姜黄素对海水吸入性肺损伤的防治作用

目的探讨姜黄素对海水吸入性肺损伤的防治作用及机制。方法 120只雄性SD大鼠随机均分为五组:空白对照组(C组)、生理盐水组(N组)、海水组(S组)、高剂量姜黄素100mg/kg组(CH组)、低剂量姜黄素20mg/kg组(CL组)。S组、CH组和CL组建立海水吸入模型。吸入海水前2d,姜黄素组每天腹腔注射姜黄素100或20mg/kg,2次/天,C、N和S组腹腔注射等容PBS溶液。测定吸入海水前(基础值,T1)、吸入海水后15 min(T2)、30 min(T3)、1h(T4)、4h(T5)、24h(T6)时PaO2、PaCO2、右肺中叶湿干重比(W/D)。T3时测定左肺下叶丙二醛(MDA)和超氧化物歧化酶(SOD)的浓度,右肺下叶组织行常规病理切片检查。结果 T2时N组,T2~T4时S、CH和CL组PaO2明显低于C组(P0.05);T2~T4时S、CH和CL组PaCO2明显高于C组(P0.05)。T2~T4时N组PaO2明显高于,PaCO2明显低于S组(P0.01)。T2、T3时CH组PaO2明显高于S组(P0.05)。S、CH和CL组MDA明显高于C组(P0.05);N组和CH组MDA明显低于,SOD明显高于S组(P0.05)。T2~T4时S、CH和CL组肺组织W/D明显高于C组(P0.05);T2~T4时CH组和N组肺组织W/D明显低于S组(P0.05)。海水吸入后S组出现明显的肺泡破裂,肺泡、肺间质水肿,炎细胞浸润,CH组能明显减轻肺组织损伤及炎性细胞浸润,但CL组与S组差异无统计学意义。结论预防性使用姜黄素100 mg/kg能减轻海水(4 ml/kg)吸入导致的早期(1h内)肺损伤,其机制可能与其减轻肺组织的氧化应激有关。     

七氟醚预处理对小鼠心肌缺血再灌注时环氧化酶-2表达的影响:在体和离体实验

目的 通过在体和离体实验评价七氟醚预处理对小鼠心肌缺血再灌注时环氧化酶-2(COX-2)表达的影响.方法 在体实验健康成年小鼠54只,体重约250 g,6～8周龄,采用随机数字表法,将其随机分为3组(n=18):对照组(C组)、心肌缺血再灌注组(I/R组)和七氟醚预处理组(SP组).采用结扎左冠状动脉30 min再灌注的方法制备心肌缺血再灌注模型.SP组小鼠吸入2.0％七氟醚,15min/次,共3次,间隔15 min.七氟醚预处理后90 min制备心肌缺血再灌注模型.再灌注6、9h时,取心肌组织分别采用Western blot法测定COX-2表达,采用分光光度法测定心肌caspase-3的活性.再灌注24h时测定左室舒张末压(LVEDP).细胞实验采用随机数字表法,将H9C2细胞随机分为3组,每组6皿:对照组(C组)、缺氧复氧组(H/R组)和七氟醚预处理组(SP组).H9C2细胞置于95％N2+ 5％CO2培养箱中孵育14 h后,置于95％O2+ 5％CO2培养箱中孵育3h,建立缺氧复氧模型.SP组H9C2细胞培养基中加入2.0 mmol/L七氟醚,每次孵育15 min,共3次,间隔15 min.七氟醚预处理结束后15 min建立缺氧复氧模型.复氧3h时采用Western blot法测定细胞COX-2的表达水平,采用比色法测定上清液和细胞裂解液LDH活性,计算LDH释放量.结果 在体实验:与C组比较,I/R组和SP组LVEDP升高,心肌组织caspase-3活性增强,COX-2表达上调(P＜0.01);与I/R组比较,SP组LVEDP降低,心肌组织caspase-3活性减弱,COX-2表达下调(P＜0.01).细胞实验结果:与C组比较,H/R组和SP组H9C2细胞COX-2表达上调,LDH释放量增加(P＜0.01);与H/R组比较,SP组H9C2细胞COX-2表达下调,LDH释放量减少(P＜0.01).结论 七氟醚预处理可通过直接下调心肌COX-2的表达,抑制细胞凋亡减轻小鼠心肌缺血再灌注损伤.     

