模拟缺血后心外膜心室肌细胞钠通道变化的研究

目的探讨钠通道在缺血性心律失常发生中的作用及其机制.方法以酶解法分离兔心外膜心室肌细胞,并采用膜片钳全细胞记录技术,观察灌流正常细胞外液后以及模拟缺血液后10、20及30 min的快钠电流(INa)的大小及动力学变化.结果心外膜细胞缺血后INa的I-V曲线上移,模拟缺血前及缺血后10、20及30 min的峰电流密度在外层细胞(n=20个细胞)依次为(12.60±2.26)、(7.46±3.45)、(5.57±2.32)、(4.87±2.70)pA/pF,缺血前后比较有显著差异(P＜0.05).缺血后失活曲线左移,失活半电压在缺血前及缺血后10、20、30 min于外层细胞(n=18个细胞)依次为(-97.38±5.42)、(-115.83±6.16)、(-122.00±5.82)、(-128.83±3.13)mV,缺血前后比较有显著差异(P＜0.05).缺血后INa灭活后的恢复减慢,但无显著性差异(P＞0.05).结论缺血时钠通道活性的变化可影响心肌兴奋性和传导性,可能为心律失常发生的机制之一.     

心肌梗死后心室肌细胞钙通道改变的实验研究

目的　探讨L型钙通道在急性心肌梗死 (AMI)后室性心律失常发生的作用及机制。　方法　开胸冠脉结扎制备兔AMI模型 ,于 1周和 2个月处死动物分离心室肌细胞 ,以膜片钳技术记录梗死及周边区心外膜细胞L型钙通道电流(ICa L)的变化。　结果　AMI兔梗死周边区心外膜细胞ICa L受到抑制 ,I V曲线上移 ,其峰值电流密度在正常对照组、梗死后 1周和 2个月分别为 (-5 5 8± 1 5 3 ) pA /pF、(-3 5 2± 0 93 ) pA/pF (n =6,与对照组比较P <0 0 5 )和 (-4 84± 1 48)pA/pF(n =11,与对照组比较P <0 0 5 ) ,但I V曲线的形态轨迹不变。其失活曲线左移 ,失活速度加快 ,半数最大失活电位分别为 (-13 1± 4 2 )mV、(-2 5 9± 7 0 )mV和 (-2 1 3± 5 6)mV ,P <0 0 5。　结论　AMI后梗死周边带心外膜细胞ICa L通道受抑制 ,可能为AMI后发生室性心律失常的机制之一 ;梗死后 2个月钙通道异常程度减轻 ,有恢复正常的趋势     

一种筛选抗心律失常药物新模型的建立

目的　建立一种细胞水平的心律失常模型 ,以用于抗心律失常药物的筛选和评价。方法　酶解法分离单个大鼠心室肌细胞 ,在细胞水平给予传统诱发心律失常药物乌头碱 ,应用膜片钳技术观察记录应用乌头碱后心肌动作电位时程 (APD)、钠电流 (INa)、L 型钙电流 (ICa L)、内向整流钾电流(IK1)及瞬时外向钾电流 (Ito)的变化。结果　应用乌头碱 1μmol·L-1使大鼠单个心室肌细胞 90 %复极化动作电位时程(APD90 )从给药前的 ( 15 0 2 3± 7 0 2 )ms延长至 ( 2 3 6 0 3±2 3 2 2 )ms(n =8,P <0 0 1)。应用奎尼丁 10 μmol·L-1后动作电位时程延长与乌头碱组比较 ,APD90 进一步延长 (n =6,P <0 0 5 ) ,但应用维拉帕米 10 μmol·L-1后被乌头碱延长的APD恢复近于正常 ,在乌头碱的作用下 ,除极电压为 0mV时ICa L从 ( 72 7 9± 178 0 ) pA增加至 ( 10 82 1± 2 2 2 2 ) pA(n =6,P <0 0 1) ;钠电流 (INa)在 - 5 0mV刺激电压下从( 2 5 4± 5 5 3 ) pA增加至 ( 45 3 0 2± 475 1) pA(n =4,P <0 0 5 ) ;IK1在 - 12 0mV的刺激电压下 ,Ik1的内向成分从( 2 0 0 7 1± 3 5 9 3 ) pA增加至 ( 2 3 17 7± 40 1 8)pA(n =10 ,P <0 0 1) ;奎尼丁、维拉帕米对乌头碱诱发的钠电流和钙电流增加有抑制的作用。结论　乌头碱使?     

