ERK1/2通路参与大鼠肠系膜动脉平滑肌细胞ETB受体上调表达

目的探讨细胞外信号调节激酶1/2(ERK1/2)信号转导通路在血管平滑肌细胞内皮素-1 B型受体(ET_B)上调中的作用。方法用大鼠肠系膜上动脉离体培养模型，以敏感的离体小血管张力描记技术记录血管张力变化，实时PCR定量ET_B受体mRNA，PhosphoELISA法测定细胞内磷酸化的ERK1/2蛋白水平。结果大鼠肠系膜上动脉培养3 h，细胞内ERK1/2蛋白磷酸化水平明显增高，培养24 h ET_B受体mRNA表达水平显著上调，选择性ET_B受体激动剂蛇毒类似物(sarafotoxin 6c，S6c)引起的收缩增强；与特异性ERK1/2通路阻滞剂SB386023共同孵育24 h，S6c引起的最大收缩E_max明显下降，ET_B受体mRNA水平也显著降低。结论ERK1/2信号转导通路参与大鼠肠系膜上动脉离体平滑肌细胞ET_B受体上调过程。     

供应结肠癌组织的动脉对缩血管物质的反应性

目的研究供应结肠癌组织的动脉对缩血管物质的反应性。方法取结肠癌患者供应癌组织的动脉(癌动脉),以供应癌周正常组织的动脉(癌周动脉)和交通事故死亡者对应部位的动脉(正常动脉)为对照,动脉切成长约1mm的动脉环,记录4种缩血管物质对动脉收缩的量效曲线。结果去甲肾上腺素(NA)引起正常动脉的最大收缩(Emax)和半最大效应浓度(pEC50)分别为97%±19%和5.94±0.17(n=5);NA收缩癌动脉的Emax和pEC50分别为74%±5%和5.54±0.21(n=5),NA收缩癌周动脉的Emax和pEC50分别为119%±11%和5.84±0.09(n=5)。NA收缩癌动脉的Emax较癌周动脉及正常动脉的Emax值降低(P<0.01,P<0.05)。内皮素-1(ET-1)强烈收缩癌周动脉,其Emax和pEC50分别为248%±34%和8.37±0.04。与癌周动脉的量效曲线比较,ET-1收缩正常动脉和癌动脉的量效曲线明显右移、压低。5-HT和S6c对正常动脉、癌动脉及癌周动脉无明显的缩血管作用。结论ET-1和NA是收缩结肠癌动脉的主要缩血管物质,癌动脉对ET-1和NA收缩血管的反应性明显低于癌周动脉和正常动脉。     

内皮素-3对犬肺动静脉的作用

阐明内皮素 3(ET 3)对肺动静脉的作用机理 .利用犬离体肺动静脉条 ,观察其张力改变 .结果可见 :①ET 3(1～ 30 μmol·L- 1)引起肺动脉舒张 (低浓度 )和收缩 (高浓度 )双向反应 ,ETB 受体激动剂IRL162 0 (1～ 30 μmol·L- 1)只引起舒张反应 ;去内皮 ,ETB 受体阻断剂IRL10 38(1μmol·L- 1)或左旋硝基精氨酸 (L NA ,10 μmol·L- 1)均使ET 3或IRL162 0所致舒张反应减弱或消失 ,ETA 受体阻断剂BQ12 3(10 μmol·L- 1)则使ET 3所致收缩反应翻转为舒张反应 ;②同浓度的ET 3和IRL162 0只引起肺静脉浓度依赖性收缩反应 ;BQ12 3可使ET 3所致收缩反应减弱 ,IRL10 38可使IRL162 0所致收缩反应减弱 ;③在BQ12 3预处理条件下给予第二剂ET 3(30 μmol·L- 1) ,肺静脉表现为舒张反应 ,吲哚美辛 (1μmol·L- 1)可使其舒张反应减弱 .本研究表明 :①存在于肺动脉平滑肌上的ETA 受体参与血管的收缩反应 ,肺动脉内皮上的ETB 受体通过释放NO参与舒张反应 ;②肺静脉平滑肌上的ETA 和ETB 受体均参与收缩反应 ,但ETB 受体所致收缩反应易脱敏 ;③在肺静脉平滑肌上可能还存在非ETA/非ETB 受体 ,通过释放舒张性PG物质参与舒张反应 .     

