大鼠心肌缺血再灌注损伤对心肌细胞膜钠泵表达的影响

目的：观察心肌缺血再灌注（MIR）损伤大鼠心肌组织内洋地黄素水平、ATP酶活性、线粒体Ca^2＋浓度以及Na^＋-K^＋-ATP酶各亚基基因表达的改变，并观察钙通道阻滞剂维拉帕米对其影响，探讨内洋地黄素在MIR损伤细胞内钙超载中的可能作用及其机制。方法：24只雄性SD大鼠随机分成3组，每组8只。假手术组：丝线穿过左冠状动脉前降支，但不结扎：缺血再灌注组（MIR组）：结扎左冠状动脉前降支30min，再灌注45min；维拉帕米组：MIR模型＋5mg／kg维拉帕米，维拉帕米于再灌注前5min经股静脉注射。取缺血区左室心肌检测心肌匀浆内洋地黄素水平、心肌细胞膜Na^＋-K^＋-ATP酶和Ca^2＋-Mg^2＋-ATP酶活性、线粒体Ca^2＋浓度；免疫组化方法检测心肌Na^＋-K^＋-ATP酶α1、α2、α3和β1亚基蛋白水平表达的改变。结果：MIR损伤时，心肌组织内洋地黄素水平明显升高，心肌细胞膜Na^＋-K^＋-ATP酶和Ca^2＋-Mg^2＋-ATP酶活性显著下降，线粒体Ca^2＋浓度升高，Na^＋-K^＋-ATP酶α1、α2、α3和β1亚基蛋白水平表达均明显下降；维拉帕米除具有降低线粒体Ca^2＋浓度外，对缺血再灌注引起的其它各项异常指标无明显改善作用。结论：MIR促进机体内洋地黄素分泌增加，后者可能通过影响心肌细胞膜上的Na^＋-K^＋-ATP酶α1、α2、α3和β1亚基基因表达，抑制Na^＋-K^＋-ATP酶活性，导致线粒体内Ca^2＋超载。     

自体血液回收技术对红细胞膜ATP酶的影响

目的 探讨血液回收技术对红细胞膜Na^＋-K^＋-ATP酶和Ca^2＋-Mg^2＋-ATP酶活性的影响。方法 将24例患者麻醉前静脉血、术野回收原血及回收红细胞血液标本采用沈茂星法测定红细胞膜Na^＋-K^＋-ATP酶和Ca^2＋-Mg^2＋-ATP酶活性,并予统计分析。结果 经血液回收技术处理后的红细胞膜Na^＋-K^＋-ATP酶和Ca^2＋-Mg^2＋-ATP酶活性与术野回收血值比较均有显著降低（P〈0．05）。结论 自体血液回收技术能使红细胞膜Na^＋-K^＋-ATP酶和Ca^2＋-Mg^2＋-ATP酶活性明显降低。     

辛伐他汀对大鼠骨骼肌线粒体膜流动性及ATP酶活性的影响

目的：观察辛伐他汀（Sim）对大鼠骨骼肌线粒体膜流动性及ATP酶活性的影响.方法：大鼠口服给予3种不同剂量Sim,酶法测定丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）及肌酸激酶（CK）活性.以1-苯氨基萘-8-磺酸（ANS）、1,6-二苯基-1,3,5-己三烯（DPH）为荧光探针标记骨骼肌线粒体膜浅层及深层,测定骨骼肌线粒体膜荧光强度（F）,荧光偏振度（P）及平均微黏度（^-η）.比色法测定Na^＋-K^＋-ATP酶和Ca^2＋-Mg^2＋-ATP酶活性.结果：给予Sim 3.6,7.2 mg·kg^-1·d^-1组,血清ALT、AST、CK和骨骼肌线粒体膜Na^＋-K^＋-ATP酶、Ca^2＋-Mg^2＋-ATP酶的活性均无显著变化.而给予Sim 14.4mg·kg^-1·d^-1,血清CK活性显著增高,骨骼肌线粒体膜Na^＋-K^＋-ATP酶、Ca^2＋-Mg^2＋-ATP酶活性则明显降低.给予Sim3.6,7.2,14.4 mg·kg-1·d-1组,ANS标记的线粒体膜浅层和DPH标记的线粒体膜深层F、P、^-η均明显降低,表明Sim可增加线粒体膜浅层及深层流动性.结论：Sim在高剂量时可导致骨骼肌细胞损伤,其机制可能与Sim能增加骨骼肌线粒体膜流动性,降低骨骼肌线粒体膜Ca^2＋-Mg^2＋-ATP酶及Na^＋-K^＋-ATP酶活性有关.     

