TREK-1双孔钾通道的药理学研究及其抗氧化损伤机制的探讨
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摘要
双孔钾通道是近年发现的一类新型钾通道超家族。目前发现的双孔钾通道共包括15个成员,可分为六类。目前研究最为广泛和深入的是TREK一类。这类双孔钾通道包括三个成员:TREK-1、TREK-2和TRAAK。
TREK-1钾通道高表达于人类中枢神经系统,在大小鼠体内也有表达。多项研究提示TREK-1通道在麻醉和神经保护两方面可能发挥作用,因而受到人们的广泛关注。研究表明,TREK-1可以被氟烷、异氟烷等挥发性麻醉剂和一些气体麻醉剂所激活。此外,作为一种非挥发性麻醉剂的水合氯醛,也可在临床使用浓度范围内开放TREK-1。这些研究提示,TREK-1可能与全身麻醉药的作用有关。但对于氯胺酮和依托咪酯等静脉麻醉剂的作用机理,尚未见报道。本实验中我们研究了这两种药物对稳定转染TREK-1的中国仓鼠卵巢细胞上钾电流的作用,探讨它们引起全麻作用的机理。
已有很多证据提示自由基介导的氧化应激是引起脑缺血再灌损伤,帕金森氏病和阿尔茨海默病的关键因素。基于以上的研究基础,我们分别在稳定转染了TREK-1双孔钾通道的中国仓鼠卵巢细胞(TREK-1/CHO)上和未转染的空白细胞上建立了三种氧化损伤模型,分别采用硝普钠、过氧化氢以及黄嘌呤/黄嘌呤氧化酶系统进行损伤,研究这类钾离子通道在氧化损伤中的作用,并进一步探讨了TREK-1在氧化损伤中的作用机制。
第一部分氯胺酮和依托咪酯对TREK-1双孔钾通道的作用
采用全细胞膜片钳技术,我们发现,氯胺酮和依托咪酯均能在临床使用浓度下开放TREK-1通道。氯胺酮可剂量依赖性地激活TREK-1/CHO细胞上的TREK-1钾通道。EC_(50)值为10±2.4μM。10μM氯胺酮可使TREK-1Ⅰ-Ⅴ曲线左移。依托咪酯亦可剂量依赖性地激活稳定转染CHO细胞上的TREK-1钾通道。EC_(50)为1.8±0.3μM。3gM依托咪酯可使TREK-1Ⅰ-Ⅴ曲线左移。氯胺酮和依托咪酯对TREK-1通道电压依赖性未见影响。
第二部分TREK-1双孔钾通道在氧化损伤中的作用
一、TREK-1双孔钾通道在硝普钠引起的氧化损伤中的作用
硝普钠分别与CHO细胞和TREK-1/CHO细胞共孵育12小时,MTT比色实验测定细胞存活率。结果发现,在0.1—5mM剂量范围内,硝普钠对两种细胞的损伤均呈现量效关系。换算为细胞存活抑制率后,CHO细胞的IC_(50)为0.42mM;TREK-1/CHO细胞的IC_(50)值为0.67mM。选择0.75mM为最佳损伤剂量。
5μM、10μM、100μM、200μM的氯胺酮可以提高TREK-1/CHO损伤组的存活率,中间两组(10μM、100μM)与损伤组相比,有显著性差异(p＜0.05)。10μM、100μM氯胺酮的保护作用可以被100μM奎尼丁部分抑制(p＜0.05)。200μM的氯胺酮可以降低CHO细胞损伤组的存活率(p＜0.05)。
5μM、10μM、20μM的依托咪酯可以提高TREK-1/CHO损伤组的存活率,后两组与损伤组相比,有显著性差异(p＜0.05)。10μM、20μM依托咪酯的保护作用可以被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,药物对其无影响。
氟烷(终浓度为0.1、0.4、0.8、3.3mM)可以提高TREK-1/CHO损伤组的存活率,中间两组(0.4、0.8 mM)与损伤组相比,有显著性差异(p＜0.05)。0.4、0.8 mM氟烷的保护作用可以被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,3.3mM的氟烷可降低细胞的存活率(p＜0.05),低浓度的药物则对其无影响。
异氟烷(终浓度为0.1、0.4、0.8、3.1mM)可以提高TREK-1/CHO损伤组的存活率,前三组与损伤组相比,有显著性差异(p＜0.05)。0.4、0.8 mM异氟烷的保护作用可被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,3.1 mM的异氟烷可降低细胞的存活率(p＜0.05),低浓度的药物则对其无影响。
Hoechst 33342染色表明,SNP可以诱导两种细胞的凋亡。TREK-1/CHO细胞损伤组与对照组相比,凋亡率从5%增加至17.2%(P＜0.05),其凋亡率增加了244%;CHO细胞损伤组与对照组相比,凋亡率从6.2%增加至29.3%(P＜0.05),其凋亡率增加了388%。两者相比,有显著性差异(P＜0.05)。
Hoechst 33342染色亦表明,对硝普钠损伤的TREK-1/CHO细胞,0.1、0.4、0.8mM氟烷可降低其凋亡比率,后两组与损伤组相比,有显著性差异(P＜0.05)。0.8mM氟烷的保护作用可以被100μM奎尼丁部分抑制(P＜0.05)。0.1、0.4、0.8mM异氟烷可降低TREK-1/CHO细胞损伤组的凋亡发生率,与损伤组相比,有显著性差异(P＜0.05)。0.4、0.8mM异氟烷的保护作用可以被100μM奎尼丁部分抑制(P＜0.05)。对CHO细胞,0.1、0.4、0.8mM氟烷和异氟烷未见保护作用。
