like receptor.

Effect of endothelin-1 (1-31) on human mesangial cell proliferation.

It was previously found that human chymase cleaves big endothelins (ETs) at the Tyr31-Gly32 bond and produces 31-amino acid ETs (1-31). In the present study, human plasma concentrations of ET-1 (1-31) and ET-1 were examined and the effect of synthetic ET-1 (1-31) on the proliferation of cultured human mesangial cells (HMCs) was investigated. The proliferative effect of ET-1 (1-31) was evaluated from the [3H]-thymidine uptake. The activity of extracellular signal-regulated kinase (ERK) and DNA binding activity of activator protein-1 were determined by using an in-gel kinase assay and gel mobility shift assay, respectively. Immunoreactive ET-1 (1-31) was detectable in plasma, but the level was slightly lower than that of ET-1. ET-1 (1-31) increased [3H]-thymidine incorporation in HMCs to a degree similar to that induced by ET-1. ET-1 (1-31) also activated ERK1/2. Inhibition of protein kinase C and ERK kinase caused a reduction of ET-1 (1-31)-induced ERK1/2 activation. The ERK1/2 activation was followed by an increase in transcription factor activator protein-1 DNA binding activity. These findings suggest that ET-1 (1-31) is a bioactive peptide in humans and ET-1 (1-31) itself stimulates HMC proliferation.

Endothelin-1[1-31], acting as an ETA-receptor selective agonist, stimulates proliferation of cultured rat zona glomerulosa cells.

Endothelin-1 (ET-1)[1-31] is a novel hypertensive peptide that mimics many of the vascular effects of the classic 21 amino acid peptide ET-1[1-21]. However, at variance with ET-1[1-21] that enhances aldosterone secretion from cultured rat zona glomerulosa (ZG) cells by acting via ETB receptors, ET-1[1-31] did not elicit such effect. Both ET-1[1-21] and ET-1[1-31] raised the proliferation rate of cultured ZG cells, the maximal effective concentration being 10(-8) M. This effect was blocked by the ETA-receptor antagonist BQ-123 and unaffected by the ETB-receptor antagonist BQ-788. Quantitative autoradiography showed that ET-1[1-21] displaced both [(125)I]PD-151242 binding to ETA receptors and [(125)I]BQ-3020 binding to ETB receptors in both rat ZG and adrenal medulla, while ET-1[1-31] displaced only [(125)I]BQ-3020 binding. The tyrosine kinase (TK) inhibitor tyrphostin-23 and the p42/p44 mitogen-activated protein kinase (MAPK) inhibitor PD-98059 abolished the proliferogenic effect of ET-1[1-31], while the protein kinase-C (PKC) inhibitor calphostin-C significantly reduced it. ET-1[1-31] (10(-8) M) stimulated TK and MAPK activity of dispersed ZG cells, an effect that was blocked by BQ-123. The stimulatory action of ET-1[1-31] on TK activity was annulled by tyrphostin-23, while that on MAPK activity was reduced by calphostin-C and abolished by either tyrphostin-23 and PD-98059. These data suggest that ET-1[1-31] is a selective agonist of the ETA-receptor subtype, and enhances proliferation of cultured rat ZG cells through the PKC- and TK-dependent activation of p42/p44 MAPK cascade.

O papel das endotelinas na fisiopatologia cardiovascular / Role of endothelins in cardiovascular patophysiology

As endotelinas são peptídeos de 21 aminoácidos produzidas em vários tecidos, conhecidas como o vasoconstritor endógeno mais potente que existe...

Morphological changes in rabbit ciliary epithelium and blood-aqueous barriers after intravitreal 10(-5) M endothelin-1.

Morphological changes in the ciliary epithelium and blood-aqueous barriers were observed with a tracer, horseradish peroxidase, after 10 microliters of 10(-5) M endothelin-1 was injected intravitreally. A decrease of intraocular pressure and an increase of anterior chamber flare were found, and the structure of the ciliary epithelium changed drastically 6-7 hr after the injection. Swelling of mitochondria appeared in both non-pigmented and pigmented epithelial cells of the ciliary body. Intercellular spaces and ciliary channels were markedly dilated in the iridial processes. Some non-pigmented epithelial cells in both iridial and ciliary processes appeared to be degenerated and necrotic. Horseradish peroxidase leaked into the posterior chamber via extremely dilated ciliary channels and necrotic cells. The dose of 10(-5) M endothelin-1 seems to be so large that a toxic action occurred. The mechanism of the effect of endothelin on intraocular pressure was also discussed.