吸入不同浓度氧化亚氮对全麻患者双腔喉罩囊内压的影响

目的 评价吸入不同浓度氧化亚氮对全麻患者双腔喉罩囊内压的影响.方法 择期全麻患者48例,ASAⅠ或Ⅱ级,年龄25～64岁,随机分为4组(n=12):C组、N1组、N2组及N3组.根据患者身高和体重选择合适型号的双腔喉罩.依次静脉注射异丙酚、瑞芬太尼、利多卡因及维库溴铵行麻醉诱导,喉罩置入成功后,套囊内注入空气,调节囊内压使其达加cm H2O(基础值),连接麻醉机行机械通气,C组、N1组、N2组及N3组分别吸入100%O2、65%O2+35%N2O、50%O2+50%N2O及35%O2+65%N2O,于吸入15、30、45、60、75、90 min时(T1-6)测定喉罩囊内压.结果 与C组比较,N1-3组T1-6时囊内压升高;与N1组比较,N2,3组T1-6时囊内压升高;与N2组比较,N3组,T1-6时囊内压升高(P<0.05).与T0时比较,C组T2-6时囊内压降低,N1组T2-6时囊内压升高,N2,3组T1-6时囊内压升高(P<0.05).N1-3组囊内压与吸入时间呈正相关(相关系数分别为0.968、0.987、0.973,P<0.05).结论 吸入N2O可使喉罩囊内压升高,呈浓度及时间依赖性.     

高温预处理对过氧化氢诱导大鼠心肌细胞线粒体金属硫蛋白表达的影响

目的 探讨高温预处理对过氧化氢(H2O2)诱导大鼠心肌细胞线粒体金属硫蛋白(MT)表达的影响.方法 采用随机数字表法,将体外培养的H9C2大鼠心肌细胞分为3组(n=6):正常对照组(C组)心肌细胞加入含血清DMEM培养基,置于37℃5％CO2培养箱中3 h;H2O2组加入含0.5mmol/L H2O2的无血清DMEM培养基,置于37℃5％CO2培养箱中孵育3h;高温预处理组(HTP组)加入含血清DMEM培养基,置于42℃恒温水浴lh,行高温预处理,然后在37℃5％CO2细胞培养箱中孵育12 h,去除DMEM培养基,随后处理同H2O2组.采用流式细胞术测定心肌细胞凋亡率;观察心肌细胞线粒体超微结构;采用Western blot法测定线粒体MT的表达.结果 与C组比较,H2O2组和HTP组细胞凋率升高,线粒体MT表达上调(P＜0.01);与H2O2组比较,HTP组细胞凋率降低,线粒体MT表达上调(P＜0.01).HTP组心肌细胞线粒体损伤较H2 O2组减轻.结论 高温预处理减轻H2O2诱导大鼠心肌细胞损伤的机制可能与上调线粒体MT表达,增强心肌内源性保护机制有关.     

微小RNA-139-3p靶向调控性别决定区相关高迁移率族盒蛋白4减轻心肌细胞氧化应激损伤

目的研究微小RNA(miR)-139-3p对H2O2诱导的大鼠心肌细胞系H9c2细胞氧化应激的影响及其可能机制。方法分别将miR-139-3p模拟物(mimics)及其阴性对照(miR-NC)、性别决定区相关高迁移率族盒蛋白4(Sox4)过表达载体(pc-Sox4)及其对照(pcDNA3.1)转染H9c2细胞, 细胞共分为6组:对照组、H2O2组、mimic组(转染mimics)、miR-NC组(转染miR-NC)、pcDNA3.1组(转染mimics和pcDNA3.1)和pc-Sox4组(转染mimics和pc-Sox4), 除对照组外的其他细胞均在转染后建立H2O2诱导的H9c2细胞氧化应激损伤模型。细胞计数试剂盒(CCK-8)法检测H9c2细胞活力;比色法检测细胞培养上清中乳酸脱氢酶(LDH)的含量;原位缺口末端标记法(TUNEL)染色检测H9c2细胞凋亡;蛋白质印迹法(Western blot)检测Sox4表达;荧光定量聚合酶链反应(PCR)检测miR-139-3p表达水平;生物信息学网站预测miR-139-3p与Sox4的互补结合位点, 双荧光素酶报告基因实验验证两者之间的靶...     

