Endothelin Receptor Subtypes Research Articles

Endothelin-B receptors mediate intimal hyperplasia in an organ culture of human saphenous vein

Objective: Although a number of pharmacologic agents have been shown to reduce intimal hyperplasia in animal models of restenosis, to date no systemic agent has conclusively been shown to be effective in humans. Recently, considerable attention has been directed towards endothelin (ET), a potent vasoconstrictor and a powerful mitogen for vascular smooth muscle cells, as a mediator of intimal hyperplasia. Endothelin-1 has been shown to be mitogenic for human saphenous vein smooth muscle cells, and expression also is elevated in human vein graft stenosis. The aim of this study was the investigation of whether ET receptor antagonists can attenuate neointima formation in a laboratory model of vein graft intimal hyperplasia and the determination of whether the effects are mediated by a specific ET receptor subtype. Methods: We used an organ culture of human saphenous vein, a well-validated model of vein graft intimal hyperplasia. Paired segments of human long saphenous vein were cultured with and without the following antagonists: bosentan, a nonselective ET receptor antagonist; BQ 123, a specific endothelin-A antagonist; or BQ 788, a specific endothelin-B (ET B) antagonist. After 14 days in the culture, the segments were fixed and processed and the sections were immunostained to facilitate the measurements of neointimal thickness with a computerized image analysis system. Results: The nonselective antagonist bosentan and the ET B selective antagonist BQ 788 significantly reduced neointima formation by 70% ( P = .001) and 50% ( P = .03), respectively, but the ET A antagonist BQ 123 had no significant effect on the reduction of neointima formation ( P = 1.0). Conclusion: The results of this study imply an important role for ET as a mediator of human vein graft intimal hyperplasia and imply further that a specific ET B antagonist may have a therapeutic potential for the prevention of vein graft stenosis. (J Vasc Surg 1998;28:695-701.)

Involvement of the endothelin receptor subtype A in neuronal pathogenesis after traumatic brain injury

Endothelin-1 (ET-1) is a 21 amino acid peptide that has been closely linked to cerebral vasospasm and more recently to oxidative stress after traumatic brain injury. In this study, we have examined the effects of the endothelin receptor subtype A antagonist, Ro 61-1790, on acute cortical neuronal injury and delayed neuronal death in the cerebellum after mild traumatic brain injury. Rats were administered Ro 61-1790 or vehicle for 24 h after injury and euthanized at 1 day, 3 days, or 7 days. Heat shock protein70 (HSP70), a marker of neuronal stress/injury, was immunolocalized in the cortex. Induction of heme oxygenase-1 (HO-1) and enhanced immunoexpression of the complement C3bi receptor, both of which are indicators of cerebellar glial reactivity, and Purkinje cell loss were evaluated in the cerebellum. There was maximal induction of HSP70 in cortical neurons at 24 h postinjury in all animals. Drug treated animals showed significantly fewer HSP70 labeled cortical neurons at this time point. There were fewer reactive glia in the cerebellum of drug treated animals as compared to vehicle controls at 3 days postinjury. However, at 7 days postinjury glial reactivity and Purkinje cell loss were similar in both groups. These findings demonstrate that Ro 61-1790, when administered for the first 24 h postinjury, limits acute neuronal injury in the cortex, transiently influences glial reactivity in the cerebellum, and does not attenuate delayed Purkinje cell death. The latter finding may reflect the duration of infusion of the drug.

Endothelin receptor subtypes in the microvillous trophoblastic membrane of early gestation and term human placentas.

The 125I-labeled endothelin-1 ([125I]ET-1) binding sites in microvillous membranes from early gestation and term human placentas were investigated. The Kds for [125I]ET-1 binding to early gestation (68 +/- 15 pmol/l) and term (45 +/- 8 pmol/l) microvilli (n = 4) were not significantly different. The density of binding sites decreased significantly, from 243 +/- 80 fmol/mg protein in early gestation microvilli to 54 +/- 10 fmol/mg protein in term microvilli. The endothelin (ET) receptor (ET-R) subtype profiles were determined by competition binding studies with unlabeled ET-1, ET-3, and selective agonists and antagonists for ETA-R and ETB-R. In early gestation placental microvilli, we observed the presence of 72%) ETB-R, (mainly ETB2-R subtype), and 28%. ETA-R. Only ETB-R (mainly the ETB2-R subtype) was present in term placental microvilli. We suggest that the ETB-R on the placental microvillous membrane is involved in specific trophoblastic functions and may play a major role in ET clearance by modulating the amounts of ETs in the maternal intervillous blood space.

