BQ123 Group Research Articles

Effect and Mechanism of Endothelin Receptor A Inhibitor BQ-123 Combined with Electroacupuncture on Tibia Cancer Pain in Rats.

Objective To research the impact and mechanism of endothelin receptor A inhibitor BQ-123 combined with electroacupuncture on tibia cancer pain in rats. Methods Sprague-Dawley (SD) rats were randomly divided into sham group (SHAM group) and bone cancer pain model group (BCP group). The behavior of SD rats was measured. The histology of the right tibia was observed by hematoxylin-eosin (HE) staining. The remaining rats were randomly divided into model, BQ-123, electroacupuncture, and BQ-123+ electroacupuncture group. Behavioral tests were performed, and mechanical pain threshold (MWT) and thermal pain threshold (TWL) were measured. The expressions of α-smooth muscle actin (αSMA), ETAR (endothelin A receptor), ETB (End of Transmission Block), P-Phosphatidylinositol 3-kinase (PI3K), and P-Protein kinase B (Akt) were detected by real-time fluorescence quantitative PCR and western blot. Results In the BCP group, bone structure was severely damaged, local tissue swelling was obvious, bone trabecula was missing, and bone cortex was discontinuous. The optical density of Glial fibrillary acidic protein (GFAP) and CD11b immunoreactive signal in BCP group was significantly increased, and most of the ETAR of endothelin receptor was comapped with NeuN, and a small part of GFAP was comapped with CD11b, but no comapped with CD11b. The AS score of BQ-123+ electroacupuncture group was significantly lower than that of BQ-123 group and electroacupuncture group (P < 0.05), whereas the MWT and TWL values were significantly higher than that of the BQ-123 group and electroacupuncture group (P < 0.05). The mRNA expression of α-SMA and ETAR in BQ-123+ electroacupuncture group was lower than that in BQ-123 and electroacupuncture group, and the protein expression of P-PI3K and P-Akt in BQ-123+ electroacupuncture group was lower as well. Conclusion BQ-123 may inhibit the activation of PI3K/Akt signal path combined with electroacupuncture to alleviate the effects of tibia cancer pain in rats.

Protective effects of endothelin-1 recepter antagonist against lung injury during one-lung ventilation in rabbits

Objective To investigate the protective effect of endothelin-1 (ET-1) recepter antagonist against lung injury during one-lung ventilation (OLV) in rabbits. Methods Fifteen big-ear Japanese rabbits (no limit of male or female) were divided into 3 groups randomly (n=5 in each group): sham group (group T), one-lung ventilation group (group O), OLV+ BQ-123 group (group OB). Two lungs were ventilated for 2.5 h in group T, and one lung was ventilated for 2.0 h followed by 0.5 h OLV in group O and group OB. About 10 min before one lung ventilation, BQ-123 (50 μg/kg) was injected through the femoral vein in group OB. Group O and group OB received the same amount of normal saline at the same time point. During the experiment, arterial blood gas analyses were done at the time of before OLV (T0), 1 h after OLV (T1), 2 h after OLV (T2) and 0.5 h after TLV (T3), as well as OI was calculated. At end of the experiment, the two lungs were removed for light microscopic evaluations, and the content of ET-1, activity of superoxide dismutase (SOD), concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were determined in lung tissures. Results As compared with group T, the content of ET-1 [(165.73±23.87) ng/L vs. (94.31±9.86) ng/L], the activity of SOD [(62.21±2.10) U/mg vs. (84.42±6.77) U/mg], the concentration of IL-6 [(20.82±1.33) ng/L vs. (7.97±1.98) ng/L] and TNF-α [(37.11±5.03) ng/L vs. (19.47±4.30) ng/L](P<0.05) in lung tissue of group O were increased, the pathological score was increased, and the OI was decreased at 2.0 hafter one-lung ventilation. As compared with group O, the content of ET-1 [(119.37±8.55) ng/L vs. (165.73±23.87) ng/L], the activity of SOD [(78.29±1.78) U/mg vs. (62.21±2.10) U/mg] and the concentrations of IL-6 [(14.13±1.26) ng/L vs. (20.82±1.33) ng/L] and TNF-α [(26.30±2.18) ng/L vs. (37.11±5.03) ng/L] in the collapsed lung tissue of group OB were decreased (P<0.05), and the pathological score decreased. In group O and OB, the content of ET-1 and concentrations of IL-6 and TNF-α in the collapsed lung were significantly higher than those in the ventilated lung, the activity of SOD was decreased and the pathological score was increased (P<0.05). Conclusion ET-1 receptor antagonist has protective effect on lung injury during one-lung ventilation in rabbits, which may be related to the reduction of ET-1 binding to receptor and the inhibition of inflammatory response and oxidative stress reaction in lung tissue. Key words: Lung injury; One-lung ventilation; Endothelin-1 recepter antagonist; Rabbit