毒蕈碱受体亚型介导逼尿肌细胞收缩与IP3关系的实验研究

目的　探讨信使分子IP3 在毒蕈碱受体亚型M3 R介导逼尿肌细胞收缩中的作用。　方法　MR非选择性激动剂 (carbachol)、拮抗剂 (atropine)及M2 R拮抗剂 (methoctramine)、M3 R拮抗剂 (4 DAMP)刺激原代培养人逼尿肌细胞 ,通过 [3 H]掺入法 ,检测磷脂酰肌醇 (PI)代谢产物 [3 H] IP含量。　结果　 [3 H] IP含量随carbachol刺激浓度增加而增加 ;10 -9、10 -8、10 -7、10 -6、10 -5、10 -4mmol/L的 4 DAMP抑制carbachol后 ,[3 H] IP含量分别为 392 6 .5 7± 2 73.2 9、2 780 .5 2± 2 11.0 9、2 4 36 .84± 15 3.6 2、1973.2 2± 16 4 .71、1372 .38± 14 1.35及 110 7.98± 92 0 .4 5cpm ,相同浓度的at ropine作用后 ,[3 H] IP含量分别为 36 0 2 .6 9± 2 80 .17、2 891.31± 2 0 7.4 5、1983.97± 14 5 .74、12 6 9.5 7± 10 5 .31、110 6 .37± 75 .2 3、92 7.5 0± 77.36cpm ;而相同浓度的methoctramine作用后 ,[3 H] IP含量分别为 4 4 6 2 .74± 36 0 .6 9、3938.6 1± 32 7.13、3315 .4 5± 2 70 .36、30 6 3.19± 2 4 6 .79、2 92 7.37± 2 2 6 .4 5及 2 836 .5 5± 2 4 1.6 3cpm ,两者之间差异有非常显著性意义 (P <0 .0 1) ,表明 4 DAMP和atropine能显著抑制carbachol诱导的代谢反应 ,而methoctram     

丙泊酚抑制过氧化氢诱导人红细胞衰亡

目的 观察过氧化氢(hydrogen peroxide,H2O2)在体外诱导人红细胞磷脂酰丝氨酸(phosphatidylserine,PS)外露及前向散射值(forward scatter,FSC)的变化,探讨丙泊酚对此的影响和机制.方法 健康成年人红细胞制成2％悬液,分为5组:对照组(C组)、H2O2组(H组)、丙泊酚10 μmol/L+H2O2组(P10+H组)、丙泊酚50 μmoL/L+ H2O2组(P50+H组)、丙泊酚100μmol/L+H2O2组(P100+H组).以离子霉素、维生素E和英脱利匹特为阳性和阴性对照.各组H2O2的反应浓度均为200μmol/L,孵育1h后用流式细胞仪检测红细胞PS标记率及FSC.所得数据用SPSS软件统计处理.结果 红细胞PS标记率H组比C组(15.20±1.01)、(1.45±0.21)(P＜0.001)明显增高,P50+H组(3.09±1.66)和P100+H组(1.68±0.28)均比H组(15.20±1.01)明显低(P＜0.001).FSCH组比C组小(1 768±9)、(1 808±26)(P=0.047),P50+H组比H组明显恢复(1 811±16)、(1 768±9)(P=0.037).结论 H2O2能诱导人红细胞衰亡,丙泊酚对其有明显抑制作用,其机制在于丙泊酚有较强的抗氧化及清除自由基功能.     