Effects of endothelin on mitogen-activated protein kinase activity and protein synthesis in isolated adult feline cardiac myocytes.

The growth-promoting effects of endothelin-1 (ET-1) were examined in adult heart cells. The activity of mitogen-activated protein kinases (MAPKs) was measured in cytosolic extracts of isolated adult feline cardiac myocytes incubated with and without ET-1. Kinase activity was assessed by phosphorylation of the exogenous substrate, myelin basic protein. ET-1 stimulated the activity of MAPK up to 4-fold, with peak activation occurring between five and ten minutes after addition of ET-1. Polyclonal antisera raised against a 14-amino acid sequence of the erk-2 gene product, a MAPK isoform, identified two major bands in cytosolic extracts of the cardiac myocytes. Partial purification of kinase activities using Mono Q anion-exchange chromatography demonstrated two major peaks of myelin basic protein kinase activity. Subsequent immunoblots of the eluted fractions demonstrated that the immunoreactive bands observed in the cytosolic extracts eluted in those fractions possessing kinase activity. Overnight pretreatment of the cardiac myocytes with 100 ng/ml pertussis toxin inhibited the ET-1 stimulated increase in MAPK activity by 50 - 70%, but did not alter stimulation by 100 nM phorbol 12-myristate 13-acetate (PMA). These data suggest that stimulation of MAPK by ET-1 may be mediated by more than one pathway. MAPK has been shown to be activated in the intracellular transmission of growth factor signals. Indicative of a growth effect in this adult heart cell model, myocytes exposed to increasing concentrations of ET-1 demonstrated a dose dependent increase in [3H]-phenylalanine incorporation into cellular protein. This response was blocked by staurosporine and partially inhibited by pretreatment with pertussis toxin, again suggesting the possible involvement of multiple early signals. These data from isolated adult cardiac myocytes further support the hypothesis that ET-1 has a role in the regulation of cardiac growth.

ET-3 is extracted by and induces potent vasoconstriction in human splanchnic and renal vasculatures.

To investigate splanchnic and renal vascular effects and elimination of endothelin-3 (ET-3), ET-3 (10 pmol.kg-1.min-1 iv for 20 min) was given to six healthy male volunteers. Arterial plasma ET-3-like immunoreactivity (ET-3-Li) increased 10-fold to 111 +/- 31 pmol/l (P < 0.01). The initial half-life of plasma ET-3-Li determined in three subjects was 1.7 +/- 0.2 min. The fractional extraction of ET-3-Li was 68 +/- 7% in the splanchnic and 63 +/- 4% in the renal vascular beds. Mean arterial blood pressure fell from 86 +/- 4 to 94 +/- 4 mmHg (10%) (P < 0.05). Splanchnic and renal blood flows fell by 43 +/- 3% (P < 0.05) and 29 +/- 4% (P < 0.05), respectively, during the infusion. Splanchnic and renal vascular resistances rose by 92 +/- 22% (P < 0.05) and 58 +/- 7% (P < 0.05). In conclusion, ET-3 infusion in humans induces splanchnic and renal vasoconstriction of similar magnitude as previously shown during endothelin-1 infusion, presumably by ETB receptor activation. Plasma ET-3 is efficiently extracted in the splanchnic and renal vascular regions.

Localization of [125I]endothelin-1 in injured aorta of rabbits.

Endothelin-1 is a potent vasoconstrictor. This study was performed to determine whether arterial injury, induced by either hypercholesterolemia or mechanical disruption of the endothelium, is associated with increased localization of endothelin-1 in the artery. The blood clearance and tissue distribution of intravenously injected [125I]endothelin-1 was evaluated in 33 rabbits--control animals (n = 7), balloon de-endothelialized animals (n = 12), cholesterol-fed animals (n = 6) and animals that had both balloon de-endothelialization and high cholesterol diet (n = 8). The blood clearance half time was less than 10 min, with slightly slower clearance in the ballooned/cholesterol-fed animals. [125I]Endothelin-1 localized in the lung (approximately 12% injected dose (ID)/organ) and kidney (approximately 8%ID/organ). [125I]Endothelin-1 localization in the injured aorta increased from the baseline level of 0.06%kgID/g to its highest level within 5 min of balloon de-endothelialization (0.2%kgID/g) and decreased to 0.11%kgID/g within one week and remained essentially unchanged through 16 weeks. The area with increased binding of [125I]endothelin-1 corresponded to the zone of arterial injury stained with Evans blue. On the other hand, the binding in the aorta did not increase with the atherogenic diet. These findings suggest that endothelin-1 accumulates in injured vessels, attaining the highest levels immediately after mechanical injury.