二、TREK-1双孔钾通道在过氧化氢引起的氧化损伤中的作用
过氧化氢分别与CHO细胞和TREK-1/CHO细胞共孵育12小时,MTT比色实验测定细胞存活率。结果发现,在0.01-0.5mM剂量范围内,过氧化氢对两种细胞的损伤均呈现量效关系。换算为细胞存活抑制率后,CHO细胞IC_(50)值为0.05mM;TREK-1/CHO细胞IC_(50)值为0.09mM。选择0.05mM为损伤剂量。
5μM、10μM、100μM、200μM的氯胺酮可以提高TREK-1/CHO损伤组的存活率,中间两组(10μM、100μM)与损伤组相比,有显著性差异(p＜0.05)。10μM、100μM的氯胺酮的保护作用可被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,200μM的氯胺酮亦可降低细胞的存活率(p＜0.05),低浓度的药物对其无影响。
5μM、10μM、20μM的依托咪酯可以提高TREK-1/CHO损伤组的存活率,后两组与损伤组相比,有显著性差异(p＜0.05)。20μM依托咪酯保护作用可被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,药物对其无影响。
氟烷(终浓度为0.1、0.4、0.8、3.3mM)可以提高TREK-1/CHO损伤组的存活率,各组与损伤组相比,有显著性差异(p＜0.05)。0.1、0.4、0.8mM氟烷的保护作用可被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,3.3mM的氟烷可降低细胞的存活率(p＜0.05),低浓度的药物则对其无影响。
异氟烷(终浓度为0.1、0.4、0.8、3.1 mM)可以提高TREK-1/CHO损伤组的存活率,中间两组(0.4、0.8mM)与损伤组相比,有显著性差异(p＜0.05)。0.8mM异氟烷的保护作用可被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,3.1mM的异氟烷可降低细胞的存活率(p＜0.05),低浓度的药物则对其无影响。
Hoechst 33342染色表明,过氧化氢可以诱导两种细胞的凋亡。TREK-1/CHO细胞损伤组与对照组相比,凋亡率从5.5%增加至15.6%(P＜0.05),其凋亡率增加了183.6%;CHO细胞损伤组与对照组相比,凋亡率从6%增加至22.3%(P＜0.05),其凋亡率增加了271.7%。两者相比,有显著性差异(P＜0.05)。
Hoechst 33342染色表明,5、10、20μM依托咪酯可分别降低TREK-1/CHO细胞的凋亡率,后两组与损伤组相比,有显著性差异(P＜0.05);10、20μM依托咪酯的保护作用可被100μM奎尼丁部分抑制(P＜0.05)。0.1、0.4、0.8mM异氟烷可降低凋亡率,后两组与损伤组相比,有显著性差异(P＜0.05)。0.4、0.8mM异氟烷的保护作用可被100μM奎尼丁部分抑制(P＜0.05)。对CHO细胞,同样浓度的依托咪酯和异氟烷未见保护作用。
三、TREK-1双孔钾通道在黄嘌呤/黄嘌呤氧化酶体系引起的氧化损伤中的作用
固定X终浓度为0.25mM,结果表明XO在2.5—10U/L剂量范围内,作用12小时,对CHO和TREK-1/CHO两种细胞的损伤呈现明显的量效关系。CHO细胞IC_(50)值为5.7 U/L;TREK-1/CHO细胞IC_(50)值为6.4 U/L,选择5 U/LXO为损伤剂量。
5μM、10μM、100μM、200μM的氯胺酮可以提高TREK-1/CHO损伤组的存活率,中间两组(10μM、100μM)与损伤组相比,有显著性差异(p＜0.05)。100μM氯胺酮的保护作用均可被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,200μM的氯胺酮可降低细胞的存活率(p＜0.05),低浓度的药物对其无影响。
1μM、5μM、10μM、20μM的依托咪酯可以提高TREK-1/CHO损伤组的存活率,后三组与损伤组相比,有显著性差异(p＜0.05)。10μM、20μM依托咪酯的保护作用可被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,药物对其无影响。
氟烷(终浓度为0.1、0.4、0.8、3.3mM)可以提高TREK-1/CHO损伤组的存活率,后三组与损伤组相比,有显著性差异(p＜0.05)。0.8、3.3mM氟烷的保护作用均可被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,3.3 mM的氟烷可降低细胞的存活率(p＜0.05),低浓度的药物则对其无影响。
异氟烷(终浓度为0.1、0.4、0.8、3.1mM)可以提高TREK-1/CHO损伤组的存活率,中间两组(0.4、0.8 mM)与损伤组相比,有显著性差异(p＜0.05)。0.4、0.8 mM异氟烷的保护作用均可被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,3.1 mM的氟烷可降低细胞的存活率(p＜0.05),低浓度的药物则对其无影响。
