内皮素-1对肾近曲小管上皮细胞形态及TGF-β_1表达的调节作用及其机制

目的:研究内皮素1(endothelin 1,ET-1)对人肾近曲小管上皮细胞形态变化及对转化生长因子β_1(transforming growth factor-β_1,TGF-β_1)表达的调节作用,以及内皮素受体A和受体B在其中所发挥的作用,阐明ET-1对肾小管上皮细胞-肌成纤维细胞转分化(TEMT)的诱导作用。方法:人近曲小管上皮细胞株(HK-2)进行体外培养,随机分为四组,一组为空白对照组,三组采用ET-1进行干预,分别加入BQ123(ETaR拮抗剂)、BQ788(ETbR拮抗剂)、BQ123+BQ788,扫描电镜观察各组细胞形态的变化,实时荧光定量PCR和Western blot检测各组细胞中TGF-β_1的mRNA和蛋白质表达水平的变化。结果:ET-1作用后,HK-2细胞形态明显不规则,胞质减少,细胞皱缩,表面微绒毛脱落,胞间连接显著减少,TGF-β_1的mRNA和蛋白表达均明显上调(P0.05,与空白对照组对比);加入ETR拮抗剂BQ123、BQ788后,细胞形态改善,TGF-β_1mRNA和蛋白表达较模型组均明显下调(P0.05),两组间对比BQ788的抑制效果更明显,但二组差异无统计学意义。结论:ET-1可导致促纤维化因子TGF-β_1显著增加,对于肾小管上皮细胞具有明显的诱导TEMT作用,阻断ET-1受体A或B均可以显著抑制ET-1诱导TEMT作用,其中ETbR拮抗剂BQ788效果更为显著。     

内皮素1及其受体拮抗剂对HSC-T6细胞内皮素受体mRNA表达的影响

目的探讨内皮素1(ET-1)及其受体拮抗剂对HSC-T6细胞内皮素受体mRNA表达的作用及其机制,进一步了解缩血管物质在门静脉高压症发病机制中的作用。方法培养的肝星状细胞HSC-T6细胞系分为7组,分别为空白对照组,ET-1组(培养瓶中加入10 nmol/L的ET-1),BQ-123组[加入1μmol/L的选择性内皮素A型受体(ETRA)拮抗剂BQ-123],BQ-788组[加入1μmol/L的选择性内皮素B型受体(ETRB)拮抗剂BQ-788],ET-1+BQ-123组(加入10 nmol/L的ET-1和1μmol/L的BQ-123),ET-1+BQ-788组(加入10 nmol/L的ET-1和1μmol/L的BQ-788),ET-1+BQ-123+BQ-788组(加入10 nmol/L的ET-1、1μmol/L的BQ-123和1μmol/L的BQ-788)。采用逆转录聚合酶链反应(RT-PCR)检测HSC-T6细胞内皮素受体mRNA的表达。结果ET-1+BQ123+BQ788组ETRA mRNA的表达与空白对照组相比差异具有统计学意义(分别为0.292±0.023和0.440±0.030,P<0.05),其余各组与之相比无明显差异(P>0.05);与ET-1组相比,ET-1+BQ788组和ET-1+BQ123+BQ788组ETRA mRNA表达低,差异具有统计学意义(分别为0.329±0.044,0.292±0.023和0.487±0.039,P<0.05];与空白对照组相比,ET-1组ETRB mRNA表达上调,但差异无统计学意义(分别为0.499±0.136和0.289±0.047,P=0.134];与ET-1组相比,ET-1+BQ788组ETRB mRNA表达明显低,差异具有统计学意义(分别为0.153±0.071和0.499±0.136,P<0.05)。结论ET-1对ETRA mRNA的表达无明显作用;ET-1本身可能会上调HSC-T6 ETRB mRNA的表达,ET-1作用于ETRA时则会抑制ETRB mRNA的表达。     