Human breast cancer cells contain a phosphoramidon-sensitive metalloproteinase which can process exogenous big endothelin-1 to endothelin-1: a proposed mitogen for human breast fibroblasts.

Endothelin-1 (ET-1) levels are elevated in human breast tumours compared with normal and benign tissues, and in the presence of insulin-like growth factor 1 (IGF-I) ET-1 is a potent mitogen for human breast fibroblasts. In this study we have examined the ability of intact human breast cancer cell lines to process exogenously added big ET-1 (1-38) to the active mature ET-1 peptide by using a specific radioimmunometric assay. In both hormome-dependent (MCF-7, T47-D) and hormone-independent (MDA-MB-231) breast cancer cell lines the putative endothelin-converting enzyme (ECE) exhibited apparent Michaelis-Menten kinetics when converting added big ET-1 to ET-1. Both basal ET-1 production and exogenously added big ET-1 to ET-1 conversion were greatly reduced in all three cell lines in response to the metalloproteinase inhibitor phosphoramidon but were insensitive to other classes of protease inhibitors. Inhibition was also observed when cells were incubated in the presence of the divalent cation chelators 1,10-phenanthroline and EDTA. In MCF-7 cells the optimal pH for the ECE activity using a saponin cell permeabilisation procedure was found to residue within a narrow range of 6.2-7.26. Our results indicate that human breast cancer cells contain a neutral phosphoramidon-sensitive metalloproteinase which can process big ET-1 to ET-1. In the breast this conversion could contribute substantially to the local extracellular levels of this proposed paracrine breast fibroblast mitogen.

Radioimmunoassay evidence that the pressor effect of big endothelin-1 is due to local conversion to endothelin-1.

Compared with endothelin-1 (ET-1), big endothelin-1 (big ET-1) is only weakly active on isolated vascular smooth muscle preparations. However, on systemic administration high doses of big ET-1 (1 nmol.kg-1) are approximately equipotent to ET-1, indicating the existence of an endothelin converting enzyme in the circulation that rapidly converts big ET-1 to ET-1. In this study arterial blood levels of big ET-1 and ET-1 immunoreactivity were measured after bolus i.v. administration of big ET-1 (1 or 3 nmol.kg-1) or ET-1 (1 nmol.kg-1) in anaesthetised male Wistar rats. In addition, the effect of phosphoramidon (10 mg.kg-1) on the pressor response to big ET-1 and its disappearance rate from the circulation were examined. After big ET-1 injection, circulating ET-1 concentrations did not exceed 2% of the big ET-1 level. Phosphoramidon reduced the pressor response to big ET-1 by 93%, but did not alter its rate of clearance from the circulation. Thus exogenous big ET-1 is converted locally in the vasculature and its disappearance from the circulation is not dependent on conversion to ET-1.

Predominance of endothelinA (ETA) receptors in ovine airway smooth muscle and their mediation of ET-1-induced contraction.