不同给药方式对P2X_1受体介导大鼠肠系膜动脉收缩反应的影响

目的研究不同方式给予α,β-MeATP对肠系膜动脉P2X1受体介导血管收缩反应的影响。方法制备大鼠离体肠系膜动脉环标本,采用非累积给药法和单浓度给药法两种方式给予α,β-MeATP,记录药物诱发的等长收缩反应。结果两种给药方式给予α,β-MeATP(10-7-10-4mol·L-1)均可使大鼠离体肠系膜动脉产生浓度依赖性收缩反应。以KCl最大收缩反应或以标本湿重标化α,β-MeATP诱发的收缩反应时,α,β-MeATP单浓度给药的收缩反应均大于非累积给药(P<0.01)。以标本湿重标化时,10-4mol·L-1浓度α,β-MeATP诱发的收缩反应分别是(0.73±0.10)g·mg-1(单浓度组)和(0.38±0.05)g·mg-1(非累积组);以KCl最大收缩反应标化时,分别是(53.17±6.0)%(单浓度组)和(36.78±5.71)%(非累积组)。在α,β-MeATP非累积给药组120mmol·L-1KCl诱发的动脉收缩反应小于单浓度给药组和NA对照组(P<0.01)。结论在大鼠肠系膜动脉,非累积给予α,β-MeATP降低P2X1受体介导的收缩反应以及高钾诱发的收缩反应,该给药方式可能导致错误的实验结论 。     

缝隙连接在苯肾上腺素缩血管中的作用

目的　探讨缝隙连接 (GJ)在苯肾上腺素 (PE)缩血管中的作用。方法　通过大鼠离体肠系膜动脉环 (superiormesentericarterialrings,MARs)试验 ,观察GJ阻断剂Hep tanol预处理对PE引起的去内皮和完整内皮MARs收缩及其量效曲线的影响。结果　Heptanol预处理可明显抑制 1 0× 10 - 7mol·L- 1PE的缩血管作用 ,并呈剂量依赖关系。 3 0× 10 - 5 mol·L- 1Heptanol抑制百分率分别为 11 70 %±3 2 5% (去内皮组 )和 15 2 4%± 7 87% (完整内皮组 ) ;3 0× 10 - 4mol·L- 1Heptanol预处理 ,抑制百分率分别为3 6 3 6%± 11 54% (去内皮组 )和 3 8 85%± 13 0 3 % (完整内皮组 ) ;同浓度Heptanol对PE致去内皮和完整内皮MAR收缩的抑制作用差异无显著性。 3 0× 10 - 5 ,1 0× 10 - 4,3 0× 10 - 4mol·L- 1Heptanol使PE的量效曲线右移 ,最大效应 (Emax)不变。结论　GJ参与介导苯肾上腺素的缩血管作用 ,GJ阻断剂Heptanol对PE缩血管的抑制作用无明显内皮依赖性 ,具易逆性特点     

内皮素A受体拮抗剂对离体大鼠心脏急性缺血性心律失常的拮抗作用(英文)

