endothelin-B Receptor Agonist Research Articles

Efficacy and Safety of Sovateltide in Patients with Acute Cerebral Ischaemic Stroke: A Randomised, Double-Blind, Placebo-Controlled, Multicentre, Phase III Clinical Trial.

Sovateltide (Tycamzzi™), an endothelin-B (ET-B) receptor agonist, increases cerebral blood flow, has anti-apoptotic activity, and promotes neural repair following cerebral ischaemic stroke. The objectives of this study were to evaluate the efficacy and safety of sovateltide in adult participants with acute cerebral ischaemic stroke. This was a randomised, double-blind, placebo-controlled, multicentre, Phase III clinical trial of sovateltide in participants with cerebral ischaemic stroke receiving standard of care (SOC) in India. Patients aged 18-78 years presenting up to 24 h after the onset of symptoms with radiologic confirmation of ischaemic stroke and a National Institutes of Health Stroke Scale score (NIHSS) of ≥ 6were enrolled. Patients with recurrent stroke, receiving endovascular therapy, or with intracranial haemorrhage were excluded. The study drug (saline or sovateltide [0.3 µg/kg] was administered intravenously in three doses at 3 ± 1 h intervals on Days 1, 3, and 6, and follow-up was 90 days). The Multivariate Imputation by Chained Equations (MICE) was used to impute the missing assessments on the endpoints. An unpaired t-test, two-way analysis of variance with Tukey's multiple comparison test, and the Chi-square test were used for the statistical analysis. The objective was to determine at Day 90 (1) the number of patients with a modified Rankin Scale score (mRS) 0-2, and (2) the number of patients with an NIHSS 0-5 at 90 days. Patients were randomised with 80 patients in the sovateltide and 78 in the control group. Patients received the investigational drug at about 18 h of stroke onset in both control and sovateltide groups. The median NIHSS at randomisation was 10.00 (95% CI 9.99-11.65) in the control group and 9.00 (95% CI 9.11-10.46) in the sovateltide group. Seventy patients completed the 90-day follow-up in the control group and 67 in the sovateltide group. The proportion of intention-to-treat (ITT) patients with mRS 0-2 score at Day 90 post-randomisation was 22.67% higher (odds ratio [OR] 2.75, 95% CI 1.37-5.57); similarly, the proportion of patients with NIHSS score of 0-5 at Day 90 was 17.05% more (OR 2.67, 95% CI 1.27-5.90) in the sovateltide group than in the control group. An improvement of ≥ 2 points on the mRS was observed in 51.28% and 72.50% of patients in the control and sovateltide groups, respectively (OR 2.50, 95% CI 1.29-4.81). Seven of 78 patients (8.97%) in the control group and 7 of 80 (8.75%) in the sovateltide group developed intracranial haemorrhage (ICH). The adverse events were not related to sovateltide. The sovateltide group had a greater number of cerebral ischaemic stroke patients with lower mRS and NIHSS scores at 90 days post-treatment than the control group. This trial supported the regulatory approval of sovateltide in India, but a multinational RESPECT-ETB trial will be conducted for US approval. Clinical Trials Registry, India (CTRI/2019/09/021373) and the United States National Library of Medicine, ClinicalTrials.gov (NCT04047563).

Sovateltide: First Approval.

Sovateltide (Tycamzzi™), a highly selective endothelin-B receptor agonist and synthetic analog of endothelin-1, is being developed by Pharmazz, Inc. as a neural progenitor cell therapeutic agent for the treatment of acute cerebral ischemic stroke (ACIS), hypoxic-ischemic encephalopathy (HIE), spinal cord injuries and Alzheimer's disease. In May 2023, sovateltide was approved in India for the treatment of cerebral ischemic stroke within 24 h of stroke onset. This article summarizes the milestones in the development of sovateltide leading to this first approval for use in patients with ACIS.