Short-term haemodynamic effects of BQ-123, a selective endothelin ETA-receptor antagonist, in chronic heart failure

Plasma concentrations of endothelin-1 (ET1), a potent vasoconstrictor peptide, are raised in patients with chronic heart failure, correlate with the symptomatic and haemodynamic severity of heart failure, and predict prognosis. The vasoconstrictor action of ET1 is mediated through two high-affinity endothelin receptor subtypes on smooth muscle, denoted ETA and ETB. ETB receptors are also present on the vascular endothelium where they mediate vasodilatation via nitric oxide and/or prostaglandins.

Endothelin receptor subtypes and their functional relevance in human small coronary arteries.

1. The potent constrictor peptide endothelin (ET) has been implicated in various cardiovascular disorders including myocardial infarction and atherosclerosis. We have investigated the nature of ET receptor subtypes present on human small coronary arteries. 2. Small coronary arteries were mounted in a wire-myograph for in vitro pharmacology. To investigate the ET receptor subtypes present in different segments of the coronary vascular tree, arteries were grouped according to internal diameter. Responses in arteries with small internal diameters (mean 316.7+/-7.9 microm; Group B) were compared to those in larger arteries (mean 586.2+/-23.1 microm; Group A). 3. ET-1 consistently and potently contracted arteries from Group A and B, with EC50 values of 1.7 (0.9-3.2) nM (n=15) and 2.3 (1.4-4.2) nM (n=14), respectively. No correlation was observed between ET-1 potency and internal diameter. The response to ET-1 was potently antagonized by the selective ET(A) receptor antagonist PD156707 in both Group A and Group B, yielding pA2 values of 8.60+/-0.12 (n=4-6) and 8.38+/-0.17 (n=4-6), respectively. Slopes from Schild regression were not significantly different from unity. 4. In contrast to ET-1, individual responses to ET-3 were variable. While all arteries from Group A responded to ET-3 (EC50 approximately 69 (23-210) nM) (n=12), no response was obtained in 5 of the 14 tested in Group B. Of those responding, many failed to reach a maximum at concentrations up to 1 microM. ET-1 was more potent than ET-3 in all arteries tested. A biphasic ET-3 response was observed in 8 arteries suggesting that a small ET(B) population was also present in some patients. The selective ET(B) receptor agonist sarafotoxin S6c had little or no effect up to 10 nM (n=4-6). 5. Responses to ET-1 and ET-3 were unaffected by removal of the endothelium in arteries from both groups suggesting a lack of functional, relaxant ET(B) receptors on endothelial cells (n=5). 6. Using autoradiography, specific high density binding of the non-selective, ET(A)/ET(B) ligand [125I]-ET-1 and selective ET(A) ligand [125I]-PD151242 was detected on the vascular smooth muscle layer of small intramyocardial coronary arteries (n=5). In contrast, little or no binding of the selective ET(B) receptor ligand [125I]-BQ3020 was observed (n=5). Similarly, [125I]-ET-1 binding to vascular smooth muscle was absent in the presence of the selective ET(A) receptor antagonist PD156707. 7. We conclude that human small epi- and intramyocardial coronary arteries express predominantly ET(A) receptors and it is these receptors which mediate ET-induced contractions. A constrictor ET(B) receptor population may exist in some patients. However, these receptors may have a limited role as contractions to ET-1 can be blocked fully by the selective ET(A) receptor antagonist PD156707.

Functional Role for Glycosylated Subtypes of Rat Endothelin Receptors

Glycosylation of endothelin (ET) receptors was found to occur in rat cerebellar and atrial membranes. Specifically, we investigated whether the ETAand ETBreceptor subtypes differed in their sensitivity to deglycosylation treatment and whether the two affinity states (nanomolar and picomolar) observed in each receptor subtype reflect differences in glycosylation states. Pretreatment of cerebellar or atrial membranes with endoglycosidase H (endo H) caused a marked decrease in the number of maximal binding sites that bind ligand with nanomolar affinity, whereas ligand affinity remained the same. The picomolar-affinity binding sites were not affected by endo H. The use of specific antagonists indicated that the receptor subtype most likely to be influenced by glycosylation is ETA.We suggest that in both cerebellar and atrial membranes, the carbohydrate chains of the ETAreceptor contribute to the binding of ligand to the nanomolar-affinity binding sites, but not to the picomolar-affinity binding sites.