目的:观察内皮素A(ET_A)受体拮抗剂PD156707和内皮素B(ET_B)受体拮抗剂IRL1038对离体大鼠心脏急性缺血性心律失常及心功能的影响,以探讨内源性内皮素(ET)是否参与急性心肌缺血所致心律失常等病理生理过程。方法:雄性SD大鼠53只,以Langendorff方式离体灌流,结扎左冠状动脉前降支(LAD)造成急性心肌缺血。观察PD156707和IRL1038对LAD结扎后急性缺血性心律失常、心功能的影响和缺血心肌超氧化物歧化酶(SOD)活性、 丙二醛(MDA)含量的变化。结果:PD156707 20-500nmol/L预处理后,剂量依赖性地改善急性缺血的离体大鼠心脏心功能,增加SOD活性,减低MDA含量,减少心律失常的发生,并减轻心律失常的严重程度。IRL1038 20-500 nmol/L预处理,对心肌缺血后心功能、SOD活性、MDA含量以及心律失常无显著影响。结论:ET_A受体拮抗剂能有效改善离体大鼠急性缺血期心脏心功能,在一定程度上增强心脏抗氧化能力,并可明显减少和减轻急性缺血性心律失常;ET_B受体拮抗剂上述作用不明显。提示内源性ET-1可能是急性缺血期参与致心律失常及心功能损害的重要因素之一,其作用很可能是由ET_A受体介导的。     

吗啡对原代培养大鼠海马神经元CCK受体mRNA表达的影响

目的探讨原代培养大鼠海马神经元上胆囊收缩素(cholecystokinin,CCK)受体CCK-AR及CCK-BR mRNA表达及吗啡对其表达的影响。方法采用新生大鼠海马神经元无血清原代培养技术,用吗啡(100μmol·L-1培养基)孵育3,6,12,18,24,36,48,72h,以及采用不同浓度吗啡(10,100,150μmol·L-1)孵育6、30h后,RT-PCR及测序方法观察CCK-AR及CCK-BR mRNA表达及吗啡对其表达的影响。结果RT-PCR结果显示,CCK-AR和CCK-BR mRNA在原代大鼠海马神经元上均有表达;CCK-AR mRNA RT-PCR扩增产物为218bp,CCK-BR mRNA RT-PCR扩增产物为444bp,经测序证实结果的可靠性;CCK-BR mRNA的表达量明显高于CCK-AR。吗啡作用后可使2种CCK受体mRNA表达上调。吗啡浓度及作用时间的不同对CCK-AR和CCK-BRmRNA表达的影响不同。结论原代大鼠海马神经元上有CCK-AR和CCK-BR,以CCK-BR为主;吗啡可使2种CCK受体mRNA表达上调。     

M受体机制在粉防己碱保护心脏缺血再灌注损伤作用中的影响

目的　探讨M受体对粉防己碱抗心脏缺血再灌注损伤作用的影响。方法　以离体豚鼠工作心脏缺血再灌注为心肌损伤模型 ,观察乙酰甲胆碱 (Mch ,0 1μmol·L-1) ,粉防己碱 (Tet,0 3μmol·L-1)的心肌保护作用。结果　对照组在缺血复灌后 2 0min ,主动脉压 (AP)、左室收缩压(LVSP)、左室压力上升最大速率 (+dp/dtmax) ,左室压力下降最大速率 (-dp/dtmax)仅分别恢复至缺血前的 6 5 6 %±3 6 9%、6 7 9%± 9 0 9%、5 8 8%± 7 6 4%、48 6 %±8 38%。Mch、Tet使AP恢复至缺血前的 80 7%±11 70 %、77 2 %± 6 96 % (P <0 0 1vscontrol) ,LVSP恢复至 97 9%± 6 32 %、93 9%± 10 1% (P <0 0 1vscon trol) ,+dp/dtmax 恢复至 91 2 %± 11 99%、80 0 3%±9 2 4% (P <0 0 1vscontrol) ,-dp/dtmax恢复至 92 13%±6 89%、79 43%± 8 83% (P <0 0 1) ;对照组复灌后心输出量 (CO)、冠脉流量 (CF)均降低 ,恢复率不足 5 0 % ;Mch、Tet能使CO、CF维持于缺血前水平的 90 %、80 %以上。给予阿托品 0 1μmol·L-1阻断心脏M受体可见Mch、Tet保护心功能的作用明显受到抑制 ,心功能各项指标恢复程度与对照组差异无显著性。结论　调节心脏M受体能影响Tet的心肌保护作用。     

