Nociceptive tolerance is improved by bradykinin receptor B1 antagonism and joint morphology is protected by both endothelin type A and bradykinin receptor B1 antagonism in a surgical model of osteoarthritis

Introduction  

Endothelin-1, a vasoconstrictor peptide, influences cartilage metabolism mainly via endothelin receptor type A (ETA). Along with the inflammatory nonapeptide vasodilator bradykinin (BK), which acts via bradykinin receptor B1 (BKB1) in chronic inflammatory conditions, these vasoactive factors potentiate joint pain and inflammation. We describe a preclinical study of the efficacy of treatment of surgically induced osteoarthritis with ETA and/or BKB1 specific peptide antagonists. We hypothesize that antagonism of both receptors will diminish osteoarthritis progress and articular nociception in a synergistic manner.     

Role of endothelin in human hypertension

Endothelin-1 (ET-1) is a pleiotropic hormone produced primarily by the endothelium. Synthesis of ET-1 is stimulated by the major signals of cardiovascular stress, such as vasoactive agents (angiotensin II, norepinephrine, vasopressin, and bradykinin), cytokines (e.g., tumor necrosis factor alpha and transforming growth factor beta), and other factors, including thrombin and mechanical stress. ET-1 induces vasoconstriction, is proinflammatory, promotes fibrosis, and has mitogenic potential, important factors in the regulation of vascular tone, arterial remodeling, and vascular injury. These effects are mediated via two receptor types, ETA and ETB. The role ET-1 plays in normal cardiovascular homeostasis and in mild essential hypertension in humans is unclear. However, certain groups of essential hypertensive patients may have ET-1-dependent hypertension, including blacks (subjects of African descent), salt-sensitive hypertensives, patients with low renin hypertension, and those with obesity and insulin resistance. ET-1 has also been implicated in severe hypertension, heart failure, atherosclerosis, and pulmonary hypertension. In all of these conditions, plasma immunoreactive ET levels are elevated and tissue ET-1 expression is increased. Accordingly, it is becoming increasingly apparent that ET-1 plays an important role in cardiovascular disease and in some forms of hypertension in humans. Data from clinical trials using combined ETA-ETB receptor blockers have already demonstrated significant blood-pressure-lowering effects. Thus, targeting the endothelin system may have important therapeutic potential in the treatment of hypertension, particularly by contributing to the prevention of target organ damage and the management of cardiovascular disease.     

Role of endothelins in congestive heart failure

Despite major advances in conventional medical therapy, patients with heart failure continue to experience significant morbidity and mortality. Endothelin-1 (ET-1) is a potent vasocontrictor and mitogenic peptide that is activated in heart failure. There is increasing experimental and clinical evidence in support of an important role of ET-1 in the pathophysiology of heart failure. Manipulation of the activity of ET-1, especially using endothelin receptor blockers, has allowed for the further elucidation of the role of this neurohormonal system and development of novel therapeutic strategies in heart failure. Published clinical studies of these agents to date have involved relatively small numbers of patients with severe heart failure, followed for a relatively short period of time, and have mainly examined surrogate endpoints. Large-scale trials that address to hard clinical outcomes are ongoing and their results forthcoming. A key question that remains concerns whether selective ETA or dual ETA-ETB receptor blockade will be more effective.     

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.     

Discovery of 4'-[(imidazol-1-yl)methyl]biphenyl-2-sulfonamides as dual endothelin/angiotensin II receptor antagonists

A series of 4'-[(imidazol-1-yl)methyl]biphenylsulfonamides has potent antagonist activity against both angiotensin II AT(1) and endothelin ET(A) receptors. Such dual-acting antagonists could have utility in the treatment of hypertension, heart failure, and other cardiovascular diseases in a broad patient population. Certain compounds in the present series are orally active in a rat model of angiotensin II-mediated hypertension.     