CT perfusion imaging of cerebral microcirculatory changes following subarachnoid hemorrhage in rabbits: Specific role of endothelin-1 receptor antagonist

BackgroundCerebral vasospasm may lead to delayed ischemic neurological deficits following subarachnoid hemorrhage (SAH). Endothelin (ET-1) is an important factor participating in cerebral vasospasm underlying SAH. We used a specific endothelin receptor antagonist, BQ123 to assess the specific role of endothelin-1 receptor antagonist in cerebral vasospasm in a rabbit model of SAH by examining plasma ET-1 levels and the principal CT perfusion (CTP) parameters pertinent to the hemodynamic status of microcirculation following SAH. Methods102 male New Zealand white rabbits were divided into control, SAH and SAH + BQ123 intervention group (BQ123 group). Rabbit SAH model was established by double hemorrhage injection of autologous blood into the cisterna magna; Aquilion ONE was used to collect cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT) which were used to evaluate cerebral microcirculation hemodynamics; Elisa was used to assess plasma ET-1 levels. Data were collected on days 1, 4, 7 and 14 following SAH, respectively. ResultsCompared with the control group, the CBF in the SAH group was significantly lower, while the MTT was significantly higher. The CBF decreased on the 4th day and reached the lowest on the 7th day. The MTT began to rise on the 4th day and peaked on the 7th day. While in the BQ123 intervention group, the CBF significantly increased while the MTT significantly decreased on the 1st and the 4th days, respectively. Compared with SAH group, plasma ET-1 levels in BQ123 group significantly increased on the earlier (1st and 4th days) but not later days (between the 7th and 14th days). In addition, the inflammatory infiltration of brain tissues in rabbits treated with BQ123 post-SAH was significantly reduced compared with SAH group. ConclusionCTP can quantify the therapeutic effect of BQ123 after SAH; Selective blockade of ET-1 endothelin receptor, BQ123 significantly improved microcirculatory perfusion along with a reduction in resultant vasogenic inflammatory responses. The effect of BQ123 on the cerebral microcirculation was lobe dependent.

The effect of endothelin receptor antagonists in the endotoxin-induced uveitis rabbit model

Purpose: To investigate the effect of Bosentan (non-selective endothelin receptor antagonist) and BQ123 (ETA receptor antagonist) on intraocular inflammation in an endotoxin-induced uveitis (EIU) rabbit model.Methods: Uveitis was induced by intravitreal injection of lipopolysaccharide (LPS). The animals were divided into 7 groups and there were six rabbits in each group (saline, saline and ethanol, bosentan, BQ123, lipopolysaccharide (LPS), bosentan and LPS, BQ123 and LPS-injected groups). Bosentan and BQ123 were applied before LPS injection. Aqueous humour was collected at 24th hour post-injections and enucleation was performed for the evaluation of histopathological changes.Results: BQ123 decreased clinical score, cell counts and protein amount more than bosentan and it was significant for cell counts (p = 0.018). Bosentan significantly diminished inflammatory reactions more than BQ123 as shown in histopathological specimens (p = 0.002).Conclusions: ETA receptor blockage is effective on uveitis treatment by its protective effect on blood aqueous barrier.

Relationship between endothelin-1 and p38 MAPK and PI3K/Akt signaling pathways during mechanical stretch-induced enhancement of adhesion of rat pulmonary microvascular endothelial cells

