Endothelin ETA Research Articles

Mechanism of protective actions of sparsentan in the kidney: lessons from studies in models of chronic kidney disease.

Simultaneous inhibition of angiotensin II AT1 and endothelin ETA receptors has emerged as a promising approach for treatment of chronic progressive kidney disease. This therapeutic approach has been advanced by the introduction of sparsentan, the first dual AT1 and ETA receptor antagonist. Sparsentan is a single molecule with high affinity for both receptors. It is US Food and Drug Administration approved for immunoglobulin A nephropathy (IgAN) and is currently being developed as a treatment for rare kidney diseases, such as focal segmental glomerulosclerosis. Clinical studies have demonstrated the efficacy and safety of sparsentan in these conditions. In parallel with clinical development, studies have been conducted to elucidate the mechanisms of action of sparsentan and its position in the context of published evidence characterizing the nephroprotective effects of dual ETA and AT1 receptor inhibition. This review summarizes this evidence, documenting beneficial anti-inflammatory, antifibrotic, and hemodynamic actions of sparsentan in the kidney and protective actions in glomerular endothelial cells, mesangial cells, the tubulointerstitium, and podocytes, thus providing the rationale for the use of sparsentan as therapy for focal segmental glomerulosclerosis and IgAN and suggesting potential benefits in other renal diseases, such as Alport syndrome.

Co-localization of the sodium-glucose co-transporter-2 channel (SGLT-2) with endothelin ETA and ETB receptors in human cardiorenal tissue.

Immunocytochemistry localised SGLT-2, ETA and ETB receptors in sections of histologically normal kidney, left ventricle from patients undergoing heart transplantation or controls. Primary antisera were visualised using fluorescent microscopy. Image analysis was used to measure intensity compared with background in adjacent control sections. As expected, SGLT-2 localised to epithelial cells of the proximal convoluted tubules, and co-localised with both ET receptor sub-types. Similarly, ETA receptors predominated in cardiomyocytes; low (compared with kidney but above background) positive staining was also detected for SGLT-2. Whether low levels of SGLT-2 have a (patho)physiological role in cardiomyocytes is not known but results suggest the effect of direct blockade of sodium (and glucose) influx via SGLT-2 inhibition in cardiomyocytes should be explored, with potential for additive effects with ETA antagonists.

Role of endothelin ETA receptors in the hypertension induced by the VEGFR-2 kinase inhibitors axitinib and lenvatinib in conscious freely-moving rats

Receptor tyrosine kinase inhibitors (RTKIs) suppress tumour growth by targeting vascular endothelial growth factor receptor 2 (VEGFR-2) which is an important mediator of angiogenesis. Here, we demonstrate that two potent RTKIs, axitinib and lenvatinib, are associated with hypertensive side effects. Doppler flowmetry was used to evaluate regional haemodynamic profiles of axitinib and lenvatinib. Male Sprague Dawley rats (350–500 g) were instrumented with Doppler flow probes (renal and mesenteric arteries and descending abdominal aorta) and catheters (jugular vein and distal abdominal aorta, via the caudal artery). Rats were dosed daily with axitinib (3 or 6 mg.kg−1) or lenvatinib (1 or 3 mg.kg−1) and regional haemodynamics were recorded over a maximum of 4 days. Both RTKIs caused significant (p < 0.05) increases in mean arterial pressure (MAP), which was accompanied by significant (p < 0.05) vasoconstriction in both the mesenteric and hindquarters vascular beds. To gain insight into the involvement of endothelin-1 (ET-1) in RTKI-mediated hypertension, we also monitored heart rate (HR) and MAP in response to axitinib or lenvatinib in animals treated with the ETA receptor selective antagonist sitaxentan (5 mg.kg−1) or the mixed ETA/ETB receptor antagonist bosentan (15 mg.kg−1) over two days. Co-treatment with bosentan or sitaxentan markedly reduced the MAP effects mediated by both RTKIs (p < 0.05). Bosentan, but not sitaxentan, also attenuated ET-1 mediated increases in HR. These data suggest that selective antagonists of ETA receptors may be appropriate to alleviate the hypertensive effects of axitinib and lenvatinib.