血红素加氧酶-1预处理对大鼠肺泡Ⅱ型上皮细胞氧化损伤的影响

目的 评价血红素加氧酶-1(HO-1)预处理对大鼠肺泡Ⅱ型上皮细胞氧化损伤的影响.方法 成年健康雄性SD大鼠,体重180～220 g,原代培养肺泡Ⅱ型上皮细胞,经鉴定后随机分为6组(n=8):对照组(C组),不给予任何药物,继续培养5 h;H2 O2组,加入0-5 mmol/L H2 O2,孵育3 h;不同浓度HO-1预处理组(H1-4组),分别加入0.01、0.10、1.00、10.00μmol/L HO-1孵育2 h,然后加入0.5 mmol/L H2 O2,孵育3 h.孵育结束后,于倒置相差显微镜下观察细胞形态,并进行肺泡Ⅱ型上皮细胞计数,测定细胞活力.结果 C组、H2～4组肺泡Ⅱ型上皮细胞大部分贴壁,呈圆形,胞质均匀,胞浆内含颗粒状物质;而H2 O2组和H1组肺泡Ⅱ型上皮细胞内可见反光增强的空泡,上清液中有较多的细胞碎片.与C组比较,H2O2组和H1组细胞计数和细胞活力降低(P＜0.05),H2～4组细胞计数和细胞活力差异无统计学意义(P＞0.05);与H2O2组和H1组比较,H2～4组细胞计数和细胞活力升高(P＜0.05);H2O2组和H1组间,H2～4组间细胞计数和细胞活力差异无统计学意义(P＞0.05).结论 0.10～10.00μmol/L HO-1预处理可减轻大鼠肺泡Ⅱ型上皮细胞氧化损伤.     

羧甲基壳聚糖抗氧化损伤诱导雪旺细胞凋亡的作用及机制研究

[目的]探讨羧甲基壳聚糖(carboxymethylated chitosan,CMCS)对氧化应激诱导雪旺细胞(schwann cells,SCs)凋亡的保护作用及作用机制。[方法]体外培养SCs,S-100免疫荧光染色鉴定。将SCs分为空白对照组、H2O2诱导组、H2O2加CMCS处理组。通过CCK-8检测不同组SCs增殖情况,流式细胞仪计数检测SCs早期凋亡率,检测各组细胞内超氧化物歧化酶(SOD)及丙二醛(MDA)的含量,Real-time PCR技术检测Bcl-2及Bax mRNA表达水平,Western blot法检测Bcl-2,Bax,Caspase-3、-9的表达水平。[结果]H2O2诱导SCs后其细胞增殖活性明显降低,加入不同浓度CMCS后增殖活性增加。流式细胞技术检测结果表明,经H2O2诱导SCs早期凋亡率增加,加入50²00μg/ml CMCS后凋亡率逐渐降低。经H2O2诱导组SCs与空白对照组比较SOD含量明显减少,MDA含量明显增加(P<0.05);加入CMCS后SOD含量增加,MDA含量减少(P<0.05)。Real-time PCR及Western blot检测结果表明经H2O2诱导SCs内Bcl-2表达降低,Bax,Caspase-3、-9表达增高,加入CMCS后Bcl-2表达恢复,Bax,Caspase-3、9表达抑制。[结论]CMCS对H2O2诱导SCs凋亡具有保护作用。     

Induction of apoptosis in malignant pleural mesothelioma cells by activation of the Fas (Apo-1/CD95) death-signal pathway