Upregulation of endothelin-1 binding in tissues of salt-loaded stroke-prone spontaneously hypertensive rats

Upon maintained on a 1% NaCl drinking solution beginning at 7 weeks of age, the stroke-prone spontaneously hypertensive rat (SHRsp) developed severe hypertension and stroke; most died by 16 weeks. The mechanism by which these diseases evolve remains unclear. Endothelin-1 (ET-1) is a potent, peptidic vasoconstrictor and is implicated in the pathogenesis of various cardiovascular, renal, and central nervous system diseases. The purpose of the present study was to compare the binding of [125I]ET-1 to the brain, heart, kidney, liver, and spleen membrane preparations of 16-week-old SHRsp and age-matched normotensive Wistar-Kyoto rats (WKY). The KD values for [125I]ET-1 binding to the corresponding tissues of the two strains were not significantly different, except in the brain (SHRsp: 17 +/- 1 pM; WKY: 24 +/- 1 pM). In contrast, the Bmax values measured in the brain, heart, kidney, and liver of SHRsp were 1.5- to 2.1-fold greater than those of their WKY counterparts. Competition of [125I]ET-1 binding to the membrane preparations by the specific ETA receptor antagonist BQ-123 or the specific ETB receptor agonist sarafotoxin S6c revealed a similar proportion of ETA and ETB receptor subtypes in the corresponding tissues of the two rat strains. These results indicate that ET-1 binding is upregulated in SHRsp and suggest that ET-1 may play a pathophysiological role in this animal model of genetic hypertension.     

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.     

Therapeutic potential of endothelin receptor antagonists for chronic proteinuric renal disease in humans

Diabetes and arterial hypertension continue to be the main causes of chronic renal failure in 2010, with a rising prevalence in part due to the worldwide obesity epidemic. Proteinuria is a main feature of chronic renal disease and mediated by defects in the glomerular filtration barrier and is as a good predictor of cardiovascular events. Indeed, chronic renal disease due to glomerulosclerosis is one of the important risk factors for the development of coronary artery disease and stroke. Glomerulosclerosis develops in response to inflammatory activation and increased growth factor production. Preclinical and first preliminary clinical studies provide strong evidence that endogenous endothelin-1 (ET-1), a 21-amino-acid peptide with strong growth-promoting and vasoconstricting properties, plays a central role in the pathogenesis of proteinuria and glomerulosclerosis via activation of its ET_A subtype receptor involving podocyte injury. These studies have not only shown that endothelin participates in the disease processes of hypertension and glomerulosclerosis but also that features of chronic renal disease such as proteinuria and glomerulosclerosis are reversible processes. Remarkably, the protective effects of endothelin receptors antagonists (ERAs) are present even on top of concomitant treatments with inhibitors of the renin–angiotensin system. This review discusses current evidence for a role of endothelin for proteinuric renal disease and podocyte injury in diabetes and arterial hypertension and reviews the current status of endothelin receptor antagonists as a potential new treatment option in renal medicine.     

Stress upregulates arterial matrix metalloproteinase expression and activity via endothelin A receptor activation

Degradation of the extracellular matrix proteins by matrix metalloproteinases (MMP) is an important regulatory step in the vascular remodeling process. Recent studies demonstrated that ETA receptors regulate cardiac MMP activity and fibrosis in DOCA-salt hypertension. However, little is known about endothelin (ET)-1 regulation of vascular MMP activity in hypertension. Thus early changes in ET-1-mediated regulation of MMP activity were measured in borderline hypertensive rats that develop impaired vasorelaxation and hypertension with chronic exposure to stress. Experiments were performed after 10 days of exposure to the behavioral stressor, air-jet stress, but before the onset of stress-induced hypertension. Study groups were 1) control (n = 8); 2) air-jet stress for 10 days (n = 8); 3) control plus ETA antagonist ABT-627 (n = 4), and 4) air-jet stress plus ETA antagonist (n = 4). MMP activity in the thoracic aorta was assessed by gelatin zymography. MMP protein and tissue ET-1 levels were evaluated by immunohistochemistry, and ET receptor density was determined by immunoblotting. Exposure to stress caused a twofold increase in plasma ET-1 levels (P < 0.05), and there was increased ET-1 staining at the tissue level. Total MMP activity and expression of MMP-2 and MMP-9 were increased in the stress group. ETA receptor antagonism prevented the increase in MMP expression and activation in the stress group. These results provide evidence that the MMP system is activated before the development of hypertension and ET-1 mediates these early events in vascular remodeling.     

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.     

