Endothelin causes contraction of human esophageal muscularis mucosae through interaction with both ETA and ETB receptors

Endothelin (ET) causes contraction of the muscularis mucosae in the guinea pig esophagus, but its role in the human esophagus remains unknown. To investigate effects of ET in the human esophagus, we measured contraction of isolated human esophageal muscularis mucosae strips caused by ET related peptides and binding of 125I-ET-1 to cell membranes prepared from the human esophageal muscularis mucosae. Autoradiography demonstrated specific binding of 125I-ET-1 to the muscularis mucosae and muscularis propria (muscularis externa) of the human esophagus. ET-1 caused tetrodotoxin and atropine-insensitive contraction of muscularis mucosae strips. In terms of the maximal tension of contraction, ET-1 and ET-2 were equal in efficacy. The relative potencies for ET related peptides to cause contraction were ET-1=ET-2>ET-3>sarafotoxin S6c (SX6c), an ETB receptor agonist. ET-1 caused contraction was mildly inhibited by BQ-123, an ETA receptor antagonist, and not by BQ-788, an ETB receptor antagonist. It was moderately inhibited by the combination of both antagonists, indicating synergistic inhibition. Furthermore, desensitization to SX6c with SX6c pretreatment failed to abolish the contractile response to ET-1, which was completely inhibited by BQ-123. These indicate the involvement of both ETA and ETB receptors in the contraction. Binding of 125I-ET-1 to cell membranes of the muscularis mucosae was saturable and specific. Analysis of dose-inhibition curves demonstrated the presence of ETA and ETB receptors. This study demonstrates that, the muscularis mucosae of the human esophagus, similar to that of the guinea pig esophagus, possesses both ETA and ETB receptors mediating muscle contraction.     

Identification of ETA and ETB binding domains using ET-derived photoprobes

Using the structure of ET-1 as a template, a series of photosensitive analogs were developed to investigate the binding domain of ETA and ETB receptors. Accordingly, a p-benzoyl-l-phenylalanine (Bpa) residue was introduced into the peptide chain following a pattern aiming at scanning N- to C-terminal portions of the molecule. Among the analogs, those containing a Bpa amino acid in position 7 ([L-Bpa7, Tyr(125I)13]hET-1) or 12 ([Nle7, L-Bpa12, Tyr(125I)13]hET-1) exhibited the capacity to activate both receptors, thus showing that residues Met-7 and Val-12 of ET-1 do not play a key role in the activation process. The binding capacity of the probes was also evaluated on transfected CHO cells overexpressing either ETA or ETB receptors. Subsequently, these photoprobes were used to label ETA and ETB receptors overexpressed in transfected CHO cells. Enzymatic digestions and chemical cleavages were then performed on ligand-receptor complexes and fragments produced by the lysis were analyzed to point out putative interaction areas on the receptors. Results showed that Phe147-Lys166, covering the second segment of EC I and the top part of TM III, contains a contact point for [Nle7, L-Bpa12, Tyr(125I)13]hET-1 on ETA receptors whereas Ile292-Trp319, spanning from the second half of the intracellular loop III up to the middle turns of TM VI, includes a residue that can interact with [L-Bpa7, Tyr(125I)13]hET-1. Moreover, upon binding of [Nle7, L-Bpa12, Tyr(125I)13]hET-1, it was observed that Thr263-Met266 (EC II) of the ETB receptor would come close with the ligand.     

Biological profiles of highly potent novel endothelin antagonists selective for the ETA receptor.

We describe novel potent endothelin (ET) antagonists that are highly potent and selective for the ETA receptor (selective to ET-1). Of the synthetic analogs based on ETA antagonist BE-18257A isolated from Streptomyces misakiensis (IC50 value for ETA receptor on porcine aortic smooth muscle cells (VSMCs); 1.4 microM), the compounds BQ-123 and BQ-153 greatly improved the binding affinity of [125I]ET-1 for ETA receptors on VSMCs (IC50; 7.3 and 8.6 nM, respectively), whereas they barely inhibited [125I]ET-1 binding to ETB receptors (nonselective with respect to isopeptides of ET family) in the cerebellar membranes (IC50; 18 and 54 microM, respectively). Associated with the increased affinity for ETA receptors, these peptides antagonized ET-1-induced constriction of isolated porcine coronary artery. However, there was a small amount of ET-1-induced vasoconstriction resistant to these antagonists, which paralleled the incomplete inhibition of [125I]ET-1 binding in the membrane of the aortic smooth muscle layer. These data suggest that the artery has both ETA and ETB receptors responsible for ET-1-induced vasoconstriction. The antagonists shifted the concentration-response curve to the right for ET-1 in the coronary artery, and increased the apparent dissociation constant in the Scatchard analysis of [125I]ET-1 binding on the VSMCs without affecting the binding capacity, indicative of the competitive antagonism for ETA receptor. In conscious rats, pretreatment with the antagonists markedly antagonized ET-1-induced sustained pressor responses in dose-dependent fashion without affecting ET-1-induced transient depressor action, suggesting that the pressor action is mediated by ETA receptors, while the depressor action is mediated by ETB receptors. In addition, pretreatment with the potent antagonists prevented ET-1-induced sudden death in mice. Thus, these potent ETA antagonists should provide a powerful tool for exploring the therapeutic uses of ETA antagonists in putative ET-1-related disorders.     