OBJECTIVE: Although well characterized in several solid tumors, the effects of Fas/Fas ligand interactions in malignant pleural mesothelioma cells have not been defined. The present study was undertaken to examine the functional status of the Fas/Fas ligand pathway in malignant pleural mesothelioma cells and to determine the feasibility of targeting this death-signal pathway for molecular intervention in patients with mesotheliomas. METHODS: Fas expression in primary normal human bronchial epithelial cells and 6 malignant pleural mesothelioma cell lines was quantified by means of flow cytometry. The caspase components of the Fas-mediated apoptotic pathway were evaluated by means of Western blot techniques. Soluble Fas ligand-mediated cytotoxicity and apoptosis were evaluated by means of MTS and TUNEL assays, respectively. Cisplatin (3 microg/mL) and lymphokine-activated killer cells were used to enhance mesothelioma sensitivity to soluble Fas ligand. An H2373 nude mouse xenograft model of malignant pleural mesothelioma was established to assess the in vivo effects of soluble Fas ligand. RESULTS: Four of 6 malignant pleural mesothelioma lines exhibited high levels of Fas expression, and 2 of 4 were inherently susceptible to soluble Fas ligand-mediated cytotoxicity (soluble Fas ligand 50% inhibitory concentration, < 15 ng/mL). Two soluble Fas ligand refractory cell lines (H2052 and H513) exhibited high levels of Fas receptor. Pretreatment with cisplatin resulted in a reduction of 50% inhibitory concentration from infinity to 4.17 +/- 0.14 ng/mL and 10.23 +/- 1.58 ng/mL, respectively. Two additional soluble Fas ligand refractory cell lines (H2595 and REN) expressed low levels of Fas. Exposure of these cells to lymphokine-activated killer cells or lymphokine-activated killer cell-conditioned medium followed by a 24-hour treatment with cisplatin resulted in a significant reduction in 50% inhibitory concentration of soluble Fas ligand and pronounced induction of apoptosis. Intraperitoneally administered soluble Fas ligand mediated regression of H2373 xenografts. CONCLUSION: The Fas/Fas ligand pathway in mesothelioma cells is either intrinsically intact or can be rendered functional with chemotherapeutic agents or immune effector cells. These preclinical data support further evaluation of strategies to enhance Fas-mediated apoptosis in mesotheliomas.     

Differential involvement of the Fas receptor/ligand system in p53-dependent apoptosis in human prostate cancer cells

BACKGROUND: The objective of this study was to characterize the involvement of the Fas receptor/ligand system in p53-dependent apoptosis in human prostate cancer cells. METHODS: The effects of adenovirus-mediated p53 gene transfer (Ad5CMV-p53) into human prostate cancer LNCaP, DU145, and PC3 cells on their growth, apoptosis and Fas receptor/ligand expression were examined by the MTT assay, DNA fragmentation assay, and Northern blot analysis, respectively. The sensitivity of these cells to an agonistic anti-Fas receptor antibody (CH11) and the effects of an antagonistic anti-Fas ligand antibody (4H9) on Ad5CMV-p53-induced apoptosis were analyzed by the MTT assay and DNA fragmentation assay. RESULTS: Ad5CMV-p53 treatment resulted in substantial growth inhibition, induction of apoptosis and up-regulation of Fas receptor as well as Fas ligand mRNA expression in LNCaP, DU145 and PC3 cells. Despite the abundant expression of Fas receptor in all of these cells, CH11 induced apoptosis only in PC3 cells. Furthermore, 4H9 partially blocked the apoptosis induced by Ad5CMV-p53 in PC3 cells, but not in LNCaP and DU145 cells. CONCLUSIONS: The Fas receptor/ligand system is differentially involved in p53-dependent apoptosis in prostate cancer cells; therefore, reintroduction of wild-type p53 into prostate cancer cells may induce apoptosis through Fas receptor/ligand interaction as well as through an alternative pathway.     

Three-dimensional comparison of peripheral benzodiazepine binding and histological findings in rat brain tumor

Experiments were undertaken to determine the in vivo utility of the mixed benzodiazepine ligand [3H]flunitrazepam and the selective peripheral benzodiazepine ligand [3H]PK 11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide] to outline the borders of rat C6 glial tumors in three dimensions. Intravenous injection of [3H]flunitrazepam resulted in a tumor/cortex ratio of radioactive densities between 2.7 and 1.5 within the first 60 minutes after injection. [3H]PK 11195 demonstrated a higher tumor/cortex ratio (5.3) than [3H]flunitrazepam. For three-dimensional studies, images were generated from thionin-stained histological sections and autoradiograms. The mixed type benzodiazepine ligand [3H]flunitrazepam was superior in showing some of the normal anatomical structures surrounding the tumor, whereas [3H]PK 11195, a specific peripheral ligand, demonstrated higher tumor/brain contrast and superior topographical correlation between histological and autoradiographic images. Implications of peripheral benzodiazepine receptor ligands for positron emission tomography are discussed.     