Calcium handling coupled to the endothelin ETA and ETB receptor-mediated vasoconstriction in resistance arteries: Differential regulation by PI3K, PKC and RhoK

Abnormal endothelin-1 (ET-1) activity is involved in the pathogenesis of vascular diseases such as essential and pulmonary arterial hypertension, coronary artery disease, and cerebrovascular disease, blockade of ET receptors having shown efficacy in clinical assays and experimental models of hypertension. Augmented Ca2+ influx and changes in Ca2+ sensitization associated with arterial vasoconstriction underlie increased systemic vascular resistance in hypertension. Since peripheral resistance arteries play a key role in blood pressure regulation, we aimed to determine here the specific Ca2+ signaling mechanisms linked to the ET receptor-mediated vasoconstriction in resistance arteries and their selective regulation by protein kinase C (PKC), Rho kinase (RhoK), the phosphatidylinositol 3-kinase (PI3K) and the mitogen-activated protein kinase (MAPK). ET-1-induced contraction was mediated by the endothelin ETA receptor with a minor contribution of vascular smooth muscle (VSM) endothelin ETB receptors. ET receptor activation elicited Ca2+ mobilization from intracellular stores, extracellular Ca2+ influx and Ca2+ sensitization associated with contraction in resistance arteries. Vasoconstriction induced by ET-1 was largely dependent on activation of canonical transient receptor potential channel 3 (TRPC3) and extracellular Ca2+ influx through nifedipine-sensitive voltage-dependent Ca2+ channels. PI3K inhibition reduced intracellular Ca2+ mobilization and Ca2+ entry without altering vasoconstriction elicited by ET-1, while PKC has dual opposite actions by enhancing Ca2+ influx associated with contraction, and by inhibiting Ca2+ release from intracellular stores. RhoK was a major determinant of the enhanced sensitivity of the contractile filaments underlying ET-1 vasoconstriction, with also a modulatory positive action on Ca2+ influx and intracellular Ca2+ release. Augmented RhoK and PKC activities are involved in vascular dysfunction in hypertension and vascular complications of insulin-resistant states, and these kinases are thus potential pharmacological targets in vascular diseases in which the ET pathway is impaired.

Progenitor-derived endothelin controls dermal sheath contraction for hair follicle regression.

Substantial follicle remodelling during the regression phase of the hair growth cycle is coordinated by the contraction of the dermal sheath smooth muscle, but how dermal-sheath-generated forces are regulated is unclear. Here, we identify spatiotemporally controlled endothelin signalling-a potent vasoconstriction-regulating pathway-as the key activating mechanism of dermal sheath contraction. Pharmacological blocking or genetic ablation of both endothelin receptors, ETA and ETB, impedes dermal sheath contraction and halts follicle regression. Epithelial progenitors at the club hair-epithelial strand bottleneck produce the endothelin ligand ET-1, which is required for follicle regression. ET signalling in dermal sheath cells and downstream contraction is dynamically regulated by cytoplasmic Ca2+ levels through cell membrane and sarcoplasmic reticulum calcium channels. Together, these findings illuminate an epithelial-mesenchymal interaction paradigm in which progenitors-destined to undergo programmed cell death-control the contraction of the surrounding sheath smooth muscle to orchestrate homeostatic tissue regression and reorganization for the next stem cell activation and regeneration cycle.

Endothelium and endothelin: regulators of arterial stiffness and fibrinolysis in ANCA-associated vasculitis

In this issue, Farrah etal. report clinical investigations in patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV). They demonstrate that endothelium-dependent vasodilatation, fibrinolytic capacity, and monocyte-dependent endothelin-1 clearance are reduced in patients with AAV, whereas circulating levels of endothelin-1 and vascular stiffness are increased. Acute infusion of an endothelin ETA receptor antagonist reduced vasoconstriction and arterial stiffness. This Commentary discusses biological insights and clinical implications of this study.