Endothelin and endothelin antagonists: Potential role in cardiovascular and renal disease

Endothelin-1 is a recently discovered peptide mainly released from endothelial cells. Hypoxia and ischemia as well as numerous factors such as angiotensin 11, thrombin and transforming growth factor ₁ stimulate the fomation of the peptide. On the other hand the synthesis of endothelin is inhibited by nitric oxide and atrial natriuretic peptide via the formation of cyclic guanosine monophosphate. Released from endothelial cells endothelin-1 mediates transient vasodilation followed by a profound and longlasting vasoconstriction. Endothelin is also a mitogen for smooth muscle proliferation. Endothelins exert their biological effects via activation of specific receptors. Two different receptors have been cloned from mammalian tissues (ET_A and ET_B receptors). On vascular smooth muscle cells both receptors mediate contractions. Endothelial cells only express ET_B receptors linked to the formation of nitric oxide and/or prostacyclin formation. Increased plasma concentrations of endothelin-1 have been described in a variety of diseases such as pulmonary hypertension, arteriosclerosis, renal failure, acute coronary syndromes, heart failure, migraine and vascular diseases.Recently an increasing number of endothelin receptor antagonists have been synthetized, which have been shown to inhibit endothelin-mediated vasoconstriction. Clinical studies are now ongoing to elucidate the pathophysiologic role of endothelin and the potential benefit of the blockade of the system in different disease states.     

Endothelin-1 in chronic renal failure and hypertension

Investigation into the role of endothelin-1 (ET-1) in renal function has revealed two major direct actions leading to the control of extracellular volume and blood pressure. These are the regulation of renal hemodynamics and glomerular filtration rate and the modulation of sodium and water excretion. In the rat remnant kidney model of chronic renal failure, ET-1 production is increased in blood vessels and renal tissues. These changes are related to an increase in preproET-1 expression and correlate with the rise in blood pressure, the development of cardiovascular hypertrophy, and the degree of renal insufficiency and injury. Selective ETA receptor blockade prevents the progression of hypertension and the vascular and renal damage, supporting a role for ET-1 in chronic renal failure progression. The increase in ET-1 production can be associated with other local mediators, including angiotensin II, transforming growth factor-beta1 and nitric oxide, the local production of which is also altered in chronic renal failure. In human patients with essential hypertension, atherosclerosis, and nephrosclerosis, plasma ET-1 levels are increased compared with patients with uncomplicated essential hypertension. Similarly, plasma ET-1 concentrations are markedly increased in patients with end-stage renal disease undergoing dialysis, and this correlates with blood pressure, suggesting that ET-1 may contribute to hypertension in these patients. The treatment of anemia in patients with renal failure with human recombinant erythropoietin increases blood pressure by accentuating the underlying endothelial dysfunction and the elevated vascular ET-1 production. Overall, these results support a role for ET-1 in hypertension and the end-organ damage associated with chronic renal failure. ETA receptor blockade may then represent a potential target for the management of hypertension and cardiovascular and renal protection.     

The endothelin axis in cancer

The endothelin axis, comprising endothelins and their receptors, has recently emerged as relevant player in tumor growth and metastasis by regulating mitogenesis, cell survival, angiogenesis, bone remodeling, stimulation of nociceptor receptor, tumor-infiltrating immune cells, epithelial-to-mesenchymal transition, invasion and metastatic dissemination. Endothelin-1 participates in the growth and progression of a variety of tumors such as prostatic, ovarian, renal, pulmonary, colorectal, cervical, breast, bladder, endometrial carcinomas, Kaposi's sarcoma, brain tumors, melanoma, and bone metastases. This review highlights key signaling pathways activated by endothelin-1 axis in cancer, since the understanding the full spectrum activated by endothelin-1 is critical for the optimal design of targeted therapies. Preliminary experimental and clinical data demonstrate that interfering with endothelin receptor by using endothelin-1 receptor antagonists alone and in combination with cytotoxic drugs or molecular inhibitors could represent a new mechanism-based antitumor strategy.     

