Endothelin systems in the brain: involvement in pathophysiological responses of damaged nerve tissues.

In addition to their potent vasoconstriction effects, endothelins (ETs) show multiple actions in various tissues including the brain. The brain contains high levels of ETs, and their production is stimulated in many brain disorders. Accumulating evidence indicates that activation of brain ET receptors is involved in several pathophysiological responses in damaged brains. In this article, the roles of brain ET systems in relation to brain disorders are reviewed. In the acute phase of stroke, prolonged vasospasm of cerebral arteries and brain edema occur, both of which aggravate brain damage. Studies using ET antagonists show that activation of ETA receptors in the brain vascular smooth muscle induces vasospasm after stroke. Brain edema is induced by increased activity of vascular permeability factors, such as vascular endothelial growth factor and matrix metalloproteinases. Activation of ETB receptors stimulates astrocytic production of these permeability factors. Increases in reactive astrocytes are observed in neurodegenerative diseases and in the chronic phase of stroke, where they facilitate the repair of damaged nerve tissues by releasing neurotrophic factors. ETs promote the induction of reactive astrocytes through ETB receptors. ETs also stimulate the production of astrocytic neurotrophic factors. Recent studies have shown high expression of ETB receptors in neural progenitors. Activation of ETB receptors in neural progenitors promotes their proliferation and migration, suggesting roles for ETB receptors in neurogenesis. Much effort has been invested in the pursuit of novel drugs to induce protection or repair of damaged nerve tissues. From these studies, the pharmacological significance of brain ET systems as a possible target of neuroprotective drugs is anticipated.

Endothelin receptors: what's new and what do we need to know?

Receptors are at the heart of how a molecule transmits a signal to a cell. Two receptor classes for endothelin (ET) are recognized, the ET(A) and ET(B) receptors. Intriguing questions have arisen in the field of ET receptor pharmacology, physiology, and function. For example, a host of pharmacological studies support the interaction of the ET(A) and ET(B) receptor in tissues (veins, arteries, bronchus, arterioles, esophagus), but yet few have been able to demonstrate direct ET(A)/ET(B) receptor interaction. Have we modeled this interaction wrong? Do we have a truly selective ET(A) receptor agonist such that we could selectively stimulate this important receptor? What can we learn from the recent phylogenic studies of the ET receptor family? Have we adequately addressed the number of biological molecules with which ET can interact to exert a biological effect? Recent mass spectrometry studies in our laboratory suggest that ET-1 interacts with other hereto unrecognized proteins. Biased ligands (ligands at the same receptor that elicit distinct signaling responses) have been discovered for other receptors. Do these exist for ET receptors and can we take advantage of this possibility in drug design? These and other questions will be posed in this minireview on topics on ET receptors.

Endothelin receptors in gastrointestinal smooth muscle.

Endothelins (ETs) are a family of peptides with 21-amino-acid residues. ET-1 was identified as a potent vasoconstrictor produced by vascular endothelial cells. Three distinct isoforms of ET, i.e. ET-1, ET-2 and ET-3, have been found to exist in a variety of tissues. ET was later found to cause contraction as well as relaxation of smooth muscle in many physiologic systems. In the gastrointestinal tract, ET causes contraction and/or relaxation of the esophagus, stomach, ileum and colon. In the hepatobiliary system, ET causes contraction of the portal vein, hepatic stellate cells, gallbladder and common bile duct. In mammalian species, two classes of ET receptors, ET(A) and ET(B), have been cloned. ET(A) receptors have higher affinities for ET-1 and ET-2 than ET-3, while ET(B) receptors have the same affinities for ET-1, ET-2 and ET-3. In the gastrointestinal system, ET causes smooth muscle contraction through interaction with ET(A) receptors, ET(B) receptors or both ET(A) and ET(B) receptors, depending on the tissues and species. In addition to contraction, ET causes smooth muscle relaxation through interaction with ET(A) receptors or ET(B) receptors. At the present time, there are no studies showing that ET causes smooth muscle relaxation through interaction with both ET(A) and ET(B) subtypes. ET induces contraction in most of the non-sphincter muscle except the fundus of the stomach. On the other hand, ET causes relaxation and contraction in the lower esophageal and internal anal sphincters. ET may play an important role in the control of human gastrointestinal motility and portal vein pressure.