The use of radioactive 7α, 17α-dimethyl-19-nortestosterone (mibolerone) in the assay of androgen receptors

Tritiated 7α, 17α-dimethyl-19-nortestosterone (DMNT; mibolerone), a synthetic androgen stable to metabolic conversion in the rat ventral prostate, is an excellent radioactive ligand for the quantitation and characterization of androgen receptors in prostate, liver, and cultured cells. DMNT is more receptor-selective than 17α-methyl-17β-hydroxy-estra-4,9,11-trien-3-one (R1881); DMNT interacts with glucocorticoid and progestin receptors much less strongly than R1881. Unlike 5α-dihydrotestosterone, DMNT does not bind tightly to testosterone-estradiol binding globulin of human serum. The hydroxylapatite-filter assay we employed can clearly distinguish between DMNT binding to androgen receptors of rat ventral prostate and interaction of DMNT with androgen binding protein of epididymides. The prostate cytosol (³H)DMNT-receptor complex sediments in two forms (4 and 8 S) in a low salt medium. In 0.4 M KCl, both the prostate cytosol and nuclear (³H)DMNT-receptor complexes migrated as 3–4 S components. The formation of both the cytosol and nuclear DMNT-receptor complexes is inhibited by antiandrogens and 17β-estradiol.     

Effects of nitrous oxide and halothane on mu and kappa opioid receptors in guinea-pig brain

The effects of two general anesthetics, nitrous oxide and halothane, and oxygen on mu and kappa opioid receptor subtypes from guinea-pig brain were investigated. mu receptor binding was defined using [3H]dihydromorphine as the ligand. Nitrous oxide (100%) and halothane (2%) decreased the [3H]dihydromorphine binding affinity (Kdair = 0.87 nM, KdN2O = 1.45 nM, Kdhalothane = 2.30 nM) without affecting the density of binding sites. A decrease in the [3H]dihydromorphine binding affinity without influence on the density of binding sites was also observed in the presence of 100% oxygen (KdO2 = 1.40 nM). kappa receptor binding was defined using [3H](-)ethylketocyclazocine as the ligand, in the presence of 100 nM D-ala2-D-leu5-enkephalin and 30 nM morphine. While 100% nitrous oxide caused a slight decrease in [3H](-)ethylketocyclazocine binding affinity (Kdair = 0.24 nM, KdN2O = 0.31 nM) and a substantial decrease in the density of binding sites (Bmaxair = 115 fmol/mg protein, BmaxN2O = 84 fmol/mg protein), halothane dramatically affected both the affinity (Kdhalothane = 0.70 nM) and density (Bmaxhalothane = 38 fmol/mg protein). Oxygen (100%) reduced [3H]dihydromorphine binding affinity. Differential effects of two anesthetics on the same receptor or distinct actions of the same anesthetic on different receptors could indicate the presence of specific targets for anesthetics at the membrane level. Conversely, effects of volatile anesthetics on opioid receptors could reflect a non-specific perturbation of the lipidic and proteinaceous components of the membranes.     

Autoradiographic localization of vasopressin and oxytocin binding sites in rat kidney

The presence of vasopressin receptors of the V1 (vascular) type and of oxytocin receptors in the rat kidney was investigated using an autoradiographical approach. Rat kidney sections were incubated with tritiated vasopressin ([3H]vasopressin, 1.5 nM) or oxytocin ([3H]oxytocin, 3 nM). The ligand selectivity of the [3H]vasopressin binding sites detected was deduced from competition experiments using one selective unlabeled ligand for V2 (antidiuretic) vasopressin receptors (1-deamino-[8-D-arginine]-vasopressin, dDAVP) and one selective unlabeled ligand for V1 receptors (des-glycineamide-[1-(beta-mercapto-beta,beta-cyclopentamethylene propionic acid]-arginine vasopressin, des(Gly(NH2)9d(CH2)5-AVP). Specific and dense [3H]vasopressin labeling was observable in the medullopapillary and cortical portions of the kidney. Specific [3H]vasopressin binding in the cortex was insensitive to the V1-selective ligand, des(Gly(NH2)9d(CH2)5-AVP, but was inhibited by dDAVP. Glomerular structures identified as such by microscopical observation of the kidney sections were specifically labeled with [3H]oxytocin and [125I]-SAR1-angiotensin II but not with [3H]vasopressin. It is concluded that V1 receptors which have been evidenced on mesangial cells in culture are not expressed in a detectable quantity on mesangial cells in situ. The specific [3H]oxytocin binding to glomeruli might reflect the presence on glomerular structures of oxytocin receptors involved in the effects of the hormone on renal hemodynamics, and possibly in some of the effects ascribed to vasopressin.     