Pericardial mesothelial cells produce endothelin-1 and possess functional endothelin ET B receptors

We investigated the endothelin production and endothelin receptor activity of pericardial mesothelial cells obtained from spontaneously hypertensive rats (SHR) and Wistar–Kyoto (WKY) rats. The pericardial mesothelial cells were maintained in vitro and the production of endothelin-1 by these cells was evaluated by using a sensitive sandwich-type enzyme immunoassay for endothelin-1 and big endothelin-1. Endothelin receptor subtypes were pharmacologically analyzed by measuring the changes of intracellular Ca 2+ concentration ([Ca 2+] i ) in pericardial mesothelial cells. Mesothelial cells from both strains produced more immunoreactive endothelin-1 than big endothelin-1. The production of immunoreactive endothelin-1 progressively increased in a culture time-dependent manner. The amount of immunoreactive endothelin-1 detected after 72 h in pericardial mesothelial cells of SHR was significantly ( P<0.05) higher than that in the cells of WKY rats (SHR: 196.7±19.1 pg/10 6 cells; WKY: 122.7±10.6 pg/10 6 cells). Endothelin-1 and endothelin-3 induced elevation of [Ca 2+] i in pericardial mesothelial cells. The selective agonist of the endothelin ET B receptor, sarafotoxin S6c, also induced elevation of [Ca 2+] i . The endothelin- and sarafotoxin S6c-induced elevations of [Ca 2+] i in pericardial mesothelial cells from SHR were greater than those in mesothelial cells from WKY rats. The endothelin ET B receptor antagonist, IRL 1038 ([Cys 11,Cys 15]endothelin-1-(11-21)), significantly inhibited the endothelin-1- and endothelin-3-induced changes in [Ca 2+] i . The endothelin ET A receptor antagonist, FR1393171 (( R)2-[( R)-2-[( S)-2-[[1-(hexahydro-1 H-azepinyl)]carbonyl]ammino-4-methylpentanoyl]amino-3-[3-(1-methyl-1 H-indoyl)]propionyl]amino-3-(2-pyridyl)propionic acid), did not affect these changes. From these results, pericardial mesothelial cells from both SHR and WKY rats shared the ability to produce endothelin-1 spontaneously in culture, although consistently greater production was detected in cultures of SHR origin. Moreover, pericardial mesothelial cells of SHR origin may have increased numbers of endothelin ET B receptors and/or a more active signal transduction system compared with those of WKY rats. These results suggest that endothelin-1 may play an important role in the pericardium in an autocrine and/or paracrine fashion.

A028 Increasing the expression of endothelin converting enzyme and endothelin receptor subtypes in human myocardium with congestive heart failure

A028 Increasing the expression of endothelin converting enzyme and endothelin receptor subtypes in human myocardium with congestive heart failure

Distribution of endothelin-1 (ET) receptors (ET A and ET B) and immunoreactive ET-1 in porcine saphenous vein–carotid artery interposition grafts

Proliferation of vascular smooth muscle cells (VSMC) is a principal event in neointima formation in saphenous vein–coronary artery bypass grafts. Since endothelin-1 (ET-1) promotes VSMC replication and ET-1 receptor antagonists inhibit neointima formation in arterial injury models, it is reasonable to propose that ET-1 may be involved in neointima formation in vein grafts. However, it is not known what alterations of ET-1 and its receptors (if any) occur in vein grafts. The objective of this study, therefore, was to investigate the distribution of ET-1 and ET-1 receptor subtypes (ET A and ET B) in porcine vein grafts. Unilateral interposition saphenous vein grafting was performed by end to end anastomosis after excision of a segment of carotid artery in Landrace pigs. One month after surgery, vein grafts, ungrafted saphenous veins and carotid arteries were excised, ET-1 immunoreactivity identified by immunocytochemistry and ET A and ET B receptor subtypes studied using autoradiography. In vein grafts, there was a greater density of ET A compared to ET B receptors in both the tunica media and neointima. ET A binding in the tunica media of ungrafted saphenous vein was greater than that in the carotid artery or vein grafts, but greater in the vein graft compared to the carotid artery. Immunoreactive ET-1 was located in endothelial cells and throughout the neointima of the vein graft. Dense ET-1 binding (to both ETA and ET B receptors) was also associated with microvessels in the adventitia within the graft. In vein grafts, there was strong ET B binding to neutrophils which were present in high numbers at the subendothelium and within the adventitia. It is concluded ET A receptors may play a role in vein graft thickening at the medial and neointimal VSMC level, whereas ET B receptors may play a role in microangiogenesis. The higher levels of ET A receptors in the tunica media of ungrafted saphenous vein relative to the carotid artery and vein graft may also render this conduit susceptible to neointima formation. These data indicate that studies on the effect of ET receptor antagonists on the pathobiology of vein graft disease is warranted.