PTHrP fragments 1-16 and 1-23 do not bind to either the ETA or the ETB endothelin receptors

Because of some isofunctional similarities with endothelin-1 (ET-1), it has been suggested that PTHrP(1-16) and PTHrP(1-23) could interact with osteoblast cells via ETA receptors. To document this interaction, we used the thoracic rat aorta and the guinea-pig lung parenchyma paradigms as ETA and ETB models, respectively. In addition, we also performed a series of competition experiments against [125I]ET-1, using transfected cells expressing the ETA or ETB receptor. So far, no agonistic nor antagonistic activities were observed in the ETA and ETB bioassays with the PTHrP fragments. Furthermore, both fragments were unable to displace [125I]ET-1 bound to cells expressing the ETA or ETB receptor.     

Characterization of endothelin receptors ETA and ETB expressed in COS cells.

The cloned cDNA genes for endothelin receptors ETA and ETB were expressed in COS cells, and the binding characteristics of the two receptors with three isopeptide ligands (ET-1, ET-2, and ET-3) were examined in detail. The results indicated that the stability of receptor-ET-1 complexes formed with ETA and ETB were significantly different from each other, while their affinities to ET-1 were similar. The preformed ETA-ET-1 complex readily dissociated upon SDS-PAGE, as did many of the other receptors so far studied, while the ETB-ET-1 complex survived SDS-PAGE when it was run at low temperature (approximately 4 degrees C). Clear differences in stability were also shown in comparative studies of acid treatment of the two types of complexes. Only the ETB-ET-1 complex was resistant to acid treatment (0.2 M acetic acid, 0.5 M NaCl), and its 50 kDa monomeric receptor-ligand complex remained intact. The ETB-ET-1 complex (50 kDa) formed at 4 degrees C on the surface of COS cells, however, was susceptible to limited proteolysis at 37 degrees C that reduced the molecular size of the complex to a distinct 35 kDa. No such size reduction was observed with the preformed ETA-ET-1 complex. The overall structure of two endothelin receptors, as deduced from the sequence of cloned cDNAs, is similar in many respects. However, the present findings demonstrate distinct differences in the biochemical nature of the two receptors, which suggest their distinct biological functions.     

High affinity interaction of endothelin-3 with recombinant ETA receptors

Pharmacological evidence has suggested that endothelin-3 (ET-3) may act via a novel form of ET receptor that is shared by ETA receptor antagonists but not by ETB receptor selective agonists. This study analyses the properties of interaction of ET-3 with recombinant bovine ETA receptor. Apparent Kd(ET-3) values as low as 50 nM were defined from [125I]ET-1 binding experiments performed at low (5 microg/ml) protein concentrations in the assays. Larger (up to 1 microM) values were artefactually obtained in experiments performed at larger protein concentrations. The three monoiodo ET-3 derivatives were synthetized. ([125I]Y14)ET-3 did not recognize ETA receptors. ([125I]Y6)ET-3 labelled 18% of [125I]ET-1 binding sites with a Kd value of 320 pM. ([125I]Y13)ET-3 labelled 44% of [125I]ET-1 binding sites with a Kd value of 130 pM. High affinity ([125I]Y6)ET-3 and ([125I]Y13)ET-3 bindings were prevented by ET-1 (Kd = 5-7 pM), ET-3 (Kd = 70-250 pM), BQ-123 (Kd = 2 nM) and FR139317 (Kd = 2 nM) but not by low concentrations of 4-AlaET-1, sarafotoxin S6c or IRL1620. The three monoiodo ET-3 derivatives bound to recombinant rat ETB receptors with a pM affinity. The results suggest that ET-3, ([125I]Y6)ET-3 and ([125I]Y13)ET-3 should not be considered as ETB receptor specific ligands.     