Acute ethanol treatment modulates delta opioid receptors in N18TG2 cells

BACKGROUND: The in vitro adaptive responses of delta opiate receptors (DOR) to chronic ethanol treatment have been well documented. The acute effects of ethanol on these receptors are not well characterized beyond its effect on ligand binding. The aim of this study was to evaluate the acute effects of clinically relevant concentrations of ethanol (50-200 mm) on the saturation binding kinetics, receptor/ligand internalization, and agonist stimulation of G-protein coupling in N18TG2 cells expressing the Flag epitope-tagged mouse DOR. METHODS: Confocal microscopy was used to localize Flag epitope-tagged DOR in N18TG2 cells. Saturation binding assays at 4 degrees C and 37 degrees C were conducted in the absence or presence of ethanol on cells not pretreated or pretreated with ethanol for 30 min at 37 degrees C. Highly specific delta agonist, DPDPE ([D-Pen2,D-Pen5]enkephalin), was used in these studies. The effect of ethanol on agonist stimulation of G-protein coupling was examined using [35S]GTPgammaS (guanosine-5'-O-(3-thio)triphosphate) binding to membranes. Agonist-mediated receptor internalization was examined using flow cytometry of cells labeled with the antiserum directed against the Flag epitope, and the ligand internalization was examined using [3H]DPDPE. RESULTS: Ethanol decreased the binding of the agonist [3H]DPDPE, and not the antagonist [3H]diprenorphine, in a dose-dependent manner. These effects were temperature-dependent. Ethanol reversibly inhibited agonist stimulation of [35S]GTPgammaS binding. In non-pretreated cells, ethanol decreased the rate of receptor/ligand internalization, but this effect was not seen in ethanol pretreated cells. Taken together, these results suggest that pretreatment of N18TG2 cells with ethanol induces compensatory mechanisms that allow the receptor to function efficiently in its presence. CONCLUSION: Acute ethanol decreased the binding, agonist-mediated functional coupling and receptor/ligand internalization in N18TG2 cells expressing epitope-tagged DOR. In these cells, 30-min pretreatment with ethanol was sufficient to reverse these effects.     

Acute Ethanol Treatment Modulates δ Opioid Receptors in N18TG2 Cells

Background: The in vitro adaptive responses of [delta] opiate receptors (DOR) to chronic ethanol treatment have been well documented. The acute effects of ethanol on these receptors are not well characterized beyond its effect on ligand binding. The aim of this study was to evaluate the acute effects of clinically relevant concentrations of ethanol (50-200 mm) on the saturation binding kinetics, receptor/ligand internalization, and agonist stimulation of G-protein coupling in N18TG2 cells expressing the Flag epitope-tagged mouse DOR.

Methods: Confocal microscopy was used to localize Flag epitope-tagged DOR in N18TG2 cells. Saturation binding assays at 4[degrees]C and 37[degrees]C were conducted in the absence or presence of ethanol on cells not pretreated or pretreated with ethanol for 30 min at 37[degrees]C. Highly specific [delta] agonist, DPDPE ([D-Pen2,D-Pen5]enkephalin), was used in these studies. The effect of ethanol on agonist stimulation of G-protein coupling was examined using [35S]GTP[gamma]S (guanosine-5'-O-(3-thio)triphosphate) binding to membranes. Agonist-mediated receptor internalization was examined using flow cytometry of cells labeled with the antiserum directed against the Flag epitope, and the ligand internalization was examined using [3H]DPDPE.

