Electroacupuncture induces antihyperalgesic effect through endothelin-B receptor in the chronic phase of a mouse model of complex regional pain syndrome type I.

Complex regional pain syndrome (CRPS) is a disorder that involves abnormal inflammation and nerve dysfunction frequently resistant to a broad range of treatments. Peripheral nerve stimulation with electroacupuncture (EA) has been widely used in different clinical conditions to control pain and inflammation; however, the use of EA in the treatment of CRPS is under investigation. In this study, we explore the effects of EA on hyperalgesia and edema induced in an animal model of chronic post-ischemia pain (CPIP model) and the possible involvement of endothelin receptor type B (ETB) in this effect. Female Swiss mice were subjected to 3 h hind paw ischemia/reperfusion CPIP model. EA treatment produced time-dependent inhibition of mechanical and cold hyperalgesia, as well as edema in CPIP mice. Peripheral administration (i.pl.) of BQ-788 (10 nmol), an ETB antagonist, prevented EA-induced antihyperalgesia while intrathecal administration prolonged EA's effect. Additionally, peripheral pre-treatment with sarafotoxin (SRTX S6c, 30 pmol, ETB agonist) increased EA anti-hyperalgesic effect. Furthermore, the expression of peripheral ETB receptors was increased after EA treatments, as measured by western blot. These results may suggest that EA's analgesic effect is synergic with ETB receptor activation in the periphery, as well as central (spinal cord) ETB receptor blockade. These data support the use of EA as a nonpharmacological approach for the management of CRPS-I, in an adjuvant manner to ETB receptor targeting drugs.

A novel neuroregenerative approach using ET(B) receptor agonist, IRL-1620, to treat CNS disorders.

Endothelin B (ET(B)) receptors present in abundance the central nervous system (CNS) have been shown to have significant implications in its development and neurogenesis. We have targeted ET(B) receptors stimulation using a highly specific agonist, IRL-1620, to treat CNS disorders. In a rat model of cerebral ischemia intravenous administration IRL-1620 significantly reduced infarct volume and improved neurological and motor functions compared to control. This improvement, in part, is due to an increase in neuroregeneration. We also investigated the role of IRL-1620 in animal models of Alzheimer's disease (AD). IRL-1620 improved learning and memory, reduced oxidative stress and increased VEGF and NGF in Abeta treated rats. IRL-1620 also improved learning and memory in an aged APP/PS1 transgenic mouse model of AD. These promising findings prompted us to initiate human studies. Successful chemistry, manufacturing and control along with mice, rat and dog toxicological studies led to completion of a human Phase I study in healthy volunteers. We found that a dose of 0.6 microg/kg of IRL-1620 can be safely administered, three times every four hours, without any adverse effect. A Phase II clinical study with IRL-1620 has been initiated in patients with cerebral ischemia and mild to moderate AD.

Nomad Biosensors: A New Multiplexed Technology for the Screening of GPCR Ligands.

Nomad Technology (Innoprot [Innovative Technologies in Biological Systems], Derio, Spain), a novel tool for multiplexing high-throughput cell-based G protein-coupled receptor (GPCR) assays, is described in this work. This new technology comprises a family of fluorescent biosensors called Nomad Biosensors that allow for the measurement of responses mediated by G proteins through their interactions with second-messenger transduction proteins. GPCRs are one of the largest protein families of receptors in eukaryotes, and their signaling mediates important physiological processes within cells. Thus, GPCRs are associated with a wide variety of diseases, and considered major targets in therapeutic research. Nomad constitutes a novel tool for unraveling the mechanism of GPCR signal transduction by simultaneously tracing different pathways. GPCR activation changes the structural folding of the biosensor and promotes its vesicularization, as well as an increase in the fluorescence intensity. Based on this technology, the MPXNomad cellular model was developed to discriminate between the Ca2+-mediated pathway and the cyclic adenosine monophosphate (cAMP)-mediated pathway. To validate this model, endothelin receptor B (ETBR) was coexpressed into the MPXNomad cell line and assessed with a specific agonist, an antagonist, and a chemical library of compounds. Nomad Technology optimizes the identification of novel GPCR ligands and enables the testing of large numbers of compounds.

Nanocurcumin accords protection against acute hypobaric hypoxia induced lung injury in rats.