Endothelin-1 activation of ETB receptors leads to a reduced cellular proliferative rate and an increased cellular footprint

Endothelin-1 (ET-1) is a vasoactive peptide which signals through two G-protein coupled receptors, endothelin receptor A (ETA) and B (ETB). We determined that ET-1 activation of its ETB receptor in stably cDNA transfected CHO cells leads to a 55% reduction in cell number by end-point cell counting and a 35% decrease in cell growth by a real-time cell-substrate impedance-based assay after 24h of cell growth. When CHO ETB cells were synchronized in the late G1 cell cycle phase, ET-1 delayed their S phase progression compared to control by 30% as determined by [(3)H]-thymidine incorporation. On the other hand, no such delay was observed during late G2/M to G1 transit when cells were treated with ET-1 after release from mitotic arrest. Using the cell-substrate impedance-based assay, we observed that ET-1 induces opposing morphological changes in CHO ETA and CHO ETB cells with ETB causing an increase in the cell footprint and ETA a decrease. Likewise, in pulmonary artery smooth muscle cells, which express both ETA and ETB receptors, ET-1 induces an ETA-dependent contraction and an ETB dependent dilation. These results are shedding light on a possible beneficial role for ETB in diseases involving ET-1 dysfunction such as pulmonary hypertension.     

Dipeptide sulfonamides as endothelin ETA/ETB receptor antagonists

Endothelin-1 (ET-1) is a potent mitogen and modulator of vascular tone. It is synthesized and released from endothelial cells and acts upon two receptor subtypes designated as ETA and ETB. In this study, a series of potent dipeptide sulfonamide dual-endothelin ETA/ETB receptor antagonists were prepared to investigate their potential benefit in vascular diseases. CGS 31398 inhibited [125I]ET-1 binding to human ETA and ETB receptors expressed in Chinese hamster ovary (CHO) cells (ETA/CHO, ETB/CHO) with respective IC50 values of 0.26 and 0.12 nM. However, in anesthetized rats, this compound markedly potentiated ET-1-induced renal vascular resistance, a response normally observed with selective ETB receptor antagonists. To determine whether species differences account for these results, a direct comparison was made between binding to rat and rabbit aortic membranes versus functional antagonism in isolated rat aortic rings. It was found that CGS 31398 had potent affinity for the ETA receptor in rat and rabbit aorta with IC50 values of 0.87 and 0.79 nM, respectively. Inhibition of ET-1-induced contractions of rat aorta by the compound was considerably weaker than expected (pKB = 6.4), while that of sarafotoxin S6c induced contraction of dog saphenous vein (100% inhibition at 100 nM) was consistent with corresponding binding data. These results suggest that although CGS 31398 is a potent dual inhibitor of ETA/ETB receptor binding, it surprisingly displays potent ETB and weak ETA receptor antagonism in functional assays.     

Localization of functional endothelin receptor signaling complexes in cardiac transverse tubules

Endothelin-1 (ET-1) is an autocrine factor in the mammalian heart important in enhancing cardiac performance, protecting against myocardial ischemia, and initiating the development of cardiac hypertrophy. The ETA receptor is a seven-transmembrane G-protein-coupled receptor whose precise subcellular localization in cardiac muscle is unknown. Here we used fluorescein ET-1 and 125I-ET-1 to provide evidence for ET-1 receptors in cardiac transverse tubules (T-tubules). Moreover, the ETA receptor and downstream effector phospholipase C-beta 1 were co-localized within T-tubules using standard immunofluorescence techniques, and protein kinase C (PKC)-epsilon-enhanced green fluorescent protein bound reversibly to T-tubules upon activation. Localized photorelease of diacylglycerol further suggested compartmentation of PKC signaling, with release at the myocyte "surface" mimicking the negative inotropic effects of bath-applied PKC activators and "deep" release mimicking the positive inotropic effect of ET-1. The functional significance of T-tubular ET-1 receptors was further tested by rendering the T-tubule lumen inaccessible to bath-applied ET-1. Such "detubulated" cardiac myocytes showed no positive inotropic response to 20 nM ET-1, despite retaining both a nearly normal twitch response to field stimulation and a robust positive inotropic response to 20 nm isoproterenol. We propose that ET-1 enhances myocyte contractility by activating ETA receptor-phospholipase C-beta 1-PKC-epsilon signaling complexes preferentially localized in cardiac T-tubules. Compartmentation of ET-1 signaling complexes may explain the discordant effects of ET-1 versus bath applied PKC activators and may contribute to both the specificity and diversity of the cardiac actions of ET-1.     

