Distinct Endothelin Research Articles

Characterization of a new endothelin receptor ligand by in vitro assays.

Following the discovery of endothelin (), functional characterization of the three endothelin (ET) isoforms (ET-1, ET-2 and ET-3) predicted that two mammalian receptor subtypes are present: the ETA receptor that is selective for ET-1, and the ETB receptor that has equal affinity for the three isoforms (). The existence of the two distinct high-affinity ET receptor subtypes has been confirmed by cloning. Unique cDNAs that code for ETA and ETB belonging to the G-protein linked heptahelical receptor superfamily are identified in human, bovine and rat tissues (, , , ). While pharmacological studies suggest that there may be more ET receptor subtypes (), no additional homologous mammalian cDNAs have been identified.

Measurement of endothelin: clinical and research use.

In 1988, investigators led by Masaki reported the isolation, sequencing and cloning from the supernatant of cultured pig endothelial cells of the most potent constrictor described to date, which they termed endothelin (ET). The ET peptide initially found consisted of a unique sequence of 21 amino acids, including four cysteine residues, forming two intramolecular disulphide bonds. This unique primary structure translated into a peculiar pharmacological action, entailing a very potent, sustained and long-lasting pressor response when ET-1 was injected intravenously. This suggested a role of ET-1 in the control of vascular tone and in the pathogenesis of several conditions characterized by excess vasoconstriction and/or cell proliferation, such as arterial and pulmonary hypertension, congestive heart failure, atherosclerosis and coronary artery disease. A worldwide recruitment of interest by investigators rapidly established that the ET isolated was the prototype of a novel family, consisting of three distinct ET isopeptides, ET-1, ET-2 and ET-3, which have very similar amino acid sequences and also remarkable similarities to the sarafotoxins, which are peptides isolated from the venom of the Israeli burrowing asp Actraspis engaddensis (Fig. 1). The three ETs are encoded by three distinct genes, and are synthesized as preproproteins of about 200 amino acids sharing a high degree of sequence homology, and a two-step processing pathway. They have different tissue distributions and thus are likely to have multiple biological actions. The major isopeptide synthesized by the human endothelium and present in greatest concentration in the blood is ET-1. However, ET-1 is predominantly released toward the vascular tunica media and therefore its concentrations are likely to be much higher in tissues, where they can be suf®ciently high to activate local receptors, than in plasma where it is in the picomolar range. In addition, ET-1 can be synthesized in cell types other than the endothelial cells, such as vascular smooth muscle cells (VSMC) and adrenocortical zona glomerulosa (ZG) cells. Therefore, it is likely that ET-1 may play a biological role far beyond that of local regulation of vascular tone. The application of molecular research techniques and the development of ET-1 receptor antagonists and of inhibitors of ET-1 biosynthesis have enabled investigators to gather a considerable amount of knowledge on the physiological and pathophysiological role of ET-1. For instance, there is strong evidence supporting a role of ET-1 in the regulation of endocrine glands, including adrenal cortex and medulla, testis, ovary, cardiac atria, pituitary, pancreatic b-cells and parathyroid. A major issue in research has been the development of speci®c assays for measurement of endothelins in blood and tissues. ET-1 has been the most widely investigated and here we focus on knowledge available on this isopeptide, the methods for its measurement and the available information on its level in pathophysiological conditions.