Understanding the molecular mechanism of endothelin ETA receptor selecting isopeptides endothelin-1 and -3

Endothelin-1 and -3 (ET1 and ET3) are potent vasoconstricting isopeptides that are involved in the pathophysiology of various diseases, such as cardiovascular and renal diseases. The two ETs exert their effects by binding to two G-protein-coupled receptors (GPCRs), the endothelin ETA receptor (ETAR) and the endothelin ETB receptor (ETBR). ETAR and ETBR reveal different preferences in the recognition of the two ETs: The binding affinity of ETAR with ET1 is much higher than that with ET3, while the binding affinities of ETBR with the two ETs are similar. Recently, the structures of ETBR with ET1 and ET3 were determined. These crystal structures provide detailed descriptions of how ETBR interacts with ET1 and ET3. However, the knowledge of how ETAR recognizes the two isopeptides is still lacking. Based on the structure of ETBR in complex with ET1, the structures of ETAR with ET1 and ET3 were modeled. Then, molecular dynamics simulations were performed to study how ETAR discriminates the two ETs. Simulation results demonstrate that ET1 has greater binding free energy with ETAR than ET3, which is in agreement with the binding affinity data of ETAR. By interaction energy analysis, the key residues of ETAR that discriminate between ET1 and ET3 are identified. Structural and dynamical analyses indicate that, compared with ET3, ET1 is more stable in binding with ETAR. Furthermore, the orientation change of W319 in the conserved CWxP motif that plays an important role in the signaling function of GPCRs was observed. Through the orientation change of this residue, the difference in the orthosteric pocket volume resulting from the binding of ET1 and ET3 on the extracellular side of ETAR leads to a conformational difference of TM6 on the intracellular side of ETAR. This study elucidates the molecular mechanism of how ETAR selects the isopeptides ET1 and ET3.

Prenatal endothelin or thromboxane receptor antagonism surpasses sympathoinhibition in improving cardiorenal malfunctions in preeclamptic rats

Current therapies for preeclampsia (PE) and its complications are limited and defective. Considering the importance of endothelin (ET) and thromboxane A2 (TXA2) signaling in PE pathophysiology, we tested the hypothesis that prenatal blockade of endothelin ETA or thromboxane TXA2 receptors favorably reprograms preeclamptic cardiovascular and renal insults. PE was induced by daily oral administration of L-NAME (50 mg/kg) to pregnant rats for 7 consecutive days starting from gestational day 14. The effects of co-exposure to atrasentan (ETA receptor blocker, 10 mg/kg/day) or terutroban (TXA2 receptor blocker, 10 mg/kg/day) on cardiovascular and renal anomalies induced by PE were assessed on gestational day 20 (GD20) and at weaning time and compared with those evoked by the sympatholytic drug α-methyldopa (α-MD, 100 mg/kg/day), a prototypic therapy for PE management. Among all drugs, terutroban was basically the most potent in ameliorating PE-evoked increments in blood pressure and decrements in creatinine clearance. Cardiorenal tissues of PE rats exhibited significant increases in ETA and TXA2 receptor expressions and these effects disappeared after treatment with atrasentan and to a lesser extent by terutroban or α-MD. Atrasentan was also the most effective in reversing the reduced ETB receptor expression in renal tissues of PE rats. Signs of histopathological damage in cardiac and renal tissues of PE rats were mostly improved by all therapies. Together, pharmacologic elimination of ETA or TXA2 receptors offers a relatively better prospect than α-MD in controlling perinatal cardiorenal irregularities sparked by PE.

POS-272 DIFFERENTIAL EFFECTS OF ENDOTHELIN ETA RECEPTOR BLOCKADE ON VASCULAR CALCIFICATION AND RENAL INJURY IN RATS WITH CHRONIC KIDNEY DISEASE

Endothelin ETA receptor blockade has been shown to reduce arterial stiffness and calcification without affecting renal function in a rat model of chronic kidney disease (CKD). In the present study, we investigated the differential effects of ETAreceptor blockade on vascular calcification and renal injury in CKD rats. CKD was induced in Wistar rats by renal mass ablation. Then, vascular calcification was induced by mineral imbalance with a calcium/phosphate-rich diet and vitamin D supplementation (Ca/P/VitD). One group of CKD+Ca/P/VitD rats was given the ETAreceptor antagonist atrasentan (10 mg/kg/day) for 6 weeks. Hemodynamic parameters, including systolic blood pressure (SBP), pulse pressure (PP) and pulse wave velocity (PWV) were determined. The thoracic aorta and the remnant kidney were harvested to assess vascular calcification and remodeling, and renal injuries. As expected, CKD+Ca/P/VitD rats developed arterial medial calcification associated with increased PWV and PP, indicating enhanced arterial stiffness. Renal injuries were comprised of mild focal and segmental glomerular sclerosis, diffused tubular atrophy and cortical and medullary interstitial fibrosis and inflammation. Although ETA receptor blockade improved vascular stiffness and calcification, unexpectedly, atrasentan aggravated renal injuries, including the tubular atrophy and the interstitial fibrosis and inflammation. In this rat model of CKD with vascular calcification, ETA receptor blockade reduced vascular stiffness and calcification, did not improve renal function due to aggravation of renal histological injuries.