非选择性内皮素受体拮抗剂CPU0213改善感染性休克小鼠/大鼠的血管活性

目的:通过非选择性内皮素受体拮抗剂CPU20213对结扎小鼠和大鼠盲肠并穿孔致感染性休克血管活性的改善,探讨其通过干预内皮素-活性氧(ET-ROS)轴心治疗感染性休克可能的作用机制。方法:结扎小鼠盲肠并穿孔,8 h后按30 mg·kg-1皮下注射(sc)CPU0213,bid×3 d。观察术后该时间段的存活率,计算腹腔渗出液重量,生命器官脏器系数,心肌、肾匀浆和血清丙二醛(MDA),谷胱甘肽过氧化物酶(GSH-PX)含量及胸主动脉血管活性;同样方法结扎大鼠盲肠,检测血浆ET-1浓度和肠系膜血管ETA和ETB受体mRNA表达。结果:模型组小鼠存活率明显降低,生命器官脏器系数明显增加,腹腔渗出液显著增多,GSH-PX活性下降,MDA含量增加,血管收缩和舒张功能及NO生物利用度明显降低;大鼠血浆ET-1浓度显著升高,肠系膜血管ETA和ETB受体mRNA表达上调;CPU20213治疗后,均有不同程度改善。结论:CPU0213通过阻断ET-ROS轴心,改善血管活性,减少腹腔渗出液,提高存活率。     

蛋白酪氨酸激酶抑制剂对脑血管平滑肌细胞Ca~(2+)池操纵性Ca~(2+)内流的影响

目的　研究蛋白酪氨酸激酶和蛋白酪氨酸磷酸酶抑制剂对牛脑血管平滑肌细胞 (CSMC)Ca2 + 池操纵性Ca2 + 内流的影响。方法　采用培养的CSMC ,在生物荧光双波长影像分析系统用Fura 2 /Am荧光探针测定单个细胞内游离Ca2 + 浓度。结果　 (1)蛋白酪氨酸激酶抑制剂 (genistein ,2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低内皮素 1(ET 1,10 -7mol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为5 6%± 2 .9%、2 5 6%± 3 9%、48 9%± 3 7% ;蛋白酪氨酸磷酸酶抑制剂 (vanadate ,2 ,4,8μmol·L-1)能浓度依赖性升高CPA刺激引起的CSMCCa2 + 内流 ,增加比率分别为8 2 %± 3 9%、18 8%± 4 9%、46 6%± 6 9% ;(2 ) genistein(2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低ATP(10 μmol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为 6 7%±2 6%、2 4 6%± 6 5 %、5 1 3 %± 6 9% ;vanadate (2 ,4,8μmol·L-1)能浓度依赖性升高ATP刺激引起的CSMCCa2 +内流 ,增加比率分别为 4 8%± 2 0 %、2 8 5 %± 4 6%、49 6%± 3 3 % ;(3 ) genistein (2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低环匹阿尼酸 (Cyclopiazonicacid ,CPA ,10 μmol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为 6 5 %± 3 0 %、2 2 5 %± 5 2 %、     

Importance of ERK1/2 in upregulation of endothelin type B receptors in cerebral arteries