Effects of endothelin-1 on capsaicin-induced nociception in mice

The influence of endothelin-1 on nociception induced by capsaicin was assessed in the mouse hindpaw. Local endothelin-1 injection (1 to 20 pmol/paw) 30 min prior to ipsilateral injection of capsaicin (0.1 μg/paw) increased, in a graded fashion, the time spent licking the injected paw. Maximal hyperalgesia was obtained with 10 pmol/paw of endothelin-1 (capsaicin-induced hindpaw licking time increased from 43±3 s to 114±7 s, n=6), but no hyperalgesia was evident following 30 pmol/paw of endothelin-1. The selective endothelin ET B receptor agonists sarafotoxin S6c (≤30 pmol/paw) and IRL 1620 (i.e., Suc[Glu 9,Ala 11,15]endothelin-1-(10–21); ≤100 pmol/paw) failed to induce hyperalgesia. Local treatment with BQ-123 (i.e., cyclo[ dTrp- dAsp-Pro- dVal-Leu] 1 nmol/paw selective endothelin ET A receptor antagonist), 10 min before endothelin-1 (10 pmol/paw), fully blocked the hyperalgesic response, whereas similar treatment with the selective endothelin ET B receptor antagonist BQ-788 (i.e., N- cis-2,6-dimethylpiperidinocarbonyl- l- γ-methylleucyl- d-1-methoxycarboyl- d-norleucine) was ineffective. Intravenous injection of bosentan (17 and 52 μmol/kg a non-peptidic mixed endothelin ET A/ET B receptor antagonist) or BMS 182874 (i.e., 5-[dimethylamino]- N-[3,4-dimethyl-5-isoxazolyl]-1-naphthalenesulphonamide; 10 and 30 μmol/kg; a non-peptidic selective endothelin ET A receptor antagonist), 1 h before endothelin-1, inhibited its hyperalgesic effect in a graded fashion and abolished the response at the higher doses. None of the antagonists modified nociception induced by capsaicin alone or the hyperalgesia induced by local injection of 5-hydroxytryptamine (5-HT; 2 nmol/paw, 30 min before capsaicin). Hyperalgesia induced by 5-HT was abolished by simultaneous injection of endothelin-1 or the endothelin ET B receptor agonist IRL 1620 (each at 30 pmol/paw). Therefore, local endothelin-1 exerts a dual influence in this model: at low doses it causes endothelin ET A receptor-mediated hyperalgesia (i.e., it potentiates capsaicin-induced nociception), whereas at higher doses it induces an anti-hyperalgesic effect against 5-HT which seems to be mediated via distinct endothelin ET (possibly ET B) receptors.

Clinical Implications of a Sandwich Enzyme Immunoassay for Big Endothelin-1

Endothelin (ET) is an endothelial-derived peptide that exerts potent vasoconstrictive activity in vivo and in vitro. An increasing body of evidence suggests that ET may have a significant role in the pathophysiological processes leading to cardiovascular disease states. Although it is not certain at this point whether ET is primarily responsible for initiating these events, sufficient evidence exists to conclude that ET is involved as a mediator or cofactor in these disease states. ET was first described by Yanagisawa et al. in 1988 (1). The pre-propeptide is cleaved to form big ET (39 amino acids) and then is further cleaved by an endothelin-converting enzyme (ECE) to form the 21-amino acid active peptide. Three pharmacologically distinct isopeptides (ET-1, ET-2, and ET-3) exist, derived from three different pre-propeptides that are encoded by separate genes (2). The ET-1 isopeptide is primarily responsible for the observed vasomotor effects. Two distinct ET receptors exist: ET-A and ET-B, the latter being associated with both vasoconstrictive and vasodilatory effects (3). In addition to its vasoactive effects, ET has been shown to affect cellular proliferation. Endothelial cells form a single-cell-thick layer that lines the entire vasculature. The continuous production and release of endothelin from the endothelial cells underscores the significant role of this peptide in the regulation of vascular tone and growth in both physiological and pathophysiological states. ET-1 has been implicated in the pathogenesis of numerous disease processes, particularly cardiovascular diseases. Significant ongoing research …

Inhibition by endothelin-1 of cholinergic nerve-mediated acetylcholine release and contraction in sheep isolated trachea.