Influence of Social Isolation Stress on Age-Related Changes in Functional Activity and Expression of Receptors of Endogenous Vasoconstrictors in Rat Aorta.

Social isolation stress was modeled by long-term isolation of 12-month-old rats in individual cages over 28 weeks. It was found that sensitization of blood vessels to the vasoconstrictor action of serotonin due to overexpression of 5HT2A-type receptor genes, as well as an imbalance in the expression level of endothelin ETA- and ETB-receptors (55 and 153%, respectively) are the early signs of vascular aging. A significant contribution to the development of age-related changes in the contractile properties of blood vessels is made by the stress component, which is manifested at the level of glucocorticoid-dependent mechanisms of regulation of gene expression. The decrease in the expression of glucocorticoid receptors caused by isolation stress leads to a decrease in the expression of the genes responsible for the synthesis of V1A-R and ATII-R and to the development of vascular hyporeactivity to the vasoconstrictor action of ATII and AVP. In the aorta of stressed rats, the α1-AR mRNA level increases by 3 times. At the same time, stress did not affect the dynamics of age-related changes in the expression of genes encoding 5HT2A-R and ETA/ETB-R.

Central endothelin ETB receptor activation reduces blood pressure and catecholaminergic activity in the olfactory bulb of deoxycorticosterone acetate-salt hypertensive rats

Endothelins regulate catecholaminergic activity in the olfactory bulb (OB) in normotensive and hypertensive animals. Administration of an endothelin ETA receptor antagonist decreases blood pressure in deoxycorticosterone acetate-salt (DOCA-salt) rats along with a reduction in tyrosine hydroxylase (TH) activity and expression. In the present work, we sought to establish the role of brain endothelin ETB receptor on blood pressure regulation and its relationship with the catecholaminergic system within the OB of DOCA-Salt rats. Sprague-Dawley male rats were divided into control and DOCA-Salt groups. Blood pressure, heart rate and TH activity as well as neuronal nitric oxide synthase (nNOS) expression were assessed following IRL-1620 (selective endothelin ETB receptor agonist) applied to be brain. IRL-1620 significantly reduced systolic, diastolic, and mean arterial pressure in DOCA-Salt hypertensive rats. It also decreased TH activity, TH total and phosphorylated forms expression as well as its mRNA in the OB of hypertensive animals. The expression of phospho-Ser1417-nNOS, which reflects nNOS activation, was significantly decreased in the of OB of DOCA-salt rats, but it was enhanced by IRL-1620. These findings suggest that DOCA-Salt hypertension depends on endogenous central endothelin ETA receptor activity, rather than on ETB, and that low endothelin ETB stimulation is essential for blood pressure elevation in this animal model. The effect of endothelin ETA receptor antagonism may also result from endothelin ETB receptor overstimulation. The present study shows that endothelin receptors are involved in the regulation of TH in the OB and that such changes are likely implicated in the hemodynamic control and sympathetic outflow.

Crystal structure of human endothelin ETB receptor in complex with sarafotoxin S6b

Sarafotoxins (SRTXs) are endothelin-like peptides extracted from snake venom. SRTXs stimulate the endothelin ETA and ETB receptors and enhance vasoconstriction, followed by left ventricular dysfunction and bronchoconstriction. SRTXs include four major isopeptides, S6a-d, with different subtype selectivities. Here, we report the crystal structure of the human ETB receptor in complex with the non-selective sarafotoxin S6b at 3.0 Å resolution. This structure reveals the similarities and differences between the binding modes of the endothelins and S6b. Moreover, molecular dynamics simulations based on the S6b-bound receptor provides structural insight into the subtype selectivity of the sarafotoxins. Our study clarifies the recognition mechanism of the endothelin-like peptide families.