This study examines the importance of mitogen-activated protein kinases (MAPKs) in upregulation of endothelin type B (ETB) receptors. Rat middle cerebral arteries (MCAs) were incubated for 24 h with or without kinase inhibitors. Vessel segments were mounted in myographs and the contractile responses to endothelin-1 (ET-1; ETA and ETB receptor agonist) and sarafotoxin 6c (S6c; ETB receptor agonist) were studied. We used real-time PCR to measure the receptor mRNA levels. An ELISA assay showed the activation of ERK1/2 kinases after 3 h. Immunohistochemistry revealed the presence of ETB receptors on the vessels. After organ culture, S6c induced vasoconstriction. Incubation with the MEK/ERK inhibitors U0126 and SB386023 diminished the contractile response to S6c. The p38 MAPK inhibitor SB239063 did not affect the S6c-induced contraction. The ET-1-induced vasoconstriction was increased after incubation with SB386023 or SB239063, while unaffected by U0126. The ETB receptor mRNA levels were diminished by SB386023 and U0126. The ETA receptor mRNA levels were unaffected. The levels of activated ERK1/2 kinases were significantly higher after 3 h of organ culture as compared to fresh vessels. The level of ETB receptor protein on the smooth muscle cells of the MCA, visualised by immunohistochemistry, was somewhat diminished by SB386023. Our results show that the ERK1/2 MAPK is important in the upregulation of contractile ETB receptors in MCA after organ culture. Since there is a similar upregulation in models of focal ischaemia and subarachnoid haemorrhage, this may be an important pathophysiological event.     

Vascular endothelin ET(B) receptor-mediated contraction requires phosphorylation of ERK1/2 proteins

In cardiovascular diseases, endothelin type B (ET(B)) receptors in arterial smooth muscle cells are upregulated. The present study revealed that organ culture of rat mesenteric artery segments enhanced endothelin ET(B) receptor-mediated contraction paralleled with increase in the receptor mRNA and protein expressions. The endothelin ET(B) receptor-mediated contraction was associated with increase in phosphorylation of extracellular regulation kinase 1 and 2 (ERK1/2) proteins and elevated levels of intracellular calcium. The elevation curve of intracellular calcium consisted of two phases: one rapid and one sustained. Inhibition of ERK1/2 phosphorylation by SB386023 or blockage of calcium channels by nifedipine significantly reduced the endothelin ET(B) receptor-mediated contraction (P<0.05) and decreased the sustained phase of intracellular calcium level, but not the rapid phase. Thus, phosphorylation of ERK1/2 proteins and elevation of intracellular calcium level are required for endothelin ET(B) receptor-mediated contraction in rat mesenteric artery.     

LPS from Porphyromonas gingivalis increases the sensitivity of contractile response mediated by endothelin-B (ETB) receptors in cultured endothelium-intact rat coronary arteries

The purpose of our study was to examine if lipopolysaccharide (LPS) from Porphyromonas gingivalis (P.g.) modifies the vasomotor responses to Endothelin-1 (ET-1) and Sarafotoxin 6c (S6c) in rat coronary arteries. The arteries were studied directly or following organ culture for 24 h in absence and presence of 2.5 EU/ml LPS. The contractile responses of coronary arteries were investigated by using the selective ETB receptor agonist S6c (1 pM–0.3 μM) and ET-1 (1 pM–0.3 μM). The functional studies demonstrated an augmented contractile response only to S6c in isolated rat coronary arteries after organ culture (with or without LPS). These contractile responses by S6c were blocked by the selective ETB receptor antagonist BQ788 in both vessel groups. The augmented contractile response to S6c was supported by immunohistochemistry, where a significant increase in fluorescence intensity for ETB receptors in smooth muscle cells was observed after organ culture. The presence of LPS in the culture medium significantly increased the sensitivity of endothelium-intact coronary artery to S6c as compared to endothelium-denuded segments. Our results showed a significant increase in both ETB receptor protein levels and S6c-induced maximal contraction in coronary arteries upon 24 h of organ culture, which was further sensitized by LPS.     