铅对大鼠海马神经元钠电流的抑制作用

目的　铅对神经系统有损害作用 ,钠通道是神经元产生和传递电信号的重要枢纽 ,故研究铅对大鼠海马CA1区神经元钠电流 (INa)的影响。方法全细胞膜片钳技术。结果　醋酸铅可浓度依赖地抑制INa,1 ,1 0 ,50和 1 0 0 μmol·L-1 醋酸铅对INa的抑制率分别为 (8.2± 0 .8) % ,(2 0 .9± 2 .6) % ,(51 .8±4.8) %和 (66 .4±5 .7) %。此外 ,它还与电压呈依赖关系 ,50 μmol·L-1 醋酸铅可使INa的激活曲线显著右移 ,但不改变斜率因子 ,还可使INa的失活曲线显著左移。结论　铅可抑制INa的激活过程 ,可促进INa的失活过程。铅改变了细胞膜的电压感应 ,这可能是铅损伤海马神经元的作用机制之一     

依布利特对兔左心室肌细胞钠离子通道跨壁异质性的影响

目的探讨依布利特抗室性心律失常作用的离子机制。方法利用酶解的方法分离出新西兰纯种大耳白兔（体重2．0～2．5kg）心外膜、心内膜和中层心室肌细胞。将各层细胞按处理试剂分为正常对照组、依布利特A（10^-7mol／L）组、依布利特B（10^-6mol／L）组：应用膜片钳全细胞记录方法,记录各层心室肌细胞钠通道电流（INa）活性的变化,并与正常对照组心肌细胞进行比较分析。结果3组间的中层心肌细胞INa电流Ⅰ-Ⅴ曲线差异有统计学意义（P〈0．05）;对照组与依布利特组的心内膜下细胞INaⅠ-Ⅴ差异有统计学意义（P〈0．01）,依布利特A、B组间无显著差异（P〉0．05）;对照绍与依布利特两组心外膜下细胞INaⅠ-Ⅴ差异有统计学意义（P〈0．05）,依布利特A、B组间无差异（P〉0．05）：依布利特两组与对照组中层心审肌细胞INa稳态失活曲线比较差异有统计学意义（P〈0．05）,依布利特A、B组间无显著差异（P〉0．05）;3组间心内膜下细胞INa稳态失活曲线差异有统计学意义（P〈0．05）;对照绀与依布利特两组间的心外膜下细胞INa稳态失活曲线差异有统计学意义（P〈0．01）,依布利特两组间无显著差异（P〉0．05）。依布利特两组与对照组内、中、外3层比较INa。失活后恢复曲线在45、95和145ms时,差异有统计学意义（P〈0．05）,依布利特两组间各层比较差异也有统计学意义（P〈0．05）。结论依布利特使INaⅠ-Ⅴ曲线及INa峰值电流密度下降,稳态失活曲线左移,失活后再恢复时间延长,且高浓度依布利特对心肌细胞钠离子通道的抑制作用强于低浓度。     

葡甘聚糖对大鼠口服荧光素钠吸收的影响

目的 :研究葡甘聚糖对大鼠口服荧光素钠药动学的影响。方法 :利用荧光素钠作为示踪药物 ,HPLC反相柱色谱分离原药 ,荧光高效液相法定量分析血清荧光素钠含量。结果 :大鼠灌胃荧光素钠溶液T12 α为 (5 .0± 0 .9)min ,T12 β为 (43± 9)min ,Tmax为 (7.5± 0 )min ,Cmax为 (192± 7)mg·L- 1,AUC为 (83± 17) μg·h- 1·L- 1。以 0 .4 %或 0 .8%的葡甘聚糖为制剂时T12 α分别为 (2 0± 6 )min和 (38± 13)min ,T12 β为 (6± 3)h和 (2 5± 12 )h ,Tmax为(19± 9)min和 (40± 16 )min ,Cmax为 (92 .3± 2 .0 )mg·L- 1和 (5 2± 3)mg·L- 1,AUC为 (6 14± 10 5 ) μg·h- 1·L- 1和 (30 1± 2 2 ) μg·h- 1·L- 1。结论 :葡甘聚糖能明显改变口服药物的药动学参数 ,如降低血药峰值浓度 ,提高AUC。     

牛磺酸镁对乌头碱致大鼠心肌细胞心律失常模型钠离子通道的影响

目的研究牛磺酸镁配合物(taurine magnesium coordi-nation compound,TMCC)对乌头碱所致大鼠心室细胞心律失常模型的钠电流变化的影响,探讨其抗心律失常的作用机制。方法酶解法分离大鼠单个心室肌细胞,应用全细胞膜片钳技术记录不同浓度TMCC及胺碘酮对正常细胞及乌头碱所致大鼠单个心室肌细胞心律失常模型INa变化。结果TMCC对正常细胞INa呈浓度依赖性抑制作用。1μmol.L-1乌头碱使钠电流从(45.56±1.96)pA/pF增加到(59.19±11.49)pA/pF(n=5,P<0.01)。24.24μmol.L-1胺碘酮使电流减小到(34.23±1.33)pA/pF(n=5,P<0.01)。TMCC(100,200,400μmol.L-1)对乌头碱所致的细胞模型INa具有恢复作用,分别恢复为(51.61±5.96)pA/pF,(40.91±6.73)pA/pF,(41.50±5.50)pA/pF。胺碘酮则使之恢复为(40.22±1.47)pA/pF。结论TMCC能恢复乌头碱增大的钠电流,作用与胺碘酮相当,TMCC对钠电流的抑制作用可能是其发挥抗心律失常的机制之一。     

