麻醉药对大鼠海马锥体神经元钠通道电流的影响及其机制
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摘要
前言
全麻原理是麻醉学最重要的基本理论之一，但全麻药作用的确切机制至今不明。一般认为吸入全麻药的作用机制较为复杂，其中可能包涵静脉全麻药的作用机制。不管吸入全麻药宏观和微观水平的作用部位如何，现在可以肯定分子水平的作用部位是在神经细胞膜。越来越多的证据表明吸入麻醉药发挥效应的部位是细胞膜上的离子通道。
在诸多的离子通道中，麻醉药对n-乙酰胆碱受体(nAChR)和r-氨基丁酸A受体(GABA_AR)通道影响的研究最多，因实验条件和药物剂量等因素不同，研究结果不一。一般认为nAChR通道不是全麻药作用的主要靶位。尽管GABA_A受体通道是大家公认的全麻药主要靶位之一，在其临床麻醉浓度主要增强GABA对该受体通道的效应，但已发现许多用对该通道作用不能解释的现象，故全麻药对其它兴奋性离子通道的作用已受到重视。在中枢神经系统(CNS)主要的兴奋性离子通道中，钠通道为主要的兴奋性电压门控型离子通道，是可兴奋细胞产生动作电位及传导神经冲动的关键部位和决定因素，虽以前的实验发现枪乌贼巨轴突模型的钠通道对全麻药不敏感，但后来的研究证明临床浓度的全麻药对鼠脑钠通道、肺泡Ⅱ型细胞钠通道及心肌细胞钠通道都有显著的抑制作用，且遵循Meyer-Overton规则。因此，全麻药对脑钠通道的影响以及此影响在全麻中的地位值得研究。本课题分三部分研究异丙酚(静脉麻醉药)、异氟醚(吸入麻醉药)和利多卡因(局部麻醉药)对急性分离的CNS钠通道的影响。
第一部分
异丙酚对大鼠海马锥体神经元钠通道电流的影响
材料和方法
选用10＊4d SD大鼠，雌雄不拘，体重20－309，由徐州医学院实验动
物中心提供。将实验大鼠快速断头，置于0－4℃氧饱和的人工脑脊液
（ACSF）中分离出海马，手工切成约500pm厚的脑片。将脑片放人孵育槽
中用 ACSF漂浮孵育 60加n，连续通以 95％ O。＋5呢 CO。维持 pH在 7·35－
7．40。采用H步酶消化法，分别用 10 InL的 ACSF配成 0．05％的 TyPsin和
0．05％的Pronase，相继在孵育精内酶解根2℃，各30 min人酶解后的脑片
冲洗2次后继续用ACSF孵育。实验时每次取二片脑片放人盛有ACSF约1
InL的小玻璃杯中，先后用尖端热处理的直径为400 pm和150pm左右的
Pasteur吸管轻轻吹打，经400目钢网过滤到细胞灌流槽内，放在倒置显微
镜上静置15－20min让细胞贴壁，用细胞外液灌洗3次后作全细胞膜片钳
记录。
在显微镜下选择合适的锥体神经元作为实验对象，用硬质有芯玻璃毛
坯管二步拉制记录电极，用微电极操纵器将电极缓慢推向细胞，当电极尖端
贴住细胞膜后轻轻吸引使之封接，当封接阻抗大于 IGfl时，负压破膜，补偿
电容电流和串联阻抗形成全细胞状态。用EPC—9膜片钳放大器在室温
（24－26T）下记录钠电流，电刺激脉冲的输出及电信号的采人均通过计算
机由商用软件Pulse 8．02来完成，经数字化处理后贮存于计算机硬盘。
实验分为7组，异丙酚h，Pro）按加人在细胞外液中的浓度广0、
30、50、100Pmol／L）对应分为Pro；。组、Pro。。组、u。组和PrO；。组，目肪乳剂
Ontralwid，Iry）按 PrO。。组和 Pro；。组同样的浓度对应为 ILPw组和 ILP；。组，
对照组灌流给药时仍用细胞外液。每组6个细胞，分别记录给药前、给药后
2 dn及冲洗后2 dn的钠通道电流变化。抑制程度以最大内向电流的减
少来计算，用与基础对照值的百分比表示。
·2·
结 果
对照组＊LPro组和 ILPl。组的峰钠电流下降率分别为 4．二 L 3．2％3．7
士46％和中3土3．二邮，差别无显著性意义（P＞o．05＊异丙酚抑制峰钠电
流的程度与浓度呈正相关k＝0·993，P＜o．0＊，PrO；。组、u。组、u。组和
u；。组分别抑制峰销电流为14．4。8．7％（P＜0．05）、43·2 t 8·8防（P＜
0．0且）、67．2士二8．二例P＜o＊二）和85．二土二一9％（P＜o＊二），1q为3二．5
卜mOVL。‘
讨 论
哪药对中枢神经钠通道的影响虽有研究报道，但使用的都是人工表
达的血AJ型脑钠通道，缺乏p亚单位的调节作用。我们采用酶消化法，
急性分离出来的含有完整钠通道的大鼠海马锥体神经元的峰钠电流（平均
3－4nd）比人工表达的D A－1型细胞（平均1－ZnA）的大，阈电位和峰电
位也比人工表达的细胞更负，提示分离细胞的钠通道更完整、功能钠通道更
多、激活和失活更迅速。与 Isom LL等报道的 p；亚单位可加速钠通道的激
活和失活，在快速反应的哺乳细胞上复合表达了p；亚单位的铀通道可使其
功能钠通道。亚单位的数目增加2－4倍、并使激活和失活向更负的膜电位
转移的结果相吻合，表明本实验模型更能完整的代表整体动物脑神经元的
铀通道。
异丙酚是一种脂溶性的药物，有实验报道溶解异丙酚的脂肪乳剂对心
肌细胞的收缩功能有一定的影响。本实验结果证实脂肪乳剂对铺通道电流
无明显影响，表明异丙酚对海马神经?