Results: Ethanol decreased the binding of the agonist [3H]DPDPE, and not the antagonist [3H]diprenorphine, in a dose-dependent manner. These effects were temperature-dependent. Ethanol reversibly inhibited agonist stimulation of [35S]GTP[gamma]S binding. In non-pretreated cells, ethanol decreased the rate of receptor/ligand internalization, but this effect was not seen in ethanol pretreated cells. Taken together, these results suggest that pretreatment of N18TG2 cells with ethanol induces compensatory mechanisms that allow the receptor to function efficiently in its presence.     

Expression of a functional asialoglycoprotein receptor in human renal proximal tubular epithelial cells

BACKGROUND: The asialoglycoprotein receptor (ASGPR) is a C lectin which binds and endocytoses serum glycoproteins. In humans, the ASGPR is shown mainly to occur in hepatocytes, but does occur extrahepatically in thyroid, in small and large intestines, and in the testis. In the kidney, there has been evidence both for and against its existence in mesangial cells. METHODS: Standard light microscopy examination of renal tissue stained with an antibody against the ASGPR was performed. The mRNA expression for the ASGPR H1 and H2 subunits in primary human renal proximal tubular epithelial cells (RPTEC), in the human proximal tubular epithelial cell line HK2, and in human renal cortex was investigated using reverse-transcribed nested polymerase chain reaction. ASGPR protein expression as well as ligand binding and uptake were also examined using confocal microscopy and flow cytometry (fluorescence-activated cell sorting). RESULTS: Light microscopy of paraffin renal biopsy sections stained with a polyclonal antibody against the ASGPR showed proximal tubular epithelial cell staining of the cytoplasm and particularly in the basolateral region. Renal cortex and RPTEC specifically have mRNA for both H1 and H2 subunits of the ASGPR, but HK2 only expresses mRNA for H1. Using a monoclonal antibody, the presence of the ASGPR in RPTEC was shown by fluorescence-activated cell sorting and immunofluorescent staining. Specific binding and uptake of fluorescein isothiocyanate labelled asialofetuin which is a specific ASGPR ligand was also demonstrated in RPTEC. CONCLUSIONS: Primary renal proximal tubular epithelial cells have a functional ASGPR, consisting of the H1 and H2 subunits, that is capable of specific ligand binding and uptake.     

Neither Fas ligand nor endotoxin is responsible for inducible peritoneal phagocyte apoptosis during sepsis/peritonitis

Regulation of the phagocyte apoptotic response appears to play a significant role in the pathophysiology of sepsis. In this regard, prior studies have shown that the onset of phagocyte apoptosis, as well as those agents that regulate it at the nidus of infection, differ significantly from those seen in circulation. The aim of this study therefore was to determine if the increase in inducible phagocyte apoptosis and caspase activities seen in the peritoneum during sepsis is due to endotoxin or Fas ligand. To study this, male C3H/HeN (endotoxin-sensitive), C3H/HeJ (endotoxin-tolerant), and C3H/HeJ-FasL(gld) (endotoxin-tolerant/FasL-deficient) mice were subjected to cecal ligation and puncture or sham operation. Twenty-four hours later, phagocytes were collected and cultured with lipopolysaccharide (LPS), then harvested for apoptosis (propidium iodide cell cycle or cell death ELISA analysis), cytokine release (ELISA), and caspase activity (fluorogenic assay) determination. The data indicate that there was a marked increase in apoptosis in LPS-stimulated phagocytes which was associated with a significant increase in caspase 3, 8, and 9 activities but a decrease in caspase 1 activity from C3H/HeN and C3H/HeJ-FasL(gld) septic mice and an increase in caspase 3 and 8 activities in phagocytes from C3H/HeJ septic mice. Furthermore, cells from septic mice, including all three strains, lost their ability to produce IL-1beta and IL-6 in response to LPS stimulation. The inability to completely suppress these changes suggests that neither endotoxin (via signaling through TLR-4 pathway) nor Fas ligand regulates the peritoneal phagocyte apoptotic responses seen during the late phase of polymicrobial sepsis/peritonitis.