Endothelin receptors in the detached retina of the pig.

Endothelin-1 (ET-1) is a potent vasoconstrictor that causes hypoperfusion of the neurosensory retina. We investigated immunohistochemically the expression of the receptors for ET-1, ET(A) and ET(B), in control and locally detached retinas of the pig. Immunoreactivity for ET(A) was expressed in the innermost retinal layers and in the outer plexiform layer in control retinas, and was additionally strongly expressed by retinal blood vessels at 7 days after detachment of the sensory retina from the pigment epithelium. Immunoreactivity for ET(B) was expressed by the innermost retinal layers, by ganglion cell somata, and by Müller glial cells in the control tissue, and was not altered in its expression after detachment. The vascular expression of ET(A) may suggest a hypoperfusion of the retina after detachment.

Receptor subtypes mediating the inotropic effects and Ca(2+) signaling induced by endothelin-1 through crosstalk with norepinephrine in canine ventricular myocardium.

In canine ventricular myocardium, endothelin-1 (ET-1) alone induced only a weak transient negative inotropic effect (NIE). However, ET-1 induced a marked sustained positive inotropic effect (PIE) subsequent to a transient NIE in the presence of norepinephrine (NE) at low concentrations (0.1 - 1 nM) and elicited a pronounced sustained NIE in the presence of NE at high concentrations (around 100 nM). Thus, the extent of beta-adrenoceptor stimulation induced by NE played a crucial role in determining the characteristics of the inotropic effects of ET-1. The characteristics of ET receptor subtypes involved in contractile regulation and Ca(2+) signaling induced by ET-1 were determined. The ET-1-induced transient NIE and decrease in Ca(2+) transients were abolished by the selective ET(A)-receptor antagonist FR319317, but not by the selective ET(B)-receptor antagonist BQ-788. The sustained PIE and the increase in Ca(2+) transients induced by ET-1 were abolished by FR319317, but not inhibited by BQ-788. In contrast, the sustained NIE of ET-1 was abolished by the non-selective ET antagonist TAK-044, markedly attenuated by FR319317, and partially inhibited by BQ-788. ET-1 alone elicited a PIE in the presence of BQ-788, which indicates that the activation of ET(B)-receptors counteracts the development of the PIE of ET-1. The current findings indicate that both ET(A) and ET(B) receptors are involved in the regulation of Ca(2+) signaling and contractility in canine ventricular myocardium.

Intracerebroventricular administration of anti-endothelin-1 IgG selectively upregulates endothelin-A and kappa opioid receptors.

Endothelin (ET) type A receptor antagonists enhance morphine-induced antinociception and restore morphine analgesia in morphine tolerant rats [Peptides 23 (2002) 1837; Peptides 24 (2003) 553]. These studies suggest that the central ET and opioid systems functionally interact. To explore this idea further, we determined the effect of i.c.v. administration of anti-ET-1 IgG (rabbit) on brain opioid receptor and ET receptor expression. Three days after implanting cannula into the lateral ventricle, male Sprague-Dawley rats were administered 10 microl (i.c.v.) of either control rabbit IgG (2.5 microg/microl) or anti-ET IgG (2.5 microg/microl) on day 1, day 3, and day 5. On day 6, animals were killed and the caudate and hippocampus collected. Anti-ET IgG had no significant effect on expression, measured by Western blots, of mu, delta or ET-B receptors, but increased kappa opioid (59%) and ET-A (33%) receptor protein expression in the caudate. [35S]-GTP-gamma-S binding assays demonstrated that anti-ET IgG decreased [D-Ala2-MePhe4, Gly-ol5]enkephalin efficacy, but not potency in the caudate. Control experiments showed that there was no detectable rabbit IgG in caudate and hippocampal samples. These results suggest that ET in the CSF negatively regulates kappa opioid and ET-A receptors in certain brain regions. These findings support the hypothesis that CSF neuropeptides have regulatory effects and further demonstrate a link between ET and the opioid receptor system.

ET--phone the pain clinic.