The mechanism of general anesthetics is an important part of anesthesiolo-gy. But the exact mechanism of general anesthetics is unclear. In general, the mechanism of inhaled anesthetics is more complex than that of intravenous general anesthetics. Some scholar thought the mechanism of intravenous anesthetics might be partly the same as that of inhaled anesthetics. No matter where general anesthetics act in macroscopic and microscopic anatomic level, it is certain that the position general anesthetics act on is on the nerve membrane in molecular level and increasing evidence had indicated that anesthetics set their sites on ion channels.
Over the past years, the two receptor - gated ion channels of nicotinic acetycholine (nACh) and -y - aminobutyric acid (GAB A) had been widely studied in so many ion channels. But the results were different because of the differences in experimental protocols and doses of general anesthetics. Generally speaking, nACh receptor channels are not the main targets of anesthetic action. Although GABAA receptor channels are commonly considered as one of the main targets the general anesthetics act on, the results could not explain all the phenomenon of general anesthesia. Therefore, much attention has been paid to other excitatory ion channels recently. Sodium channels, which play an important part in the production of action potential and the conduction of nerve pulse, are one
kind of the main excitatory voltage - gated ion channels and have been proposed as possible anesthetic targets once more because of many new experimental evidences.
The aim of the present research is to investigate the effects of general anesthetics (propofol and isoflurane) and local anesthetics (Lidocaine) on the sodium channel currents of brain in rats.
Part One Effects of Propofol on the Sodium Channel Currents of Hippocampal Pyramidal Neurons in Rats
Materials and Methods
SD rats aged 10 ~ 14 days, male and female, weighed from 20 ~30 grams. Heads sheared, Hippocampus were detached and put into 0 ~4TI artificial cerebral spinal fluid (ACSF) . Then they were cut into 500 jxm thick brain slices . The slices were put into incubate chamber full of ACSF and the fluid were saturated by mixed gas of 95% 02 and 5% C02 to maintain the pH at 7. 35 ~ 7.40 for Ih. 0.05%Trypsin and 0.05% Pronase were administered into the chamber one after another to yield enzymolysis ( 30 min , 32癈 ). One of the hippocampus slice was gently transferred into a piece of glasses full of 1ml ACSF, lightly blown with two Pasteru straws which were given heat treatment and whose diameters were 400fjun and 150{jutn, and then filtered into perfusion chamber fixed on an inverted microscope. The whole - cell patch clamp record began after 15 ~ 20min and perfusion 3 times with extracellular fluid.