Objective To evaluate the relationship between endothelin-1 (ET-1) and p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathways during mechanical stretch-induced enhancement of adhesion of rat pulmonary microvascular endothelial cells (PMVECs). Methods Rat PMVECs were seeded in the culture plate at a density of 0.5×105 cells/ml (2 ml/well) and divided into 5 groups (n=24 each) using a random number table: control group (group C), mechanical stretch group (group MS), mechanical stretch plus specific PI3K inhibitor LY294002 group (LY group), mechanical stretch plus specific p38 MAPK inhibitor SB203580 group (SB group), and mechanical stretch plus selective ETA receptor blocker BQ123 group (BQ group). Cells were exposed to 20% cyclic stretch at 0.3 Hz for 4 h using a sine wave.In LY, SB and BQ groups, LY294002, SB203580 and BQ123 at the final concentration of 10 μmol/L were added, respectively, after mechanical stretch, cells were incubated for 10 min, and then extracted and purified rat polymorphonuclear neutrophil leukocytes (PMNs, 5×105 cells/well) were added and co-incubated with PMVECs for 30 min and then washed out.The concentrations of ET-1 and interleukin-6 (IL-6) in the culture medium were determined using enzyme-linked immunosorbent assay.The expression of phosphorylated p38 MAPK (p-p38 MAPK) and phosphorylated Akt (p-Akt) was detected by Western blot.Adhesion of PMNs was measured by immuno-histochemistry, and the adhesion rate was calculated.The expression of P-selectin mRNA was detected using real-time polymerase chain reaction. Results Compared with group C, the concentrations of IL-6 and ET-1 in the culture medium were significantly increased, the expression of p-p38 MAPK, p-Akt and P-selectin mRNA was up-regulated, and the adhesion rate of PMNs was increased in the other four groups (P<0.05). Compared with group MS, the concentration of IL-6 in the culture medium was significantly decreased, the expression of p-Akt and P-selectin mRNA was down-regulated, and the adhesion rate of PMNs was decreased in LY, SB and BQ groups, the concentration of ET-1 in the culture medium was significantly decreased in group BQ, and the expression of p-p38 MAPK was significantly down-regulated in SB and BQ groups (P<0.05). Conclusion The signaling mechanism underlying ET-1-mediated enhancement of rat PMVEC adhesion may be related to activating p38 MAPK and PI3K/Akt signaling pathways. Key words: Endothelin-1; p38 Mitogen-activated protein kinases; 1-Phosphatidylinositol 3-kinase; Protein-serine-threonine kinases; Lung; Endothelial cells; Cell adhesion

Role of endothelin in microvascular dysfunction following percutaneous coronary intervention for non-ST elevation acute coronary syndromes: a single-centre randomised controlled trial

ObjectivesPercutaneous coronary intervention (PCI) for acute coronary syndromes frequently fails to restore myocardial perfusion despite establishing epicardial vessel patency. Endothelin-1 (ET-1) is a potent vasoconstrictor, and its expression is increased...

Beneficial effects of melatonin and BQ-123 on the rat testis damage caused by cigarette smoke.

Several studies have demonstrated that cigarette smoke has detrimental effects on testicular function. However, it is unknown whether melatonin or BQ-123 has beneficial effects on the rat testis damage caused by cigarette smoke. The aim of the present study was to investigate the beneficial effects of melatonin or BQ-123 on the testicular damage caused by cigarette smoke. Twenty Wistar rats were randomly divided into 4 equal groups: control group (n = 5), cigarette smoke group (n = 5), melatonin group (n = 5), and BQ-123 group (n = 5). At the end of 4 weeks, all the rats were sacrificed for histopathological evaluation and subsequent stereological analysis. The optical fractionator counting method, the most efficient and unbiased method, was used to estimate the total number of spermatogonia and spermatocytes. All the control testes demonstrated complete spermatogenesis. There was a significant decrease in the germ cells of rats exposed to cigarette smoke for 4 weeks. After the application of melatonin or BQ-123, the total number of spermatogonia and spermatocytes in the testes was significantly higher. Based on these findings, melatonin and BQ-123 are able to minimize the degenerative effects of cigarette smoke by increasing the germ cell count.

The protective effects of endothelin-A receptor antagonist BQ-123 in pentylenetetrazole-induced seizure in rats

Endothelin-1 has been shown to increase neuronal activity and glutaminergic synaptic transmission by endothelin-A receptors (ETAR) in the nucleus tractus solitarius neurons that play an important role in epileptic seizures. Therefore, BQ-123 as an ETAR antagonist might attenuate neuronal excitability and glutaminergic synaptic transmission. The main purpose of the present study is to investigate the protective effect of acute BQ-123 treatment against pentylenetetrazole (PTZ)-induced tonic-clonic seizures. Wistar albino rats were divided into three groups: control, PTZ, and PTZ + BQ-123 groups. BQ-123 (3 mg/kg, intravenously) was administered for 15 min before injecting with PTZ (50 mg/kg, intraperitoneally). We determined a delay resulting from BQ-123 in "duration of the seizure onset." "Number of rats with major seizure" also decreased according to scoring with video camera in PTZ + BQ-123 group. In BQ-123-treated group, there were eight rats without a major seizure, but only one rat had a delayed major seizure. The brain tissue glutathione peroxidase activity was significantly decreased in the PTZ and PTZ + BQ-123 groups. According to the results of the control group, there was a significant increase in the protein carbonyl levels of the PTZ group and a significant increase in the nitric oxide levels of the PTZ + BQ-123 group. Histological examination showed an increase in the number of neuronal hyperchromatic nucleus especially in hippocampal gyrus dentatus region of BQ-123-treated group. We concluded that BQ-123 impeded the formation and spread of seizure to a great degree. The beneficial effects of BQ-123 were comparatively supported with biochemical parameters and histological examinations.