1. The relative roles of ETA and ETB receptor activation on cholinergic nerve-mediated contraction and acetylcholine (ACh) release were examined in sheep isolated tracheal smooth muscle. 2. Electrical field stimulation (EFS; 90 V, 0.5 ms duration, 1 Hz, 10 s train) applied to sheep isolated tracheal smooth muscle strips induced monophasic contractile responses that were abolished by either 1 microM tetrodotoxin or 0.1 microM atropine, but were insensitive to 10 microM hexamethonium and 100 microM L-NAME. Thus, EFS-induced contractions resulted from the spasmogenic actions of ACh released from parasympathetic, postganglionic nerves. 3. As expected, sheep isolated tracheal smooth muscle preparations did not contract in response to the ETB receptor-selective agonist, sarafotoxin S6c (0.1-100 nM). However, sarafotoxin S6c caused a concentration-dependent and transient inhibition of EFS-induced contractions. The inhibitory effect induced by a maximally effective concentration of sarafotoxin S6c (10 nM; 72.1 +/- 5.7%, n = 6) was abolished in the presence of the ETB receptor-selective antagonist BQ-788 (1 microM). Contractile responses to exogenously administered ACh (10 nM-0.3 mM) were not inhibited by sarafotoxin S6c (1 or 10 nM; n = 7). 4. In contrast to sarafotoxin S6c, endothelin-1 induced marked contractions in sheep isolated tracheal smooth muscle. These contractions were inhibited by BQ-123, consistent with an ETA receptor-mediated response. In the presence of BQ-123 (3 microM), endothelin-1 produced a concentration-dependent inhibition of EFS-induced contractions (30 nM endothelin-1, 68.9 +/- 10.2% inhibition, n = 5). These responses were inhibited by 1 microM BQ-788, indicative of an ETB receptor-mediated process. Endothelin-1 was about 3 fold less potent than sarafotoxin S6c. 5. EFS (90 V, 0.5 ms duration, 1 Hz, 15 min train) induced the release of endogenous ACh (1.94 +/- 0.28 pmol mg-1 tissue, n = 12), as assayed by h.p.l.c. with electrochemical detection. EFS-induced release of ACh was inhibited to a similar extent by 100 nM endothelin-1 (47 +/- 4%, n = 9) and 10 nM sarafotoxin S6c (46 +/- 9%, n = 3). These effects of endothelin-1 on ACh release were inhibited by 1 microM BQ-788 alone (n = 4), by BQ-788 in the presence of 3 microM BQ-123 (n = 4), but not by 3 microM BQ-123 alone (n = 5). 6. In summary, sheep isolated tracheal smooth muscle contains two anatomically and functionally distinct endothelin receptor populations. ETA receptors located on airway smooth muscle mediate contraction, whereas ETB receptors appear to exist on cholinergic nerves that innervate tracheal smooth muscle cells and mediate inhibition of ACh release. The inhibitory effect of ETB receptor stimulation on cholinergic neurotransmission is in stark contrast to the enhancing effects hitherto described in the airways.

Pharmacological evidence for the presence of three distinct functional endothelin receptor subtypes in the rabbit lateral saphenous vein.

1. Contraction of the rabbit isolated saphenous vein is mediated by a heterogeneous endothelin (ET) receptor population. This study has characterized these receptor subtypes by use of several pharmacologically distinct ET receptor agonists and antagonists. 2. ET-1, ET-3, sarafotoxin S6c (STXc) and [Ala3,11]ET-1 produced biphasic, concentration-dependent contractions of the saphenous vein, responses which were best fitted by a two-site model comprised of a high (pM) and a low (nM) affinity site. In contrast, IRL 1620 only recognized one of these sites. ET(16-21) was devoid of contractile activity. ET-1, ET-3 and STXc were equipotent at the high affinity site (pD2s of 12.0 +/- 0.2, 12.2 +/- 0.2 and 12.3 +/- 0.3) indicating that this site had the characteristics of an ETB receptor. In contrast, the low affinity site had the functional characteristics of an ETc receptor since the pD2s for ET-3 (10.2 +/- 0.3) and STXc (10.6 +/- 0.3) were significantly greater than that for ET-1 (9.1 +/- 0.1). These contractile responses were insensitive to BQ-123, confirming that ETA receptors were not involved in mediating this effect. 3. SB 209670 differentially antagonized the high affinity phases of the isopeptide concentration-response curves in a fashion dependent on the competing agonist: relative to the KB obtained against STXc (0.15 nM). SB 209670 was 10 fold less potent when ET-1 was used as the competing agonist. This differential effect was not evident at the low affinity site (KB = 38 nM). SB 209670 produced parallel, concentration-dependent rightward shifts in the concentration-response curve to STXc Ro 47-0203 was approximately 1 to 2 orders of magnitude less potent than SB 209670 at inhibiting the high affinity component of the concentration-response curve to STXc, whereas BQ-788 and Ro 46-2005 were approximately 3 orders of magnitude less potent than SB 209670. In addition to RES-701 and BQ-123, the high affinity site was insensitive to PD 142893 suggesting that it may represent an ETB2 receptor. Ro 47-0203 and SB 209670 were equipotent at inhibiting the low affinity component of the STXc concentration-response curve. Although Ro 46-2005, BQ-788, PD 142893 and RES-701 produced significant antagonism at the low affinity site, they were at least ten fold less potent than SB 209670. 4. ET-1, ET-3 and STXc produced endothelium-dependent vasorelaxation in the precontracted saphenous vein. Antagonist IC50s were approximated as being: SB 209670, 3 nM; BQ-788 and RES 701,300 nM; Ro 46-2005 and PD 142893, 3 microM; BQ-123, > 10 .M, consistent with vasorelaxation being mediated by an ETB1 receptor.5. In summary, three pharmacologically distinct ET receptor subtypes have been identified in the rabbits aphenous vein. Two contractile receptors are present on the vascular smooth muscle, a high affinity site with the characteristics of an ETB2 receptor and a distinct lower affinity site with the characteristics of an ETc receptor. In addition, an ETBI receptor is present on the endothelium which mediates the vasodilator actions of this peptide family.