氯丙嗪和尼莫地平对锰致大鼠神经毒性的影响

目的观察氯丙嗪（CPZ）和尼莫地平（NIMO）对锰致大鼠脑纹状体谷氨酰胺合成酶（GS）、磷酸活化的谷氨酰胺酶（PAG）、脑皮质Na^＋-K^＋-ATP酶、Ca^2＋-ATP酶活力和脑丙二醛（MDA）含量的影响。方法32只大鼠按体重随机分成4组,第1组大鼠为对照组,腹腔注射0.9％的氯化钠;第2组为染锰组,腹腔注射0.9％的氯化钠;第3和4组为预处理干预组,分别腹腔注射14.1μmol·kg^-1CPZ和2.4mmol·kg^-1NIMO。腹腔注射后2h,第1组大鼠皮下注射0.9％的氯化钠,第2-4组皮下注射200μmol·kg^-1的氯化锰。给大鼠连续注射4周,每周5d。最后一次染毒后24h,将大鼠处死,断头取出脑纹状体和大脑皮质,测定脑纹状体GS、PAG和脑皮质Na^＋-K^＋-ATP酶、Ca^2＋-ATP酶活力、MDA含量。结果与对照组比较,单纯染锰组大鼠脑纹状体GS活力明显降低,PAG活力明显升高;脑皮质Na^＋-K^＋-ATP酶、Ca^2＋-ATP酶的活力明显降低,MDA含量增加。与单纯染锰组比较,CPZ处理组大鼠脑纹状体GS活力升高;脑皮质Na^＋-K^＋-ATP酶和Ca^2＋-ATP酶活力上升,MDA含量下降;NIMO预处理组大鼠脑纹状体GS的活力升高,PAG的活力降低。脑皮质Na^＋-K^＋-ATP酶和Ca^2＋-ATP酶活力上升。结论锰可致大鼠脑Gs、Na^＋-K^＋-ATPase酶和Ca^2＋-ATP酶活力降低,PAG活力升高,MDA含量增加。CPZ和NIMO对锰所致神经毒性有一定的拮抗作用。     

异丙酚对缺血再灌注损伤大鼠海马线粒体能量代谢、ATP酶活性及自由基系统的影响

目的：观察异丙酚对缺血再灌注损伤大鼠海马线粒体能量代谢、ATP酶活性、脂质过氧化及超微结构的影响。方法：雄性Wistar大鼠30只，随机分为假手术组、缺血再灌注对照组和缺血再灌注异丙酚处理组。采用大鼠全脑缺血再灌注损伤模型。全脑缺血10min再灌注60min时，断头处死大鼠。检测海马线粒体三磷酸腺苷(ATP)、丙二醛(MDA)含量及Na^ -K^ -ATP酶、Ca^2 -ATP酶、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)活性并观察线粒体超微结构的改变。结果：缺血再灌注后海马线粒体的ATP含量及Na^ -K^ -ATP酶、Ca^2 -ATP酶、SOD、GSH活性均有不同程度的降低，MDA含量增高，线粒体超微结构亦发生明显损害；麻醉相关剂量的异丙酚可使ATP含量、Na^ -K^ -ATP酶、Ca^2 -ATP酶、SOD、GSH活性有不同程度的恢复，并降低MDA的含量，减轻线粒体损伤的程度。结论：异丙酚对脑缺血再灌注损伤的保护作用可能与其抑制线粒体脂质过氧化反应，清除自由基，保持线粒体结构的完整性，促进线粒体ATP含量和ATP酶活性的恢复有关。     