内皮素受体在雄激素非依赖性前列腺癌中的作用

目的 研究雄激素非依赖性前列腺癌株PC3中内皮素(ET)受体表达及受体阻断后PC3细胞的凋亡情况。方法 RT-PCR法测定PC3中ET受体的表达以及采用受体拮抗剂干预后其表达的变化,通过流式细胞仪和透射电镜研究干预后PC3细胞凋亡的情况。结果 PC3中ETA表达较高,而ETB表达非常弱。ETA受体拮抗剂干预后,ETA表达明显减少,随干预浓度的增加,PC3细胞凋亡的比例逐渐增加;而ETB受体拮抗剂干预后无明显改变。结论 PC3中ET-1的作用主要通过ETA受体介导,ETB受体是静止的。ETA受体阻断后,表达减少,PC3细胞出现凋亡,并呈剂量依赖关系。     

内皮素1诱导肝脏血管收缩的受体研究

本研究探讨内皮素 (ET)对肝脏循环的影响与ETα和ETβ两种受体在这种调节过程中的作用。材料与方法1.实验动物与试剂 :选择体重为 2 5 0～ 30 0 g的SD大鼠作为实验动物。内皮素 1(ET 1) (Sigma公司 ) ,BQ12 3,BQ788和BQ30 2 0为一组人工合成的内皮素衍生物 ,其中BQ12 3是ETα受体拮抗剂 ,BQ788是ETβ受体拮抗剂 ,BQ30 2 0是ETβ受体激活剂。离体肝脏立即放入灌注箱中 ,门静脉插管与一个循环灌注系统连接 ,通过测定每分钟肝静脉流出量观察门静脉系统循环量 ,用ml·min-1·g-1肝组织表达。在肝脏灌注稳定15min后开始加入试剂并观…     

内皮素受体拮抗剂对肝硬化、门静脉高压大鼠内皮素受体mRNA表达的影响

内皮素(ET) 1是强力缩血管物质,在门静脉压力升高和维持中起重要作用[1] ,ET 1通过与其受体结合而发挥作用。ET受体至少可分为两种亚型,即ETA受体(ETRA)和ETB受体(ETRB) ,其中ETRB根据介导的舒张和收缩作用又分为ETRB1和ETRB2 [2 ] 。本实验旨在观察CCl4诱导的肝硬化、门静脉高压大鼠门静脉压力、肝组织ET受体mRNA表达的变化以及ETA受体拮抗剂(BQ 12 3 )、ETB受体拮抗剂(BQ 788)及两者联合应用对研究结果的影响。一、材料与方法1.实验对象:雄性SD大鼠,体重3 70～40 0 g ,参照文献[3 ]方法制备肝硬化、门静脉高压模型…     

缺氧诱导因子1α调控内皮素1表达在高糖诱导肾小管上皮细胞转分化中的作用

糖尿病肾病(DN)是终末期肾脏病(ESRD)的主要原因,也是一类以进行性肾间质纤维化(RIF)为特征的疾病.研究表明在DN状态下,肾脏固有细胞如系膜细胞、肾小管上皮细胞均能发生表型转换而表现间充质细胞特性参与RIF[1].缺氧诱导因子1α(HIF-1α)和其下游基因内皮素1 (ET-1)均能导致上皮细胞转分化(EMT)[2-3].但二者是否参与DN肾小管上皮细胞转分化(TEMT)及其相互作用并不清楚.本研究观察高糖环境下HIF-1α、ET-1、α-SMA和E-cadherin的表达情况,以及HIF-1α siRNA转染和ET-1特异性A受体拮抗剂BQ123对其的影响,旨在探讨HIF-1α和ET-1在高糖诱导TEMT中的作用.     