Endothelin ETA- and ETB-Receptor-Mediated Inhibition of Noradrenaline Release From Isolated Rat Stomach

We examined the effect of endothelin-1, endothelin-3 and sarafotoxin S6c on the release of noradrenaline from gastric sympathetic nerve terminals using an isolated, vascularly perfused rat stomach. The release of noradrenaline evoked by electrical stimulation of the gastric postganglionic sympathetic nerves (at 2.5 Hz for 1 min) was inhibited by endothelin-1 (10-10 – 10-8 M), endothelin-3 (10-9 – 10-8 M) and sarafotoxin S6c (a highly selective agonist of endothelin ETB receptors) (10-9 – 10-8 M) in a concentration-dependent manner; the inhibitory potencies were as follows: endothelin-1 > endothelin-3 > sarafotoxin S6c. The inhibitory effect of endothelin-1 (3 × 10-9 M) on noradrenaline release was abolished by BQ-123 (a selective antagonist of endothelin ETA receptors) in a dose-dependent manner (10-7 and 10-6 M), but not influenced by BQ-788 (a selective antagonist of endothelin ETB receptors) (10-7 and 10-6 M). The endothelin-1-induced inhibition of noradrenaline release was attenuated by pretreatment with pertussis toxin (10 μg/animal, i.v., 4 days before experiments), but not influenced by indomethacin (3 × 10-6 M). These results indicate that endothelin ETA and ETB receptors located on the sympathetic nerve terminals play a role in the inhibition of noradrenaline release from the rat stomach: endothelin ETA receptor-mediated inhibition is carried out by pertussis toxin-sensitive and indomethacin-insensitive mechanisms.

Interactions between the complement and endothelin systems in normal pregnancy and following placental ischemia

Preeclampsia is characterized by new onset hypertension and fetal growth restriction and is associated with aberrant activation of the innate immune complement system and stressed or ischemic placenta. Previous studies have suggested a role for both endothelin and complement system activation products in new onset hypertension in pregnancy, but inter-relationships of the pathways are unclear. We hypothesized that complement activation following placental ischemia stimulates the endothelin pathway to cause hypertension and impair fetal growth. The Reduced Uterine Perfusion Pressure (RUPP) model results in hypertension and fetal growth restriction in a pregnant rat due to placental ischemia caused by mechanical obstruction of blood flow to uterus and placenta. The effect of inhibitor of complement activation soluble Complement Receptor 1 (sCR1) and endothelin A receptor (ETA) antagonist atrasentan on hypertension, fetal weight, complement activation (systemic circulating C3a and local C3 placental deposition) and endothelin [circulating endothelin and message for preproendothelin (PPE), ETA and endothelin B receptor (ETB) in placenta] in the RUPP rat model were determined. Following placental ischemia, sCR1 attenuated hypertension but increased message for PPE and ETA in placenta, suggesting complement activation causes hypertension via an endothelin independent pathway. With ETA antagonism the placental ischemia-induced increase in circulating C3a was unaffected despite inhibition of hypertension, indicating systemic C3a alone is not sufficient. In normal pregnancy, inhibiting complement activation increased plasma endothelin but not placental PPE message. Atrasentan treatment increased fetal weight, circulating endothelin and placental ETA message, and unexpectedly increased local complement activation in placenta (C3 deposition) but not C3a in circulation, suggesting endothelin controls local placental complement activation in normal pregnancy. Atrasentan also significantly decreased message for endogenous complement regulators Crry and CD55 in placenta and kidney in normal pregnancy. Results of our study indicate that complement/endothelin interactions differ in pregnancies complicated with placental ischemia vs normal pregnancy, as well as locally vs systemically. These data clearly illustrate the complex interplay between complement and endothelin indicating that perturbations of either pathway may affect pregnancy outcomes.

Involvement of Endothelin Receptors in Peripheral Sensory Neuropathy Induced by Oxaliplatin in Mice.