Expressions of endothelin receptor subtype proteins and mRNA in human liver

Expressions of endothelin receptor subtype proteins and mRNA in human liver

Depressor responses to endothelin-1 into the superior colliculus of rats: predominant role of endothelin ET(B) receptors.

We used in vitro autoradiography to identify the endothelin-1 receptor subtype(s) in the superficial gray layer of the superior colliculus of rats. These studies showed dense binding of (3-[125I]iodotyrosyl13)-[Ala11,15]Ac-endothelin-1-(6-2 1) (BQ3020) (for endothelin ETB receptors), while tissues incubated with [125I]( N-(hexahydro-1-azepinyl)carbonyl)L-Leu(1-Me)D-Trp-D-Tyr (PD151242) (for endothelin ETA receptors) had low binding. In addition, we examined the effects of the endothelin receptor antagonists, (R)-2-[(R)-2-[(S)-2-[[1-hexahydro-1H-azepinyl)]carbonyl]amino-4-++ +methylpentanoyl]-amino-3-(2-pyridyl)propionic acid (FR139317) (endothelin ETA receptor-selective), (+)-(1S,2R,3S)-3-(2-carboxymethoxy-4-methoxyphenyl)-1-(3,4-methylenediox yphenyl)-5-(prop-1-yloxy)indane-2-carboxylic acid (SB209670) (endothelin ETA/ETB receptor non-selective) and N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-metLeu-D-1-m ethoxycarbonylTrp-D-Nle (BQ-788) (endothelin ETB receptor-selective antagonist) on the responses following administration of endothelin-1 into the superficial layer of the superior colliculus. Endothelin-1 microinjected into this nucleus causes decreases in blood pressure (control, 109 +/- 6 mmHg, n = 6; endothelin-1, 72 +/- 5 mmHg, n = 6). These effects were greatly reduced by pre-administration into the superior colliculus of BQ-788 (5 nmol/rat) or SB209670 (3 nmol/rat) (94 +/- 5 and 98 +/- 6%, respectively) but were not affected by FR139317 (5 nmol/rat) (6 +/- 3%). Therefore, together with autoradiography, our functional data showed that endothelin ETB receptors are the predominant mediators of depressor responses induced by endothelin-1 injected into the superior colliculus of the rat.

Biochemical and molecular endothelin responses to morphine sulfate infusion in conscious newborn piglets