1. Autoradiographic studies were conducted to investigate the receptor subtypes for endothelin-1 (ET-1) that were present in the ovine respiratory tract. In addition, the receptor subtypes mediating contraction of airway smooth muscle and the possible involvement of extracellular Ca2+ and inositol phosphate generation in intracellular signal transduction were assessed. 2. Specific [125I]-ET-1 binding in ovine trachea increased in a time- and concentration-dependent manner. Autoradiographic studies demonstrated that significant binding was associated with airway smooth muscle, although higher densities of specific binding were associated with submucosal glands and with cells immediately below the epithelial basement membrane (lamina propria). The ETA receptor-selective antagonist, BQ 123 (1 microM), virtually abolished specific binding to airway smooth muscle. Quantitative analyses of autoradiographic data describing the time-dependence of specific [125I]-ET-1 binding in ovine airway smooth muscle in the presence and absence of BQ 123 or sarafotoxin S6c, revealed a homogeneous population of ETA receptors. BQ 123 (1 microM) also abolished specific binding to structures associated with submucosal glands, whereas the ETB receptor selective agonist, sarafotoxin S6c (100 nM) had little effect on this binding, indicating the predominance of ETA receptors at these sites. In contrast, ETB receptors predominated in the lamina propria, since sarafotoxin S6c abolished specific binding in this tissue. 3. High levels of specific [125I]-ET-1 binding were also detected in the alveoli and in the walls of blood vessels and small airways in ovine peripheral lung. Specific binding associated with alveoli was reduced to similar extents by BQ 123 (1 MicroM; 54%) and sarafotoxin S6c (100 nM; 40%), suggesting the coexistence of both ETA and ETB receptors in approximately equal proportions in this tissue. In contrast,specific binding to blood vessels and to peripheral bronchial smooth muscle was abolished in the presence of BQ 123 (1 MicroM), but was unaffected by sarafotoxin S6c, indicating the presence of only ETA receptors at these sites.4. ET-1 caused concentration-dependent contractions of ovine tracheal smooth muscle which were inhibited in the presence of BQ 123 (1 MicroM). ET-1 also caused concentration-dependent contraction of ovine lung parenchyma strips. In contrast, the ETB receptor-selective agonists, sarafotoxin S6c and BQ 3020, were virtually inactive as spasmogens in both tracheal smooth muscle and lung strip preparations.Thus contraction was mediated by ETA receptors in ovine tracheal smooth muscle and this is consistent with binding and autoradiographic data demonstrating a homogeneous population of these binding sites in this tissue. Contraction of parenchymal lung strip preparations to ET-1 was mediated via non-ETB receptors, presumably ETA receptors, with contributions to this response perhaps coming from airway and vascular smooth muscle and from alveolar wall contractile cells.5. ET-1-induced contraction of tracheal smooth muscle was not significantly altered in the presence of indomethacin (5 MicroM), indicating that cyclo-oxygenase metabolites of arachidonic acid were not involved in this response. Contraction induced by ET-1 was virtually abolished in Ca2+-free medium containing 0.1 mM EGTA, indicating that this response was dependent upon the influx of extracellular Ca2 .Contraction was inhibited by about 50% in the presence of nicardipine (1 MicroM), indicating that a significant component of this response was mediated via the activation of L-type Ca2+ channels.6. ET-1 caused poorly defined increases in the accumulation of intracellular inositol phosphates in ovine tracheal smooth muscle. The maximal response to ET-1 was less than 20% of that to the cholinoceptor agonist, carbachol. Furthermore, sarafotoxin S6c was inactive. These data, when taken together with the results of autoradiographic and contraction studies, indicate that ovine airway smooth muscle contraction in response to ET-1 is mediated via ETA receptors which are linked to the influx of extracellular Ca2+, partly through voltage-dependent channels. ETB receptors also exist in the lamina propria of ovine trachea and in peripheral alveoli, perhaps residing in vascular endothelial cells.

Identification of a 65 kDa endothelin receptor in bovine cardiac sarcolemmal vesicles.

Endothelin-1, an endothelial cell-derived vasoconstrictor peptide, also exerts a potent positive inotropic effect on cardiac tissue. Characterization of specific binding of endothelin-1 to bovine cardiac sarcolemmal vesicles is reported. In the presence of 1 mM CaCl2, the observed binding for 125I-endothelin-1 had a Kd of 6.2 nM with an observed Bmax of 14 pmol/mg sarcolemmal protein. In the presence of 1 mM EDTA (and no added Ca2+) Bmax was reduced to 9 pmol/mg sarcolemmal protein while the Kd remained unchanged. Binding affinity for sarafotoxin S6b was at least one order of magnitude less than for endothelin-1. 125I-Endothelin-1 covalently cross-linked to a sarcolemmal protein with an apparent molecular weight of 65 kDa. Site-directed polyclonal antibodies to a sequence located on the third extramembranal segment of a previously cloned endothelin ETA receptor from bovine lung were produced. Using Western blot analysis, the site-directed polyclonal antibody recognized a sarcolemmal protein at 65 kDa. We conclude that sarcolemmal membranes from bovine ventricular myocardium contain an endothelin binding site and that it is a protein with an apparent molecular weight of 65 kDa.

Effects of endothelin-1 on arterial pressure: a comparison of intravenous and intra-arterial administration in conscious rabbits.

1. Intravenous endothelin-1 releases endothelium-derived relaxing factor (EDRF) and prostacyclin from the lungs, and substantial pulmonary clearance is claimed. Since these vaso-active substances could alter the haemodynamic effects of endothelin-1, the effects of boluses of endothelin-1 injected intravenously and into the left ventricle (intra-arterial) were compared, measuring arterial pressure and heart rate in conscious male New Zealand rabbits. The pulmonary clearance of endothelin-1-like immunoreactivity (Et-1LI) and initial plasma half-life were measured in anaesthetized rabbits during and after intravenous infusion of endothelin-1. 2. The effects of intravenous and intra-arterial routes of administration were not significantly different. Arterial pressure decreased (intravenous: 21.6, intra-arterial: 24.2 mmHg, P = 0.12, n = 10, t-test, 9 d.f.), then increased (intravenous: 21.2, intra-arterial: 16.4 mmHg, P = 0.33), while heart rate increased (intravenous: 66, intra-arterial: 53 beats/min, P = 0.09), then decreased (intravenous: 50, intra-arterial: 54 beats/min, P = 0.30). 3. Arterial and venous plasma levels of Et-1LI were not significantly different, venous levels increasing from 30 +/- 3 pg/mL (12 +/- 1 pmol/L) at 1 pmol/kg per min to 770 +/- 78 pg/mL (308 +/- 31 pmol/L) at 16 pmol/kg per min (n = 5). Mean initial plasma half-life was 0.6 min (range: 0.25-1.1 min). 4. It was concluded that there is no significant net pulmonary clearance of exogenously administered endothelin-1 in the conscious rabbit, and that any vaso-active factors released in the lungs by endothelin-1 do not have a significant effect on systemic arterial pressure.