肥胖儿童高血压发病因素有机理的研究

目的：探讨儿童肥胖引起高血压的机制。方法：测定104例12－16岁儿童的血脂、胰岛素水平,Na^ -K^ -ATP酶和Ca^2 -ATP酶活性。结果：在正常血压肥胖组（ONT）和高血压肥胖组（OHT）,空腹胰岛素（Ins）显著升高,面NNa^ -K^ -ATP酶和Ca^2 -ATP酶活性显著下降,三者在ONT和OHT组之间也有显著性差异,在OHT组Ins升高,两酶活性下降,Pearson相关和多元回归分析显示,在OHT组,Ins和Ca^2 -ATP酶与收缩压（SBP）显著相关,Na^ -K^ -ATP酶与舒张压9DBP）显著相关。结论：高胰岛素血症、Na^ -K^ -ATP酶和Ca^2 -ATP酶活性降低可能在肥胖儿童高血压的发病机制中起重要作用。     

灯盏花素对缺血再灌注沙土鼠海马ATP含量变化和兴奋性氨基酸释放的影响

目的 研究灯盏花素对缺血再灌注沙土鼠海马ATP含量、ATP酶活性和兴奋性氨基酸释放的影响。方法 脑缺血10min建立沙土鼠缺血再灌注模型。90只沙土鼠分为假手术组、脑缺血组和灯盏花素组。测定脑缺血后和再灌注1、12和24h海马ATP含量、ATP酶活性和兴奋性氨基酸含量。结果 脑缺血组,ATP、Na^＋-K^＋ATP酶和Ca^2＋-ATP酶含量明显下降（P〈0．01）,谷氨酸（Glu）、天冬氨酸（Asp）、γ-氨基丁酸（GABA）含量明显增加（P〈0．01）,谷氨酰胺（Gln）含量在缺血时降低,再灌注后增加（P〈0．01）。灯盏花素组,ATP、Na^＋-K^＋-ATP酶和Ca^2＋-ATP酶含量高于脑缺血组（P〈0．01）。Glu、Asp、GABA含量明显低于脑缺血组（P〈0．01）,Gln含量高于脑缺血组（P〈0．01）。结论 灯盏花素明显增加脑缺血及再灌注后ATP含量和ATP酶活性,降低兴奋性氨基酸释放,可减轻脑缺血再灌注损伤。     

缺血预处理减轻心肌缺血再灌注诱导的Na+-K+-ATP酶异常表达

目的：探讨Na^＋ -K^＋ -ATP酶在心肌缺血预处理保护机制中的作用。方法：以前降支结扎30min后复灌60min制备心肌缺血再灌注模型，连续3次缺血5min再灌注10min制备缺血预处理模型。以毛花甙丙抑制Na^＋ -K^＋ -ATP酶功能，观察心脏收缩和舒张功能、心肌Na^＋ -K^＋ -ATP酶功能，免疫组化法检测Na^＋ -K^＋ -ATPα1、α2、α3及β1亚基的蛋白表达。结果：心肌缺血再灌注显著抑制心脏舒缩功能、Na^＋ -K^＋ -ATP酶活性以及α1、α2、α3及β1亚基的蛋白原位表达；缺血预处理可减轻心肌缺血再灌注诱导的Na^＋ -K^＋ -ATP酶异常，保护心脏功能；毛花甙丙可维持心脏舒缩功能，但显著抑制Na^＋ -K^＋ -ATP酶和蛋白表达。结论：缺血预处理能减轻心肌缺血再灌注诱导的Na^＋ -K^＋ -ATP酶异常，抑制Na^＋ -K^＋ -ATP酶功能导致缺血预处理保护作用丧失，维持Na^＋ -K^＋ -ATP酶功能是缺血预处理心肌保护重要机制之一。     