Abstract 88: A Multicentric, Randomized, Controlled Phase III Study With Sovateltide (Tycamzzi™) Improving Outcomes In Patients With Acute Cerebral Ischemic Stroke

Sovateltide (Tycamzzi™), a selective endothelin-B receptor agonist, has antiapoptotic activity, protects neural mitochondria, and produces neurogenesis. It showed significant safety and efficacy in preclinical and clinical phase I and II studies. A prospective, multicentric, randomized phase III study was conducted in acute cerebral ischemic stroke (ACIS) patients aged 18 through 78 years. Patients with radiologic confirmation of ischemic stroke could be enrolled if presenting up to 24 hours with NIHSS score of greater than 5. Patients with intracranial hemorrhage and those receiving endovascular therapy were excluded. Sovateltide was administered in three doses, each dose of 0.3 μg/kg, as an intravenous bolus at an interval of 3 hours ± 1 hour on day 1, day 3, and day 6 (total dose/day: 0.9 μg/kg). The primary objectives were to determine the neurological outcome based on the mRS score, NIHSS score, and BI scale score from day 1 through day 90. A total of 158 patients with ACIS were enrolled, of which 137 completed a 90-day follow-up. Patients received saline (n=70, 62% male) or sovateltide (n=67, 66% male) at 16.85 ± 0.74 and 17.40 ± 0.67 hours (mean ± SEM) of stroke onset, respectively. The baseline characteristics and SOC in both cohorts were similar. ASPECTS mean value was similar in the control (7.44) and sovateltide (7.61) groups. A significantly greater number of patients in the sovateltide group had an improvement of mRS of ≥2 points (p=0.004) and NIHSS of ≥6 points (p=0.033) vs. baseline at 90 days of treatment. However, an improvement in BI (change of ≥40 points vs. baseline) missed significance (p=0.063). At 90 days, mRS (p=0.007) and NIHSS (p=0.003) were significantly lower, while BI (p=0.011) and EuroQol-EQ-5D (p=0.0055) were significantly higher in the sovateltide compared to the control group. Sovateltide is safe, well-tolerated, and significantly improved neurological outcomes in ACIS patients and may prove to be a novel, effective agent for its treatment.

Abstract 14: A Randomized Study to Determine Efficacy of Sovateltide (PMZ-2010) in Patients With Acute Cerebral Ischemic Stroke

Sovateltide (IRL-1620, PMZ-1620), an endothelin-B receptor agonist, administered intravenously following acute cerebral ischemic stroke increased cerebral blood flow, had anti-apoptotic activity and produced neurovascular remodeling. Its safety and tolerability were confirmed in healthy human volunteers (CTRI/2016/11/007509). A prospective, multi-centric, randomized, double-blind, parallel, controlled clinical study to compare efficacy of sovateltide with standard treatment in patients with acute cerebral ischemic stroke (CTRI/2017/11/010654; NCT04046484) was conducted in 40 patients, of which 36 completed 90-day follow-up. Patients received saline (n=18; 11 male and 7 female) or sovateltide (n=18; 15 male and 3 female) within 24 hours of onset of stroke. Standard treatment in both cohorts was similar. Mean age in saline and sovateltide cohorts was 58.34±1.73 and 49.83±2.79 years, respectively. Number of patients receiving investigational drug within 20 hours of onset of stroke were 14/18 in saline and 10/18 in sovateltide cohorts. National Institute of Health Stroke Scale (NIHSS), modified Rankin Scale (mRS) and Barthel Index (BI) on day 6 when compared to day 1 showed P values of 0.0495, 0.0859 and 0.3948, respectively in saline cohort, whereas the P value in sovateltide cohort was less than 0.0001 of all three indices in sovateltide cohort. An improvement of 6 or more in NIHSS at 90 day of treatment was 57% in sovateltide and 43% patients in saline cohort (P=0.2714; Odds ratio 2.275). mRS improvement of 2 or more was observed in 60% and 40% patients in sovateltide and saline cohorts, respectively (P=0.0519; Odds ratio 5.25). BI improvement of 40 or more was 64% and 36% in sovateltide and saline cohorts, respectively (P=0.0112; Odds ratio 12.44). Number of patients with full recovery achieving NIHSS score of 0 and BI of 100 were significantly (P<0.05) more in sovateltide cohort compared to saline. Hemodynamic, biochemical or hematological parameters were not affected and no incidence of drug related adverse event with sovateltide was reported. Sovateltide (PMZ-1620) has the potential to an effective first-in-class drug that promotes quicker recovery and improves neurological outcome in acute ischemic stroke patients.