前列腺雄激素调节基因:一个新的雄激素非依赖性前列腺癌治疗的潜在靶点(英文)

目的:初步研究前列腺雄激素调节基因(PAR)与雄激素—雄激素受体信号转导通路的关系及其在前列腺癌细胞恶性转化过程中的作用,探讨通过抑制 PAR 基因治疗雄激素非依赖性前列腺癌的可能性。方法:用 RT-PCR 检测 LNCaP、PC3细胞中 PAR 基因 mRNA 表达水平的差异。分别用 RT-PCR 检测双氢睾酮对LNCaP、PC3及稳定转染了 pcDNA3-AR 的 PC3细胞株 PC3-AR 的 PAR 基因 mRNA 表达的调节作用,并观察这一调节作用是否可被雄激素受体拮抗剂氟他胺阻断。进一步用 RNA 干扰技术下调 PC3细胞 PAR 的表达,用细胞计数、软琼脂克隆形成实验、流式细胞术研究 PAR 基囚表达下调对 PC3细胞生长的抑制作用。结果:PC3细胞 PAR 基因 mRNA 的表达是 LNCaP 细胞的3倍;双氢睾酮可调节 LNCaP 和 PC3-AR 细胞株PAR 基因 mRNA 表达水平,此种对 PAR 表达的调节作用可被氟他胺阻断:双氢睾酮对 PC3细胞 PAR 基因mRNA 表达无明显影响。RNA 干扰可抑制 PC3细胞 PAR 基因表达,使细胞增殖受抑制,细胞周期阻滞于G_2-M 期,凋亡增加。结论:PAR 可能是雄激素—雄激素受体信号转导通路下游的与雄激素非依赖性前列腺癌恶性表型密切相关的癌基因,有望成为其基冈和药物治疗的靶点。     

内皮素-1及其受体拮抗剂对HSC-T6肝星状细胞收缩作用机制的体外研究

目的研究内皮素及其受体拮抗剂对肝星状细胞(hepatic stellate cell,HSC)的收缩作用及其作用的受体机制。方法采用胶原晶格法(collagen lattice)观察ET-1及其受体拮抗剂BQ-123、BQ-788和选择性ETB受体协同剂IRL-1620对肝星状细胞收缩的影响。结果与空白对照组相比,ET-1组和ET-1+BQ-788组收缩面积百分比显著降低(0·504±0·082vs0·848±0·038,P<0·05;0·498±0·102vs0·848±0·038,P<0·05),而ET-1+BQ-123组则无明显差异(0·865±0·025vs0·848±0·038,P>0·05);与空白对照组相比,ET-110nmol/L+IRL-16201μmol/L组收缩面积百分比显著降低(0·429±0·117vs0·758±0·036,P<0·05),而ET-1100pmol/L+IRL-16201μmol/L组与空白对照组无差异(0·724±0·061vs0·758±0·036,P>0·05)。结论内皮素-1引起肝星状细胞的收缩是由内皮素受体A介导的,选择性内皮素受体A拮抗剂BQ-123可以抑制内皮素-1介导的肝星状细胞的收缩,而内皮素B受体拮抗剂则无此作用;选择性内皮素B受体协同剂IRL-1620能抑制较低浓度的ET-1引起的肝星状细胞的收缩。     

表皮生长因子对PC-3细胞内皮素-1及其受体mRNA表达的影响

目的:探讨表皮生长因子(EGF)对激素非依赖性前列腺癌(HRPC)PC-3细胞中内皮素1(ET-1)及其受体mRNA表达的影响。方法:EGF作用不同时间(0、8、16、24、32、48h)后,RT-PCR法测定PC-3细胞中ET-1及其受体ETAR mRNA、ETBR mRNA表达;EGF干预24h后,RT-PCR法测定ET-1及其受体ETAR mRNA、ETBR mRNA表达变化。结果:在PC-3细胞中可检测到ET-1及ETAR mRNA表达,但无ETBR mRNA表达;EGF可上调ET-1及ETAR mRNA表达,与对照组比较,差异具有显著性;ET-1及ETAR mRNA表达随EGF干预时间增加而增加,EGF作用不同时间对PC-3细胞ET-1、ETAR mRNA表达的影响不同,差异具有显著性(P<0.05)。结论:EGF可上调PC-3细胞中ET-1及ETAR mRNA表达,为HRPC的治疗提供了分子生物学基础。     