地奥心血康对大鼠心肌缺血再灌注损伤的干预作用

目的 观察地奥心血康（DK）对大鼠心肌缺血再灌注损伤的影响。方法 48只Wistar大白鼠随机分为正常对照组、模型对照组与DK组。前2组每日灌胃0.5％CMC 10mL/kg，DK组每日灌胃DK 70mg／kg，共10天。末次给药后24h结扎大鼠冠状动脉左前降支30min，再灌注90min复制大鼠心肌缺血再灌注损伤模型，观察心律失常评分、心肌梗死面积、心功能、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）、心肌Na^＋-K^＋-ATP酶和Ca^2＋-ATP酶活性及丙二醛（MDA）含量等的变化。结果 与模型对照组相比，DK组心律失常评分明显降低，心肌梗死面积明显缩小，心功能明显改善，SOD、CAT及GSH-Px、Na^＋-K^＋-ATP酶、Ca^2＋-ATP酶活性显著升高、MDA含量显著降低。结论 DK对大鼠心肌缺血再灌注损伤有保护作用。其机制可能与清除自由基，抑制脂质过氧化有关。     

Ⅱ型糖尿病肾病和红细胞膜ATP酶活性的关系

目的 探讨Ⅱ型糖尿病患红细胞膜Na^K^ -ATP酶和Ca^2 Mg^2 -ATP酶活性改变参与糖尿病肾病的可能机制。方法 按Reinila制膜法测定了25例无糖尿病肾病和57例糖尿病肾病患红细胞膜Na^ K^ -ATP酶和Ca^2 Mg^2 -ATP酶含量,并与35名正常人作对照。结果 糖尿病无肾病组和肾病组红细胞膜Na^ -K^ -ATP酶和Ca^2 Mg^2 -ATP酶活性均显地低于正常人组（P＜0．01）,糖尿病肾病组与无肾病组比较差异亦有显性（P＜0．05）。结论 Ⅱ型糖尿病肾病的发生和发展与红细胞膜Na^2 K^ -ATP酶和Ca^2 Mg^2 -ATP酶活性有密切的关系。     

Effect of diphenolic laxatives on Na+-K+-activated ATPase and cyclic nucleotide content of rat colon mucosa in vivo

1. The effect of bisacodyl and oxyphenisation on the Na+-K+- and Mg2+-activated ATPase and on the mucosa levels of cAMP and cGMP was investigated in transporting ligated loops of the rat colon in acute studies and in chronic feeding experiments 2. The specific activity of the Na+-K+-ATPase was lowered in both types of experiments, concomitantly with a reduction in net sodium absorption. The specific activity of the Mg2+-activated ATPase was unaffected. 3. The cAMP content per mg protein was elevated and the cGMP content decreased in the acute experiments in which the effect on transport was most marked. The content of cyclic nucleotides returned to normal within 2 h whereas absorption, Na+-K+-ATPase specific activity and the mucosal potential difference were still significantly depressed at that time. In chronic experiments with bisacodyl, cAMP was not affected and cGMP was increased in colon loops exhibiting reduced absorption. 4. The results indicate that the inhibition of the Na+-K+-activated ATPase by diphenolic laxatives may play a role in the inhibition of intestinal fluid absorption caused by these compounds. The increase of cAMP in acute experiments could point to a cAMP-mediated stimulation of secretory processes under this condition.     

青龙衣多糖对H22型肿瘤细胞的ATPase活性影响的研究

目的 研究青龙衣多糖对H22型肿瘤细胞的ATPase活性的影响。方法 按照测定无机磷含量的方法对H22肿瘤细胞的Ca^2＋-ATPase、Na^＋K^＋-ATPase和Mg^2＋-ATPase活性进行了测定。结果 发现青龙衣多糖可以降低ATPase的活性，除青龙衣多糖中、高剂量组显著抑制Ca^2＋-ATPase活性。高、中、低剂量组都显著降低Na^＋-K^＋-ATPase和Mg^2＋-ATPase活性（P〈0．05）。结论 ATPase活性降低导致了膜电位下降。细胞容积降低．可能引起细胞凋亡发生．Ca^2＋．调节凋亡发生．Mg^2＋调控ATPase的活性,可能是青龙衣多糖抗肿瘤的机制之一。     