Decline in oxygen availability experienced under hypobaric hypoxia (HH) mediates imbalance in lung fluid clearance and is a causative agent of acute lung injury. Here, we investigate the pathological events behind acute HH mediated lung injury and assess the therapeutic efficacy of nanocurcumin in its amelioration. We assess the protective efficacy of nanotized curcumin (nanocurcumin) in ameliorating HH induced lung injury and compare to curcumin. Rats exposed to acute HH (6, 12, 24, 48 and 72 h) were subjected to histopathology, blood-gas analysis and clinical biochemistry, cytokine response and redox damage. HH induced lung injury was analysed using markers of lung injury due to pulmonary vasoconstriction (ET-1/2/3 and endothelin receptors A and B) and trans-vascular fluid balance mediator (Na+/K+ ATPase). The protective efficacy of nanocurcumin was analysed by examination of Akt/Erk signalling cascade by western blot. HH induced lung injury was associated with discrete changes in blood analytes, differential circulatory cytokine response and severe pulmonary redox damages. Up-regulation of ET-1/2/3 and its receptors along with down-regulation of Na+/K+ ATPase confirmed defective pulmonary fluid clearance which promoted edema formation. Nanocurcumin treatment prevented lung edema formation and restored expression levels of ET-1/2/3 and its receptors while restoring the blood analytes, circulatory cytokines and pulmonary redox status better than curcumin. Modulation in Akt/Erk signalling pathway in rat lungs under HH confirmed the protective efficacy of nanocurcumin.

Glytan decreases portal pressure via mesentery vasoconstriction in portal hypertensive rats.

AIM: To investigate the effects of Glytan on splanchnic hemodynamics and its reduction of portal pressure in portal hypertensive rats. METHODS: Glytan (Ganluotong in Chinese), is composed of salvianolic acid B and diammonium glycyrrhizinate. Portal hypertension (PHT) was induced in the rats by common bile duct ligation (BDL). Hemodynamic studies were performed using the colored microsphere method. Radioimmunoassay (RIA) was used to determine endothelin (ET)-1 levels in the mesenteric circulation. Western blotting methods were used to investigate the effect of Glytan on ET A receptor (ETAR), ET B receptor (ETBR), endothelial NO synthase (eNOS), G-protein-coupled receptor kinase (GRK)2, and ß-arrestin 2 expression in the mesentery. The mRNA of ETAR and ETBR was determined using real-time polymerase chain reaction. RESULTS: Treatment with Glytan reduced portal pressure (PP) and portal territory blood flow (PTBF) and increased both mean arterial pressure (MAP) and splanchnic vascular resistance (SVR). Especially at 4 wk, PP decreased by about 40%, while MAP increased by 13%, SVR increased by 12%, and PTBF decreased by about 21%. The effect of blood flow reduction was greatest in the mesentery (about 33%) at 4 wk. The mesenteric circulation ET-1 levels of BDL rats were lower and negatively correlated with PP at 4 wk. Glytan can increase mesenteric ET-1 content and inhibit ETBR, eNOS, GRK2, and ß-arrestin 2 expression in the mesentery. Moreover, Glytan showed no effect on the expression of ETAR protein and mRNA. CONCLUSION: The decreased PP and PTBF observed after Glytan treatment were related to increased mesenteric vasoconstriction and increased receptor sensitivity to vasoconstrictor.

Early MEK1/2 inhibition after global cerebral ischemia in rats reduces brain damage and improves outcome by preventing delayed vasoconstrictor receptor upregulation.

BACKGROUND: Global cerebral ischemia following cardiac arrest is associated with increased cerebral vasoconstriction and decreased cerebral blood flow, contributing to delayed neuronal cell death and neurological detriments in affected patients. We hypothesize that upregulation of contractile ETB and 5-HT1B receptors, previously demonstrated in cerebral arteries after experimental global ischemia, are a key mechanism behind insufficient perfusion of the post-ischemic brain, proposing blockade of this receptor upregulation as a novel target for prevention of cerebral hypoperfusion and delayed neuronal cell death after global cerebral ischemia. The aim was to characterize the time-course of receptor upregulation and associated neuronal damage after global ischemia and investigate whether treatment with the MEK1/2 inhibitor U0126 can prevent cerebrovascular receptor upregulation and thereby improve functional outcome after global cerebral ischemia. Incomplete global cerebral ischemia was induced in Wistar rats and the time-course of enhanced contractile responses and the effect of U0126 in cerebral arteries were studied by wire myography and the neuronal cell death by TUNEL. The expression of ETB and 5-HT1B receptors was determined by immunofluorescence. RESULTS: Enhanced vasoconstriction peaked in fore- and midbrain arteries 3 days after ischemia. Neuronal cell death appeared initially in the hippocampus 3 days after ischemia and gradually increased until 7 days post-ischemia. Treatment with U0126 normalised cerebrovascular ETB and 5-HT1B receptor expression and contractile function, reduced hippocampal cell death and improved survival rate compared to vehicle treated animals. CONCLUSIONS: Excessive cerebrovascular expression of contractile ETB and 5-HT1B receptors is a delayed response to global cerebral ischemia peaking 3 days after the insult, which likely contributes to the development of delayed neuronal damage. The enhanced cerebrovascular contractility can be prevented by treatment with the MEK1/2 inhibitor U0126, diminishes neuronal damage and improves survival rate, suggesting MEK1/2 inhibition as a novel strategy for early treatment of neurological consequences following global cerebral ischemia.