负载BMP-2掺锶磷酸钙复合材料对成骨细胞增殖及功能的影响

目的探索负载BMP-2掺锶磷酸钙复合材料对成骨细胞增殖及功能的影响。方法获取SD大鼠成骨细胞,随机分为空白对照组(Con组)、磷酸钙组(CPC组)和复合材料组(BSCPC组);培养基中分别添加安慰剂、磷酸钙和负载BMP-2掺锶磷酸钙共培养一段时间,通过CCK-8法检测成骨细胞的增殖情况,碱性磷酸酶(alkalinephosphatase,ALP)及茜素红染色观察细胞的功能状态,蛋白电泳观察骨保护素(osteoprotegerin,OPG)、核因子κB受体活化因子(receptor activator of nuclear factor-KB,RANK)及核因子κB受体活化因子配体(ligand of receptor activator of nuclear factor-κB,RANKL)蛋白的表达情况。结果共培养1、3、5和7 d,和Con组比较,CPC组和BSCPC组的成骨细胞数目明显增加(P0.05),且以BSCPC组成骨细胞数目最多(P0.05);共培养14 d及21 d,和Con组比较,CPC组和BSCPC组的成骨细胞的矿化能力及ALP活性明显增加(P0.05),且以BSCPC组细胞钙化能力最强及ALP活性最高(P0.05);共培养7 d,和Con组比较,CPC组和BSCPC组的成骨细胞的OPG表达明显增加,而RANK及RANKL蛋白表达明显降低(P0.05),且以BSCPC组的成骨细胞蛋白OPG、RANK及RANKL蛋白表达量改善最为显著(P0.05)。结论负载BMP-2掺锶磷酸钙复合材料促进成骨细胞增殖分化和改善细胞活性和功能。     

Role of interactions among endothelin-1, CD44, and hyaluronic acid in mouse mesangial cell proliferation

Background. Endothelin-1 (ET-1) is involved in the progression of nephritis. Glomerular CD44 expression and the accumulation of its ligand hyaluronic acid (HA) both increase in nephritis. HA induces proliferation in many types of cells. We studied possible interactions among ET-1, CD44, and HA in mesangial cell (MC) proliferation, which contributes to the progression of nephritis. Methods. ET-1, CD44, proliferating cell nuclear antigen (PCNA), and HA immunohistochemistry were studied in renal lesions in MRL/MpJ-lpr/lpr (lpr/lpr) mice. The effects of ET-1, an endothelin-converting enzyme inhibitor, phosphoramidon, and an endothelin type-A (ETA) receptor antagonist, BQ-123, on CD44 expression and on HA-induced proliferation were studied in MC cultures. Results. Glomerular expression of ET-1 and CD44, and HA accumulation increased in nephritic lpr/lpr mice. Colocalization of areas positive for ET-1, CD44, and HA was observed, in part together with PCNA-positive nuclei in the glomeruli of the lpr/lpr mouse. ET-1 was secreted into MC culture medium. Phosphoramidon and BQ-123 suppressed the CD44 expression and HA-induced proliferation in MC culture, while the addition of ET-1 reversed the suppression of CD44 expression and the suppression of HA-induced proliferation by phosphoramidon. Conclusions. These results indicate that endogenously secreted ET-1 stimulates CD44 expression and HA-induced DNA synthesis via the ETA receptor. The increase in CD44 caused by ET-1 may be responsible for its stimulation of HA-induced DNA synthesis in MCs. These findings support the concept that interactions among ET-1, CD44, and HA promote MC proliferation, resulting in the progression of nephritis in lpr/lpr mice. Received: August 17, 2000 / Accepted: September 28, 2000     

Basal release of endothelin-1 and the influence of the ETB receptor on single vessel hydraulic permeability