葡萄糖酸镁对大鼠心肌缺血再灌注损伤的保护作用

目的:观察葡萄糖酸镁对心肌缺血再灌注损伤的影响,探讨葡萄糖酸镁心肌保护作用及其机理。方法:采用改进了的整体大鼠急性心肌缺血再灌注损伤模型,检测组织内Na+- K+- ATPase活力及丙二醛(MDA)和Ca2 +含量,并测定由ADP诱导的血小板5min最大聚集率AGG (M )。结果:与假手术组相比,缺血再灌注组心肌组织内Na+-K+ -ATPase活力显著下降(P <0 .0 1) ,MDA和Ca2 +含量明显升高(P<0 .0 1) ,AGG(M)升高(P <0 .0 5 )。与缺血再灌注组比较,葡萄糖酸镁3个不同剂量保护组心肌组织内Na+ -K+ -ATPase活力明显升高(P <0 .0 1) ,MDA和Ca2 +含量明显降低(P <0 .0 5或P <0 .0 1) ,AGG(M)也明显降低(P <0 .0 1) ,且各作用均随用药剂量的增加而更加显著。结论:葡萄糖酸镁对心肌缺血再灌注损伤有保护作用,其保护作用机制与增加Na+ -K+- ATPase活性、减轻钙超载和抑制脂质过氧化反应以及血小板聚集有关。     

黄芩苷对大鼠心肌缺血再灌注损伤的保护作用(英文)

目的：研究中药有效成分黄芩苷(baicalin)对大鼠心肌缺血再灌注损伤的作用及其机制。方法：雄性Wistar大鼠随机分为4组,假手术组、对照组和黄芩苷50,100 mg·kg~(-1)组。结扎大鼠左冠状动脉前降支30 min后松开结扎线120 min复制心肌缺血-再灌注损伤模型,以多道生理记录仪持续记录左室压力变化速率(±dp/ dt_(max))和左室舒张末期压(LVEDP)的变化。检测心肌丙二醛(MDA)含量、超氧化物歧化酶(SOD)、Na~+-K~+-ATP酶和Ca~(2+)-ATP酶活性以及血清乳酸脱氢酶(LDH)和磷酸肌酸激酶(CK)含量,以透射电镜观察心肌超微结构的改变。结果：与假手术组比较,对照组大鼠左室±dp/dt_(max)明显降低(P＜0.01),而LVEDP明显升高(P＜0.01)。静脉注射黄芩苷50,100 mg·kg~(-1)可使降低的±dp/dt_(max)明显升高(P＜0.05,P＜0.01),升高的LVEDP显著降低(P＜0.05,P＜0.01)。黄芩苷组血清CK和LDH含量分别是(72±19)kU·L~(-1),(64±15)kU·L~(-1)和(1 365±209)U·L~(-1),(1 124±169)U·L~(-1),均明显低于对照组[(90±23)U·L~(-1)和(1 826±123)U·L~(-1),P＜0.01]。对照组大鼠心肌SOD,Na~+-K~+-ATP酶和Ca~(2+)-ATP酶活性均明显低于假手术组,而降低的酶活性可被预先给予黄芩苷所升高(P＜0.05,P＜0.01)。此外,黄芩苷还可降低缺血心肌MDA含量,改善心肌超微结构。结论：黄芩苷通过清除氧自由基,抗脂质过氧化,改善心肌ATP酶活性而保护心肌缺血再灌注损伤。     

Effect of cinnamon, clove and some of their constituents on the Na(+)-K(+)-ATPase activity and alanine absorption in the rat jejunum.

The effect of a water extract of some spices on the in vitro activity of the rat jejunal Na(+)-K(+)-ATPase was investigated. Extracts of nutmeg, cinnamon, clove, cumin, coriander, turmeric and caraway all inhibited the ATPase, while anise seed and white pepper exerted no significant effects. The extracts of clove and cinnamon had the most potent inhibitory effect on the intestinal ATPase as compared to extracts of other spices. They also inhibited the in vitro Na(+)-K(+)-ATPase activity in a crude kidney homogenate and the activity of an isolated dog kidney Na(+)-K(+)-ATPase. The alcoholic extract of cinnamon, compared to the aqueous extract, had a stronger inhibitory action on the jejunal enzyme and a lower IC(50) value, which was not significantly different from the one observed with cinnamaldehyde, the major volatile oil present cinnamon, suggesting that in alcoholic extracts cinnamaldehyde is the major inhibitory component. The IC(50) values of eugenol, aqueous clove extract and ethanolic clove extract all fell within the same range and were not significantly different from each other, suggesting that eugenol is the major inhibitory component in both alcoholic and aqueous extracts. Based on the IC(50) values, the order of sensitivity of the enzyme to the spices extracts is as follows: isolated dog kidney ATPase>rat kidney ATPase>rat intestine ATPase. The aqueous extracts of clove and cinnamon also significantly lowered the absorption of alanine from the rat intestine. It was concluded that the active principle(s) in clove and cinnamon can permeate the membrane of the enterocytes and inhibit the Na(+)-K(+)-ATPase that provides the driving force for many transport processes.     