Efficacy of the specific endothelin a receptor antagonist zibotentan (ZD4054) in colorectal cancer: a preclinical study.

Endothelin 1 (ET-1) is overexpressed in cancer, contributing to disease progression. We previously showed that ET-1 stimulated proliferative, migratory, and contractile tumorigenic effects via the ET(A) receptor. Here, for the first time, we evaluate zibotentan, a specific ET(A) receptor antagonist, in the setting of colorectal cancer, in cellular models. Pharmacologic characteristics were further determined in patient tissues. Colorectal cancer lines (n = 4) and fibroblast strains (n = 6), isolated from uninvolved areas of colorectal cancer specimens, were exposed to ET-1 and/or ET(A)/(B) receptor antagonists. Proliferation (methylene blue), migration (scratch wounds), and contraction (gel lattices) were assessed. Receptor distribution and binding characteristics (K(d), B(max)) were determined using autoradiography on tissue sections and homogenates and cytospun cells, supported by immunohistochemistry. Proliferation was inhibited by ET(A) (zibotentan > BQ123; P < 0.05), migration by ET(B) > ET(A), and contraction by combined ET(A) and ET(B) antagonism. Intense ET-1 stromal binding correlated with fibroblasts and endothelial cells. Colorectal cancer lines and fibroblasts revealed high density and affinity ET-1 binding (B(max) = 2.435 fmol/1 × 10(6) cells, K(d) = 367.7 pmol/L; B(max) = 3.03 fmol/1 × 10(6) cells, K(d) = 213.6 pmol/L). In cancer tissues, ET(A) receptor antagonists (zibotentan; BQ123) reduced ET-1 binding more effectively (IC(50): 0.1-10 µmol/L) than ET(B) receptor antagonist BQ788 (∼IC(50), 1 mmol/L). ET-1 stimulated cancer-contributory processes. Its localization to tumor stroma, with greatest binding/affinity to fibroblasts, implicates these cells in tumor progression. ET(A) receptor upregulation in cancer tissues and its role in tumorigenic processes show the receptor's importance in therapeutic targeting. Zibotentan, the most specific ET(A) receptor antagonist available, showed the greatest inhibition of ET-1 binding. With its known safety profile, we provide evidence for zibotentan's potential role as adjuvant therapy in colorectal cancer.

Prunellae spica extract contains antagonists for human endothelin receptors.

Endothelin-1 (ET-1) is a powerful vasoconstrictor that contributes to blood pressure elevation. The biological effects of ETs are mediated by two receptors, namely, endothelin type A receptor (ET(A)R) and endothelin type B receptor (ET(B)R). Chinese herbal medicines (CHM) with antagonist activity for these two receptors were screened by establishing stable clones of CHO-K1 cells expressing high levels of human ET(A)R and ET(B)R, namely CHO-ET(A)R and CHO-ET(B)R.The aqueous extract of Prunellae Spica (P1) inhibited the binding of (125)I-ET-1 to ET(A)R and ET(B)R in CHO-ET(A)R and CHO-ET(B)R cells, respectively. P1 suppressed the ET-1-induced mobilization of intracellular Ca(2+) . Through the alcohol fractionation of P1, the antagonists of human ET(A)R and ET(B)R were found to belong to different, separable ingredients and the antagonist of ET(A)R is more soluble in alcohol. The two antagonists were also effective in the test on human primary cells, HASMC and HUVEC. P1 successfully prevented the development of ET-1-associated hypertension in rats without further purification. These results indicate the presence of anti-hypertensive ingredients in P. Spica extract, at least through the inactivation of ET(A)R and/or ET(B)R.

Effects of selenium supplementation on expression of endothelin-1 and its receptors in pulmonary microvascular endothelial cells from chick embryos.