Recent findings by Khodorova et al. demonstrate that the vasoconstrictor endothelin-1 plays an important role in certain nociceptive behaviors in an animal model of pain, through activation of sensory neurons. Endothelin-1 might also have the unexpected capacity to release an opioid from surrounding keratinocytes and thereby inhibit the pain response. Such results suggest that, in the periphery, there are important interactions between sensory nerve terminals and surrounding cells, and that glia and keratinocytes could modulate the perception of environmental stimuli to a greater extent than previously considered.

Differential expression of endothelin receptors in regenerating spinal motor neurons in mice.

On day 4 after sciatic nerve crush injury, expression and localization of endothelin receptors ET(A) and ET(B) in the lumbar spinal cord were examined. Immunohistochemical staining with antibodies to ET(A) and ET(B) receptors showed cytoplasmic distribution of ET(A) receptors in motor neurons, whereas ET(B) receptors were localized in the perinuclear region. On the injured side of the lumbar spinal cord, when compared to contralateral, results demonstrated an up-regulation of ET(B) and a down-regulation of ET(A) receptors expression at the level of both mRNA and protein. These results suggest that ET(B) receptors may play a role in the regeneration of axotomized motor neurons.

Endothelin-1 receptor subtypes expression and binding in a perfused rat model of myocardial infarction.

Endothelin-1 (ET-1) pathophysiologic actions are mediated via binding with two receptor subtypes, ET(A) and ET(B). Release of ET-1 from endocardial endothelial cells and cardiac myocytes can modulate heart tissue necrosis and alterations. This study investigates the remodeling processes in Sprague-Dawley rats of myocardial infarction (MI) induced by ligating the left anterior descending coronary artery. Histological studies were done on cell type distribution using cell specific markers and Western blot analysis to localize ET-1 receptor subtypes and assess their expression post-MI. In addition, the binding kinetics of ET-1 with its receptors in heart perfusion, inlet via the aortic lumen and effluent outlet via the right atrium, between two animal model-subgroups were done: (1) sham-operated, and sham-operated-CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate)-treated; and (2) MI-operated, and MI-operated-CHAPS-treated. Effluent ET-1 concentration was plotted vs. time using a physical model for 1:1 ligand-receptor binding at coronary endothelium and myocytes. First order impulse function was used to calculate the affinity constants. In MI hearts, fluorescence activity increased for ET(A) vs. ET(B) across areas of the muscle compared to normal hearts. Western blotting showed upregulation of ET(A) and ET(B) receptors in MI compared with normal hearts. Results of ET-1 binding affinity post-MI indicated drastic reduction in spite the upregulation of ET(B) on coronary endothelium. Furthermore, substantial affinity increase was observed between ET-1 binding with ET(A) at the myocyte site. These findings stipulate that during 1 month post-MI some biochemical and hormonal effects could alter ET-1 receptor subtype(s) regulation and pharmacodynamics thus predisposing to cardiac hypertrophy and mitogenesis.

International Union of Pharmacology. XXIX. Update on endothelin receptor nomenclature.

In mammals, the endothelin (ET) family comprises three endogenous isoforms, ET-1, ET-2, and ET-3. ET-1 is the principal isoform in the human cardiovascular system and remains the most potent and long-lasting constrictor of human vessels discovered. In humans, endothelins mediate their actions via only two receptor types that have been cloned and classified as the ET(A) and ET(B) receptors in the first NC-IUPHAR (International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification) report on nomenclature in 1994. This report was compiled before the discovery of the majority of endothelin receptor antagonists (particularly nonpeptides) currently used in the characterization of receptors and now updated in the present review. Endothelin receptors continue to be classified according to their rank order of potency for the three endogenous isoforms of endothelin. A selective ET(A) receptor agonist has not been discovered, but highly selective antagonists include peptides (BQ123, cyclo-[D-Asp-L-Pro-D-Val-L-Leu-D-Trp-]; FR139317, N- [(hexahydro-1-azepinyl)carbonyl]L-Leu(1-Me)D-Trp-3 (2-pyridyl)-D-Ala) and the generally more potent nonpeptides, such as PD156707, SB234551, L754142, A127722, and TBC11251. Sarafotoxin S6c, BQ3020 ([Ala(11,15)]Ac-ET-1((6-21))), and IRL1620 [Suc-(Glu(9), Ala(11,15))-ET-1((8-21))] are widely used synthetic ET(B) receptor agonists. A limited number of peptide (BQ788) and nonpeptide (A192621) ET(B) antagonists have also been developed. They are generally less potent than ET(A) antagonists and display lower selectivity (usually only 1 to 2 orders of magnitude) for the ET(B) receptor. Radioligands highly selective for either ET(A) ((125)I-PD151242, (125)I-PD164333, and (3)H-BQ123) or ET(B) receptors ((125)I-BQ3020 and (125)I-IRL1620) have further consolidated classification into only these two types, with no strong molecular or pharmacological evidence to support the existence of further receptors in mammals.