Identification of a novel endothelin receptor in Xenopus laevis liver.

Membranes prepared from Xenopus liver displayed high density of high affinity endothelin (ET) binding sites. These sites have the same affinity for [125I] ET-1 and [125I] ET-3. Scatchard analysis of the specific binding from saturation binding experiments revealed an apparent dissociation constant (Kd) of 93.1 and 70.9 pM and maximum binding (Bmax) of 602 and 651 fmol/mg protein for [125I] ET-1 and [125I] ET-3, respectively. Competition binding experiments using [125I] ET-1 and unlabelled ET-1, ET-3, S6c, and BQ123 indicated that ET-1 and ET-3 were the most potent in displacing [125I] ET-1 binding from these membranes (IC50 1 and 0.3 nM, respectively), whereas S6c BQ123, selective for ETB and ETA receptors, respectively, did not have any inhibitory effect up to 1 microM. These data clearly indicate that the ET receptors present in Xenopus liver membranes belong to a new subtype of ET receptor. Because it resembled mammalian ETB receptors in its affinities for ET-1 and ET-3, we propose that this receptor be called the ETBX receptor.

Clearance of circulating endothelin-1 by ETB receptors in rats.

Exogenous endothelin (ET) is rapidly cleared from the circulation. We investigated which ET receptor subtypes (ETA and ETB) participate in ET-1 clearance. Following an intravenous (i.v.) bolus dose of [125I]ET-1 in anesthetized rats, radioactivity was rapidly cleared from the circulation and trapped by the lungs, kidneys and liver. Tissue distribution of the radioactivity was significantly inhibited in the lungs and kidneys, but not in the liver by infusion of the ETB antagonist BQ-788 (0.1 mg/kg/min i.v.), and the ET-1 clearance rate was reduced, while the ETA antagonist BQ-123 had no such effect. Furthermore, in isolated perfused rat lungs, about 80% of bolus-injected [125I]ET-1 was retained by the lungs after one passage. The retention of ET-1 was significantly inhibited by infusion of 1 microM BQ-788, but not BQ-123. These results suggest that ETB receptors play an important role in the clearance of ET-1.

Differential inhibition of the pressor effects of natural pro-endothelins by phosphoramidon in rats.

The vasopressor responses of three natural endothelins (ET-1, ET-2, ET-3) and their precursors, pro-ETs, were studied in a pithed rat preparation, which allows the profile and the potency of vasoconstrictor agents to be determined in the absence of central control of the cardiovascular system. ET-1 was found to be 4- and 8-fold more potent in raising blood pressure than ET-2 and ET-3, respectively. The immediate precursors of these isopeptides, h-pro-ET-1 (human), p-pro-ET-1 (porcine), pro-ET-2 and pro-ET-3, produced significantly smaller pressor responses than their respective ETs, when measured either as peaks or as areas under the time-effect curve. Hence, the bioavailability of h-pro-ET-1, p-pro-ET-1 and pro-ET-2, assessed on the basis of these two parameters, was approximately 50% of that of their corresponding ET, whereas the bioavailability of pro-ET-3 was only 25% that of ET-3. Phosphoramidon inhibits metallopeptidases, the enzymes that convert pro-ETs to ETs. The approximate i.v. doses of phosphoramidon reducing by 50% the pressor effects of the pro-ETs were 2.5, 0.625, less than 2.5 (this dose produced 75% inhibition) and 5 mg/kg i.v. for h-pro-ET-1, p-pro-ET-1, pro-ET-2 and pro-ET-3, respectively. In conclusion, these results indicate that the rat may have more than one pro-ET converting enzyme, each specific for one of the natural pro-ETs studied, although the alternative explanation, that there is a single enzyme with different affinities for these pro-ETs, cannot be entirely dismissed.