3、4-二羟基苯乙酮对大鼠肺内动脉平滑肌细胞钾电流的影响

目的　研究 3、4 二羟基苯乙酮 (3、4 Dihydroxyace tophenone,DHAP)对钾通道的作用。 方法　建立大鼠肺内动脉平滑肌全细胞膜片钳方法并观察 3、4 二羟基苯乙酮对大鼠肺内动脉平滑肌细胞钾离子通道的作用。结果　①将平滑肌细胞钳制在 - 4 0mV ,给予从 - 30mV复极化至 +6 0mV、阶跃电压为 10mV、脉冲时间是 30 0ms的去极化刺激10次时 ,可记录到时间及电压依赖性的延迟整流钾电流。②在指令电位 +5 0mV、+6 0mV时 ,细胞外给予 10 -6mol·L-1DHAP 5min后 ,可使该电流由用药前 (12 97± 2 4 5 )、(16 83± 3 2 8)pA/pF ,增加至 (18 2 9± 2 6 3)、(2 2 90±3 2 1)pA/pF ,增加百分比为 4 1%和 36 % (P <0 0 5 ,n =6 )。结论　DHAP可能通过开放钾通道而舒张肺内动脉平滑肌细胞 ,这可能是其降低肺动脉高压的重要机制之一。     

三氯化铝对大鼠海马CA1区锥体细胞钠电流的影响

目的　为了进一步明确铝及含铝化合物对神经系统的损伤及其作用机制。方法　采用全细胞膜片钳技术研究三氯化铝 (AlCl3)对急性分离的大鼠海马CA1区神经元钠通道的影响。结果　AlCl3对钠电流有明显的抑制作用 ,且呈浓度依赖性 ,10 0 0μmol·L- 1AlCl3给药前后钠电流激活曲线Vh 分别为- (5 1.3± 6 .0 )mV和 - (4 7.5± 5 .4 )mV (P <0 .0 5 ) ,k值分别为 - (8.1± 2 .3)mV和 - (8.6± 3.2 )mV(P >0 .0 5 ) ,给药前后钠电流失活曲线Vh 分别为- (6 7.4± 5 .5 )mV和 - (71.4± 4 .4 )mV(P <0 .0 5 ) ,k值分别为 (6 .1± 1.1)mV和 (6 .8± 1.2 )mV (P >0 .0 5 )。结论　AlCl3对大鼠海马CA1区神经元钠通道有抑制作用 ,这可能是铝引起中枢神经系统损伤的机制之一     

花生四烯酸对兔心室肌细胞电压门控钠通道的影响

目的研究花生四烯酸(AA)对兔心室肌细胞电压门控钠通道(VGSC)的影响。方法酶解法分离兔心室肌细胞,采用标准全细胞膜片钳技术记录电压门控钠通道电流(INa)。结果AA可浓度依赖性抑制INa,使INa的I-U曲线上移,但其激活电位、电位峰值和反转电位保持不变;AA使INa稳态失活曲线左移,恢复曲线右移;但对INa的抑制作用不具有明显的频率依赖性。结论AA对INa具有浓度依赖性抑制作用,主要是通过抑制失活和失活后恢复过程而发挥作用。因此AA对INa的抑制作用可能是AA抗心律失常,保护心肌的作用机制之一。     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

---

Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.     

Alzheimer’s disease – current trends in basic and clinical research. Highlights from the 16th ECNP

Advances in the molecular biological knowledge of neuronal nicotinic acetylcholine receptors (nAChRs) have led to a growing interest by the pharmaceutical industry in the development of novel compounds that selectively modulate nAChR function. The ability of (-)-nicotine, an activator of nAChRs, to enhance attentional aspects of cognition in animals and humans, to exert neuroprotective and anxiolytic-like effects, and presumably to mediate the negative correlation between smoking and Alzheimer's (and Parkinson's) Disease, has focused interest on the potential therapeutic utility of modulators of nAChR function for treatment of some of the deficits associated with these progressive, neurodegenerative conditions. Numerous compounds are known which activate nAChRs and which might serve as lead compounds toward the development of such agents. The pharmacologic diversity of neuronal nAChR subtypes suggests the possibility of developing selective compounds which would have more favourable side-effect profiles than existing agents. This broader class of agents, collectively called cholinergic channel modulators (ChCMs), is anticipated to encompass compounds which would have more favourable side-effect profiles than existing agents, which generally exhibit low selectivity. This selectivity may be achieved by preferentially activating some subtypes of nAChRs (i.e., Cholinergic Channel Activators, ChCAs) or inhibiting the function of other subtypes (Cholinergic Channel Inhibitors, ChCIs). An overview of the biology of nAChRs and the rationale for the use of ChCMs for the treatment of dementia related to neurodegenerative diseases are presented, followed by a discussion of lead compounds and compounds under consideration for clinical evaluation.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.