从以上结果可以得出以下结论:
1.氯胺酮和依托咪酯可以在临床使用浓度下开放TREK-1,该作用可能是两种药物发挥麻醉作用的机制之一,提示TREK-1通道可能是全身静脉麻醉剂的靶点。
2.与CHO细胞比较,转染和高表达TREK-1双孔钾通道的CHO细胞具有较强的抗氧化损伤能力。
3.全身静脉麻醉剂氯胺酮和依托咪酯以及挥发性麻醉剂氟烷和异氟烷能够进一步加强TREK-1/CHO细胞的抗氧化损伤能力,可以使细胞的存活率提高,凋亡率降低。
4.非选择性TREK-1阻断剂奎尼丁可部分抑制TREK-1的激动剂引起的细胞保护作用。以上结果表明TREK-1双孔钾通道与抗氧化损伤有关,并证明它是脑保护作用的又一个药物新靶点。
Background and purpose
TREK-1 is a two-pore-domain potassium channel expressed highly in the human and mammalian central nervous systems and has been proposed to play an important role in general anesthesia.Previous studies have shown that TREK-1 can be activated by several anesthetic agents such as halothane,nitrious oxide, choloroform.However,whether ketamine and etomide affect TREK-1 channel is not characterized.Here we investigated the action of ketamine and etomidate on TREK-1 channel activity.Moreover,TREK-1 has also been proposed to play an important role in neuroprotection.Considering the importance of oxidative stress and damage induced by free radicals in many diseases such as Alzheimer's disease, Parkinson's disease,we thus investigated the possible role of TREK-1 in oxidative stress and damage induced by SNP,H_2O_2 and X/XO system.
Methods
Membrane currents were recorded with the use of whole-cell voltage-clamp recordings in Chinese hamster ovary(CHO) cells stably expressing TREK-1.Cell viability was detected by MTT method.Apoptosis were detected by Hoechst33342 staining with fluorescent microscopy.
Results
Both ketamine and etomidate can enhance the currents passed in Chinese hamster ovary(CHO) cells stably expressing TREK-1.Clinically relevant concentrations of ketamine increased outward currents with an EC_(50) of 10μM, whereas etomidate can enhance the channel activity with an EC_(50) of 1.8μM.In the study of CHO and TREK-1/CHO cells after exposure to SNP,H_2O_2 and X/XO system respectively,we find that TREK-1/CHO cells have higher viability rate than CHO cells.As TREK-1 openers,ketamine,etomidate,halothane and isoflurane can improve the cell viability of CHO cells stably expressing TREK-1 after exposure to SNP,H_2O_2 and X/XO.No similar effects were found in nontransfected CHO cells.