Anti-apoptotic activity of ETB receptor agonist, IRL-1620, protects neural cells in rats with cerebral ischemia

Endothelin-B receptor agonist, IRL-1620, provides significant neuroprotection following cerebral ischemia in rats. Whether this neuroprotection is due to inhibition of apoptosis is unknown. IRL-1620-treated rats following permanent middle cerebral artery occlusion (MCAO) showed significant improvement in neurological and motor functions along with a decrease in infarct volume at 24 h (−81.3%) and day 7 (−73.0%) compared to vehicle group. Cerebral blood flow (CBF) significantly improved in IRL-1620-treated animals compared to vehicle by day 7 post MCAO. IRL-1620-treated rats showed an increase in phospho-Akt and decrease in Bad level 7 h post-occlusion compared to vehicle, while Akt and Bad expression was similar in cerebral hemispheres at 24 h post-MCAO. The phospho-Bad level was lower in vehicle- but not in IRL-1620-treated rats at 24 h. Anti-apoptotic Bcl-2 expression decreased, while pro-apoptotic Bax expression increased in vehicle-treated MCAO rats, these changes were attenuated (P < 0.01) by IRL-1620. Mitochondrial membrane-bound Bax intensity significantly decreased in IRL-1620 compared to vehicle-treated MCAO rats. IRL-1620 treatment reduced (P < 0.001) the number of TUNEL-positive cells compared to vehicle at 24 h and day 7 post MCAO. The results demonstrate that IRL-1620 is neuroprotective and attenuates neural damage following cerebral ischemia in rats by increasing CBF and reducing apoptosis.

IRL-1620, an Endothelin-B Receptor Agonist, Enhanced Radiation Induced Reduction in Tumor Volume in Dalton’s Lymphoma Ascites Tumor Model

ETB receptor agonist, IRL-1620 (or SPI-1620) presently in US Phase 1 clinical trial, has been demonstrated to selectively and transiently increase tumor blood flow. The present study was conducted to determine the effect of IRL-1620 on radiation therapy in tumor bearing mice inoculated with Dalton's Lymphoma Ascites cells. Tumors were allowed to grow for 30 days to a size of 1.10-1.29 cm3 before starting the treatment. The animals with or without IRL-1620 treatment were exposed to radiation (4 Gy/dose) on every alternate day for a total of 5 doses. Tumor volume was determined twice every week till the end of study. Radiation alone did not affect the tumor volume; however, animals treated with IRL-1620 followed by radiation produced a significant (64%) reduction in tumor volume. Survival of mice improved from 0/10 at 56 days after tumor inoculation in vehicle plus radiation group to 6/10 at 70 days in IRL-1620 (9 nmol/kg) plus radiation group. It is concluded that IRL-1620 improves the efficacy of radiation treatment in tumor bearing mice. (These findings have been earlier presented as an abstract ).

Endothelin/endothelin-B receptor signals regulate ventricle-directed interkinetic nuclear migration of cerebral cortical neural progenitors

We determined the expression profile of ∼300 G protein-coupled receptors (GPCRs) in embryonic cortical neural progenitor cells (NPCs) and identified a number of highly expressed GPCRs, among which endothelin-B receptor (ETB-R) was expressed at the highest level. We also revealed that endothelins (ETs) were predominantly expressed in CD31-positive endothelial cells of the embryonic cerebral cortex. Activation of ETB-R induced NPC assembly in vitro by promoting fibronectin-dependent-motility and N-cadherin-associated cell contact. NPC assembly also required a Rho-family GTPase(s) and phosphatidylinositol-3-kinase. In the embryonic cerebral cortex, a specific ETB-R agonist, IRL-1620, accelerated interkinetic nuclear migration (INM) of NPCs toward the ventricular wall (VW) ex vivo. Conversely, a specific ETB-R antagonist, BQ788, slowed INM, thereby inducing mislocalization of phospho-histone H3-positive M-phase nuclei in the ventricular zone (VZ) and decreasing the number of Tuj1-positive newborn neurons. Our results suggest that ETB-R-mediated assembly signals drive INM that precedes neurogenesis.