BACKGROUND: Endothelin-1 (ET-1) has a direct permeability decreasing effect on the microvasculature. The present study was designed to test the hypothesis that this effect is mediated via the endothelin B (ETB) receptor located on the microvascular endothelium and to determine whether basal microvascular permeability is dependent on constitutive release of ET-1. To isolate the direct effect of ET-1, experiments were conducted under conditions in which hydraulic and oncotic pressures were controlled. METHODS: Postcapillary venules in the rat mesentery were perfused in situ, and paired measurements of hydraulic permeability (Lp) were obtained using the modified Landis micro-occlusion method. Lp measured after a 15-minute perfusion with the ETB receptor blocker BQ-788 (1 micromol/L) was compared with measures of Lp obtained after perfusion with a combined mixture of BQ-788 and ET-1 (80 pmol/L) (n = 6). In addition, the effect of basal endogenous ET-1 was tested by measuring the effects of BQ-788 perfusion on Lp (n = 6). RESULTS: Units for Lp are mean +/- SE x 10(-8) cm x s(-1) cm H2O(-1). ETB receptor blockade prevented any decrease in Lp induced by ET-1 (BQ-788 alone = 7.9 +/- 0.7; BQ-788 + ET-1 = 8.2 +/- 0.8;p = 0.5). Under basal conditions and in the absence of exogenous ET-1, ETB receptor blockade led to a significant increase in Lp from 6.8 +/- 0.9 to 9.7 +/- 1.2 (p = 0.001). Conclusion: Decreases in microvascular permeability in single postcapillary venules after the administration of ET-1 are mediated via the ETB receptor. Constitutive release of ET-1 from the microvascular endothelium also plays a role in maintaining basal levels of permeability. These findings suggest important roles for ET-1 in maintaining and modulating microvascular permeability.     

Impaired response of the denervated kidney to endothelin receptor blockade in normotensive and spontaneously hypertensive rats

BACKGROUND: As yet, there are only limited data available on the exact role of endothelin (ET) acting through endothelin-A (ETA) receptors in renal sodium and water regulation and the potential functional implications of an interaction of the renal ET system with renal nerves in normotensive and spontaneously hypertensive rats. METHODS: Experiments were carried out in 64 male conscious spontaneously hypertensive rats and in 56 normotensive Wistar-Kyoto (WKY) rats. Bilateral renal denervation (BRD) was performed in 32 spontaneously hypertensive rats and 28 WKY rats 7 days before the experiments. The ETA receptor antagonist, BQ-123 (16.4 nmol/kg x min intravenously) or the endothelin-B (ETB) receptor antagonist, BQ-788 (25 nmol/kg x min intravenously) were infused at a rate of 25 microL/min for 50 minutes. RESULTS: Renal papillary ET-1 concentration in intact spontaneously hypertensive rats was 67.8% lower than in intact WKY rats (154 +/- 40 fmol/mg protein vs. 478 +/- 62 fmol/mg protein, P < 0.01). BRD decreased papillary ET-1 by 73.5% in WKY rats to 127 +/- 19 fmol/mg protein (P < 0.001), but had no effect in spontaneously hypertensive rats (122 +/- 37 fmol/mg protein). BRD, BQ-123, or BQ-788 did not affect glomerular filtration rate (GFR) or renal blood flow (RBF) in any of the groups. In intact WKY, BQ-123 decreased urine flow rate (V) from 4.65 +/- 0.44 microL/min.100 g body weight to 2.44 +/- 0.35 microL/min.100 g body weight (P < 0.01), urinary excretion of sodium (UNaV) from 238.2 +/- 27.4 to 100.2 +/- 17.0 (P < 0.01) and potassium (UKV) from 532.1 +/- 62.6 nmol/min.100 g body weight to 243.0 +/- 34.2 nmol/min.100 g body weight (P < 0.001), whereas BQ-788 decreased only V and UNaV. In renal denervated WKY, BQ-123 or BQ-788 did not alter V, UNaV, or UKV. In intact spontaneously hypertensive rats BQ-123 but not BQ-788 decreased V from 3.94 +/- 0.48 microL/min.100 g body weight to 2.55 +/- 0.44 microL/min.100 g body weight (P < 0.05). In renal denervated spontaneously hypertensive rats neither BQ-123 nor BQ-788 affected V, UNaV, or UKV. CONCLUSION: An interaction between ET and renal nerves is involved in the control of renal function. Moreover, renal nerves participate in the regulation of ET-1 production within the kidney. Finally, decreased synthesis of ET-1 in the renal papilla of spontaneously hypertensive rats may contribute to development and/or maintenance of hypertension due to modulation of renal excretory function.     