Hypoglycemic effects of steroidal sapogenins isolated from Jamaican bitter yam, Dioscorea polygonoides.

In this study, three steroidal sapogenins (Delta3 diosgenin, diosgenin, and pennogenin) and the phytosterols, stigmasterol and beta-sitosterol were isolated from Jamaican bitter yam, Dioscorea polygonoides. Their effects on fasting blood glucose and intestinal amylase and ATPases in streptozotocin-induced diabetic rats were studied. The diabetic rats (fed supplemented and unsupplemented diets) lost weight significantly compared to the normal group. There was a significant increase in the activity of alpha-amylase in the proximal region of the small intestinal mucosa of diabetic rats fed sapogenin extract or commercial diosgenin. However, this did not result in increased fasting blood glucose. Instead, supplementation of the diet with bitter yam sapogenin extract significantly decreased fasting blood glucose compared to the diabetic group. Supplementation of the diet with bitter yam sapogenin extract or commercial diosgenin significantly reduced Na+-K+-ATPase activity in all three regions compared to the diabetic control group. Commercial diosgenin supplementation resulted in a significant increase in Ca2+ ATPase activity in proximal region compared to the diabetic control and bitter yam sapogenin extract groups. The effect of bitter yam sapogenin extract or commercial diosgenin on intestinal Na+-K+-ATPase activity could account for their hypoglycemic properties. However, there was adverse effect on the body weight.     

Contribution of lipid dynamics on the inhibition of bovine brain synaptosomal Na+-K+-ATPase activity induced by 4-hydroxy-2-nonenal

The effects of lipid hydroperoxide degradation products, such as 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA), on bovine brain synaptosomal ATPase activities and their membrane lipid organization were examined. When the synaptosomes were treated with HNE, this resulted in the decrease of Na(+)-K(+)-ATPase activity with the loss of sulfhydryl (SH) groups in the membrane proteins. In contrast, MDA treatment of the synaptosomes did not induce an appreciable decrease in the ATPase activity or a loss of SH groups. The decreases in ATPase activity and SH content by treatment with HNE were also observed, as a Na+-K+-ATPase preparation was used in place of the synaptosomes. On the other hand, HNE had very little effect on synaptosomal Ca2+- and Mg2+-ATPase activities. The results of the kinetic analysis of the Na+-K+-ATPase activity indicated that the decrease in the activity by HNE-modification is due to a decreased affinity for the substrate. ATP completely protected the ATPase from the HNE attack. Modification of the synaptosomes with HNE caused a decrease in the membrane lipid fluidity near the lipid/water interface, not the lipid layer interior. In addition, it was found that there is a good relationship between the lipid fluidity and the Na+-K+-ATPase activity under the presence of various concentrations of HNE, suggesting that the lipid dynamics are closely related to HNE-induced inhibition of the ATPase activity. On the other hand, MDA did not induce change in the membrane lipid fluidity. HNE and MDA are mainly incorporated into the lipid and protein fractions in the synaptosomal membranes, respectively. Based on these results, we proposed a possible mechanism of HNE-induced inhibition of synaptosomal Na+-K+-ATPase activity associated with alterations in the membrane lipid organization.     