Endothelin receptor-mediated attenuation of carcinoma-induced nociception is opioid-dependent in mice

Author(s): Quang, Phuong Ngoc | Advisor(s): Damsky, Caroline; Schmidt, Brian L | Abstract: Cancer pain is a frequent and disabling consequence for many patients yet the mechanism of cancer pain remains unknown largely due to its complex etiology. The objective of this thesis work was to investigate the potential of a peptide produced by carcinoma cells, endothelin, as a novel target for cancer pain management. This study provided evidence in support of Hypothesis 1: Oral squamous carcinoma cells produce endogenous opioids in response to changes in endothelin-1 signaling. Effects of endothelin-1 (ET-1) signaling on secretion of opioids by oral squamous cell carcinoma (SCC) were studied in vitro. Oral SCC cell line HSC-3 produces abundant ET-1 that can act in both an autocrine or paracrine manner. In the untreated oral SCC cells, endogenous opioids were detected in both cultured media and cell lysates. When treated with Endothelin-A receptor antagonist, secreted levels of abeta-endorphin and leu-enkephalin were increased; whereas treatment with Endothelin-B receptor agonist increased production of only abeta-endorphin. This apparent regulation of endogenous opioid levels by endothelin receptor drugs in vitro procured evidence suggesting that Hypothesis 2: Endothelin-A receptor antagonist and Endothelin-B receptor agonist attenuate carcinoma-induced nociception in cancer animals through regulation of endogenous opioids. Endothelin-A (ET-AR) antagonism has been demonstrated previously to attenuate carcinoma-mediated hyperalgesia in an orthotopic cancer pain mouse model. In this study, effects of ET-BR agonist in vivo were evaluated and determined to also result in significant increase in paw withdrawal thresholds, indicating attenuation of cancer-induced nociception. When peripheral amu- or adelta-opioid receptor antagonists were administered following ET-AR antagonism, nociceptive attenuation was completely reversed. Attenuation from ET-BR agonism was likewise reversed upon administration of amu-opioid receptor antagonist. Combined results demonstrate that endogenous opioids are the likely mediators responsible for attenuation of carcinoma-induced nociception with either ET-AR antagonist or ET-BR agonist.These novel findings suggest that there exist innate modulation of pain by SCCs involving endogenous opioids that can be exploited through manipulation of endothelin activity in the cancer micro-environment. Regulation of ET-AR or ET-BR signaling may be targets for future innovations for cancer pain management.

Natriuretic response to renal medullary endothelin B receptor activation is blunted in chronic angiotensin II‐infused rats

Endothelin B (ETB) receptors, highly expressed in the renal medulla, function to promote sodium and water excretion such that the impairment of ETB receptor function results in salt‐dependent hypertension. Since chronic angiotensin (Ang) II also results in salt‐dependent hypertension, we hypothesized that renal medullary ETB receptor function is impaired in Ang II‐dependent hypertension. We compared the diuretic and natriuretic response to intramedullary infusion of ETB receptor agonist sarafotoxin 6c (S6c) in rats treated with Ang II (65 ng/min s.c.) or vehicle for 14 days. Rats were anesthetized and renal perfusion pressure maintained at a normotensive level in Ang II rats via an adjustable clamp placed around aorta. Urine was collected during 2 consecutive 20 min periods for baseline collection followed by 4 additional 20 min periods of intramedullary saline or S6c (0.45 μg/kg/h) infusion. Renal medullary blood flow (MBF) was determined by laser‐doppler flowmetry. In the vehicle group, S6c significantly increased urine flow (Δ8.4+1.7 μL/min; p&lt;0.01) and sodium excretion (Δ0.79+0.24 μmol/min; p&lt;0.01). However, Ang II rats did not display an increase of urine flow (Δ0.7+0.7 μL/min) and sodium excretion (Δ‐0.06+0.04 μmol/min) during S6c infusion. S6c did not affect MBF in either group. These data demonstrate that renal medullary ETB receptor function is impaired in Ang II‐dependent hypertension.

Endothelin B Receptors on Vascular Smooth Muscle Cells of the Human Umbilical Vein Mediate Vasoconstriction

Objective: To test the hypothesis that smooth muscle cells of the human umbilical vein have vasoconstricting endothelin B (ETB) receptors. Methods: In strip preparations of human umbilical veins isometric tension after exposure to the selective ETB receptor agonist sarafotoxin S6c (S6c) was compared to the tension before S6c exposure (set as 100%). Results: In intact preparations S6c induced vasoconstriction only at the highest concentration applied (10^–8M; 149.5 ± 12.5 vs. 100%, p < 0.05). In contrast, in endothelium-denuded preparations S6c induced vasoconstriction already at the lowest S6c concentration investigated (10^–11M; 111.7 ± 4.3 vs. 100%, p < 0.05). Conclusions: The vasoconstricting effect of S6c in endothelium-denuded human umbilical vein preparations points to smooth muscle cell ETB receptor activation.