Evidence for two endothelin Et(A) receptor subtypes in rabbit arteriolar smooth muscle.

1. Effects of endothelin-1 (Et-1) were studied on membrane currents in choroidal arteriolar smooth muscle by using perforated patch-clamp recordings. 2. Et-1 (10 nM) activated oscillatory Ca(2+)-activated Cl(-)-currents (I(Cl(Ca))) which could not be reversed by washing out. 3. Currents through L-type Ca(2+) channels were resolved in a divalent free medium (I(Ca(L)Na)). Et-1 reduced I(Ca(L)Na) by 75 +/- 7% within 30 s and this effect faded over 5 min, when the depression remained constant. On washing out Et-1, I(Ca(L)Na) almost completely recovered within 10 s. 4. BQ123 (1 microM), a peptide Et(A) receptor blocker, prevented the activation of I(Cl(Ca)), but failed to inhibit I(Cl(Ca)) transients once they had been initiated. In contrast, BQ123 not only prevented but also reversed the inhibition of I(Ca(L)Na) by Et-1. BQ788 (1 microM), an Et(B) receptor antagonist, did not prevent the activation of I(Cl(Ca)) or the inhibition of I(Ca(L)Na) by Et-1. 5. ABT-627 (10 nM), a non-peptide Et(A) receptor antagonist also blocked the activation of I(Cl(Ca)). However, on I(Ca(L)Na), ABT-627 (10 nM) mimicked the action of Et-1 an effect blocked by BQ123 suggesting that ABT-627 acted as an agonist. 6. The data are consistent with choroidal arteriolar smooth muscle cells having two types of Et(A) receptor, one where BQ123 is an antagonist and ABT-627 an agonist, where ligands dissociate freely and this receptor is coupled to inhibition of L-type Ca(2+) channels. In the other, BQ123 and ABT-627 are both antagonists and with Et-1 the receptor converts to a high affinity state producing the classical irreversible activation I(Cl(Ca)).

Enhanced expression of endothelin receptor subtypes in cirrhotic rat liver.

BACKGROUND/AIMS: A number of vasoactive substances have been implicated as potential mediators of intrahepatic portal hypertension. Endothelin (ET)-1 has been suggested to be involved in the regulation of hepatic microcirculation and development of portal hypertension. The aim of this study was to clarify the localization of two subtypes of ET receptors, ET A (ETAR) and B receptors (ETBR), in normal rat liver, and how the receptor expressions are altered in CCl4-induced cirrhotic rat liver. METHODS: Liver specimens were examined immunohistochemically after reacting with anti-ETAR and anti-ETBR rabbit polyclonal antibodies. Immunogold staining was also performed using the same antibodies, and examined under light and electron microscopy. RESULTS: In normal rat liver, immunohistochemistry revealed expression of ETAR and ETBR on the hepatic sinusoidal lining cells. By immunogold electron microscopy, electron-dense gold particles indicating the presence of ETARs were localized mainly on hepatic stellate cells (HSCs) and to a lesser extent on sinusoidal endothelial cells (SECs), while ETBRs were expressed equally intensely on HSCs and SECs. In cirrhotic animals, both ETAR and ETBR increased significantly on HSCs, while there were no significant increases in either receptor on SECs. CONCLUSIONS: In the normal state, HSCs possess both ETARs and ETBRs, while SECs mainly possess ETBRs. In cirrhosis, endothelins may exert more intense effects on HSCs via the enhanced ETARs and ETBRs, causing an increase in hepatic sinusoidal microvascular tone.