Collecting duct-specific endothelin B receptor knockout increases ENaC activity

Collecting duct (CD)-derived endothelin-1 (ET-1) acting via endothelin B (ETB) receptors promotes Na(+) excretion. Compromise of ET-1 signaling or ETB receptors in the CD cause sodium retention and increase blood pressure. Activity of the epithelial Na(+) channel (ENaC) is limiting for Na(+) reabsorption in the CD. To test for ETB receptor regulation of ENaC, we combined patch-clamp electrophysiology with CD-specific knockout (KO) of endothelin receptors. We also tested how ET-1 signaling via specific endothelin receptors influences ENaC activity under differing dietary Na(+) regimens. ET-1 significantly decreased ENaC open probability in CD isolated from wild-type (WT) and CD ETA KO mice but not CD ETB KO and CD ETA/B KO mice. ENaC activity in WT and CD ETA but not CD ETB and CD ETA/B KO mice was inversely related to dietary Na(+) intake. ENaC activity in CD ETB and CD ETA/B KO mice tended to be elevated under all dietary Na(+) regimens compared with WT and CD ETA KO mice, reaching significance with high (2%) Na(+) feeding. These results show that the bulk of ET-1 inhibition of ENaC activity is mediated by the ETB receptor. In addition, they could explain the Na(+) retention and elevated blood pressure observed in CD ET-1 KO, CD ETB KO, and CD ETA/B KO mice consistent with ENaC regulation by ET-1 via ETB receptors contributing to the antihypertensive and natriuretic effects of the local endothelin system in the mammalian CD.     

A novel radioligand [125I]BQ-3020 selective for endothelin (ETB) receptors.

A linear endothelin (ET) analog, N-acetyl-LeuMetAspLysGluAlaValTyrPheAlaHisLeu-AspIleIleTrp (BQ-3020), is highly selective for ETB receptors. BQ-3020 displaces [125I]ET-1 binding to ETB receptors (nonselective to ET isopeptides) in porcine cerebellar membranes (IC50: 0.2nM) at a concentration 4,700 times lower than that to ETA receptors (selective to ET-1) on aortic vascular smooth muscle cells (VSMC) (IC50: 940nM). BQ-3020 as well as ET-1 and ET-3 elicits vasoconstriction in the rabbit pulmonary artery. The ETA antagonist BQ-123 failed to inhibit this BQ-3020-induced vasoconstriction. Furthermore, BQ-3020 elicits endothelium-dependent vasodilation. These data indicate that BQ-3020 has ETB agonistic activity. The radioligand [125I]BQ-3020 binds to cerebellar membranes at single high affinity sites (Kd = 34.4pM), whereas it scarcely binds to VSMC. [125I]BQ-3020 binding to the cerebellum was displaced by BQ-3020, ET-1 and ET-3 in a nonselective manner (IC50: 0.07-0.17nM). However, the binding of [125I]BQ-3020 was insensitive to the ETA antagonist BQ-123 and other bioactive peptides. Both [125I]ET-1 and [125I]BQ-3020 show slow onset and offset binding kinetics to ETB receptors. These data indicate that the radioligand [125I]BQ-3020 selectively labels ETB receptors and that the slow binding kinetics of ET-1 are dependent on the peptide sequence from Leu6 to Trp21, but not on the structure formed by its two disulfide bridges.     

Endothelin-1[1-31] acts as a selective ETA-receptor agonist in the rat adrenal cortex