ERK1/2 Inhibition Attenuates Cerebral Blood Flow Reduction and Abolishes ETB and 5-HT1B Receptor Upregulation after Subarachnoid Hemorrhage in Rat

Upregulation of endothelin B (ET(B)) and 5-hydroxytryptamine 1B (5-HT(1B)) receptors via transcription has been found after experimental subarachnoid hemorrhage (SAH), and this is associated with enhanced phosphorylation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK1/2). In the present study, we hypothesized that inhibition of ERK1/2 alters the ET(B) and 5-HT(1B) receptor upregulation and at the same time prevents the sustained cerebral blood flow (CBF) reduction associated with SAH. The ERK1/2 inhibitor SB386023-b was injected intracisternally in conjunction with and after the induced SAH in rats. At 2 days after the SAH, cerebral arteries were harvested for quantitative real-time polymerase chain reaction, immunohistochemistry and analysis of contractile responses to endothelin-1 (ET-1; ET(A) and ET(B) receptor agonist) and 5-carboxamidotryptamine (5-CT; 5-HT1 receptor agonist) in a sensitive myograph. To investigate if ERK1/2 inhibition had an influence on the local and global CBF after SAH, an autoradiographic technique was used. At 48 h after induced SAH, global and regional CBF were reduced by 50%. This reduction was prevented by treatment with SB386023-b. The ERK1/2 inhibition also decreased the maximum contraction elicited by application of ET-1 and 5-CT in cerebral arteries compared with SAH. In parallel, ERK1/2 inhibition downregulated ET(B) and 5-HT(1B) receptor messenger ribonucleic acid and protein levels compared with the SAH. Cerebral ischemia after SAH involves vasoconstriction and subsequent reduction in the CBF. The results suggest that ERK1/2 inhibition might be a potential treatment for the prevention of cerebral vasospasm and ischemia associated with SAH.

Local Injection of a Selective Endothelin-B Receptor Agonist Inhibits Endothelin-1-Induced Pain-Like Behavior and Excitation of Nociceptors in a Naloxone-Sensitive Manner

We showed previously that subcutaneous injection of the injury-associated peptide mediator endothelin-1 (ET-1) into the rat plantar hindpaw produces pain behavior and selective excitation of nociceptors, both through activation of ET(A) receptors likely on nociceptive terminals. The potential role of ET(B) receptor activation in these actions of ET-1-has not been examined. Therefore, in these experiments, we studied the effect of blocking or activating ET(B) receptors on ET-1-induced hindpaw flinching and excitation of nociceptors in rats. An ET(B) receptor-selective antagonist, BQ-788 (3 mm), coinjected with ET-1 (200 microm) reduced the time-to-peak of flinching and significantly enhanced the average maximal flinch frequency (MFF). In contrast, coinjection of an ET(B) receptor selective agonist, IRL-1620 (100 or 200 microm), with ET-1 reduced the average MFF and the average total number of flinches. Interestingly, this unexpected inhibitory effect of IRL-1620 was prevented by the nonselective opioid receptor antagonist naloxone (2.75 mm). To confirm these inhibitory actions, we studied the effects of IRL-1620 on ET-1-induced spike responses in single, physiologically characterized nociceptive C-fibers. IRL-1620 suppressed spike responses to ET-1 in all (n = 12) C-units, with mean and maximum response frequencies of 0.08 +/- 0.02 and 1.5 +/- 0.4 impulses/sec versus 0.32 +/- 0.07 and 4.17 +/- 0.17 impulses/sec for ET-1 alone. In additional support of the behavioral results, coinjection of naloxone (2.75 mm) completely prevented this inhibitory action of IRL-1620. These results establish that ET(B) receptor activation inhibits ET-1-induced pain behavior and nociception in a naloxone-sensitive manner and point to a previously unrecognized dual modulation of acute nociceptive signaling by ET(A) and ET(B) receptors in cutaneous tissues.

Increased coronary effects of stimulation of endothelin-B receptor in experimental hypercholesterolemia.