内皮素-1及其受体在良性前列腺增生中的表达研究

目的探讨内皮素-1及其受体(ETAR与ETBR)在良性前列腺增生(BPH)移行区组织中表达的意义。方法采用免疫组化及Western blot技术检测5例正常前列腺组织(NP)与16例BPH组织中内皮素-1及其受体表达情况。结果BPH移行区组织中内皮素-1阳性面积为(77936.16±85291.33)μm2,ETAR积分吸光度为316.6±65.2,明显高于正常前列腺组织(阳性面积75.68±110.85μm2,ETAR积分吸光度为140.2±64.8),而2组ETBR的表达(积分吸光度分别为81.4±31.8,105.0±45.5)差异无统计学意义。结论BPH移行区组织中内皮素-1及其ETAR表达上调,在BPH的病理生理过程中可能产生重要作用。     

Augmentation of endothelial function by endothelin antagonism in human saphenous vein conduits

OBJECT: Cerebral revascularization with saphenous vein (SV) conduits is used in the management of hard-to-treat lesions that require deliberate arterial occlusion and in selected patients with occlusive vascular disease. Endothelial dysfunction is thought to contribute to acute perioperative vasospasm and chronic graft atherosclerosis. In the present study the authors examined the contribution of the potent vasoconstrictor endothelin-1 (ET-1) to endothelial dysfunction in human SVs. METHODS: The effects of an ET(A/B) receptor antagonist (bosentan), an ET(A) receptor antagonist (BQ-123), and an ET(B) receptor antagonist (BQ-788) on in vitro endothelium-dependent and -independent responses were studied in human SVs. Vascular segments were obtained in 34 patients who had undergone revascularization procedures, and isometric dose-response curves (DRCs) were constructed using the isolated tissue bath procedure as follows: 1) cumulative DRCs to norepinephrine; and 2) DRCs to acetylcholine (ACh) and sodium nitroprusside in the absence and presence of bosentan, BQ-123, or BQ-788. Maximal vasodilatory responses and sensitivity were compared between groups. In the presence of bosentan (Experiment 1) and BQ-123 or BQ-788 (Experiment 2), ACh responses were significantly augmented (percent maximum relaxation values: 7+/-2 [control] compared with 17+/-3 [bosentan], p < 0.002 [Experiment 1]; and 12+/-2 [control] compared with 29+/-2 [BQ-123] and 25+/-2 [BQ-788], p < 0.003 and p < 0.002, respectively [Experiment 2]). The sensitivity of SVs to ACh was unaffected by treatment. These beneficial effects were specific for the endothelium. CONCLUSIONS: Blockade of ET receptors significantly improves endothelial function in SVs. Furthermore, these effects appear to be independently and maximally mediated by antagonism of either ET(A) or ET(B) receptors. Interventions aimed at improving endothelial function may serve to counter perioperative vasospasm and impede atherosclerosis in SVs used for revascularization procedures.     

Crosstalk between the angiotensin and endothelin system in the cerebrovasculature after experimental induced subarachnoid hemorrhage