Sodium currents were recorded using the whole - cell configuration of the patch - clamp recording technique, using a standard patch - clamp amplifier (EPC - 9, HEKA, German) controlled by commercially available software (pulse8.02) on a standard personal computer. Currents were digitized and recorded to hard disk.
According to the concentrations( uM) of propofol (Pro) and correspondent
concentrations of intralipid (ILP) , the study was divided into 7 groups, which were Pro10, Pro30, Pro50, Pro100, ILP50, ILP100 and Control group. Each group contained 6 cells, and the sodium channel currents were recorded before drug administration, 2 min after administration and 2 min after washout, respectively. Suppression was calculated as the reduction of the maximum inward current, expressed as percentage of control.
Results
The peak sodium currents of the Con group, ILP50 and ILP100 were de-creasedby4.2 ±3.2% ,5.7 ±4. 6%and4. 3 ±3. 1% , respectively ( P > 0.05) ;Propofol showed a concentration -relative effect on the peak sodium current suppression( r = 0. 993, P <0. 01) , the suppression rates of Pro10, Pro30, Pro50and Pro100werel4.4±8.7%(P<0.05) ,43. 2 ±8. 8% ( P <0. 01 ) , 67.2±18.1%(P<0.01)and 85.1±14.9%(P<0.01, respect
全麻原理是麻醉学最重要的基本理论之一，但全麻药作用的确切机制至今不明。一般认为吸入全麻药的作用机制较为复杂，其中可能包涵静脉全麻药的作用机制。不管吸入全麻药宏观和微观水平的作用部位如何，现在可以肯定分子水平的作用部位是在神经细胞膜。越来越多的证据表明吸入麻醉药发挥效应的部位是细胞膜上的离子通道。
在诸多的离子通道中，麻醉药对n-乙酰胆碱受体(nAChR)和r-氨基丁酸A受体(GABA_AR)通道影响的研究最多，因实验条件和药物剂量等因素不同，研究结果不一。一般认为nAChR通道不是全麻药作用的主要靶位。尽管GABA_A受体通道是大家公认的全麻药主要靶位之一，在其临床麻醉浓度主要增强GABA对该受体通道的效应，但已发现许多用对该通道作用不能解释的现象，故全麻药对其它兴奋性离子通道的作用已受到重视。在中枢神经系统(CNS)主要的兴奋性离子通道中，钠通道为主要的兴奋性电压门控型离子通道，是可兴奋细胞产生动作电位及传导神经冲动的关键部位和决定因素，虽以前的实验发现枪乌贼巨轴突模型的钠通道对全麻药不敏感，但后来的研究证明临床浓度的全麻药对鼠脑钠通道、肺泡Ⅱ型细胞钠通道及心肌细胞钠通道都有显著的抑制作用，且遵循Meyer-Overton规则。因此，全麻药对脑钠通道的影响以及此影响在全麻中的地位值得研究。本课题分三部分研究异丙酚(静脉麻醉药)、异氟醚(吸入麻醉药)和利多卡因(局部麻醉药)对急性分离的CNS钠通道的影响。
第一部分
异丙酚对大鼠海马锥体神经元钠通道电流的影响
材料和方法
选用10＊4d SD大鼠，雌雄不拘，体重20－309，由徐州医学院实验动
物中心提供。将实验大鼠快速断头，置于0－4℃氧饱和的人工脑脊液
（ACSF）中分离出海马，手工切成约500pm厚的脑片。将脑片放人孵育槽
中用 ACSF漂浮孵育 60加n，连续通以 95％ O。＋5呢 CO。维持 pH在 7·35－
7．40。采用H步酶消化法，分别用 10 InL的 ACSF配成 0．05％的 TyPsin和
0．05％的Pronase，相继在孵育精内酶解根2℃，各30 min人酶解后的脑片
冲洗2次后继续用ACSF孵育。实验时每次取二片脑片放人盛有ACSF约1
InL的小玻璃杯中，先后用尖端热处理的直径为400 pm和150pm左右的
Pasteur吸管轻轻吹打，经400目钢网过滤到细胞灌流槽内，放在倒置显微
镜上静置15－20min让细胞贴壁，用细胞外液灌洗3次后作全细胞膜片钳
记录。
在显微镜下选择合适的锥体神经元作为实验对象，用硬质有芯玻璃毛
坯管二步拉制记录电极，用微电极操纵器将电极缓慢推向细胞，当电极尖端
贴住细胞膜后轻轻吸引使之封接，当封接阻抗大于 IGfl时，负压破膜，补偿
电容电流和串联阻抗形成全细胞状态。用EPC—9膜片钳放大器在室温
（24－26T）下记录钠电流，电刺激脉冲的输出及电信号的采人均通过计算
机由商用软件Pulse 8．02来完成，经数字化处理后贮存于计算机硬盘。