Vasoconstriction in response to endothelin-1 has been shown to be primarily related to its effects on the endothelin-A receptor. Experimental hypercholesterolemia is associated with an increase in coronary vasoconstrictor response to endothelin-1 in vivo, although the relative contributions of subtypes of endothelin receptor in this model remain unknown. To test the hypothesis that there is an increase in coronary vasoconstriction in response to stimulation of endothelin-B receptor in hypercholesterolemia, which might be related to attenuation of activity of endothelin-derived relaxing factor. We infused 5 ng/kg/min sarafotoxin, a specific endothelin-B receptor agonist, or 50 micrograms/kg/min NG-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of nitric oxide synthase, into the left anterior descending coronary arteries of pigs before and after feeding them a cholesterol-rich diet for 10 weeks. There was a significant increase in serum level of cholesterol. After 10 weeks, infusion of sarafotoxin resulted in an accentuated decrease in coronary blood flow (CBF) compared with baseline (decreases by 60 +/- 7 versus 34 +/- 6%, P < 0.05). There was no significant difference between the effects on diameter of coronary arteries for the two time periods. The effect of L-NMMA on CBF was attenuated after 10 weeks (by 5 +/- 10.1 versus 45.6 +/- 4.7%, P < 0.05). Endothelin-receptor status of epicardial coronary arteries remained unchanged. Sarafotoxin and L-NMMA were co-infused at the above-mentioned doses into normolipidemic animals; the decrease in CBF in response to this co-infusion was comparable to the decrease observed with sarafotoxin alone in hypercholesterolemic animals (decreases of 67 +/- 5 versus 60 +/- 7, NS). The present results demonstrate that selective stimulation of the endothelin-B receptor increases coronary vasoconstriction in experimental hypercholesterolemia, primarily at the level of the microvasculature. These findings may be related to the attenuation of activity of endothelin-derived relaxing factor in this model, and support the hypothesis that endothelin-B receptor plays a role in the regulation of coronary vascular tone in pathophysiologic states.

Endothelins increase tyrosine phosphorylation of astrocytic focal adhesion kinase and paxillin accompanied by their association with cytoskeletal components

Astrocytic endothelin receptors are involved in the appearance of activated astrocytes upon injury of the brain [Ishikawa N. et al. (1997) Eur. J. Neurosci. 9, 895–901; Koyama Y. et al. (1999) Glia 26, 268–271]. To clarify signal transduction triggered by endothelin receptors, we examined the effects of endothelins on protein tyrosine phosphorylation in cultured rat astrocytes. Endothelin-1 (1 nM) increased tyrosine phosphorylation of focal adhesion kinase and paxillin. The tyrosine phosphorylation was also induced by endothelin-1 (1 nM) and Ala 1,3,11,15-endothelin-1 (10 nM), an endothelin-B receptor agonist. BQ788 (100 nM), an endothelin-B receptor antagonist, inhibited the effects of endothelin-3. Orthovanadate (VO 4 3−), a tyrosine phosphatase inhibitor, but not bradykinin (1 μM), angiotensin II (100 nM), A23187 (5 μM) and phorbol 12-myristate 13-acetate (100 nM), increased tyrosine phosphorylation of focal adhesion kinase and paxillin. The tyrosine phosphorylation by endothelin-3 was not prevented by pertussis toxin, Ca 2+ chelation, protein kinase C inhibitors (calphostin C and staurosporine) or wortmannin. Immunocytochemical staining showed that endothelin-3 and VO 4 3− induced redistribution of focal adhesion kinase and paxillin to focal adhesions concomitant with stress fiber formation in dibutyryl cyclic-AMP-treated astrocytes. Treatment with endothelin-3 and VO 4 3− increased focal adhesion kinase and paxillin associated with astrocytic cytoskeletal fraction. In the presence of cytochalasin B, an actin disrupting agent, endothelin-3 and VO 4 3− did not phosphorylate focal adhesion kinase and paxillin. Application of cytochalasin B after treatment with endothelin-3 and VO 4 3− stimulated dephosphorylation of focal adhesion kinase and paxillin. These results suggest that the associations of focal adhesion kinase and paxillin with cytoskeletal components are required in the endothelin-induced tyrosine phosphorylation of the astrocytic proteins.

Modulation of contractile force by endothelin receptors in porcine myocardial trabeculae.