Endothelin-1 Release during the Early Phase of Reperfusion Is a Mediator of Myocardial Reperfusion Injury

Purpose: In acute myocardial infarction, left ventricular (LV) unloading reduces endothelin-1 (ET-1) release. We tested that endogenous ET-1 released during acute myocardial infarction might mediate ischemia/reperfusion (I/R) injury by stimulating increased intracellular calcium concentration, [Ca²⁺]_i, and apoptosis. Methods: Rabbits were subjected to 1 h of coronary artery occlusion followed by 3 h of reperfusion. Unloading was initiated 15 min prior to reperfusion and was maintained during reperfusion. The control group was subjected to reperfusion. Animals were treated with ET-1 receptor antagonist BQ123. In parallel, isolated rabbit cardiomyocytes subjected to simulated I/R with or without ET-1 or BQ123, intracellular Ca²⁺ and cell death were assessed with flow cytometry. Results: LV unloading prior to reperfusion reduced myocardial ET-1 release at 2 h of reperfusion. Infarct size was reduced in unloaded and BQ123 groups versus controls. LV unloading and BQ123 treatment reduced the percentage of apoptotic cells associated with increases in Bcl-2 protein levels in ischemic regions. BQ123 reduced both ET-1-induced [Ca²⁺]_i increase and cell death for myocytes subjected to stimulated I/R. Conclusion: We propose that components of reperfusion injury involve ET-1 release which stimulates calcium overload and apoptosis. Intravenous ET-1 receptor blockade prior to reperfusion may be a protective adjunct to reperfusion therapy in acute myocardial infarction patients.

Neuroprotection afforded by antagonists of endothelin-1 receptors in experimental stroke

Endothelin-1 (ET-1) is involved on the development of cerebral edema in acute ischemic stroke. As edema is a therapeutic target in cerebral ischemia, our aim was to study the effect of antagonists for ET-1 receptors (Clazosentan® and BQ-788, specific antagonists for receptors A and B, respectively) on the development of edema, infarct volume and sensorial-motor deficits in rats subjected to ischemia by occlusion of the middle cerebral artery (MCAO). We used Wistar rats (280–320 g) submitted to ischemia by intraluminal transient (90 min) MCAO. After ischemia, rats were randomized into 4 groups (n = 6) treated with; 1) control group (saline), 2) Clazosentan® group (10 mg/kg iv), 3) BQ-788 group (3 mg/kg iv), and 4) combined treatment (Clazosentan® 10 mg/kg plus BQ-788 3 mg/kg iv). We observed that rats treated with Clazosentan® showed a reduction of edema, measured by MRI, at 72 h (hours) and at day 7 (both p < 0.0001), and a decrease in the serum levels of ET-1 at 72 h (p < 0.0001) and at day 7 (p = 0.009). The combined treatment also induced a reduction of edema at 24 h (p = 0.004), 72 h (p < 0.0001) and at day 7 (p < 0.0001), a reduction on infarct volume, measured by MRI, at 24 and 72 h, and at day 7 (all p < 0.01), and a better sensorimotor recovery at 24 and 72 h, and at day 7 (all p < 0.01). Moreover, Clazosentan® induced a decrease in AQP4 expression, while BQ-788 induced an increase in AQP9 expression. These results suggest that antagonists for ET-1 receptors may be a good therapeutic target for cerebral ischemia.