Endothelin receptor subtype antagonist activity of S-0139 in various isolated rabbit and canine arteries.

Vascular responses to endothelin peptides have been proposed to be mainly mediated via subtypes of the endothelin receptor, endothelin ET(A1), endothelin ET(B1), and endothelin ET(B2). The antagonist activity of 27-O-3-[2-(3-carboxy-acryloylamino)-5-hydroxyphenyl]acryloyloxy myricerone, sodium salt (S-0139) at these endothelin receptor subtypes was evaluated using isolated rabbit femoral, pulmonary, and mesenteric arteries. S-0139 competitively antagonized the endothelin-1-induced contraction mediated by the endothelin ET(A1) receptor in endothelium-denuded rabbit femoral arteries with a pA(2) value of 8.6+/-0.1. Endothelin ET(B2) receptor-mediated contraction induced by sarafotoxin S6c in endothelium-denuded rabbit pulmonary arteries was also inhibited by S-0139 with a pA(2) value of 5.6+/-0. 1. The pA(2) value of S-0139 for the endothelin ET(B1) receptor, evaluated from the endothelin-3-induced relaxant response in endothelium-intact rabbit mesenteric arteries, was 6.2+/-0.2. In isolated canine basilar, coronary, mesenteric and renal arteries, endothelin-1 caused concentration-dependent contractions with EC(50) values of 0.49+/-0.07, 0.61+/-0.25, 0.92+/-0.21 and 1.18+/-0.24 nM, respectively. S-0139 antagonized the endothelin-1-induced contraction in these arteries with pA(2) values of 8.0+/-0.1, 7. 6+/-0.2, 7.6+/-0.2 and 7.6+/-0.1, respectively. These results suggest that S-0139 is a potent and selective endothelin ET(A1) receptor antagonist, and that the contractions induced by endothelin-1 in canine basilar, coronary, mesenteric and renal arteries are mediated mainly via the endothelin ET(A1) receptor subtype.

Age-related changes in contractile responses of rabbit lower urinary tract to endothelin.

PURPOSE: As there are significant amounts of endothelin (ET) receptors in the mammalian urinary tract, we investigated the pharmacological properties and localization of ET receptors in the rabbit lower urinary tract as a function of age. MATERIALS AND METHODS: The characteristics of ET receptors in bladder dome, trigone and urethra of 6 weeks and 6 months old male rabbits were determined using muscle bath and autoradiographic techniques. RESULTS: ET-1 produces significant contractile responses in smooth muscle strips from bladder dome, trigone, and urethra in both 6 weeks and 6 months old rabbits. Although there was no significant difference in the maximum contractile response of urethral muscle strips to ET-1 between 6 weeks and 6 months old rabbits, the maximum responses to ET-1 were higher in both bladder dome and trigone of 6 weeks than 6 months old rabbits. A selective ETA receptor antagonist, BQ 610, shifted the concentration response curve to ET-1 to the right without decreasing maximal contractile responses in all regions from both age groups, whereas a selective ETB receptor antagonist, IRL 1038, had no significant effect on the contractile response in these tissues. Autoradiographic studies indicate that both ET receptor subtypes are expressed in bladder dome, trigone, and urethra with the ETA subtype being located only in the smooth muscle layers and the ETB subtype being located in both the urothelial and smooth muscle layers. CONCLUSION: Our data indicate the presence of region- and age-dependent differences in the contractile properties of ET receptors in the male rabbit lower urinary tract. Although both ETA and ETB receptor subtypes are present in the smooth muscle layers, the ETA receptor is the sub-type that is primarily involved in the mediation of contractions.

ET-receptor subtypes: roles in regional renal vascular actions of exogenous and endogenous endothelins in anesthetized rabbits.