The aim of the study was to determine possible inotropic effects mediated by endothelin-A and endothelin-B receptors in porcine myocardial trabeculae from right atria and left ventricles. Isolated trabeculae were paced at 1.5 Hz in tissue baths, and changes in isometric contractile force upon exposure to agonist were studied. Endothelin-1 and endothelin-3 had a strong and potent positive inotropic effect in all trabeculae. In all atrial and in some ventricular trabeculae this effect was preceded by a transient negative inotropic effect at 10(-7) M. The endothelin-B receptor agonist IRL 1620 had a positive inotropic effect in some of the ventricular trabeculae, and no negative inotropic effect. Another endothelin-B receptor agonist, sarafotoxin S6c, had no positive inotropic effect, but induced a transient negative inotropic effect in some atrial trabeculae at 10(-7) M. In atrial trabeculae the preincubation with the endothelin-A receptor antagonist FR139317 (10(-6) M) decreased significantly (P<0.01) the maximum positive inotropic responses and abolished negative inotropic responses to endothelin-1. In conclusion, both endothelin-A and endothelin-B receptors may have the potential to mediate both positive and negative inotropic responses, but a positive inotropic effect mediated mainly via endothelin-A receptors seems to predominate.

Role of NO in endothelium-dependent relaxation of rabbit basilar artery in situ

The purpose of this study was to investigate the possible involvement of endothelium-derived hyperpoiarizing factor in endothelium-dependent relaxation of the cerebral vasculature by testing the effectiveness of NO synthase inhibitors at inhibiting endothelium-dependent relaxation in the rabbit basilar artery. Acetylcholine (1.0pM) and 0.1/0.2pM sarafotoxin S6c, an endothelinB receptor agonist, relaxed serotonin constricted basilar artery in situ by 100% and 70%, respectively. Nc-monomethyl-L-arginine (L-NMMA; 0.1 mM) and 0.3 mM Nc-nitro-L-arginine (L-NNA), NO synthase Inhibitors, decreased the 1.0 pM acetylcholine- and 0.1/0.2 pM sarafotoxin S6c-induced relaxations by 75% and 45%, respectively. Unexpectedly, the relaxations were abolished by the combination of L-NMMA plus L-NNA. Furthermore, L-arginine (1.0 mM), but not D-arginine, restored the relaxations. Sodium nitroprusside-induced relaxation was also inhibited by L-NMMA plus L-NNA, and the inhibition was reversed by L-arginine. KCI constricted vessels only minimally relaxed in response to sodium nitroprusside, acetylcholine, and sarafotoxin S6c. These results demonstrate that combined NO synthase inhibitors more effectively inhibit endothelium-dependent relaxation than a single inhibitor. The mechanism underlying the greater inhibition due to the combined NO synthase inhibitors may result from both decreased NO release and secondary effects caused by decreased NO release, such as membrane depolarization. The results further suggest that caution should be used with respect to suggestions of the involvement of endothelium-derived hyperpoiarizing factor in endothelium-dependent relaxation based upon the partial inhibitory effects of NO synthase inhibitors. [Neurol Res 2000; 22: 204—208]

Altered endothelin receptor subtype expression in hepatic injury after ischemia/reperfusion.

This study was performed to determine whether ischemia/reperfusion (I/R) injury in rat liver results in alterations in endothelin receptor expression. Hepatic ischemia was produced in rats for 60 min followed by 6 or 24 h reperfusion. Portal inflow pressure was increased (7.38+/-0.60 mmHg) at 24 hours after reperfusion. Serum ALT increased significantly at both 6 and 24 h (6 h; 258.3+/-74.3, 24 h; 243.1+/-74.8 IU/L). Portal vascular response to an endothelin-B receptor agonist (IRL 1620) was significantly increased in the I/R livers compared to control and this was potentiated by L-NAME. IRL 1620 also caused LDH release from I/R livers but not controls. LDH release after IRL 1620 in I/R livers correlated with increased portal pressure response. To determine whether the altered response might be the result of altered endothelin receptor expression, livers were harvested after reperfusion and total endothelin binding sites were determined by competitive binding with ET-1. Proportion of endothelin receptor subtypes (ET(A)/ET(B)) was determined using the ET(A) antagonist BQ-610 (1 microM) and ET(B) agonist IRL-1620 (100 nM). There were no significant changes in Kd but Bmax for endothelin-1 was decreased in I/R group especially non-ischemic lobe at 24 h. ET(A) receptors were significantly decreased whereas ET(B) receptors were increased. These changes were more pronounced at 24 h after reperfusion than at 6 h. Interestingly, the changes in ET receptors was observed identically both in ischemic and non-ischemic lobes (ischemic lobe ET(A) 41.9%, ET(B) 51%; non-ischemic lobe ET(A) 38.8%, ET(B) 49.5%). These results indicate that the major functional endothelin receptor subtype upregulated in I/R is the ET(B) receptor and that this upregulation may contribute to microvascular dysregulation and hepatic injury.