实验分为7组，异丙酚h，Pro）按加人在细胞外液中的浓度广0、
30、50、100Pmol／L）对应分为Pro；。组、Pro。。组、u。组和PrO；。组，目肪乳剂
Ontralwid，Iry）按 PrO。。组和 Pro；。组同样的浓度对应为 ILPw组和 ILP；。组，
对照组灌流给药时仍用细胞外液。每组6个细胞，分别记录给药前、给药后
2 dn及冲洗后2 dn的钠通道电流变化。抑制程度以最大内向电流的减
少来计算，用与基础对照值的百分比表示。
·2·
结 果
对照组＊LPro组和 ILPl。组的峰钠电流下降率分别为 4．二 L 3．2％3．7
士46％和中3土3．二邮，差别无显著性意义（P＞o．05＊异丙酚抑制峰钠电
流的程度与浓度呈正相关k＝0·993，P＜o．0＊，PrO；。组、u。组、u。组和
u；。组分别抑制峰销电流为14．4。8．7％（P＜0．05）、43·2 t 8·8防（P＜
0．0且）、67．2士二8．二例P＜o＊二）和85．二土二一9％（P＜o＊二），1q为3二．5
卜mOVL。‘
讨 论
哪药对中枢神经钠通道的影响虽有研究报道，但使用的都是人工表
达的血AJ型脑钠通道，缺乏p亚单位的调节作用。我们采用酶消化法，
急性分离出来的含有完整钠通道的大鼠海马锥体神经元的峰钠电流（平均
3－4nd）比人工表达的D A－1型细胞（平均1－ZnA）的大，阈电位和峰电
位也比人工表达的细胞更负，提示分离细胞的钠通道更完整、功能钠通道更
多、激活和失活更迅速。与 Isom LL等报道的 p；亚单位可加速钠通道的激
活和失活，在快速反应的哺乳细胞上复合表达了p；亚单位的铀通道可使其
功能钠通道。亚单位的数目增加2－4倍、并使激活和失活向更负的膜电位
转移的结果相吻合，表明本实验模型更能完整的代表整体动物脑神经元的
铀通道。
异丙酚是一种脂溶性的药物，有实验报道溶解异丙酚的脂肪乳剂对心
肌细胞的收缩功能有一定的影响。本实验结果证实脂肪乳剂对铺通道电流
无明显影响，表明异丙酚对海马神经?
The mechanism of general anesthetics is an important part of anesthesiolo-gy. But the exact mechanism of general anesthetics is unclear. In general, the mechanism of inhaled anesthetics is more complex than that of intravenous general anesthetics. Some scholar thought the mechanism of intravenous anesthetics might be partly the same as that of inhaled anesthetics. No matter where general anesthetics act in macroscopic and microscopic anatomic level, it is certain that the position general anesthetics act on is on the nerve membrane in molecular level and increasing evidence had indicated that anesthetics set their sites on ion channels.
Over the past years, the two receptor - gated ion channels of nicotinic acetycholine (nACh) and -y - aminobutyric acid (GAB A) had been widely studied in so many ion channels. But the results were different because of the differences in experimental protocols and doses of general anesthetics. Generally speaking, nACh receptor channels are not the main targets of anesthetic action. Although GABAA receptor channels are commonly considered as one of the main targets the general anesthetics act on, the results could not explain all the phenomenon of general anesthesia. Therefore, much attention has been paid to other excitatory ion channels recently. Sodium channels, which play an important part in the production of action potential and the conduction of nerve pulse, are one
kind of the main excitatory voltage - gated ion channels and have been proposed as possible anesthetic targets once more because of many new experimental evidences.