Conclusion
1.Ketamine and etomidate can open the TREK-1 channel in clinically relevant concentrations,which suggested that TREK-1 channel may play a role in the anesthetic process of the two intravenous general anesthetics.
2.Overexpression of TREK-1 potassium channel in the Chinese hamster ovary cells have an anti-oxidative effect,which indicted that TREK-1 channel might play an important role in the anti-oxidative effect.
3.The study demonstrated that halothane and isoflurane,which can open the TREK-1 channel,can enhance the anti-oxidative ability of TREK-1/CHO cells. These two drugs may play a neuroprotective role,at least in part,by opening TREK-1 channel.Meanwhile,ketamine and etomidate,which can also activate TREK-1 channel,can enhance the anti-oxidative ability of TREK-1/CHO cells.The four anesthetics can increase the cell viability and reduce the apoptosis rate.
4.Quinidine can inhibit the anti-oxidative effect by TREK-1 openers. Conclusively,it suggested that TREK-1 channel might play a role in anti-oxidation.
TREK-1钾通道高表达于人类中枢神经系统,在大小鼠体内也有表达。多项研究提示TREK-1通道在麻醉和神经保护两方面可能发挥作用,因而受到人们的广泛关注。研究表明,TREK-1可以被氟烷、异氟烷等挥发性麻醉剂和一些气体麻醉剂所激活。此外,作为一种非挥发性麻醉剂的水合氯醛,也可在临床使用浓度范围内开放TREK-1。这些研究提示,TREK-1可能与全身麻醉药的作用有关。但对于氯胺酮和依托咪酯等静脉麻醉剂的作用机理,尚未见报道。本实验中我们研究了这两种药物对稳定转染TREK-1的中国仓鼠卵巢细胞上钾电流的作用,探讨它们引起全麻作用的机理。
已有很多证据提示自由基介导的氧化应激是引起脑缺血再灌损伤,帕金森氏病和阿尔茨海默病的关键因素。基于以上的研究基础,我们分别在稳定转染了TREK-1双孔钾通道的中国仓鼠卵巢细胞(TREK-1/CHO)上和未转染的空白细胞上建立了三种氧化损伤模型,分别采用硝普钠、过氧化氢以及黄嘌呤/黄嘌呤氧化酶系统进行损伤,研究这类钾离子通道在氧化损伤中的作用,并进一步探讨了TREK-1在氧化损伤中的作用机制。
第一部分氯胺酮和依托咪酯对TREK-1双孔钾通道的作用
采用全细胞膜片钳技术,我们发现,氯胺酮和依托咪酯均能在临床使用浓度下开放TREK-1通道。氯胺酮可剂量依赖性地激活TREK-1/CHO细胞上的TREK-1钾通道。EC_(50)值为10±2.4μM。10μM氯胺酮可使TREK-1Ⅰ-Ⅴ曲线左移。依托咪酯亦可剂量依赖性地激活稳定转染CHO细胞上的TREK-1钾通道。EC_(50)为1.8±0.3μM。3gM依托咪酯可使TREK-1Ⅰ-Ⅴ曲线左移。氯胺酮和依托咪酯对TREK-1通道电压依赖性未见影响。
第二部分TREK-1双孔钾通道在氧化损伤中的作用
一、TREK-1双孔钾通道在硝普钠引起的氧化损伤中的作用
硝普钠分别与CHO细胞和TREK-1/CHO细胞共孵育12小时,MTT比色实验测定细胞存活率。结果发现,在0.1—5mM剂量范围内,硝普钠对两种细胞的损伤均呈现量效关系。换算为细胞存活抑制率后,CHO细胞的IC_(50)为0.42mM;TREK-1/CHO细胞的IC_(50)值为0.67mM。选择0.75mM为最佳损伤剂量。
5μM、10μM、100μM、200μM的氯胺酮可以提高TREK-1/CHO损伤组的存活率,中间两组(10μM、100μM)与损伤组相比,有显著性差异(p＜0.05)。10μM、100μM氯胺酮的保护作用可以被100μM奎尼丁部分抑制(p＜0.05)。200μM的氯胺酮可以降低CHO细胞损伤组的存活率(p＜0.05)。
5μM、10μM、20μM的依托咪酯可以提高TREK-1/CHO损伤组的存活率,后两组与损伤组相比,有显著性差异(p＜0.05)。10μM、20μM依托咪酯的保护作用可以被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,药物对其无影响。