The objective of this study was to evaluate the effects of supplemental selenium (Se) on expression of endothelin-1 (ET-1) and its receptors in cultured chick embryos pulmonary microvascular endothelial cells (PMVECs). To accomplish this, PMVECs were treated in Se-deficient or Se-supplement (12, 24, 50, 100 ng/ml) culture medium for 48 h. Low Se medium was achieved by reducing serum concentrations and the essential growth factors were added. After the incubation, the effects of supplemental Se on ET-1 and its receptors gene expression were assessed by quantitative real-time PCR (qRT-PCR). Compared with the control group, our results showed that among the different concentrations of Se supplement, the levels of ET-1 gene expression treated with both the moderate Se doses (24, 50 ng/ml, P < 0.01, P < 0.01, respectively) and the high doses (100 ng/ml, P < 0.05) were noticeably decreased, the low-dose group (12 ng/ml), which showed no changes. Meanwhile, Se supplement (24, 50, 100 ng/ml) was found to be effective in reducing the expression levels of ETA (P < 0.01, P < 0.05, P < 0.05, respectively) in cultured PMVECs grown in low Se medium. However, there were no significant changes (P > 0.05) in ETB mRNA levels during the cell proliferation. These observations indicated that Se may play both direct and indirect role in the regulation of ET-1 and its receptors gene expression and their production in avian PMVECs. Se supplement decreases in ET-1 and ETA production in Se-deficient PMVECs may partly explain the mechanism of the protective effects of the Se on the cardiovascular system.

Endothelin B receptors contribute to retinal ganglion cell loss in a rat model of glaucoma.

Glaucoma is an optic neuropathy, commonly associated with elevated intraocular pressure (IOP) characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells which could lead to loss of vision. Endothelin-1 (ET-1) is a 21-amino acid vasoactive peptide that plays a key role in the pathogenesis of glaucoma; however, the receptors mediating these effects have not been defined. In the current study, endothelin B (ET(B)) receptor expression was assessed in vivo, in the Morrison's ocular hypertension model of glaucoma in rats. Elevation of IOP in Brown Norway rats produced increased expression of ET(B) receptors in the retina, mainly in retinal ganglion cells (RGCs), nerve fiber layer (NFL), and also in the inner plexiform layer (IPL) and inner nuclear layer (INL). To determine the role of ET(B) receptors in neurodegeneration, Wistar-Kyoto wild type (WT) and ET(B) receptor-deficient (KO) rats were subjected to retrograde labeling with Fluoro-Gold (FG), following which IOP was elevated in one eye while the contralateral eye served as control. IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in KO rats. In addition, degenerative changes in the optic nerve were greatly reduced in KO rats compared to those in WT rats. Taken together, elevated intraocular pressure mediated increase in ET(B) receptor expression and its activation may contribute to a decrease in RGC survival as seen in glaucoma. These findings raise the possibility of using endothelin receptor antagonists as neuroprotective agents for the treatment of glaucoma.

Endothelin receptor expression in idiopathic pulmonary arterial hypertension: effect of bosentan and epoprostenol treatment.

Endothelin receptor antagonists are used to treat idiopathic pulmonary arterial hypertension (IPAH), but human pulmonary arterial endothelin receptor expression is not well defined. We hypothesised that disease and treatment would modify normal receptor distribution in pulmonary resistance arteries of children. Using immunohistochemistry and semiquantitative analysis, we investigated endothelin receptor subtypes A and B (ET(A) and ET(B), respectively), and endothelial nitric oxide synthase (eNOS) expression in peripheral pulmonary arteries of tissue from untreated children with IPAH (n=7), following extended combined bosentan and epoprostenol therapy (n=5) and from normal subjects (n=5). Clinical, haemodynamic and pathological abnormalities were severe and advanced in all IPAH cases. ET(A) was detected in pulmonary arterial endothelial cells of all normal and diseased tissue and cultured cells. Endothelial ET(A), ET(B) and eNOS expression was reduced in patent, plexiform and dilatation lesions of untreated cases, but in treated cases, ET(A) and ET(B) were normal and eNOS increased. In smooth muscle, ET(A) expression was reduced in treated cases but ET(B) expression increased in all arteries of both treated and untreated cases. In summary, ET(A) is expressed on human pulmonary arterial endothelium. In IPAH, combination treatment with bosentan and epoprostenol had a more marked influence on endothelin receptor expression of endothelial than smooth muscle cells.

Differential endothelin receptor expression and function in rat myometrial cells and leiomyoma ELT3 cells.