Under physiologic conditions, losartan showed a dose-dependent antagonistic effect to the endothelin-1 (ET-1)-mediated vasoconstriction. This reduced vasoconstriction was abolished after preincubation with an endothelin B₁ receptor (ET(B₁)-receptor) antagonist. Also, an increased ET(B₁)-receptor-dependent relaxation to sarafotoxin S6c (S6c; an ET(B₁)-receptor agonist) was detected by preincubation with losartan. Investigations after experimental induced subarachnoid hemorrhage (SAH) are still missing. Therefore, we analyzed losartan in a further pathological setup. Cerebral vasospasm was induced by a modified double hemorrhage model. Rats were sacrificed on day 3 and isometric force of basilar artery ring segments was measured. Parallel to physiological conditions, after SAH, the ET-1-induced vasoconstriction was decreased by preincubation with losartan. This reduced contraction has been abolished after preincubation with BQ-788, an ET(B₁)-receptor antagonist. In precontracted vessels, ET-1 induced a higher vasorelaxation under losartan and the endothelin A receptor (ET(A)-receptor) antagonist BQ-123. After SAH, losartan caused a modulatory effect on the ET(B₁)-receptor-dependent vasorelaxation. It further induced an upregulation of the NO pathway. Under losartan, the formerly known loss of the ET(B₁)-receptor vasomotor function was abolished and a significantly increased relaxation, accompanied with an enhanced sensitivity of the ET(B₁)-receptor, has been detected. Also, the dose-dependent antagonistic effect to the ET-1-induced contraction can be effected by angiotensin II type 1 receptor (AT₁-receptor) antagonism due to losartan directly via the ET(B₁)-receptor.     

Selective endothelin B receptor blockade does not influence BNP-induced natriuresis in man

Brain natriuretic peptide (BNP) and endothelin-1 (ET-1) both exhibit natriuretic activity within the human kidney. Furthermore, they both act partly through activation of the endothelial nitric oxide pathway. Since ET-1 may cause vasodilation and natriuresis via stimulation of the ET-B receptor, the aim of the present study was to investigate whether renal ET-B receptors participate in the renal actions of BNP. In this placebo-controlled, crossover study, we infused BNP (4 pmol/kg/min) or placebo (i.v.) for 1 h, with or without co-infusion of the ET-B receptor antagonist BQ-788 (50 nmol/min) for 15 min on 4 separate days, in 10 healthy subjects (mean age 54+/-6 years.). During infusion, we measured effective renal plasma flow (ERPF), and glomerular filtration rate (GFR) using PAH/inulin clearance. Cardiac output was measured before and after infusion, using echocardiography. Blood pressure and heart rate (HR) were monitored as well. Urine and plasma samples were taken every hour to measure diuresis, natriuresis, cyclic 3',5' guanosine monophosphate, and ET-1 levels. BNP with or without ET-B receptor blockade increased natriuresis and diuresis. In addition, BNP alone increased GFR and filtered load, without changing ERPF. BQ-788 infusion did not affect renal hemodynamics or natriuresis. Neither BNP nor BQ-788 altered cardiac output, blood pressure, and heart rate. In conclusion, the present study shows that selective ET-B receptor blockade has no effect on the BNP-induced natriuresis and glomerular filtration rate.     

Endothelin- and sarafotoxin-induced receptor-mediated calcium mobilization in a clonal murine osteoblast-like cell line, MC3T3-E1/B

Previous studies have demonstrated that, in osteoblast-like MC3T3-E1 cells, various endothelin peptides and their homologous sarafotoxins generate prostaglandin E₂ (PGE₂) release through an ET_A receptor subtype. In this study, biphasic Ca²⁺ signals elicited with endothelin (ET)-1, ET-2, ET-3, β-ET, S6a1, and S6b (ET/S6) were measured by microspectrofluorimetric methods in cell suspensions loaded with Fura-2 acetoxymethylester (Fura-2 AM). Phospholipase C (PLC)-dependent calcium activation mechanisms seem to be involved. We found evidence of Ca²⁺ release from thapsigargin-sensitive and non-thapsigargin-sensitive intracellular Ca²⁺ stores as well as Ca²⁺ transmembrane inflow through multiple voltage-independent and Ni²⁺-sensitive cation channels. Using an ET_A receptor antagonist, BQ-123, we showed that this receptor was coupled to Ca²⁺ mobilization. All agonists tested, except S6c (an ET_B-receptor-specific agonist) induced receptor desensitization. Our results demonstrate that the ET/S6-induced Ca²⁺ signaling pathway is mediated via an ET_A-receptor subtype in MC3T3-E1/B cells.