氟烷(终浓度为0.1、0.4、0.8、3.3mM)可以提高TREK-1/CHO损伤组的存活率,中间两组(0.4、0.8 mM)与损伤组相比,有显著性差异(p＜0.05)。0.4、0.8 mM氟烷的保护作用可以被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,3.3mM的氟烷可降低细胞的存活率(p＜0.05),低浓度的药物则对其无影响。
异氟烷(终浓度为0.1、0.4、0.8、3.1mM)可以提高TREK-1/CHO损伤组的存活率,前三组与损伤组相比,有显著性差异(p＜0.05)。0.4、0.8 mM异氟烷的保护作用可被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,3.1 mM的异氟烷可降低细胞的存活率(p＜0.05),低浓度的药物则对其无影响。
Hoechst 33342染色表明,SNP可以诱导两种细胞的凋亡。TREK-1/CHO细胞损伤组与对照组相比,凋亡率从5%增加至17.2%(P＜0.05),其凋亡率增加了244%;CHO细胞损伤组与对照组相比,凋亡率从6.2%增加至29.3%(P＜0.05),其凋亡率增加了388%。两者相比,有显著性差异(P＜0.05)。
Hoechst 33342染色亦表明,对硝普钠损伤的TREK-1/CHO细胞,0.1、0.4、0.8mM氟烷可降低其凋亡比率,后两组与损伤组相比,有显著性差异(P＜0.05)。0.8mM氟烷的保护作用可以被100μM奎尼丁部分抑制(P＜0.05)。0.1、0.4、0.8mM异氟烷可降低TREK-1/CHO细胞损伤组的凋亡发生率,与损伤组相比,有显著性差异(P＜0.05)。0.4、0.8mM异氟烷的保护作用可以被100μM奎尼丁部分抑制(P＜0.05)。对CHO细胞,0.1、0.4、0.8mM氟烷和异氟烷未见保护作用。
二、TREK-1双孔钾通道在过氧化氢引起的氧化损伤中的作用
过氧化氢分别与CHO细胞和TREK-1/CHO细胞共孵育12小时,MTT比色实验测定细胞存活率。结果发现,在0.01-0.5mM剂量范围内,过氧化氢对两种细胞的损伤均呈现量效关系。换算为细胞存活抑制率后,CHO细胞IC_(50)值为0.05mM;TREK-1/CHO细胞IC_(50)值为0.09mM。选择0.05mM为损伤剂量。
5μM、10μM、100μM、200μM的氯胺酮可以提高TREK-1/CHO损伤组的存活率,中间两组(10μM、100μM)与损伤组相比,有显著性差异(p＜0.05)。10μM、100μM的氯胺酮的保护作用可被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,200μM的氯胺酮亦可降低细胞的存活率(p＜0.05),低浓度的药物对其无影响。
5μM、10μM、20μM的依托咪酯可以提高TREK-1/CHO损伤组的存活率,后两组与损伤组相比,有显著性差异(p＜0.05)。20μM依托咪酯保护作用可被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,药物对其无影响。
氟烷(终浓度为0.1、0.4、0.8、3.3mM)可以提高TREK-1/CHO损伤组的存活率,各组与损伤组相比,有显著性差异(p＜0.05)。0.1、0.4、0.8mM氟烷的保护作用可被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,3.3mM的氟烷可降低细胞的存活率(p＜0.05),低浓度的药物则对其无影响。
异氟烷(终浓度为0.1、0.4、0.8、3.1 mM)可以提高TREK-1/CHO损伤组的存活率,中间两组(0.4、0.8mM)与损伤组相比,有显著性差异(p＜0.05)。0.8mM异氟烷的保护作用可被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,3.1mM的异氟烷可降低细胞的存活率(p＜0.05),低浓度的药物则对其无影响。
Hoechst 33342染色表明,过氧化氢可以诱导两种细胞的凋亡。TREK-1/CHO细胞损伤组与对照组相比,凋亡率从5.5%增加至15.6%(P＜0.05),其凋亡率增加了183.6%;CHO细胞损伤组与对照组相比,凋亡率从6%增加至22.3%(P＜0.05),其凋亡率增加了271.7%。两者相比,有显著性差异(P＜0.05)。
Hoechst 33342染色表明,5、10、20μM依托咪酯可分别降低TREK-1/CHO细胞的凋亡率,后两组与损伤组相比,有显著性差异(P＜0.05);10、20μM依托咪酯的保护作用可被100μM奎尼丁部分抑制(P＜0.05)。0.1、0.4、0.8mM异氟烷可降低凋亡率,后两组与损伤组相比,有显著性差异(P＜0.05)。0.4、0.8mM异氟烷的保护作用可被100μM奎尼丁部分抑制(P＜0.05)。对CHO细胞,同样浓度的依托咪酯和异氟烷未见保护作用。
三、TREK-1双孔钾通道在黄嘌呤/黄嘌呤氧化酶体系引起的氧化损伤中的作用