Endothelin-1 (ET-1) is a 21-amino acid residue (ET-1[1-21]) hypertensive peptide, which together with its receptor subtypes A and B (ETA and ETB) is expressed in the rat adrenal cortex, where it stimulates steroid-hormone (aldosterone and corticosterone) secretion through the ETB receptor and the growth (proliferative activity) of the zona glomerulosa (ZG) through the ETA receptor. ET-1[1-21] is generated from bigET-1 by the endothelin-converting enzyme (ECE-1). However, recent evidence indicates the existence of an alternative chymase-mediated biosynthetic pathway leading to the production of an ET-1[1-31] peptide, which was found to reproduce the ETA receptor-mediated vascular effects of ET-1[1-21]. We found that ET-1[1-21], but not ET-1[1-31], concentration-dependently raised steroid secretion from dispersed rat adrenocortical cells, its effect being blocked by the ETB-receptor selective antagonist BQ-788. Both ET-1s concentration-dependently increased the number of "S-phase" cells (as detected by the 5-bromo-2'-deoxyuridine immunocytochemical method) in capsule-ZG strips within a 240 min incubation. The ZG proliferogenic action of both ET-1s was blocked by the ETA-receptor antagonist BQ-123, and ET-1[1-31] was found to be significantly more potent than ET-1[1-21]. Autoradiography showed that in the rat adrenal ET-1[1-21] displaced the binding of selective ligands to both ETA ([125I]PD-151242) and ETB receptors ([125I]BQ-3020), while ET-1[1-31] eliminates only the binding to ETA receptors. Collectively, our findings provide strong evidence that ET-1[1-31] acts in the rat adrenal glands as a selective ETA-receptor agonist, mainly involved in the stimulation of ZG proliferative activity.     

ET-1 receptor gene expression and distribution in L1 and L2 cells from hypertensive sheep pulmonary artery

We examined gene and surface expression and activity of the endothelin (ET)-1 receptors (ETA and ETB) in subendothelial (L1) and inner medial (L2) cells from the main pulmonary artery of sheep with continuous air embolization (CAE)-induced chronic pulmonary hypertension (CPH). According to quantitative real-time RT-PCR, basal gene expression of both receptors was significantly higher in L2 than L1 cells, and hypertensive L2 cells showed significantly higher gene expression of ETB than controls. Expression of both genes in hypertensive L1 cells was similar to controls. Fluorescence-activated cell sorter analysis confirmed the increased distribution of ET(B) in hypertensive L2 cells. Although only the ETA receptors in control L2 cells showed significant binding of [125I]-labeled ET-1 at 1 h, both receptors bound ET-1 to hypertensive cells. Exposure to exogenous ET-1 for 18 h revealed that only the L2 cells internalized ET-1, and internalization by hypertensive L2 cells was significantly reduced when compared with controls. Treatment with ETA (BQ-610) and ETB (BQ-788) receptor antagonists demonstrated that both receptors contributed to internalization of ET-1 in control L2 cells, whereas in hypertensive cells only when both receptor antagonists were used in combination was significant suppression of ET-1 internalization found. We conclude that in sheep receiving CAE, alterations in ETB receptors in cells of the L2 layer may contribute to the maintenance of CPH via alterations in their expression, distribution, and activity.     

Endothelin receptors in human parathyroid gland.

We studied whether specific receptors for endothelins (ETs) exist in human parathyroid tissues and whether ETs may have any effect on secretion of PTH from parathyroid cells. Binding studies using [125I]ET-1 to the parathyroid membranes obtained from patients with hyperparathyroidism (2 adenomas, 2 hyperplasias) revealed that ET-1 competitively inhibited the binding of [125I]ET-1 to the membranes (the apparent Kd: 62 +/- 18 pM), whereas ET-3 showed biphasic and less steep inhibition curve than ET-1 in all tissue membranes examined. Northern blot analysis of poly(A)+ RNA from the parathyroid adenoma clearly demonstrated gene expression of both ETA and ETB receptors as well as preproET-1. ET-1 inhibited basal PTH secretion from dispersed adenoma cells more potently than ET-3. The present study clearly demonstrates the presence of both ETA and ETB receptor subtypes in human parathyroid tissues through which ETs may modulate PTH secretion in an autocrine and/or paracrine manner.     

A potent and specific agonist, Suc-[Glu9,Ala11,15]-endothelin-1(8-21), IRL 1620, for the ETB receptor.

A series of C-terminal linear peptides of endothelin (ET)-1 and their N alpha-succinyl (Suc) analogs were synthesized and their binding affinities for the two subtypes of ET receptor, ETA and ETB, in porcine lung membranes were examined. Among the synthetic analogs, Suc-[Glu9,Ala11,15]-ET-1(8-21), IRL 1620, was the most potent and specific ligand for the ETB receptor (KiETA/KiETB approximately equal to 120,000) as judged by the Ki values for ETA (1.9 microM) and ETB (16 pM) receptors. IRL 1620 was 60 times more selective for the ETB receptor than ET-3 (KiETA/KiETB approximately equal to 1,900). IRL 1620 (10(-9)-10(-7) M) induced contractions of the guinea pig trachea with a comparable potency to those of ET-1 or ET-3, suggesting that IRL 1620 is a potent ETB receptor agonist.     