Impact of Superoxide Dismutase Mimetic AEOL 10150 on the Endothelin System of Fischer 344 Rats

Endothelin-1 is a potent vasoconstrictor and mitogenic peptide involved in the regulation of vasomotor tone and maintenance of blood pressure. Oxidative stress activates the endothelin system, and is implicated in pulmonary and cardiovascular diseases including hypertension, congestive heart failure, and atherosclerosis. Superoxide dismutase mimetics designed with the aim of treating diseases that involve reactive oxygen species in their pathophysiology may exert a hypotensive effect, but effects on the endothelin system are unknown. Our objective was to determine the effect of the superoxide dismutase mimetic AEOL 10150 on the basal endothelin system in vivo. Male Fischer-344 rats were injected subcutaneously with 0, 2 or 5 mg/kg body weight of AEOL 10150 in saline. Plasma oxidative stress markers and endothelins (bigET-1, ET-1, ET-2, ET-3) as well as lung and heart endothelin/nitric oxide system gene expressions were measured using HPLC-Coularray, HPLC-Fluorescence and RT-PCR respectively. AEOL 10150 reduced (p<0.05) the circulating levels of isoprostane (-25%) and 3-nitrotyrosine (-50%) measured in plasma 2h and 24h after treatment, confirming delivery of a physiologically-relevant dose and the potent antioxidant activity of the drug. The reduction in markers of oxidative stress coincided with sustained 24h decrease (p<0.05) of plasma levels of ET-1 (-50%) and ET-3 (-10%). Expression of preproET-1 and endothelin converting enzyme-1 mRNA were not altered significantly in the lungs. However preproET-1 (not significant) and ECE-1 mRNA (p<0.05) were increased (10–25%) in the heart. Changes in the lungs included decrease (p<0.05) of mRNA for the ET-1 clearance receptor ET_B and the vasoconstriction-signaling ET_A receptor (-30%), and an early surge of inducible nitric oxide synthase expression followed by sustained decrease (-40% after 24 hours). The results indicate that interception of the endogenous physiological flux of reactive nitrogen species and reactive oxygen species in rats impacts the endothelin/nitric oxide system, supporting a homeostatic relationship between those systems.     

Brain endothelial barrier passage by monocytes is controlled by the endothelin system

Homeostasis of the brain is dependent on the blood-brain barrier (BBB). This barrier tightly regulates the exchange of essential nutrients and limits the free flow of immune cells into the CNS. Perturbations of BBB function and the loss of its immune quiescence are hallmarks of a variety of brain diseases, including multiple sclerosis (MS), vascular dementia, and stroke. In particular, diapedesis of monocytes and subsequent trafficking of monocyte-derived macrophages into the brain are key mediators of demyelination and axonal damage in MS. Endothelin-1 (ET-1) is considered as a potent pro-inflammatory peptide and has been implicated in the development of cardiovascular diseases. Here, we studied the role of different components of the endothelin system, i.e., ET-1, its type B receptor (ET(B)) and endothelin-converting enzyme-1 (ECE-1) in monocyte diapedesis of a human brain endothelial cell barrier. Our pharmacological inhibitory and specific gene knockdown studies point to a regulatory function of these proteins in transendothelial passage of monocytes. Results from this study suggest that the endothelin system is a putative target within the brain for anti-inflammatory treatment in neurological diseases.     

Nonselective ETA/ETB-receptor blockade increases systemic blood pressure of Bio 14.6 cardiomyopathic hamsters

To examine the role of endothelin ETA and ETB receptors in congestive heart failure due to cardiomyopathy, the effect of chronic treatment with selective ETA- and ETB-receptor antagonists (atrasentan and A-192621, respectively), alone and in combination, was assessed on functional and biochemical parameters of 52-week-old Bio 14.6 cardiomyopathic hamsters. Compared with control animals, cardiomyopathic hamsters treated for 9 weeks with atrasentan showed no variation in MAP; however, selective ETB- and combined nonselective ETA- and ETB-receptor antagonists increased systemic blood pressure. After selective ETB-receptor blockade, plasma endothelin levels were augmented. Importantly, this increase was highly enhanced (more than 8-fold) by concomitant ETA-receptor antagonism. Furthermore, the left ventricle:body weight ratio of cardiomyopathic hamsters treated with A-192621, alone or in combination with atrasentan, was significantly increased. On the other hand, decreased left ventricular end-diastolic pressure was observed in cardiomyopathic hamsters after selective ETA- or combined nonselective ETA/ETB-receptor antagonism, while only selective ETA-receptor blockade reduced left ventricular endothelin levels. Our results suggest that, in congestive heart failure, ETB receptors are essential to limit circulating endothelin levels, which may argue for improved cardiac benefits after long-term treatment with highly selective ETA-receptor antagonists.     