Contractile actions of endothelins in rat gastric body: evidence for receptor subtypes and involvement of prostaglandin E 2

Endothelin-1 produced a phasic contraction in the longitudinal muscle preparation isolated from the rat gastric body, but produced a sustained contraction in the circular muscle preparation. Indomethacin, a cyclo-oxygenase inhibitor, decreased the endothelin-1-induced contraction of the longitudinal preparation, but did not affect the endothelin-1-induced contractions of the circular muscle. In the absence of indomethacin, the maximal contractile tension ( E max) and the concentration producing a half-maximal contraction (EC 50) induced by endothelin-3 in the longitudinal muscle preparations were smaller than those for endothelin-1, whereas in the circular muscle preparations there were no significant differences between the values (EC 50, E max) for endothelin-1 and endothelin-3. In the presence of indomethacin, endothelin-3-induced contraction of the longitudinal muscle preparation is more potent than that of endothelin-1. SC-19220, a prostaglandin E 2 receptor antagonist, significantly decreased endothelin-1-induced contraction of the longitudinal preparation. Prostaglandin E 2 produced a concentration-dependent contraction in the longitudinal preparation, but had no effects in the circular muscle preparation. Endothelin-1 (10 −8 M) significantly increased the release of immunoassayable prostaglandin E 2 from rat gastric smooth muscle. These results point to the existence of distinct endothelin receptor subtypes in the smooth muscle of rat gastric body, and a potential role of endothelin-1 in regulating gastric motility. Moreover, one of the endothelin receptor subtypes is related to the production of prostaglandin E 2.

Endothelins: homeostatic and compensatory actions in the circulatory and endocrine systems.

I. Introduction ENDOTHELIN (ET) is a potent vasoconstrictive peptide which was originally isolated from the conditioned medium of cultured endothelial cells (1). It comprises 21 aminoacid residues, including four cysteine residues that form two disulfide bonds (see Fig. 1). ET elicits a sustained pressor response when administrated intravenously, which suggests its importance in blood pressure maintenance and generation of vasospasm. Accordingly, many investigators have been interested in this peptide. Analysis of ET genes revealed the existence of three distinct ET genes that encode different mature ET sequences, designated endothelin-1 (ET-1), ET-2, and ET-3 (Fig. 1) (2). Two and six amino acid residues of ET-1 are replaced in ET-2 and ET-3, respectively. There are no species differences among isoforms of human, porcine, rat, bovine, or dog. Interestingly, the structure of snake venom sarafotoxins are very similar to those of ETs. In endothelial cells, ET-1 is predominant, but ET-2 and ET-3 are not dete...

Functionally Distinct Endothelin B Receptors in Vascular Endothelium and Smooth Muscle

IRL 1620 {Suc-[Glu 9,Ala 11,15]-ET-1 (8-21)} (0.1 nM - 1μM), a novel ET B-selective endothelin (ET) agonist, produced endothelium-dependent relaxations in precontracted rabbit mesenteric artery (2 nM, EC 50) and endothelium-independent contractions in porcine coronary artery (18 nM, EC 50). ET-3 (0.1 nM-10 nM) produced qualitatively similar responses in the two tissues. The maximal contractions induced by IRL 1620 or ET-3 were substantially smaller (<20%) than that produced by ET-1. BQ-123 (1 μM), an ET A receptor antagonist, inhibited responses to ET-1 without affecting IRL 1620- or ET-3-induced responses in either tissue. Thus functionally distinct ET B receptors mediating vasodilator and vasoconstrictor effects are located on the vascular endothelium and smooth muscle, respectively. The overall effect of ET B receptor activation on vascular tone is tissue-specific and presumably reflects differing receptor distribution at the two sites.