The aim of this study was to evaluate the participation of the endothelin ETA and ETB receptors and the effects of bosentan in oxaliplatin-induced peripheral sensory neuropathy (OIN) in mice. Adult male Swiss mice received 1mg/kg of oxaliplatin intravenously, twice a week for 5weeks. Dorsal root ganglia (DRG) and spinal cords were removed for evaluation of the endothelin ETA and ETB receptor expression. Afterwards, selective (BQ-123 and BQ-788; 10nmol in 30μL, intraplantarly) and non-selective (bosentan, 100mg/kg, orally) antagonists were administered in order to evaluate the involvement of the endothelin receptors in OIN. Mechanical and thermal nociception tests were performed once a week for 56days. Oxaliplatin induced mechanical and thermal hypersensitivity and increased the endothelin ETA receptor expression in both the DRG and spinal cord (P < 0.05). Endothelin ETB receptor expression was increased in the DRG (P < 0.05) but not in the spinal cord. Both endothelin ETA and ETB receptor selective antagonists partially prevented mechanical hyperalgesia in mice with OIN (P < 0.05). Moreover, bosentan prevented mechanical and thermal hypersensitivity in oxaliplatin-treated mice (P < 0.05). In conclusion, both endothelin ETA and ETB receptors seem to be involved in the OIN in mice and they should be considered possible targets for the management of this clinical feature.

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 ETA 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 ETA 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 ETA antagonist sitaxentan did not influence the aggregation state of these receptors.

Distortion of KB estimates of endothelin-1 ETA and ETB receptor antagonists in pulmonary arteries: Possible role of an endothelin-1 clearance mechanism.

Dual endothelin ETA and ETB receptor antagonists are approved therapy for pulmonary artery hypertension (PAH). We hypothesized that ETB receptor‐mediated clearance of endothelin‐1 at specific vascular sites may compromise this targeted therapy. Concentration‐response curves (CRC) to endothelin‐1 or the ETB agonist sarafotoxin S6c were constructed, with endothelin receptor antagonists, in various rat and mouse isolated arteries using wire myography or in rat isolated trachea. In rat small mesenteric arteries, bosentan displaced endothelin‐1 CRC competitively indicative of ETA receptor antagonism. In rat small pulmonary arteries, bosentan 10 μmol L−1 left‐shifted the endothelin‐1 CRC, demonstrating potentiation consistent with antagonism of an ETB receptor‐mediated endothelin‐1 clearance mechanism. Removal of endothelium or L‐NAME did not alter the EC 50 or Emax of endothelin‐1 nor increase the antagonism by BQ788. In the presence of BQ788 and L‐NAME, bosentan displayed ETA receptor antagonism. In rat trachea (ETB), bosentan was a competitive ETB antagonist against endothelin‐1 or sarafotoxin S6c. Modeling showed the importance of dual receptor antagonism where the potency ratio of ETA to ETB antagonism is close to unity. In conclusion, the rat pulmonary artery is an example of a special vascular bed where the resistance to antagonism of endothelin‐1 constriction by ET dual antagonists, such as bosentan or the ETB antagonist BQ788, is possibly due to the competition of potentiation of endothelin‐1 by blockade of ETB‐mediated endothelin‐1 clearance located on smooth muscle and antagonism of ETA‐ and ETB‐mediated contraction. This conclusion may have direct application for the efficacy of endothelin‐1 antagonists for treating PAH.

Blockade of endothelin receptors reduces tumor-induced ongoing pain and evoked hypersensitivity in a rat model of facial carcinoma induced pain

Pain reported by patients with head and neck cancer is characterized as persistent pain with mechanical allodynia. Pain management is inadequate for many patients, highlighting the need for improved therapies. We examined the hypothesis that the mixed endothelin ETA and ETB receptor antagonist, bosentan, reduces tumor-induced ongoing pain and evoked hypersensitivity in a rat model of facial cancer pain. Facial cancer was induced by inoculating a suspension of Walker-256 cells into the rat's right vibrissal pad. Tumor-bearing rats developed heat and tactile hypersensitivity along with increased spontaneous grooming behavior. Systemic morphine (2.5mg/kg, s.c.) blocked tumor-induced thermal and tactile hypersensitivity, with a lower dose (0.625mg/kg, s.c.) effective only against thermal hypersensitivity. Systemic bosentan blocked tumor-induced thermal hypersensitivity only at a high (300mg/kg, p.o.) dose, but failed to modify tactile hypersensitivity. Co-administration of the low doses of bosentan and morphine resulted in improved reduction of the tumor-induced heat and tactile hypersensitivity compared to either dose alone. Bosentan (100mg/kg, p.o.) reduced spontaneous grooming and induced conditioned place preference (CPP) selectively in tumor-bearing rats, suggesting that bosentan reduces tumor-induced ongoing pain at a lower dose than required to block tumor-induced hypersensitivity. This study provides evidence that endothelins may mediate tumor-induced ongoing pain and thermal hypersensitivity. In addition, bosentan enhanced morphine's effects on blocking tumor-induced heat and tactile hypersensitivity indicating that endothelin antagonists may be beneficial therapeutic targets that can be used to manage cancer-induced facial pain with opioid-sparing effects.