LPS from Porphyromonas gingivalis increases the sensitivity of contractile response mediated by endothelin-B (ET(B)) receptors in cultured endothelium-intact rat coronary arteries

The purpose of our study was to examine if lipopolysaccharide (LPS) from Porphyromonas gingivalis (P.g.) modifies the vasomotor responses to Endothelin-1 (ET-1) and Sarafotoxin 6c (S6c) in rat coronary arteries. The arteries were studied directly or following organ culture for 24 h in absence and presence of 2.5EU/ml LPS. The contractile responses of coronary arteries were investigated by using the selective ETB receptor agonist S6c (1 pM-0.3 μM) and ET-1 (1 pM-0.3 μM). The functional studies demonstrated an augmented contractile response only to S6c in isolated rat coronary arteries after organ culture (with or without LPS). These contractile responses by S6c were blocked by the selective ETB receptor antagonist BQ788 in both vessel groups. The augmented contractile response to S6c was supported by immunohistochemistry, where a significant increase in fluorescence intensity for ETB receptors in smooth muscle cells was observed after organ culture. The presence of LPS in the culture medium significantly increased the sensitivity of endothelium-intact coronary artery to S6c as compared to endothelium-denuded segments. Our results showed a significant increase in both ETB receptor protein levels and S6c-induced maximal contraction in coronary arteries upon 24 h of organ culture, which was further sensitized by LPS.     

Role of mitogen-activated protein kinases in endothelin ETB receptor up-regulation after organ culture of rat mesenteric artery

Organ culture of isolated arteries results in increased levels of endothelin ET(B) (ET(B)) receptor mRNA and in enhanced ET(B) receptor mediated contraction. The present study was designed to pinpoint the mitogen-activated protein kinase (MAPK) subtype involved in up-regulation of ET(B) receptors after organ culture of rat mesenteric arteries. Western blot and selective antibodies towards constitutional and phosphorylated MAPKs revealed the appearance of phosphorylated MAPK of the extracellular signal-regulated kinases (ERK) 1/2 type at 3 h of organ culture. The functional ET(B) receptor and its mRNA expression were up-regulated after 24 h of organ culture. Following incubation with the MEK 1/2 specific inhibitor SB408039 or the raf inhibitor SB386023b the up-regulation was attenuated both for ET(B) receptor responses and in ET(B) receptor mRNA expression in the vessel segments. Neither Western blot nor myograph or mRNA analysis showed involvement of the other MAPKs studied. Our results suggest that the ERK1/2 MAPKs are involved in the endothelin ET(B) receptor up-regulation following organ culture.     

螺内酯对高糖及醛固酮诱导的肾小球系膜细胞CTGF表达的影响

目的通过研究螺内酯(SPI)对高糖(HG)和醛固酮(ALD)刺激的体外培养的大鼠肾小球系膜细胞(MsC)结缔组织生长因子(CTGF)mRNA和蛋白表达的影响,探讨SPI的肾脏保护机制。方法将大鼠MsC分为:A组,正常对照组(5.6 mmol.L-1)、B组,高糖组(30 mmol.L-1)、C组,HG+SPI干预组(10-9,10-8,10-7mol.L-1)、D组,ALD组(5.6 mmol.L-1葡萄糖+10-7mol.L-1ALD)、E组,ALD+SPI干预组(10-9,10-8,10-7mol.L-1)。酶联免疫法检测上清液中CTGF蛋白的含量,RT-RCR法检测MsC CTGFmRNA表达。结果①正常糖浓度的培养基中可测到MsCCTGF mRNA和蛋白表达;②与A组比较,B组和D组MsCCTGF mRNA表达和上清液CTGF水平明显升高,差异有显著性;③与B组比较,C组中经10-8,10-7mol.L-1SPI干预MsC CTGF mRNA表达和上清液CTGF水平明显降低,P<0.05,但10-9mol.L-1SPI干预MsC CTGF mRNA表达和上清液CTGF水平无变化,P>0.05;④与D组比较,E组MsCCTGFmRNA表达和上清液CTGF水平明显降低,P<0.05。结论 SPI可抑制HG和ALD刺激的大鼠MsC CTGFmRNA表达和蛋白合成,该作用可能是其抗纤维化和防治肾损害的机制之一。