固定X终浓度为0.25mM,结果表明XO在2.5—10U/L剂量范围内,作用12小时,对CHO和TREK-1/CHO两种细胞的损伤呈现明显的量效关系。CHO细胞IC_(50)值为5.7 U/L;TREK-1/CHO细胞IC_(50)值为6.4 U/L,选择5 U/LXO为损伤剂量。
5μM、10μM、100μM、200μM的氯胺酮可以提高TREK-1/CHO损伤组的存活率,中间两组(10μM、100μM)与损伤组相比,有显著性差异(p＜0.05)。100μM氯胺酮的保护作用均可被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,200μM的氯胺酮可降低细胞的存活率(p＜0.05),低浓度的药物对其无影响。
1μM、5μM、10μM、20μM的依托咪酯可以提高TREK-1/CHO损伤组的存活率,后三组与损伤组相比,有显著性差异(p＜0.05)。10μM、20μM依托咪酯的保护作用可被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,药物对其无影响。
氟烷(终浓度为0.1、0.4、0.8、3.3mM)可以提高TREK-1/CHO损伤组的存活率,后三组与损伤组相比,有显著性差异(p＜0.05)。0.8、3.3mM氟烷的保护作用均可被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,3.3 mM的氟烷可降低细胞的存活率(p＜0.05),低浓度的药物则对其无影响。
异氟烷(终浓度为0.1、0.4、0.8、3.1mM)可以提高TREK-1/CHO损伤组的存活率,中间两组(0.4、0.8 mM)与损伤组相比,有显著性差异(p＜0.05)。0.4、0.8 mM异氟烷的保护作用均可被100μM奎尼丁部分抑制(p＜0.05)。对CHO细胞,3.1 mM的氟烷可降低细胞的存活率(p＜0.05),低浓度的药物则对其无影响。
从以上结果可以得出以下结论:
1.氯胺酮和依托咪酯可以在临床使用浓度下开放TREK-1,该作用可能是两种药物发挥麻醉作用的机制之一,提示TREK-1通道可能是全身静脉麻醉剂的靶点。
2.与CHO细胞比较,转染和高表达TREK-1双孔钾通道的CHO细胞具有较强的抗氧化损伤能力。
3.全身静脉麻醉剂氯胺酮和依托咪酯以及挥发性麻醉剂氟烷和异氟烷能够进一步加强TREK-1/CHO细胞的抗氧化损伤能力,可以使细胞的存活率提高,凋亡率降低。
4.非选择性TREK-1阻断剂奎尼丁可部分抑制TREK-1的激动剂引起的细胞保护作用。以上结果表明TREK-1双孔钾通道与抗氧化损伤有关,并证明它是脑保护作用的又一个药物新靶点。
Background and purpose
TREK-1 is a two-pore-domain potassium channel expressed highly in the human and mammalian central nervous systems and has been proposed to play an important role in general anesthesia.Previous studies have shown that TREK-1 can be activated by several anesthetic agents such as halothane,nitrious oxide, choloroform.However,whether ketamine and etomide affect TREK-1 channel is not characterized.Here we investigated the action of ketamine and etomidate on TREK-1 channel activity.Moreover,TREK-1 has also been proposed to play an important role in neuroprotection.Considering the importance of oxidative stress and damage induced by free radicals in many diseases such as Alzheimer's disease, Parkinson's disease,we thus investigated the possible role of TREK-1 in oxidative stress and damage induced by SNP,H_2O_2 and X/XO system.
Methods
Membrane currents were recorded with the use of whole-cell voltage-clamp recordings in Chinese hamster ovary(CHO) cells stably expressing TREK-1.Cell viability was detected by MTT method.Apoptosis were detected by Hoechst33342 staining with fluorescent microscopy.