Role of endothelin ET B receptors in bronchoconstrictor and vasoconstrictor responses in guinea pigs

In anesthesized and ventilated guinea-pigs, intravenous injections of endothelin (ET)-1 (0.5 nmol/kg), ET-3 (0.5 nmol/kg), and [Ala 1,3,11,15]ET-1 (20 nmol/kg), an ET B-selective receptor agonist, induced bronchoconstrictor and transient vasoconstrictor responses. Only the ET-1-induced transient vasoconstriction was followed by a secondary sustained pressor response. The ET A-selective receptor antagonists, BQ-123 (1 mg/kg i.v.), attenuated only the sustained pressor response. These results indicate that the bronchoconstrictor and transient vasoconstrictor responses to endothelins in guinea-pigs aer mediated by ET B receptors, whilst the sustained pressor response is mediated by ET A receptors. The thromboxane A 2 receptor antagonist, L-670,596 (0.5 mg/kg, i.v.) and a high dose of BQ-123 (30 mg/kg i.v.) abolished the bronchoconstriction only without affecting the transient pressor response to endothelin isopeptides. These results suggest that the ET B-mediated bronchoconstriction depends on thromboxane A 2 formation. The different sensitivity of these ET B-mediated transient responses to BQ-123 suggests the possible existence of distinct ET B receptor subtypes.

Vasoconstriction in the rat kidney induced by endothelin-1 is blocked by PD 145065.

We have previously shown that the receptors mediating the renal and systemic vasoconstrictor effects of endothelin-1 (ET-1) are of two distinct endothelin receptor subtypes. Here, we evaluate the effect of PD 145065, a nonselective endothelin receptor antagonist, on the renal vasoconstrictor effects of ET-1 in the rat kidney. ET-1 induced concentration-dependent increases in perfusion pressure in the isolated perfused kidney of the rat. The ETA receptor-selective antagonists BQ-123 (10 microM) and FR 139317 (10 microM) lowered the ET-1-induced rise in perfusion pressure by 57% and 61%, respectively, at 3 x 10(-10) M ET-1. By comparison, the ET-1-induced vasoconstriction was fully antagonized (96% inhibition) by PD 145065 (10 microM). In the anesthetized rat, ET-1 produced a dose-dependent increase in mean arterial pressure, which was attenuated by PD 145065. The initial depressor response to ET-1 was completely blocked by PD 145065, as were the reduction in renal blood flow and increase in renal vascular resistance induced by ET-1. These results suggest that both the ETA and a non-ETA receptor subtype play an important role in mediating the vasoconstrictor effects of ET-1 in the rat kidney.

Pharmacological evidence for distinct endothelin receptors in guinea-pig bronchus and aorta.

The ETA receptor antagonist, BQ-123 (1 microM) potently antagonized endothelin-1 (ET-1) concentration-response curves in guinea-pig aorta (pKB = 7.1). However, 10 microM BQ-123 was without effect on ET-1-induced contractions in guinea-pig bronchus. The ETB-selective agonist, sarafotoxin S6c did not contract the aorta but was a potent and effective contractile agonist in the bronchus. BQ-123 (10 microM) was without effect on sarafotoxin S6c-induced contractions in the bronchus. These data provide evidence for distinct endothelin receptors in guinea-pig aorta and bronchus, which appear to be predominantly of the ETA and non-ETA, perhaps ETB, subtypes, respectively.

Different distribution of endothelin receptor subtypes in pulmonary tissues revealed by the novel selective ligands BQ-123 and [Ala 1,3,11,15]ET-1

We have demonstrated the different distribution of two distinct endothelin (ET) receptor subtypes in porcine pulmonary tissues using a radioligand binding assay. The clear differentiation of the subtypes was made possible by the discovery of two compounds, BQ-123 and [Ala1,3,11,15]ET-1 (4AlaET-1), that are highly selective for ETA and ETB receptors, respectively. In the bronchus and lung parenchyma, BQ-123 inhibited 65% and 30% of [125I]ET-1 binding on the sensitive sites, while 4AlaET-1 displaced 25% and 60%, respectively. The combination of the two compounds completely inhibited ET-1 binding in both tissues. An autoradiographic study of [125I]ET-1 binding using BQ-123 and 4AlaET-1 also supported the different localization of two ET receptor subtypes in pulmonary tissues. In particular, the blood vessels and bronchi are rich in ETA, but the lung parenchyma is rich in ETB.