Protective effects of endothelin-1 receptor antagonist (BQ-123) against lung ischemia-reperfusion injury on the model of acute pulmonary embolism in rabbits

Objective To explore the protective effects of endothelin-1 ( ET-1 ) receptor antagonist BQ-123 against ischemia-reperfusion injury after thrombolysis in acute pulmonary embolism.Methods Twenty-four big-ear Japanese rabbits (no limit of male or female) were divided into 4 groups randomly ( n =6 in each group):sham group ( sham-operated group),PE group ( pulmonary embolism group),UK group (thrombolysis group),BQ group ( UK + BQ-123 group).We detected the levels of ET-1 in the venous blood plasma and mean pulmonary arterial pressure (MPAP) before and 0.5,2,4 h after embolization.The ratio of lung tissue wet/dry (W/D),the superoxide dismutase (SOD) activity and malondialdehyde (MDA) content at the end of the experiment were measured.Results As compared with sham group [(18.92 ±0.63) mm Hg(1 mm Hg=0.133 kPa) ],the MPAP in PE group (19.01 ±0.60) mm Hg,UK group (20.14 ±0.85) mm Hg,and BQ group ( 19.79 ±0.78) mm Hg was increased significantly at 0.5 h after embolization (P ＜0.05).There was no significant difference in MPAP between BQ group and UK group at 2 h and 4 h after embolization.The levels of plasma ET-1 in BQ group were reduced more significantly than UK group at 4 h after embolization.As compared with sham group,the W/D and MDA content were increased,but SOD activity was reduced in PE group and UK group at the end of the experiment,and the same results were obtained between UK group and PE group (P ＜ 0.05).The W/D ratio and MDA content were lower,and SOD activity was higher in BQ group than in UK group (P ＜0.05).Conclusion Lung ischemia-repeffusion injury appeared in thrombolysis after acute pulmonary embolism,and BQ-123 may exert the antagonistic effets. Key words: Acute pulmonary embolism; Lung ischemia; Reperfusion injury; Endothelia; Endothelia receptor antagonist

Effects of BQ-788 on amitriptyline-induced cardiovascular toxicity

We investigated both the effects of the endothelin type B (ETB) receptor antagonist, BQ-788, on amitriptyline-induced cardiotoxicity and the role of ETB receptors on amitriptyline-induced cardiovascular depression. Male Wistar rats were anaesthetized with urethane/chloralose. Mean arterial pressure (MAP), heart rate (HR) and QRS duration were recorded. Toxicity was induced by amitriptyline infusion (0.94 mg/kg per min) until the 50% inhibition of MAP. In the first protocol, 5% dextrose or BQ-788 bolus was administered to control or experimental group animals, respectively. In the second protocol, after incubation with BQ-788 or 5% dextrose, amitriptyline was infused. Amitriptyline caused a significant decrease in MAP, prolonged QRS duration and decreased HR for both the groups. BQ-788 administration improved MAP (5, 10 and 15 min), shortened the prolonged QRS (5 and 10 minutes) and increased HR (5, 10 and 15 min) compared with dextrose group. While all the amitriptyline-infused rats survived in BQ-788 group, all the amitriptyline-infused rats died within 20 min in dextrose group. In the second protocol, BQ-788 incubation did not cause any statistically significant change in amitriptyline-induced cardiovascular depression. BQ-788 may have beneficial effects in amitriptyline-induced cardiovascular changes via a physiologic antagonism. ETB receptor antagonists may be the promising antidotes for the cardiovascular toxicity with hypotension and bradycardia.

Regulatory effect of endothelin on the expression of transformation growth factor-beta 1 and phosphorylated Smad 3 in A375 cells in vitro.

Objective To investigate the effects of endothelin-1 (ET-1) and endothelin-3 (ET-3) on the expression of transformation growth factor-beta 1 (TGF-β1) and phosphorylation of Smad 3 in malignant melanoma cell line, A375. Methods Cultured A375 cells were classified into 5 groups, i.e. control group (no stimulation), ET-1 group (stimulated with ET-1), ET-1 + BQ123 group (treated with ET-1 and BQ123), ET-1 + BQ788 group (treated with ET-1 and BQ788), ET-3 group (stimulated with ET-3), to receive different stimulation. The working concentrations were 0, 0.1, 1, 10, 100 nmol/L for ET-1 and ET-3, 10 μmol/L for BQ123 and BQ788. After another 12- and 24-hour culture, ELISA, RT-PCR and Western blot were used to detect the expression of TGF-β1 protein and mRNA as well as phosphorylated Smad 3 (P-Smad 3). Results The expression of TGF-β1 in A375 cells was up-regulated by ET-1, but down-regulated by ET-3, and both of the effects were in a concentration-dependent manner. Under the stimulation with ET-1 and ET-3 of 100 nmol/L, the level of TGF-β1 reached 1289.38 ± 89.42 ng/L per 10~5 cells and 85.09 ± 9.37 ng/L per 10~5 cells, respectively, significantly different from that in unstimulated cells (both P< 0.05). BQ123 signifi-cantly blocked the up-regulatory effect of ET-1 on the expression TGF-pi protein(P < 0.05), but BQ788 had no significant influence on the effect, so was the case with TGF-β1 mRNA. Western blot revealed that ET-1 significantly elevated the expression of P-Smad 3 in A375 cells (P < 0.05), and the elevation was significantly inhibited by BQ123, but not by BQ788. The expression of P-Smad 3 was statistically decreased by ET-3 in A375 cells (P< 0.05). Conclusions The expression of TGF-β1 could be enhanced by ET-1, but suppressed by ET-3. It is likely that endothelin receptor A mediates the phosphorylation of Smad 3 induced by ET-1. Key words: Melanoma; Endothelins; Receptor; endothelin A; Transforming growth factor-beta 1