The aim of the present research is to investigate the effects of general anesthetics (propofol and isoflurane) and local anesthetics (Lidocaine) on the sodium channel currents of brain in rats.
Part One Effects of Propofol on the Sodium Channel Currents of Hippocampal Pyramidal Neurons in Rats
Materials and Methods
SD rats aged 10 ~ 14 days, male and female, weighed from 20 ~30 grams. Heads sheared, Hippocampus were detached and put into 0 ~4TI artificial cerebral spinal fluid (ACSF) . Then they were cut into 500 jxm thick brain slices . The slices were put into incubate chamber full of ACSF and the fluid were saturated by mixed gas of 95% 02 and 5% C02 to maintain the pH at 7. 35 ~ 7.40 for Ih. 0.05%Trypsin and 0.05% Pronase were administered into the chamber one after another to yield enzymolysis ( 30 min , 32癈 ). One of the hippocampus slice was gently transferred into a piece of glasses full of 1ml ACSF, lightly blown with two Pasteru straws which were given heat treatment and whose diameters were 400fjun and 150{jutn, and then filtered into perfusion chamber fixed on an inverted microscope. The whole - cell patch clamp record began after 15 ~ 20min and perfusion 3 times with extracellular fluid.
Sodium currents were recorded using the whole - cell configuration of the patch - clamp recording technique, using a standard patch - clamp amplifier (EPC - 9, HEKA, German) controlled by commercially available software (pulse8.02) on a standard personal computer. Currents were digitized and recorded to hard disk.
According to the concentrations( uM) of propofol (Pro) and correspondent
concentrations of intralipid (ILP) , the study was divided into 7 groups, which were Pro10, Pro30, Pro50, Pro100, ILP50, ILP100 and Control group. Each group contained 6 cells, and the sodium channel currents were recorded before drug administration, 2 min after administration and 2 min after washout, respectively. Suppression was calculated as the reduction of the maximum inward current, expressed as percentage of control.
Results
The peak sodium currents of the Con group, ILP50 and ILP100 were de-creasedby4.2 ±3.2% ,5.7 ±4. 6%and4. 3 ±3. 1% , respectively ( P > 0.05) ;Propofol showed a concentration -relative effect on the peak sodium current suppression( r = 0. 993, P <0. 01) , the suppression rates of Pro10, Pro30, Pro50and Pro100werel4.4±8.7%(P<0.05) ,43. 2 ±8. 8% ( P <0. 01 ) , 67.2±18.1%(P<0.01)and 85.1±14.9%(P<0.01, respect
引文
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regulation. Physiol. Rev., 1997, 77: 359-96.
13. Urban BW. Differential effects of gaseous and volatile anesthetics on sodium and potassium channels. British Journal of Anaesthesia, 1993, 71: 25-38.
14. Rehberg B, Xiao YH, Duch DS. Central nervous system sodium channels are significantly suppressed at clinical concentrations of volatile anesthetics. Anesthesiology, 1996, 84: 607-13.
15. Molliex S, Dureuil B, Aubier M. Halothane decreases Na, K-ATPase, and Na channel activity in alveolar type Ⅱ cells. Anesthesiology, 1998, 88: 1606-13.
16. Peduto VA, Concas A, Santoro G, et al. Biochemical and electrophysiologic evidence that propofol enhances GABAergic transmission in the rat brain. Anesthesiology, 1991, 75: 1000-9.
17. Simpson VJ, Blednov Y. Propofol produces differences in behavior but not chloride channel function between selected lines of mice. Anesth Analg, 1996, 82: 327-31.
18．孟晶，原颖新，戴体俊．一叶秋碱和荷包牡丹碱催醒作用的实验研究．徐州医学院学报，2001，21：345-7．
19. Yamakura T, Sakimura K, Shimoji K, et al. Effects of propofol on various AMPA-, Kainate-, and NMDA-selective glutamate receptor channels expressed in Xenopus oocytes. Neurosci Lett, 1995, 188: 187-90.
20. Rehberg B, Duch DS. Suppression of central nervous system sodium channels by propofol. Anesthesiology, 1999, 91: 512-20.