Endothelin: localization, synthesis, activity, and receptor types in human penile corpus cavernosum.

The localization, synthesis, and activity of endothelin and the receptor types mediating its effects in penile corpus cavernosum were investigated in whole tissue and in cultured cells derived from this tissue. With immunocytochemistry, utilizing an antiendothelin 1 (ET-1) monoclonal antibody, endothelin-like immunoreactivity was localized intensely in the endothelium and to a lesser degree in the trabecular smooth muscle. Human corpus cavernosum endothelial cells in culture expressed preproendothelin 1 mRNA, as determined by Northern blot analysis. Significant amounts of endothelin-like immunoreactivity were measured by radioimmunoassay in the supernatants of corpus cavernosum endothelial cells in culture. Endothelins are potent constrictors and caused long-lasting contractions of corporeal strips in organ chambers. Equilibrium binding analysis of endothelins to their receptor sites revealed high-affinity, specific, and saturable binding of labeled endothelins to corporeal membranes. Competition binding experiments demonstrated receptors with high affinity for ET-1 and -2 and low affinity for ET-3 and another, less abundant, set of receptors with high affinity for ET-1, -2, and -3. Affinity labeling of endothelins to corporeal membranes, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, revealed that ET-1 and -2 cross-linked specifically to three different molecular mass components (75, 52, and 34 kDa). ET-3 bound only to the 34-kDa component. It is concluded that human corpus cavernosum endothelium has the ability to synthesize and release endothelin, that endothelins contract corporeal smooth muscle, and that at least two distinct endothelin receptors may exist and are differentiated by their affinity for ET-3.

Ala 1,3,11,15]endothelin-1 analogs with ET B agonistic activity

A linear peptide analog of endothelin (ET)-1, [Ala 1,3,11,15]ET-1 (4AlaET-1), and its truncated peptide analogs were synthesized to study the structural requirements of ET-1 for the recognition of ETs-nonselective ET B receptors. ET-1 exhibited sub-nanomolar binding to two distinct ET receptor subtypes (ET A and ET B), but 4AlaET-1 bound to ET B with an affinity 1,700 times higher than that seen during binding to ET A. The truncated linear peptides 4AlaET-1(6–21), 4AlaET-1(8–21) and N-acetyl-4AlaET-1(10–21) still had high affinity for ET B, whereas 4AlaET-1(6–20) and 4AlaET-1(11–21) displayed remarkably reduced affinity for ET B. Therefore, ET-1 requires the Glu 10-Trp 21 sequence for ET B binding, but not the disulfide bridges. These ET B-binding peptides elicit endothelium-dependent vasorelaxation of porcine pulmonary arteries in parallel with the binding affinity for ET B, suggesting that they are ET B agonists.

Binding Characteristics of Endothelin Isoforms (ET-1, ET-2, and ET-3) in Vascular Smooth Muscle Cells

The existence of distinct endothelin (ET) receptor subtypes has been reported in several tissues. In the present study, we investigated the binding characteristics of the three endothelin isoforms to cultured rat aortic smooth muscle cells. [125I]ET-1, [125I]ET-2, and [125I]ET-3 bound to an apparent single class of binding sites with apparent dissociation constants (Kd) of 111, 123, and 1410 pM and binding capacities (Bmax) of 54.1, 46.0, and 7.9 fmol/10(6) cells, respectively. The binding of the three radiolabeled endothelin isoforms was equally inhibited by ET-1 and ET-2. ET-3 was more effective in competing with [125I]ET-3 than with [125I]ET-1 or [125I]ET-2. In contrast to ET-1 and ET-2, the binding of ET-3 was reversible. Furthermore, 18 h of pre-exposure of the cells to 1 nM ET-1 or ET-2 decreased the ET-1 binding capacity, whereas ET-3 (10 nM) was ineffective. ET-3 binding characteristics were not affected by pretreatment of the cells with any of the endothelin isoforms. These results suggest the presence of two distinct endothelin receptor subtypes in rat aortic smooth muscle cells. The ET-1 and ET-2 preferring receptor (80-85%), sensitive to downregulation or internalization, elicits an irreversible binding. The second subtype (15-20%) binds the three endothelin isoforms with the same affinity in a reversible manner, and is insensitive to downregulation or internalization.