Endothelin: 20 years from discovery to therapy

Since its identification as an endothelial cell-derived vasoconstrictor peptide in 1988, endothelin-1, the predominant member of the endothelin peptide family, has received considerable interest in basic medical science and in clinical medicine, which is reflected by more than 20 000 scientific publications on endothelin research in the past 20 years. The story of endothelin is unique as the gene sequences of endothelin receptors and the first receptor antagonists became available within only 4 years of the identification of the peptide sequence.The first clinical study in patients with congestive heart failure was published only 3 years thereafter. Yet, despite convincing experimental evidence of a pathogenetic role for endothelin in development, cell function, and disease, many initial clinical studies on endothelin antagonism were negative. In many of these studies, study designs or patient selection were inadequate. Today, for diseases such as pulmonary hypertension, endothelin antagonist treatment has become reality in clinical medicine, and ongoing clinical studies are evaluating additional indications, such as renal disease and cancer. Twenty years after the discovery of endothelin, its inhibitors have finally arrived in the clinical arena and are now providing us with new options to treat disease and prolong the lives of patients. Possible future indications include resistant arterial hypertension, proteinuric renal disease, cancer, and connective tissue diseases.     

Endothelin-1 directly modulates its own secretion: studies utilising the cell immunoblot technique

Endothelin-1 is an important factor in vasoregulation and circulating levels of the peptide are increased in a number of cardiovascular disorders. However, control of endothelin-1 secretion is only sketchily understood. The possibility that endothelin-1 influences its own release was investigated. A cell immunoblot method, which can detect local secretion of peptide from individual human vascular endothelial cells, was employed. Cells were dispersed onto a protein-binding membrane. Endothelin-1 in cells or secreted and adhering to the protein-binding membrane outside the cells was detected using immunohistochemical techniques. The numbers of cells that contained endothelin-1 and secreted endothelin-1 were counted after the cells had been incubated in control conditions, or with added endothelin-1, angiotensin-II, or endothelin receptor antagonists, bosentan and BQ788. Endothelin-1 and angiotensin-II increased the numbers of cells that secreted endothelin-1. On the other hand, bosentan and BQ788 caused a reduction in the numbers of endothelin-1-secreting cells. These results indicate that human endothelial cells contain a pathway by which endothelin-1 induces its own release. The receptor antagonists, bosentan and BQ788, inhibited basal secretion of endothelin-1.     

Number and brightness analysis to study spatio-temporal distribution of the angiotensin II AT1 and the endothelin-1 ETA receptors: Influence of ligand binding

The angiotensin II AT₁ and the endothelin 1 ET_A receptors play a crucial role in the pathogenesis of cardiovascular diseases like hypertension, heart failure, stroke, pulmonary hypertension, and cardiac hypertrophy. Both receptors are members of the rhodopsion-like superfamily of G protein-coupled receptors which can exist as monomers, dimers, and higher order aggregates.Recently, oligomerization of these two receptors have been described by several biophysical methods based mainly on luminescence and fluorescence energy transfer. Since this oligomerization can occur either spontaneously or it can be induced by ligand-binding, the aim of this work was to address whether the oligomerization of these receptors occurs upon ligand-binding. For this purpose the Number and Brightness analysis, a method that allows the identification, localization, and quantification of protein aggregates in the plasma membrane of a single cell, was used. An advantage of this method is that it is not limited to certain dyes specially required for Fluorescence Resonance Energy Transfer measurements.Our results showed that stably transfected angiotensin II AT₁ receptors and transiently transfected endothelin 1 ET_A receptors, were found as monomeric, dimeric, and tetrameric receptor aggregates. Interestingly, the binding of antihypertensive agents like losartan and BQ123, earlier suggested to be inverse agonists, significantly increased the proportion of monomers and reduced the occurrence of dimers on the cell membrane; while the kown endothelin 1 ET_A antagonist sitaxentan did not influence the aggregation state of these receptors.     

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.