Intracoronary endothelin receptor blockade improves endothelial function in patients with coronary artery disease

Endothelin (ET)-1 receptor blockade improves endothelial function in the forearm of patients with atherosclerosis. The aim was to investigate whether intracoronary ET receptor blockade improves coronary endothelial function and increases blood flow in patients with coronary artery disease. Ten patients received a 60-minute infusion of either the selective ETA receptor antagonist BQ123 (40 nmol/min, n = 6) or BQ123 + the ETB receptor antagonist BQ788 (40 nmol/min, n = 4). In all patients, substance P, an endothelium-dependent vasodilator, did not increase baseline coronary flow reserve with thermodilution (CFRThermo) (0.71 +/- 0.14 s during NaCl versus 0.59 +/- 0.14 s during substance P) or baseline quantitative coronary angiography (QCA) (2.74 +/- 0.16 mm versus 2.83 +/- 0.20 mm). After ET receptor blockade, however, the response to substance P was significantly improved as determined both by CFRThermo (0.62 +/- 0.14 s during NaCl versus 0.48 +/- 0.10 s during substance P, p < 0.05) and by QCA (2.70 +/- 0.18 mm versus 2.85 +/- 0.19 mm, p < 0.05). In addition, ET blockade increased blood flow in all patients by 16% +/- 10% (n = 10, p < 0.05) and in the BQ123 group by 22% +/- 16% (n = 6, p < 0.05). Furthermore, ETA blockade increased blood flow significantly more than did dual ETA/ETB blockade (p < 0.05). These findings indicate that ET receptor blockade may be a new therapeutic strategy to improve coronary vascular function in patients with coronary artery disease.

The Endothelin-Converting Enzyme-1/Endothelin-1 Pathway Plays a Critical Role in Inflammation-Associated Premature Delivery in a Mouse Model

Premature delivery occurs in 12% of all births and accounts for nearly half of long-term morbidity. Current therapeutic approaches to preterm delivery are ineffective and present serious risks to both mother and fetus. The single most common cause of preterm birth is infection. Previous in vitro investigations have shown that endothelin-1 (ET-1) is induced by inflammatory cytokines and that it increases myometrial smooth muscle tone. Furthermore, we have previously shown that both the endothelin-converting enzyme-1 (ECE-1) inhibitor, phosphoramidon, as well as a novel ET-1 receptor A antagonist synthesized by our group, control premature delivery in a mouse model of inflammation-associated preterm delivery. In the current work, we show that levels of both ET-1 and ECE-1 are increased in gestational tissues in E16.5 mice induced to deliver prematurely after lipopolysaccharide administration. We also show that premature delivery is controlled by treatment with the selective endothelin receptor A antagonist BQ-123 in a dose-dependent manner. Finally, we show here for the first time that premature delivery can be controlled using RNA silencing, by hydrodynamic transfection of E15 mice with ECE-1 RNAi. Taken together, these data support a critical role for the ECE-1/ET-1 system in inflammation-associated premature delivery. The ability to control premature delivery by antagonizing or silencing the ECE-1/ET-1 system offers a novel approach to an unmet clinical need.