Uterine leiomyoma are the most common benign tumors of the myometrium. We previously identified endothelin (ET)-1 as a proliferative and antiapoptotic factor in Eker rat-derived leiomyoma (ELT3) cells. A major role of ETB receptor in the prosurvival effect was revealed. Here we investigated, in ELT3 and myometrial cells, the respective contribution of ETA and ETB in the proliferative effect of ET-1. In myometrial cells, binding experiments show that ETA is almost exclusively expressed and stimulates phospholipase C (PLC) activity and ERK1/2 phosphorylation and proliferation. In ELT3 cells, ETB is expressed at about the same level as ETA, and the two receptors are differently coupled to Gi protein. The ETB agonist, sarafotoxin S6c, stimulates PLC activity 60% less than ET-1 but is as potent as ET-1 to increase ERK1/2 phosphorylation and induce proliferation. However, the ability of ETA to activate ERK1/2 is observed after ETB desensitization. Although ETA and ETB antagonists partially reduce ET-1 stimulated PLC activity, they are without effect on ET-1-induced ERK1/2 phosphorylation and proliferation. Only the simultaneous use of ETA and ETB antagonists reduces ET-1-triggered ERK1/2 activation. These unconventional properties of ETRs may reveal the existence of functional ETA-ETB heterodimers. Finally, treatment of ELT3 cells with ETB but not ETA-directed small interfering RNA reduces the proliferative effect of ET-1. All the data obtained in ELT3 cells strengthen the relation between ETB overexpression, which decreases the ETA to ETB ratio, and the ability of leiomyoma cells to highly proliferate and resist apoptosis.

Cerebrovascular characterization of clazosentan, the first nonpeptide endothelin receptor antagonist shown to be clinically effective for the treatment of cerebral vasospasm. Part II: effect on endothelin(B) receptor-mediated relaxation.

OBJECT: The disturbed balance between nitric oxide and endothelin (ET)-1 in the cerebrovasculature seems to play a major role in the development of cerebral vasospasm after subarachnoid hemorrhage. Endothelin-1 represents the contractile part in this balance. In addition to the prevailing ET(A) receptor-dependent contractile effect, ET-1 also has ET(B) receptor-mediated vasodilatory attributes. The aim of the present study was to define the actual selectivity of clazosentan, the first putative highly ET(A) receptor-selective antagonist clinically proven to be effective in the treatment of vasospasm in the cerebrovasculature. METHODS: Rat basilar artery ring segments with endothelial function were used for the measurement of isometric force. Concentration effect curves were constructed by cumulative application of sarafotoxin S6c, ET-1, or big ET-1 in the presence or absence of clazosentan (10(-9) to 10(-6) M) after a precontraction was induced by prostaglandin F2alpha. The inhibition by clazosentan was estimated by the value of the affinity constant (pA2). The relaxation induced by sarafotoxin S6c, ET-1, and big ET-1 was inhibited in a competitive manner by clazosentan, yielding pA2 values of 7.1, 6.7, and 6.5, respectively. The selectivity to the ET(A) receptor in the cerebrovascular system was approximately two logarithmic units. CONCLUSIONS: The present investigation shows a competitive inhibition of ET(B) receptor-mediated relaxation in cerebral vessels by clazosentan in therapeutically relevant concentrations. Thus, additional clinical trials should be undertaken to evaluate clazosentan concentrations in cerebrospinal fluid. Furthermore, the present data may be taken to describe the pharmacological properties for an ET receptor antagonist specifically tailored for the treatment of pathological conditions of impaired cerebral blood flow.

Changes of brain endothelin levels and peripheral endothelin receptors by chronic cigarette smoke in spontaneously hypertensive rats.

The present study was conducted to evaluate the contribution of endothelin (ET) to the pharmacodynamic response to chronic cigarette smoke in spontaneously hypertensive rats (SHR). The contribution of ET was studied consequent to the hemodynamic response following 8 weeks of cigarette smoke by determining the changes in tissue ET-1 content and ET receptors. The blood pressure (BP) at the early phase of smoking and the heart rate (HR) 24 h later were apparently reduced in SHR, while the HR at the early phase was transiently elevated in normotensive Wistar Kyoto (WKY) rats. Tissue ET-1 levels in the hypothalamus, striatum, and cortex of SHR were higher than those in WKY rats, and these higher levels in SHR were reduced by exposure to chronic cigarette smoke. The ET-1 contents in the medulla oblongata and midbrain of both strains were clearly increased by smoke exposure, although the levels of SHR and WKY rats were not different. In addition, the immunoreactivity of the ET type A receptor in the adrenal glands and type B receptor in the kidneys of SHR showed a different response to smoke exposure as compared to WKY rats. Our present findings suggest that the changes of ETs may relate to the pharmacodynamic effects of chronic cigarette smoke.