Results
Both ketamine and etomidate can enhance the currents passed in Chinese hamster ovary(CHO) cells stably expressing TREK-1.Clinically relevant concentrations of ketamine increased outward currents with an EC_(50) of 10μM, whereas etomidate can enhance the channel activity with an EC_(50) of 1.8μM.In the study of CHO and TREK-1/CHO cells after exposure to SNP,H_2O_2 and X/XO system respectively,we find that TREK-1/CHO cells have higher viability rate than CHO cells.As TREK-1 openers,ketamine,etomidate,halothane and isoflurane can improve the cell viability of CHO cells stably expressing TREK-1 after exposure to SNP,H_2O_2 and X/XO.No similar effects were found in nontransfected CHO cells.
Conclusion
1.Ketamine and etomidate can open the TREK-1 channel in clinically relevant concentrations,which suggested that TREK-1 channel may play a role in the anesthetic process of the two intravenous general anesthetics.
2.Overexpression of TREK-1 potassium channel in the Chinese hamster ovary cells have an anti-oxidative effect,which indicted that TREK-1 channel might play an important role in the anti-oxidative effect.
3.The study demonstrated that halothane and isoflurane,which can open the TREK-1 channel,can enhance the anti-oxidative ability of TREK-1/CHO cells. These two drugs may play a neuroprotective role,at least in part,by opening TREK-1 channel.Meanwhile,ketamine and etomidate,which can also activate TREK-1 channel,can enhance the anti-oxidative ability of TREK-1/CHO cells.The four anesthetics can increase the cell viability and reduce the apoptosis rate.
4.Quinidine can inhibit the anti-oxidative effect by TREK-1 openers. Conclusively,it suggested that TREK-1 channel might play a role in anti-oxidation.
引文
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11.Patel,A.J.et al.Lipid and mechano-gated 2P domain K~+ channels.Curr.Opin.Cell Biol.2001,13:422-428.
12.Maingret,F.et al.TREK-1 is a heat-activated background K~+ channel.EMBO J.2000,19:2483-2491.
13 Maingret,F.et al.Mechano- or acid stimulation,two interactive modes of activation of the TREK-1 potassium channel.J.Biol.Chem.1999,274:26691-26696.
14 Honore',E.et al.An intracellular proton sensor commands lipid- and mechano-gating of the KC channel TREK-1.EMBO J.2002,21:2968-2976.
15 Lesage,F.et al.Human TREK2,a 2P domain mechanosensitive K~+ channel with multiple regulations by polyunsaturated fatty acids,lysophospholipids,and Gs,Gi,and Gq protein-coupled receptors.J.Biol.Chem.2000,275:28398-28405.
16.Kim,Y.et al.Synergistic interaction and the role of C-terminus in the activation of TRAAK K~+ channels by pressure,free fatty acids and alkali.Pflugers Arch.2001,442:64-72.
17.Kim,Y.et al.Localization of TREK-2 K~+ channel domains that regulate channel kinetics and sensitivity to pressure,fatty acids and phi.Pflugers Arch.2001,442:952-960.
18.Fink,M.et al.A neuronal two P domain K~+ channel activated by arachidonic acid and polyunsaturated fatty acid.EMBO J.1998,17:3297-3308.
19.Maingret,E et al.Molecular basis of the voltage-dependent gating of TREK-1,a mechano-sensitive K~+ channel.Biochem.Biophys.Res.Commun.2002,292:339-346.
20.Duprat,F.et al.The neuroprotective agent riluzole activates the two P domain K~+ channels TREK-1 and TRAAK.Mol.Pharmacol.2000,57:906-912.
21.Meadows,H.et al.The neuroprotective agent sipatrigine(BW619C89) potently inhibits the human tandem pore-domain K~+ channels TREK-1 and TRAAK.Brain Res.2001,892:94-101.
23.Maingret,E et al.Lysophospholipids open the two P domain mechano-gated K~+ channels TREK-1 and TRAAK.J.Biol.Chem.2000,275:10128-10133.
24.Enyeart,J.J.et al.An ACTH- and ATP-regulated background K~+ channel in adrenocortical cells is TREK-1.J.Biol.Chem.2002,277:49186-49199.
25.Punke,M.A.et al.Inhibition of human TREK-1 channels by bupivacaine.Anesth.Analg.2003,96:1665-1673.