Receptor subtype mediating the action of circulating endothelin on glucose metabolism and hemodynamics in perfused rat liver

The subtype of endothelin receptor that mediates metabolic and hemodynamic effects of circulating endothelin was explored using perfused rat liver. Infusion of endothelin (ET)-1 or ET-3 into the portal vein at a concentration of 0.3 nM increased glucose and lactate output and decreased perfusion flow, although ET-3 was less effective than ET-1. The metabolic effects of ET-1 were observed even under costant-flow perfusion. Infusion of either sarafotoxin S6b or S6c, an ET A- or ET B-receptor agonist, mimicked the actions of ET-1 to an equal extent. The flow reduction and glucose production induced by ET-1 were partly attenuated by the ET A-receptor antagonist BQ485. By contrast, ET B-receptor antagonist BQ788 enhanced glucose production caused by ET-1 and ET-3 without affecting the hemodynamic change. The effects of ET-1 and ET-3 were almost totally inhibited by the combination of BQ485 and BQ788. These results suggest that both ET A and ET B receptors are involved in the metabolic and hemodynamic effects of circulating endothelin in rat liver, while the ET A-receptor-mediated action appears to be dominant.

Endothelin induces dopamine release from rat striatum via endothelin-B receptors

The aim of the present study was to determine whether local administration of endothelin induces the release of dopamine in the rat striatum and to characterize and localize endothelin receptors in this brain region. Local injection of endothelin-1 (10 pmol) into the ventral striatum of urethane-anaesthetized rats caused an increase of 8 μM in the extracellular concentration of dopamine as measured by in vivo chronoamperometry. The peak increase in dopamine concentration occurred within 5 min of endothelin injection. Injection of the selective endothelin-B receptor agonist [Ala 1,3,11,15]endothelin-1 (10 pmol) also caused an increase in extracellular dopamine concentration, suggesting that endothelin is acting at the endothelin-B receptor to elicit its effect. In rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway, the response to local injection of endothelin-1 (10 pmol) was significantly inhibited on the lesioned side as compared to the non-lesioned side. In contrast, pretreatment of the rats with the N-methyl- d-aspartate receptor antagonist dizocilpine maleate (5 mg/kg, i.p.) or the nitric oxide synthase inhibitor N G-nitro- l-arginine (3 mg/kg, i.p.) did not alter the endothelin-induced release of dopamine. In binding studies, addition of endothelin-1 displaced [ 125I]endothelin-1 with a K i of 220 pM. The endothelin-B receptor antagonist BQ788 displaced [ 125I]endothelin-1 with a K i of 120 nM, whereas the endothelin-A receptor antagonist BQ123 produced only a 25% displacement at 10 μM, suggesting that endothelin receptors in the striatum are of the endothelin-B subtype. In rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal dopamine system, [ 125I]endothelin-1 binding was reduced by 53% in lesioned striatum compared to non-lesioned striatum, with no difference in the K d . These data provide evidence that endothelin acts on a homogeneous population of endothelin-B receptors within the striatum to cause the release of dopamine and that a significant proportion of these receptors is located on dopaminergic neurons.

Endothelin-1, Delayed Rectifier K Channels, and Pulmonary Arterial Smooth Muscle

Sarafotoxin S6c [STXS6c; a selective endothelin-B (ETB) receptor agonist] causes constriction of isolated pulmonary arteries. In perforated-patch experiments on pulmonary arterial myocytes, ET-1 and STXS6c induced a gradual inhibition of the delayed rectifier K current (IKV), the profile of which resembled that carried by Kv1.5. Reverse-transcriptase polymerase chain reaction (RT-PCR) experiments revealed mRNA encoding this channel, and immunolocalization experiments demonstrated expression of the channel protein in pulmonary arterial smooth muscle. It is tempting to speculate that ETB receptor coupling to Kv1.5 may be implicated in contraction after stimulation of these receptors.