Role of Nitric Oxide in Regulation of Retinal Blood Flow in Response to Hyperoxia in Cats

To investigate whether nitric oxide (NO) regulates retinal circulation during and after induction of hyperoxia in cats. Hyperoxia was induced for 10 minutes with 100% oxygen. The vessel diameter and blood velocity were measured simultaneously in second-order retinal arterioles by laser Doppler velocimetry; the retinal blood flow (RBF) and wall shear rate (WSR) were calculated during and after hyperoxia. PBS, L-NAME, D-NAME, BQ-123, BQ-788, and 7-nitroindazole (7-NI) were administered before induction of hyperoxia. In the PBS group, vessel diameter, blood velocity, and RBF decreased during hyperoxia and returned to baseline within 10 minutes after hyperoxia ended. WSR decreased transiently and then returned to baseline by the delayed constriction of retinal arterioles during hyperoxia. In the l-NAME and BQ-788 groups, the decreases in RBF during hyperoxia did not differ from those in the PBS group. However, the recovery of RBF after hyperoxia ended was attenuated significantly until 20 minutes after hyperoxia ended in both groups compared with the PBS group (P < 0.05). In the BQ-123 group, the intravitreous injection of BQ-123 caused less reduction of blood velocity and RBF during hyperoxia compared with that in the PBS group, whereas the RBF immediately returned to baseline after hyperoxia. D-NAME and 7-NI did not affect RBF in response to hyperoxia. The current results indicate that NO contributes to RBF recovery after hyperoxia, probably through the action of endothelial NOS via the ETB receptor in the vascular endothelium of the retinal arterioles, suggesting that the RBF response to hyperoxia may be used to evaluate the endothelial function of the retinal arterioles.

ENDOTHELIN B RECEPTORS PRESERVE RENAL BLOOD FLOW IN A NORMOTENSIVE MODEL OF ENDOTOXIN-INDUCED ACUTE KIDNEY DYSFUNCTION

The aim was to investigate the role of endothelin 1 receptor subtypes in the early renal response to lipopolysaccharide (LPS) during normotensive endotoxemia with acute kidney dysfunction. Endotoxemia was induced in thiobutabarbital-anesthetized rats (n = 9 per group) by infusion of LPS (dosage, 1 mg/kg per hour i.v.). The study groups (1) sham-saline, (2) LPS-saline, (3) LPS-BQ123, (4) LPS-BQ788 and (5) LPS-BQ123 + BQ788 received isotonic saline, the ETA receptor antagonist BQ-123 (dosage, 30 nmol/kg per minute i.v.), and/or the ETB receptor antagonist BQ-788 (dosage, 30 nmol/kg per minute i.v.) before and during 2 h of LPS infusion. Renal clearance measurements, renal blood flow (RBF), and cortical and outer medullary perfusion (laser-Doppler flowmetry) and oxygen tension (Clark-type microelectrodes) were analyzed throughout. Before LPS administration, there were no significant differences between groups in glomerular filtration rate (GFR), RBF, or in cortical (CLDF) and outer medullary perfusion. However, mean arterial pressure (MAP) was elevated in LPS-BQ788 group compared with LPS-BQ123 + BQ788 group (P < 0.05). In saline-treated rats, endotoxin induced an approximate 35% reduction in GFR (P < 0.05), without significant effects on MAP, RBF, or on CLDF and cortical PO2. In addition, LPS increased outer medullary perfusion and PO2 (P < 0.05). The fractional urinary excretion rates of sodium, potassium, and water were not significantly different in LPS-saline group compared with sham-saline group. Neither selective nor combined ETA and ETB receptor blockade improved GFR. In BQ-788-infused rats, endotoxin produced marked reductions in RBF (-18% +/- 4% [P < 0.05]) and CLDF (-18% +/- 2% [P < 0.05]). Similarly, endotoxin decreased RBF (-14% +/- 3% [P < 0.05]) and CLDF (-10% +/- 2% [P < 0.05]) in LPS-BQ123 + BQ788 group. Endotoxin reduced MAP (-22% +/- 4% [P < 0.05]) in BQ-123-treated rats but did not significantly influence MAP in other groups. We conclude that in early normotensive endotoxemia, ETB receptors exert a renal vasodilator influence and contribute to maintain normal RBF.