21. Kaneda M, Nakamura H, Akaike N. Mechanical and enzymatic isolation of mammalian CNS neurons. Neurosci Res, 1988, 5: 299-304.
22. Isom LL, Scheuru T, Brownstein AB, et al. Functional co-expression of the β_1 and type Ⅱ A α subunits of sodium channels in a mammalian cell line. J Biol Chem, 1995, 270: 3306-12.
23. Cook DJ, Housmans PR. Mechanism of the negative inotropic effect of propofol in isolated ferret ventricular myocardium. Anesthesiology, 1994, 80: 859-71.
24. Smith C, McEwan AI, Jhaveri R, et al. The interaction of fentanyl on the
Cp_(50) of propofol for loss of consciousness and skin incision. Anesthesiology, 1994, 81: 820-8.
25. Servin F, Desmonts JM, Haberer JP, et al. Pharmocokinetics and protein binding of propofol in patients with cirrhosis. Anesthesiology, 1988, 69: 887-91.
26. Dutta S, Ebling WF. Formulation-dependent brain and lung distribution kinetics of propofol in rats. Anesthesiology, 1998, 89: 678-85.
27. Shyr MH, Tsai TH, Tan PPC, et al. Concentration and regional distribution of propofol in brain and spinal cord during propofol anesthesia in the rat. Neurosci Lett, 1995, 184: 212-5.
28. Ratnakumari L, Vysotskaya TN, Duch DS, Hemmings HC. Differential effects of anesthetic and nonanesthetic cyelobutanes on neuronal voltage-gated sodium channels. Anesthesiology, 2000, 92: 529-41.
29．刘俊杰，赵俊主编．现代麻醉学，第1版，北京：人民卫生出版社，1987，205-8．
30. Berde CB, Strichartz GR. Local anesthetics. In Miller RD(ed): Anesthesia, 5th edition, Churchill Livingstone, 2000, 511.
31. Catterall WA. Common models of drug action on Na~+ channels: local anesthetics, antiarrhythmics and anticonvulsants. Trends Pharmacol. Sci., 1987, 8: 57-65.
32. Catterall WA. Structure and function of voltage-gated ion channels. Annu Rev Biochem, 1995, 64: 493-531.
33. Ragsdale D, Mcphee JC, Scheuer T. Molecular determinants of state-dependent block of Na~+ channels by local anesthetics. Science, 1994, 265: 1724-8.
34．俞卫锋主编．麻醉与复苏新论，第1版，上海：第二军医大学出版社，2001，256．
2. Evers AS, Steinbach JH. Supersensitive sites in the central nervous system: Anesthetics block brain nicotinic receptors. Anesthesiology, 1997, 86: 760-8.
3. Eckenhoff RG. Amino acid resolution of halothane binding sites in serum albumin. J Biol Chem, 1996, 271: 15521-8.
4. Franks NP, Lieb WR. Anaesthetics set their sites on ion channels. Nature, 1997, 389: 334-5.
5. Flood P, Krasowski MD. Intravenous anesthetics differentially modulate ligand-gated ion channels. Anesthesiology, 2000, 92: 1418-25.
6. Li XS, Czajkowski C, Pearce RA. Rapid and direct modulation of GABA_A receptors by halothane. Anesthesiology, 2000, 92: 1366-75.
7. Raines DE, Zachriah VT. Isoflurane increases the apparent agonist affinity of the nicotinic acetylcholine receptor. Anesthesiology, 1999, 90: 135-46.
8. Banks MI, Pearce RA. Dual actions of volatile anesthetics on GABA_A IPSCS: Dissociation of blocking and prolonging effects. Anesthesiology, 1999, 90: 120-34.
9. Neumahr S, Hapfelmeier G, Scheller M. Dual action of isoflurane on the γ-aminobutyric acid (GABA)-mediated currents through recombinant α_1β_2γ_(2L)-GABA_A-receptor channels. Anesth Analg, 2000, 90: 1184-90.
10. Franks NP, Dickinson R, Sousa SLM, et al. How does xenon produce anaesthesia? Nature, 1998, 396: 324.
11. Fozzard HA, Hanck DA. Structure and function of voltage-dependent sodium channels: Comparison of brain Ⅱ and cardiac isoforms. Physiol. Rev., 1996, 76: 887-926.