The biochemical and molecular endothelin-1 (ET-1)responses to high dose morphine sulfate infusion were studied in conscious newborn piglets(n = 6) that received a loading dose of 100 µg/kg over 5 min followed by acontinuous i.v. infusion dose of 100 µg·kg–1·h–1 for 4 h. The controlgroup (n = 6) received equivalent volume loading and infusion doses of 5% dextrose.Blood samples were drawn serially from the femoral artery and sagittal sinus vein before (0),during (30 min, 1, 2, 3, and 4 h), and post (1 and 2 h) infusion. Five micrograms oftotal RNA obtained from brainstem tissue homogenates was analyzed by reverse transcriptase –polymerase chain reaction (RT-PCR). The amounts of mRNA encoding ET-1, and endothelinreceptor subtypes ETA and ETB, were semiquantitated using densitometricscanning. Morphine infusion resulted in elevated respiratory rate and mean arterial bloodpressure, with no effect on arterial pH, PO2, and O2saturation. Compared with the control group, morphine induced significant elevations in plasmaET-1 levels following the bolus dose (systemic: 13.2 ± 3.6 vs. 8.6 ± 2.2 pg/mL, p &lt; 0.05;sagittal sinus vein: 13.7 ± 3.4 vs. 8.2 ± 0.9 pg/mL, p &lt; 0.01). These effects lasted up to2 h after discontinuation of morphine infusion (systemic: 14.5 ± 3.4 to 18.7 ± 5.7 pg/mL vs.7.5 ± 0.8 to 9.4 ± 3.2 pg/mL, p &lt; 0.05 to p &lt; 0.01; sagittal sinus vein: 14.8 ± 2.7to 17.6 ± 2.8 pg/mL vs. 7.5 ± 1.4 to 9.4 ± 3.4 pg/mL, p &lt; 0.05 to p &lt; 0.01). TheRT-PCR assay showed a twofold (p &lt; 0.02) upregulation in ET-1 and a threefold(p &lt; 0.007) upregulation in ETA receptor mRNA expression in the brainstemof morphine-treated animals. In contrast, there was a threefold (p &lt; 0.0001)downregulation of the ETB receptor mRNA expression. The rapid and sustainedelevations in systemic arterial and sagittal sinus venous ET-1 levels suggest a role for ET-1 in themorphine-induced excitatory responses observed in newborn piglets. Upregulation ofETA receptors and downregulation of ETB receptors in the brainstem withhigh doses of morphine may indicate possible effects on cerebral vascular tone. Key words: morphine sulfate, endothelin-1, reverse transcriptase –polymerase chain reaction.

Prostaglandin endoperoxide synthase-2 contributes to the endothelin/sarafotoxin-induced prostaglandin E2 synthesis in mouse osteoblastic cells (MC3T3-E1): evidence for a protein tyrosine kinase-signaling pathway and involvement of protein kinase C.

Endothelin (ET) peptides are potent growth factors binding to G protein-coupled receptors. Sarafotoxins (S6) isolated from Atractaspis engaddensis are highly homologous to endothelins. In this study, we have investigated the effects of endothelin/sarafotoxin peptides on the prostaglandin synthesizing system in an osteoblast-like cell line, MC3T3-E1. ET-1, ET-2, beta-ET, and S6b rapidly stimulated prostaglandin E2 production within 5 min, whereas ET-3, S6a, and S6c did not. ET-1, ET-2, beta-ET, S6b, and S6a induced prostaglandin synthesis after 3 h of incubation. Antagonizing these effects with BQ-123, PD 142893, BQ-788, and S6c suggests signaling through an ET(A) receptor subtype in osteoblasts. Long-term prostaglandin synthesis was blocked by NS-398, and reduced to short-term levels by cycloheximide and actinomycin D, indicating induction of PGHS-2. There was only minor enhancement of cAMP accumulation by the agonists, which had no effect on prostaglandin synthesis. Induction of PGHS-2 was furthermore demonstrated by Northern blot analysis of PGHS-2 messenger RNA. Depletion of protein kinase C with TPA largely blunted the response. Genistein, an inhibitor of protein tyrosine kinases, also blocked long-term prostaglandin E2 formation. We conclude that in osteoblast-like MC3T3-E1 cells, ET-1, ET-2, beta-ET, S6b, and S6a peptides induce PGHS-2 through a protein tyrosine kinase-dependent and protein kinase C-dependent pathway, signaling through ET(A) receptor occupancy.

Structural studies on endothelin receptor subtype B specific agonist IRL 1620 [suc-[Glu9, Ala11,15]ET-1(8-21)] and its analogs with dipalmitoyl phosphatidylcholine vesicles by NMR spectroscopy.

IRL 1620 ¿suc-[Glu9,Ala11,15]ET-1(8-21)¿ is a potent and specific agonist for the ET(B) receptor. Five analogs of IRL 1620 were synthesized in this study. These were all C-terminal linear peptides of endothelin 1 (ET-1) comprising 14 amino acid residues and exhibiting highly potent ET(B) receptor binding affinities. The peptides consisted of three pairs and each component of the pairs differed from its partner in only the 18th residue, i.e. Asp was replaced by Gly. The replacements resulted in more than a 10-fold increase in affinity to the ET(A) receptor. The structures of these peptides were investigated in the presence of phospholipid vesicles (dipalmitoyl phosphatidylcholine) by NMR spectroscopy. By the replacement of Asp by a less bulky Gly, the C-terminal tripeptide region folded back toward the helical region, making it shorter than the Asp-substituted peptide helical region. Such a folded conformational feature may explain the increased binding affinity to ET(A) receptor.