Evidence for Distinct Endothelin Receptors in the Pulmonary Vascular Bed in vivo

The present study was undertaken to investigate the effects of endothelin (ET) isopeptides on the pulmonary vascular bed of the intact, spontaneously breathing cat under conditions of constant pulmonary blood flow and left atrial pressure. When pulmonary vasomotor tone was actively increased by intralobar infusion of U46619, intralobar bolus injections of ET-1 (1 micrograms), ET-2 (1 micrograms), and ET-3 (3 micrograms) produced marked reductions in pulmonary and systemic vascular resistances. The pulmonary vasodilator response to each ET isopeptide was not altered by atropine (1 mg/kg i.v.), indomethacin (2.5 mg/kg i.v.), or ICI 118551 (1 mg/kg i.v.), but was significantly inhibited by an intra-arterial (i.a.) infusion of glybenclamide at 5 mg/kg. This dose of glybenclamide significantly inhibited the decrease in lobar arterial and systemic arterial pressures in response to intralobar injection of pinacidil (30 and 100 micrograms), whereas the pulmonary vasodilator responses to acetylcholine (0.03 and 0.1 micrograms) and prostaglandin I2 (0.1 and 0.3 micrograms) were not altered. The systemic vasodilator response to each ET isopeptide was not changed by glybenclamide or by the other blocking agents studied. The present data demonstrate for the first time that ET-1, ET-2, and ET-3 dilate the pulmonary vascular bed in vivo. The present data suggest that the pulmonary vasodilator response to ET isopeptides depends, in part, on activation of potassium channels and is mediated differently from the systemic vasodilator response to these substances. Contrary to earlier work, the present data indicate the pulmonary vascular response to ET isopeptides depends on the pre-existing level of pulmonary vasomotor tone. Furthermore, the present data suggest that in the lung ET-1, ET-2, and ET-3 may serve as endogenous agonists for potassium channels, a newly described vasodilator mechanism in the pulmonary vascular bed of intact adult animals.

Ca 2+ signaling by distinct endothelin peptides in glomerular mesangial cells

Ca 2+ signaling by peptides of the endothelin (ET) gene family was studied in cultured glomerular mesangial cells. In addition to the increase in cytosolic free [Ca 2+] ([Ca 2+] i) previously described for ET-1, we also observed that ET-2, ET-3, and sarafotoxin S6b generate similar [Ca 2+] i waveforms but with dissimilar potencies and kinetics. The prepro form of ET-1 was inactive, suggesting that mature ET peptides are constrained in an inactive conformation within the preproET species. ET isopeptides caused both release of Ca 2+ from intracellular stores and Ca 2+ influx via a voltage- and dihydropyridine-insensitive pathway. ET-mediated Ca 2+ influx was independent of the increase in [Ca 2+] i. Activation of protein kinase C inhibited ET-induced Ca 2+ signaling, whereas addition of ET to protein kinase C-depleted cells resulted in enhanced [Ca 2+] i waveforms. Mesangial cells also demonstrated a marked adaptive desensitization response to ET. These data demonstrate that Ca 2+ signaling is a common response to different ET peptides in glomerular mesangial cells and that activation of protein kinase C down-regulates these Ca 2+ signals.

Differential activities of two distinct endothelin family peptides on ileum and coronary artery

A synthesized mouse vasoactive intestinal contractor peptide, which belongs to a novel member of the endothelin family, induced a prolonged contraction in mouse ileum as well as porcine coronary artery in vitro. Studies comparing the effects of vasoactive intestinal contractor and endothelin on different tissues revealed that the maximum ileum contraction of vasoactive intestinal contractor was much higher than that of endothelin in both guinea pig and mouse systems, but that the vasoconstriction activity of vasoactive intestinal contractor was weaker than that of endothelin in porcine artery. These resuts show that vasoactive intestinal contractor might be a novel gastrointestinal hormone.