Myocardial ischemia-reperfusion enhances transcriptional expression of endothelin-1 and vasoconstrictor ETB receptors via the protein kinase MEK-ERK1/2 signaling pathway in rat.

BackgroundCoronary artery remodelling and vasospasm is a complication of acute myocardial ischemia and reperfusion. The underlying mechanisms are complex, but the vasoconstrictor peptide endothelin-1 is suggested to have an important role. This study aimed to determine whether the expression of endothelin-1 and its receptors are regulated in the myocardium and in coronary arteries after experimental ischemia-reperfusion. Furthermore, we evaluated whether treatment with a specific MEK1/2 inhibitor, U0126, modified the expression and function of these proteins.Methods and findingsSprague-Dawley rats were randomly divided into three groups: sham-operated, ischemia-reperfusion with vehicle treatment and ischemia-reperfusion with U0126 treatment. Ischemia was induced by ligating the left anterior descending coronary artery for 30 minutes followed by reperfusion. U0126 was administered before ischemia and repeated 6 hours after start of reperfusion. The contractile properties of isolated coronary arteries to endothelin-1 and sarafotoxin 6c were evaluated using wire-myography. The gene expression of endothelin-1 and endothelin receptors were measured using qPCR. Distribution and localization of proteins (pERK1/2, prepro-endothelin-1, endothelin-1, and endothelin ETA and ETB receptors) were analysed by Western blot and immunohistochemistry. We found that pERK1/2 was significantly augmented in the ischemic area 3 hours after ischemia-reperfusion; this correlated with increased ETB receptor and ET-1 gene expressions in ischemic myocardium and in coronary arteries. ETB receptor-mediated vasoconstriction was observed to be increased in coronary arteries 24 hours after ischemia-reperfusion. Treatment with U0126 reduced pERK1/2, expression of ET-1 and ETB receptor, and ETB receptor-mediated vasoconstriction.ConclusionsThese findings suggest that the MEK-ERK1/2 signaling pathway is important for regulating endothelin-1 and ETB receptors in myocardium and coronary arteries after ischemia-reperfusion in the ischemic region. Inhibition of the MEK-ERK1/2 pathway may provide a novel target for reducing ischemia-reperfusion damage in the heart.

Endothelin-1 and its receptors on haemorrhoidal tissue: a potential site for therapeutic intervention.

Haemorrhoids is a common anorectal condition affecting millions worldwide. We have studied the effect of endothelin-1 (ET-1) and the role of endothelin ETA and ETB receptors in haemorrhoid tissue. Protein expression of ET-1, ETA and ETB receptors were compared between haemorrhoids and normal rectal submucosa using Western blot analysis, with the localization of proteins determined by autoradiography and immunohistochemistry. Effects of ET-1 and sarafotoxin 6a on human colonic and rectal arteries and veins was assessed by wire myography and the involvement of receptor subtypes established by selective antagonists. Dense binding of [125 I]-ET-1 to haemorrhoidal sections was reduced by selective receptor antagonists. A higher density of ETB than ETA receptors was found in haemorrhoidal, than in control rectal tissue and confirmed by Western blot analysis. ETA and ETB receptors were localized to smooth muscle of haemorrhoidal arteries and veins, with ETB receptors on the endothelium. Human colonic and rectal arteries and veins were similarly sensitive to ET-1 and affected by the ETA selective antagonist, but sarafotoxin S6a-induced contractions were more pronounced in veins and antagonized by a selective ETB receptor antagonist. ETA and ETB receptors are present in human haemorrhoids with ETB receptors predominating. ETA receptors are activated by ET-1 to mediate a contraction in arteries and veins, but the latter are selectively activated by sarafotoxin S6a - a response that involves ETB receptors at low concentrations. Selective ETB agonists may have therapeutic potential to reduce congestion of the haemorrhoidal venous sinusoids.