Assessment of endothelin receptor subtype-mediated increases of [Ca2+]i in distinct rat cell types using fluorimetric imaging.

Activation of endothelin (ET) receptor subtypes by various agonists causes an increase in [Ca2+]i in different cell types. This effect can be readily monitored in a 96-well plate format by detecting 1-s fluorescence changes of cell-permeant, Ca(2+)-sensitive dyes (e.g., Calcium Green-1 AM) using a fluorimetric imaging plate reader. This device was used to assess the ET receptor subtypes in primary cultures of rat mixed neocortical neuronal/glial cells and aortic smooth-muscle cells. Pharmacologic experiments with several ET receptor agonists and antagonists indicated that the ETA receptor subtype was functionally responsive in the smooth-muscle cells and that the ETB receptor subtype had a similar role in the mixed neuronal/glial cells.

Down-regulation of endothelin-B receptor sites in cavernosal tissue of hypercholesterolaemic rabbits.

To investigate the density and distribution of endothelin-1 (ET-1) and endothelin receptor subtypes in cavernosal tissue of healthy New Zealand White (NZW) rabbits (controls) and to assess any changes in a genetic model of hypercholesterolaemia (the Watanabe rabbit). Penises were excised from six hypercholesterolaemic (HC) rabbits 6 months after birth. Low- and high-resolution autoradiography was performed on cavernosal sections using radioligands for ET-1, endothelin A (ETA) and endothelin B (ETB) receptors, and the autoradiographs analysed densitometrically. The results were compared with those from six age-matched control rabbits. Immunohistochemical localization of ET-1-like immunoreactivity was also performed on adjacent cavernosal sections. ET-1, ETA and ETB receptor binding sites were primarily localized to the smooth muscle cells of the corpus cavernosum and the endothelium lining the cavernosal spaces. There was a significant decrease in ETB receptor binding sites in cavernosal tissue from HC rabbits when compared to age-matched healthy controls. The findings suggest that ET-1 may have a role in the pathophysiology of erectile dysfunction in HC. These effects may partly be due to enhanced vasoconstrictor actions and smooth muscle cell proliferation, consequent on a reduction in endothelial ETB receptors.

Endothelin receptor A is expressed and mediates the [Ca2+]i mobilization of cells in human ciliary smooth muscle, ciliary nonpigmented epithelium, and trabecular meshwork

PURPOSE. To identify which endothelin receptor subtype is expressed and is functional in the human ciliary body and trabecular meshwork, tissues that regulate aqueous humor dynamics. METHODS. Immunocytochemistry was used to characterize the primary culture cells of normal human ocular cells. Endothelin receptor gene expression was probed with reverse transcription of polymerase chain reaction (RT-PCR). Intracellular calcium ([Ca 2+ ] i) mobilization was measured with video image microscopy using Fura-2AM as a fluorescent probe. RESULTS. Identities of primary cultures, human ciliary smooth muscle (HCSM), ciliary nonpigmented epithelial (HCE), and trabecular meshwork (HTM) cells were confirmed by immuno-cytochemistry, using cell-specific markers and observing typical cell morphologies. The presence of endothelin receptor A (ET A) was detected with RT-PCR in all three types of cells. The mRNA phenotype was verified with restriction enzyme BamHI digestion. No ET B receptor subtype expression was detected with RT-PCR under the cell culture conditions used. The [Ca 2+ ] i of HCSM cells was increased from 57 ± 7 nM to 328 ± 108 nM (n = 23; mean ± SE; P < 0.05) by 1 nM endothelin-1 (ET-1). In HCE cells, [Ca 2+ ] i increased from 40 ± 3 nM to 90 ± 10 nM (n = 55) (P < 0.001) with the same concentration of ET-1. Similarly, ET-1 (1 nM) increased the [Ca 2+ ] i from 51 ± 6 nM to 185 ± 47 nM (n = 19) (P < 0.001) in the HTM cells. The agonist for ET B, S6c, had no effect on [Ca 2+ ] i transients in all three cell types. No ET B receptor expression was detected in these cell types under the experimental and culture conditions. CONCLUSION. ET A receptor is expressed and is possibly responsible for mediating the signal for [Ca 2+ ] i mobilization by ET-1 in human ciliary smooth muscle, ciliary nonpigmented epithelial cells, and trabecular meshwork cells.