Different distribution of endothelin receptor subtypes in pulmonary tissues revealed by the novel selective ligands BQ-123 and [Ala1,3,11,15]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.     

Venous smooth muscle contains vasoconstrictor ETB-like receptors.

Two endothelin (ET) receptor subtypes have been identified to date: the ETA receptor which preferentially binds ET-1 over ET-3, and the ETB receptor which is non-selective. This study characterized the ET receptor subtypes present in several vascular smooth muscle preparations using standard in vitro techniques. In all but one of the arteries tested, ET-3 was significantly less potent than ET-1. In contrast, the potency of ET-3 was very similar to that of ET-1 in all of the veins. The selective ETA receptor antagonist BQ-123 blunted the ET-1 contractions in rabbit carotid artery, but not in saphenous vein. The selective ETB receptor ligand sarafotoxin S6c contracted the rabbit saphenous vein, but not the carotid artery. These data suggest that vascular smooth muscle cells express ETA and ETB receptors. Stimulation of either receptor subtype can result in force development.     

Effects of chronic cold exposure on the endothelin system.

Cold temperatures have adverse effects on the human cardiovascular system. Endothelin (ET)-1 is a potent vasoconstrictor. We hypothesized that cold exposure increases ET-1 production and upregulates ET type A (ETA) receptors. The aim of this study was to determine the effect of cold exposure on regulation of the ET system. Four groups of rats (6-7 rats/group) were used: three groups were exposed to moderate cold (6.7 +/- 2 degrees C) for 1, 3, and 5 wk, respectively, and the remaining group was maintained at room temperature (25 degrees C) and served as control. Cold exposure significantly increased ET-1 levels in the heart, mesenteric arteries, renal cortex, and renal medulla. Cold exposure increased ETA receptor protein expression in the heart and renal cortex. ET type B (ETB) receptor expression, however, was decreased significantly in the heart and renal medulla of cold-exposed rats. Cold exposure significantly increased the ratio of ETA to ETB receptors in the heart. An additional four groups of rats (3 rats/group) were used to localize changes in ETA and ETB receptors at 1, 3, and 5 wk of cold exposure. Immunohistochemical analysis showed an increase in ETA, but a decrease in ETB, receptor immunoreactivity in cardiomyocytes of cold-exposed rats. Increased ETA receptor immunoreactivity was also found in vascular smooth muscle cells of cold-exposed rats. Cold exposure increased ETA receptor immunoreactivity in tubule epithelial cells in the renal cortex but decreased ETB receptor immunoreactivity in tubule epithelial cells in the renal medulla. Therefore, cold exposure increased ET-1 production, upregulated ETA receptors, and downregulated ETB receptors.     

Effect of chronic endothelin receptor antagonism on cerebrovascular function in type 2 diabetes

Diabetes increases the risk of stroke and contributes to poor clinical outcomes in this patient population. Myogenic tone of the cerebral vasculature, including basilar arteries, plays a key role in controlling cerebral blood flow. Increased myogenic tone is ameliorated with ET receptor antagonism in Type 1 diabetes. However, the role of endothelin-1 (ET-1) and its receptors in cerebrovascular dysfunction in Type 2 diabetes, a common comorbidity in stroke patients, remains poorly elucidated. Therefore, we hypothesized that 1) cerebrovascular dysfunction occurs in the Goto-Kakizaki (GK) model of Type 2 diabetes, and 2) pharmacological antagonism of ETA receptors ameliorates, while ETB receptor blockade augments vascular dysfunction. GK or control rats were treated with antagonists to either ETA (atrasentan, 5 mg.kg(-1).day(-1)) or ETB (A-192621, 15 or 30 mg.kg(-1).day(-1)) receptors for 4 wk and vascular function of basilar arteries was assessed using a wire myograph. GK rats exhibited increased sensitivity to ET-1. ET(A) receptor antagonism caused a rightward shift, indicating decreased sensitivity in diabetes, while it increased sensitivity to ET-1 in control rats. Endothelium-dependent relaxation was impaired in diabetes. ETA receptor blockade restored relaxation to control values in the GK animals with no significant effect in Wistar rats and ETB blockade with 30 mg.kg(-1).day(-1) A-192621 caused paradoxical constriction in diabetes. These studies demonstrate that cerebrovascular dysfunction occurs and may contribute to altered regulation of myogenic tone and cerebral blood flow in diabetes. While ETA receptors mediate vascular dysfunction, ETB receptors display differential effects. These results underscore the importance of ETA/ETB receptor balance and interactions in cerebrovascular dysfunction in diabetes.