A novel cultured tissue model of rat aorta: VSMC proliferation mechanism in relationship to atherosclerosis

Development of a cultured tissue experimental model of rat aorta was explored in order to study mechanism of vascular smooth muscle (VSMC) proliferation. This particular model has potential with regard to amelioration of atherosclerosis and other vascular diseases in comparison to whole animal and cell culture models. The aorta segments of rats were divided into 4 experimental groups: the injured endothelium, injured endothelium plus BQ123, without injured endothelium and without injured endothelium plus BQ123. Each of group was subdivided into a further 2 subgroups and cultured with 20% serum and with serum-free DMEM. Each group cultured in vitro for 5, 8 and 13 days respectively. The control group was not cultured in vitro. Bromodeoxyuridine (BrDU 8 × 10 − 4 mol/l) was added into the cultured medium of all groups, 24 h prior to harvesting. These segments were fixed in 4% paraformaldehyde for paraffin slice used to HE and immunocytochemical staining and other aorta segments were used to detect the expressions of hypertension-related gene-1 (HRG-1) and smooth muscle 22 alpha (SM22α) by RT-PCR. ET-1 content in the supernatant was detected with radioimmunology. Proliferous VSMC can be observed on artery segments cultured in vitro, and conspicuous plaques were developed on model vascular wall cultured for 13 days. Labeled cells increased with an increase in culture time but were not seen in the control group. A greater number of labeled cells were observed in injured endothelium group cultured in 20% serum DMEM. Hyperplasia was inhibited after BQ123 was added into the medium, suggesting that serum and ET-1 are important factors that lead to VSMC proliferation. Expressions of HRG-1 and SM22α were decreased while the aorta segments were cultured in vitro, minimum or even absent mRNA expressions of HRG-1 and SM22α were detected in injured endothelium cultured in 20% serum DMEM and increased in injured endothelium plus BQ123 group cultured. ET-1 content in the supernatant increased in injured endothelium cultured in 20% serum DMEM. These results show that the phenotypic transform and VSMC proliferation on cultured artery segments were related not only to serum culture, but also to ET-1 secreting. ET-1 and serum may be the main factors of contributing to the proliferation and phenotypic transform. This model provides a favorable experimental platform for research into the mechanism of vascular proliferous diseases as well as its prevention and treatment.

Effect and mechanism of salvianolic acid B in attenuating elevated portal pressure in a rat model of portal hypertension induced by endothelin-1

To investigate the effects of salvianolic acid B (SA-B) on portal hypertension induced by endothelin-1 in rats. Twenty-eight Sprague-Dawley rats were randomly divided into four groups: ET-1 group, ET-1+SA-B group, ET-1+ET(A)R blocker (BQ-123) group and ET-1+ET(B)R blocker (BQ-788) group. The rats of ET-1+SA-B group underwent intragastrical administration of salvianolic acid B for five days before ET-1 injection, while in three other groups' drinking water was given. In BQ-123 group or BQ-788 group, an intravenous injection of BQ-123 or BQ-788 via femoral vein was administered 30 minutes prior to ET-1 injection. Then changes of portal pressure, cervical artery pressure and heart rate were monitored continuously. After ET-1 injection, the portal pressure of all rats in the ET-1 group increased significantly, while slightly in groups that pretreated with SA-B, BQ-123 or BQ-788. SA-B can attenuate the elevated portal pressure induced by ET-1 with effect similar to ETR blocker.

Arrhythmogenic Action of Endothelin-11–31 Through Conversion to Endothelin-11–21

Endothelin (ET)-1(1-21) is known to play an important role in the pathogenesis of acute ischemic arrhythmia. In the present study, we attempted to determine whether administration of ET-1(1-31) would result in arrhythmia in perfused isolated rat hearts. Forty-eight Sprague-Dawley rats weighing approximately 250-350 g were randomized into 6 groups. Heart was isolated and perfused in a Langendorff mode. The effects of ET-1(1-31) on arrhythmia, heart rate, coronary flow, and heart function were analyzed. Perfusion with 1 nM ET-1(1-31) resulted in frequent ventricular ectopic beats (VEBs) and ventricular tachycardia (VT). Overall VEB was 128.0 (approximately 66.0-1015.0), and the arrhythmia score (AS) was 2.18 +/- 0.87; both were significantly higher than those of the control group (P < 0.01). Pretreatment with perfusion of 10 nM of the ETA-receptor antagonist BQ(123) markedly attenuated the occurrence of VEB and VT induced by ET-1(1-31). AS in 10 nM BQ123 group was significantly lower than that in 1 nM ET-1(1-31) group (P < 0.01). The arrhythmia induced by 1 nM ET-1(1-31) was partially but significantly reduced by phosphoramidon (1 microM), a neutral endopeptidase/ET-converting enzyme inhibitor. ET-1(1-31) per se caused arrhythmia in perfused isolated rat hearts. This arrhythmogenic action is in part mediated by ET(A) receptor and may be attributed mainly to the conversion of ET-1(1-31) to ET-1(1-21.).