Mechanism of action of endothelins on adrenocortical cells.

Endothelins (ETs) play a pivotal role in the control of various endocrine and neuroendocrine tissues. In this review, we discuss the involvement of ETs as possible regulators of steroid secretion and we describe the mechanism of action of ETs on adrenocortical cells. The occurrence of ETs has been demonstrated in the human, porcine and rat adrenal gland. In humans, immunohistochemical and biochemical techniques have reported that ETs are localized exclusively in the cortex but the presence of ETs has also been detected in pheochromocytomas. In vitro studies have shown that ETs stimulate aldosterone secretion by adrenal tissues in various mammalian and amphibian animal models. The receptor subtype involved in the corticotropic action of ETs clearly differs among the various vertebrate species studied. In rat, the effect of ETs is mediated through an ET(B) receptor subtype while, in frog, an ET(A) receptor is implicated in the stimulatory action of ETs. In human adrenocortical cells, both ET(A) and ET(B) receptor subtypes are involved in the corticotropic effect of ETs. Activation of adrenal receptors causes an elevation of inositol trisphosphates associated with an increase in cytosolic calcium concentration. In addition, ETs induce an elevation of prostaglandin E2 (PGE2) and prostacyclin PGI2 production in the adrenal tissue, indicating that prostanoids may act as second messengers of ETs. It thus appears that ETs present in the adrenal gland may act as paracrine factors to stimulate the secretory activity of adrenocortical cells.

Identification of Endothelin Receptor Subtypes in Rat Ciliary Body Using Subtype-Selective Ligands

The endothelins are important vasoactive ocular peptides and there is some evidence that they may modulate intraocular pressure. We investigated the existence and localization of endothelin receptor subtypes using subtype selective ligands in rat ciliary body. Scatchard transformation of saturation binding experiments revealed that the KDand Bmaxfor [125I]ET-1 and [125I]ET-3 to membranes from ciliary body were 41.7±9 pM and 236±20 fmol mg−1protein and 37.8±0.4 pM and 160±2.0 fmol mg−1protein, respectively. Competitive experiments in the presence of cyclic pentapeptide BQ123 (selective for ETA receptors) and BQ3020 (selective for ETB receptors), demonstrated the existence of ETA and ETB receptors in a ratio of 35:65. Cross-linking of [125I]ET-1 and [125I]ET-3 to ciliary body membranes resulted in the labeling of two bands with apparent molecular masses of 52 and 34 kDa, suggesting that ETA and ETB receptors have similar molecular mass. The 34 Kda band is a proteolytic degradation product of the 52 Kda band. Autoradiographic results show that specific [125I]ET-1 binding sites, displaced by BQ123 and BQ3020, are localized to the ciliary epithelium, supporting the idea that ETA and ETB subtype receptors exist in this tissue.

Quantification of mRNA levels of endothelin receptor subtypes and preproEndothelin-1 in renal needle biopsies by competitive reverse transcriptase polymerase chain reaction

The vasoconstrictive peptide endothelin-1 (ET-1) is an autocrine/paracrine peptide of putative pathophysiological importance in renal transplant medicine. The aim of the present study was to develop a method for analysis of gene expression of the renal endothelin system in humans. Only small amounts of tissue are available from renal cortical needle biopsies. Thus, in the present study we developed a quantitative assay based on the competitive reverse transcriptase polymerase chain reaction (RT-PCR) technology. We quantified endothelin A (ET(A)) and B (ET(B)) receptor subtype mRNAs and preproET-1 mRNA levels in renal cortex biopsies obtained before nephrectomy of healthy kidney donors. Mean (+/- SEM) mRNA levels of the ET(A) and ET(B) receptor subtypes in 26 living donors were 212 +/- 23 and 368 +/- 56 amol/microg total RNA, respectively. The preproET-1 mRNA level in 19 living donors was 213 +/- 28 amol/microg total RNA. The inter-assay coefficient of variation (CV) for the assay was 10%; the intra-assay CV was 6-13%. The competitive RT-PCR assay described provides an accurate tool for gene expression investigation of the human endothelin system in renal cortical needle biopsies.