The roles of endothelin (ET)-receptor subtypes, in the regional renal vascular effects of exogenous and endogenous ETs, were examined in pentobarbitone-anesthetized rabbits. The effects of renal arterial infusion of ET-1 (0.05-12.8 ng/kg/min) and the ET(B)-agonist [Ala1,3,11,15]-ET-1 (12.5-800 ng/kg/min) were compared. We then tested the effects of the ET(A)-antagonist BQ610 and the ET(B)-antagonist BQ788 (both 200 microg/kg plus 100 microg/kg/h, i.v.) on basal hemodynamics and on responses to renal arterial ET-1. Both ET-1 and [Ala1,3,11,15]-ET-1 dose-dependently reduced total renal blood flow (RBF) and cortical blood flow (CBF), but not medullary blood flow (MBF). ET-1 was 34-fold more potent than [Ala1,3,11,15-ET-1. BQ610 reduced mean arterial pressure (MAP; 14%), and increased RBF (21%) and CBF (12%), but not MBF. BQ788 increased MAP (13%), and reduced RBF (29%) and CBF (15%) but not MBF. Coadministration of both agents increased RBF (18%) and CBF (9%), without significantly affecting MAP. Neither antagonist (alone or combined) significantly affected responses to renal arterial ET-1. We conclude that the predominant renal vascular effects of exogenous and endogenous ETs are cortical vasoconstriction, but not at vascular sites controlling MBF. ET(A)-receptors contribute to the renal vasoconstrictor effects of endogenous ETs. ET(B2)-like receptors appear to contribute to the vasoconstrictor effects of [Ala1,3,11,15]-ET-1.

New developments in heart failure: role of endothelin and the use of endothelin receptor antagonists.

Despite conventional therapy, there is still much room for improvement in the prognosis of patients with chronic systolic heart failure. Evidence supports a role for endothelin-1 (ET-1), a potent vasoconstrictor, in the pathophysiology of heart failure. Given its potentially deleterious effects, the optimal treatment of heart failure may need to include efforts directed toward antagonizing this hormone. In support of this notion, the use of ET receptor antagonists produces a number of beneficial effects in heart failure, including both improvements in hemodynamics and reductions in the levels of other vasoconstricting neurohormones. There are at least 2 receptors for ET-1 (the ET-A and ET-B receptor), and the effects of ET-1 binding differ depending on the receptor involved. It is still unclear whether blockade of the ET-A receptor alone or the combined blockade of both the ET-A and ET-B receptors will be most efficacious as a therapeutic strategy. Long-term benefits have been achieved with the use of a mixed ET-A/B receptor antagonist, when added to standard triple-drug therapy, in patients with severe heart failure. We await the results of ongoing trials to determine if these agents will fulfill the promise of adding substantial incremental benefit to the treatment of the disease.

Characterization of a novel porcine endothelin(B) receptor splice variant.

Screening of porcine cerebellum cDNA library with porcine endothelin(B) (ET(B)) receptor cDNA revealed a novel ET(B) receptor cDNA that is distinctly different from the wild-type ET(B) receptor in length and the amino acid sequence at the C-terminal end. This sequence appears to represent alternate splicing of the carboxy terminal end of ET(B) receptor, resulting in a polypeptide of 429 amino acids in length, which is 14 amino acids shorter than the wild-type porcine ET(B) receptor. Characterization of the wild-type and alternately spliced ET(B) receptors expressed in COS cells revealed that both receptors displayed very similar binding [apparent dissociation constant (K(d)) and maximum binding (B(max)) for (125)I-ET-1 were 71 pM and 1.6 pmol/mg protein for wild-type and 81 pM and 1.2 pmol/mg protein for splice variant ET(B) receptors] as well as functional properties. These data suggest that the differences in the amino acids at the C-terminal end had no effect on binding or functional coupling of these alternately spliced ET(B) receptors.

Endothelin receptor antagonists. Promising new agents in the management of cardiovascular disorders.

Since its discovery in 1988, endothelin (ET) has been widely implicated in the pathophysiology of cardiovascular disease. ET antagonists have favourable effects in experimental models of these conditions and have proved useful in elucidating the role of the ET system. Orally acting ET antagonists appear very promising in clinical trials, particularly in patients with chronic heart failure and hypertension, but more information on the roles of the ET receptor subtypes in health and disease is required so that an informed choice can be made between the use of endothelin-A (ET-A) receptor-selective and nonselective receptor antagonists.