参附注射液对大鼠肝缺血再灌注损伤时ATP酶的影响

目的:研究参附注射液对肝缺血再灌注损伤大鼠的保护作用及可能机制。方法:将36只大鼠随机分为参附注射液(SF)组与生理盐水对照(NS)组。肝脏缺血15min,分别于再灌注后1、3、24h取肝组织测定Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶水平,并行酶组织化学染色,观察Ca2+-ATP酶的活性变化。结果:再灌注1、3h后,SF组肝组织中Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶水平显著高于NS组;但在24h时,2组数据无显著性差异(P>0.05)。结论:参附注射液可以通过保护ATP酶发挥对大鼠肝缺血再灌注损伤早期保护作用。     

Intestinal Na+-K+-ATPase activity and molecular events downstream of interferon-gamma receptor stimulation

This study evaluated the effects of human interferon-gamma (IFN-gamma) on Na(+)-K(+)-ATPase activity and the intracellular signaling pathways involved in human intestinal epithelial Caco-2 cells. Na(+)-K(+)-ATPase activity was determined as the difference between total and ouabain-sensitive ATPase. p38 MAP kinase activity was analyzed by Western blotting using the p38 MAP kinase assay kit. Total and phosphorylated STAT1 protein levels were detected using the PhosphoPlus Stat1. IFN-gamma decreased Na(+)-K(+)-ATPase activity in a time- and concentration-dependent manner. The IFN-gamma-induced decrease in Na(+)-K(+)-ATPase activity was accompanied by no changes in the abundance of alpha(1) subunit Na(+)-K(+)-ATPase. Downregulation of protein kinase C (PKC) with phorbol-12,13-dibutyrate (PDBu) prevented the inhibitory effect of IFN-gamma on Na(+)-K(+)-ATPase activity. Inhibition of Raf-1, mitogen-activated protein kinase kinase (MAPKK/MEK), p38 MAPK and STAT1 with, respectively, GW 5074, PD 98059, SB 203580 and epigallocatechin gallate prevented inhibition of Na(+)-K(+)-ATPase activity by IFN-gamma. Treatment with IFN-gamma markedly increased the expression of total and phospho-STAT1, this being accompanied by activation of p38 MAPK. Activation of phospho-STAT1 by IFN-gamma was almost abolished by epigallocatechin gallate and markedly reduced by SB 203580, but insensitive to downregulation of PKC. The increase in short circuit current (I(sc)) by 1.0 and 2.5 micrograms ml(-1) amphotericin B was markedly attenuated in IFN-gamma-treated cells. However, the inhibitory effect of PDBu on the amphotericin B-induced increase in I(sc) was of similar magnitude in vehicle- and IFN-gamma-treated cells. It is concluded that IFN-gamma markedly attenuates Na(+)-K(+)-ATPase activity. The transduction mechanisms set into motion by IFN-gamma involve the activation of PKC downstream STAT1 phosphorylation and Raf-1, MEK, ERK2 and p38 MAPK pathways, in a complex sequence of events.     

Effect of chlordecone on pH and temperature dependent substrate activation kinetics of rat brain synaptosomal ATPases

Chlordecone, a polycyclic chlorinated insecticide known as Kepone, inhibited the activities of (Na+-K+)ATPase and Mg2+-ATPase in rat brain synaptosomes. Altered pH and specific activity curves for both enzymes demonstrated significant inhibition by chlordecone in buffered acidic, neutral and alkaline pH ranges. Noncompetitive inhibition with respect to activation by ATP in the case of (Na+-K+)ATPase was indicated by altered Vmax values with no significant change in Km values at any pH studied, except at pH 9.5. Mg2+-ATPase was inhibited uncompetitively as evidenced by altered Vmax and Km values. The activities of both ATPase were decreased in the presence of chlordecone at higher temperatures. Activation energy (delta E) values were found to be decreased significantly in the presence of chlordecone at 37 degrees. Arrhenius plots of both ATPases preincubated with chlordecone were found to be nonlinear. In the presence of chlordecone, Vmax was decreased without significant change in Km values for (Na+-K+)ATPase at all temperatures, suggesting a noncompetitive type of inhibition. In the case of Mg2+-ATPase, similar noncompetitive type inhibition was obtained at 27 degrees but not at 32 and 37 degrees. The kinetic data in general suggest that the chlordecone inhibited (Na+-K+)ATPase noncompetitively and Mg2+-ATPase uncompetitively at all pHs and temperatures studied. The present data suggest that inhibition of (Na+-K+)ATPase and Mg2+-ATPase, the two membrane-bound enzymes in synaptosomes, by chlordecone is temperature dependent and pH independent.