12. Garty H, Palmer LG. Epithelial sodium channels: function, structure, and
regulation. Physiol. Rev., 1997, 77: 359-96.
13. Urban BW. Differential effects of gaseous and volatile anesthetics on sodium and potassium channels. British Journal of Anaesthesia, 1993, 71: 25-38.
14. Rehberg B, Xiao YH, Duch DS. Central nervous system sodium channels are significantly suppressed at clinical concentrations of volatile anesthetics. Anesthesiology, 1996, 84: 607-13.
15. Molliex S, Dureuil B, Aubier M. Halothane decreases Na, K-ATPase, and Na channel activity in alveolar type Ⅱ cells. Anesthesiology, 1998, 88: 1606-13.
16. Peduto VA, Concas A, Santoro G, et al. Biochemical and electrophysiologic evidence that propofol enhances GABAergic transmission in the rat brain. Anesthesiology, 1991, 75: 1000-9.
17. Simpson VJ, Blednov Y. Propofol produces differences in behavior but not chloride channel function between selected lines of mice. Anesth Analg, 1996, 82: 327-31.
18．孟晶，原颖新，戴体俊．一叶秋碱和荷包牡丹碱催醒作用的实验研究．徐州医学院学报，2001，21：345-7．
19. Yamakura T, Sakimura K, Shimoji K, et al. Effects of propofol on various AMPA-, Kainate-, and NMDA-selective glutamate receptor channels expressed in Xenopus oocytes. Neurosci Lett, 1995, 188: 187-90.
20. Rehberg B, Duch DS. Suppression of central nervous system sodium channels by propofol. Anesthesiology, 1999, 91: 512-20.
21. Kaneda M, Nakamura H, Akaike N. Mechanical and enzymatic isolation of mammalian CNS neurons. Neurosci Res, 1988, 5: 299-304.
22. Isom LL, Scheuru T, Brownstein AB, et al. Functional co-expression of the β_1 and type Ⅱ A α subunits of sodium channels in a mammalian cell line. J Biol Chem, 1995, 270: 3306-12.
23. Cook DJ, Housmans PR. Mechanism of the negative inotropic effect of propofol in isolated ferret ventricular myocardium. Anesthesiology, 1994, 80: 859-71.
24. Smith C, McEwan AI, Jhaveri R, et al. The interaction of fentanyl on the
Cp_(50) of propofol for loss of consciousness and skin incision. Anesthesiology, 1994, 81: 820-8.
25. Servin F, Desmonts JM, Haberer JP, et al. Pharmocokinetics and protein binding of propofol in patients with cirrhosis. Anesthesiology, 1988, 69: 887-91.
26. Dutta S, Ebling WF. Formulation-dependent brain and lung distribution kinetics of propofol in rats. Anesthesiology, 1998, 89: 678-85.
27. Shyr MH, Tsai TH, Tan PPC, et al. Concentration and regional distribution of propofol in brain and spinal cord during propofol anesthesia in the rat. Neurosci Lett, 1995, 184: 212-5.
28. Ratnakumari L, Vysotskaya TN, Duch DS, Hemmings HC. Differential effects of anesthetic and nonanesthetic cyelobutanes on neuronal voltage-gated sodium channels. Anesthesiology, 2000, 92: 529-41.
29．刘俊杰，赵俊主编．现代麻醉学，第1版，北京：人民卫生出版社，1987，205-8．
30. Berde CB, Strichartz GR. Local anesthetics. In Miller RD(ed): Anesthesia, 5th edition, Churchill Livingstone, 2000, 511.
31. Catterall WA. Common models of drug action on Na~+ channels: local anesthetics, antiarrhythmics and anticonvulsants. Trends Pharmacol. Sci., 1987, 8: 57-65.
32. Catterall WA. Structure and function of voltage-gated ion channels. Annu Rev Biochem, 1995, 64: 493-531.
33. Ragsdale D, Mcphee JC, Scheuer T. Molecular determinants of state-dependent block of Na~+ channels by local anesthetics. Science, 1994, 265: 1724-8.
34．俞卫锋主编．麻醉与复苏新论，第1版，上海：第二军医大学出版社，2001，256．