26.Bockenhauer,D.et al.KCNK2:reversible conversion of a hippocampal potassium leak into a voltage-dependent channel.Nat.Neurosci.2001,4:486-491.
27.Koh,S.D.et al.TREK-1 regulation by nitric oxide and cGMP-dependent protein kinase.J. Biol.Chem.2001,276:44338-44346.
28.Miller,P.et al.Acute hypoxia occludes hTREK-1 modulation:re-evaluation of the potential role of tandem P domain K~+ channels in central neuroprotection.J.Physiol.2003,548:31-37.
29.Patel,A.J.et al.Inhalational anaesthetics activate two-poredomain background K~+channels.Nat.Neurosci.1999,2:422-426.
30.Harinath,S.and Sikdar,S.K.Trichloroethanol enhances the activity of recombinant human TREK-1 and TRAAK channels.Neuropharmacology.2004,46:750-760.
31.Winegar,B.D.et al.Volatile general anesthetics produce hyperpolarization of Aplysia neurons by activation of a discrete population of baseline potassium channels.Anesthesiology.1996,85:889-900.
32 Franks,N.E et al.How does xenon produce anaesthesia? Nature 1998,396,324.
33.Jevtovic-Todorovic,V.et al.Nitrous oxide(laughing gas) is an NMDA antagonist,neuroprotectant and neurotoxin.Nat.Med.1998,4:460-463.
34.Gruss,M.et al.Two:pore-domain K~+ channels are a novel target for the anesthetic gases xenon,nitrous oxide,and cyclopropane.Mol.Pharmacol.2004,65:443-452.
35.Xing,Y.et al.The use of the potassium channel activator riluzole to test whether potassium channels mediate the capacity of isoflurane to produce immobility.Anesth.Analg.2003,97:1020-1024.
36.Mantz,J.et al.Anesthetic properties of riluzole(54274 RP),a new inhibitor of glutamate neurotransmission.Anesthesiology.1992,76:844-848.
37.Heurteaux,C.et al.TREK-l,a K~+ channel involved in neuroprotection and general anesthesia.EMBO J.2004,23:2684-2695.
38.Franks,N.R and Lieb,W.R.Molecular and cellular mechanisms of general anaesthesia.Nature.1994,367:607-614.
39.Patel,A.J.and Honore',E.Anesthetic-sensitive 2P domain K~+ channels.Anesthesiology.2001,95:1013-1025
40.Gray,A.T.et al.TOK1 is a volatile anesthetic stimulated K~+ channel.Anesthesiology.1998,88:1076-1084.
41.Franks, N.P. and Lieb, W.R. Volatile general anaesthetics activate a novel neuronal K~+ current. Nature 1988, 333: 662-664.
42.Gray, A.T. et al. Volatile anesthetics activate the human tandem pore domain baseline K~+ channel KCNK5.Anesthesiology. 2000, 92: 1722-1730.
43.Talley, E.M. and Bayliss, D.A. Modulation of TASK-1 (Kcnk3) and TASK-3 (Kcnk9) potassium channels: volatile anesthetics and neurotransmitters share a molecular site of action. J. Biol. Chem. 2002, 277: 17733-17742
44. Sirois, J.E. et al. The TASK-1 two-pore domain K~+ channel is a molecular substrate for neuronal effects of inhalational anesthetics. J. Neurosci. 2000, 20: 6347-6354.
45. Meadows, H.J. and Randall, A.D. Functional characterization of human TASK-3, anacid-sensitive two-pore domain potassium channel. Neuropharmacology. 2001, 40: 551—559.
46. Gerstin, K.M. et al. Mutation of KCNK5 or Kir3.2 potassium channels in mice does not change minimum alveolar anesthetic concentration. Anesth. Analg. 2003, 96: 1345-1349.
47. Lauritzen, 1. et al. Poly-unsaturated fatty acids are potent neuroprotectors. EMBO J. 2000,19: 1784-1793.
48. Lang-Lazdunski, L. et al. Linolenic acid prevents neuronal cell death and paraplegia after transient spinal cord ischemia in rats. J. Vasc. Surg. 2003, 38: 564-575.
49. Blondeau, N. et al. Polyunsaturated fatty acids induce ischemic and epileptic tolerance.Neuroscience. 2002, 109: 231-241.
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1.方允中,郑荣梁。 《自由基生物学的理论与应用》
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1.Nicholas R Franks and Eric Honore.The TREK K_(2p) channels and their role in general anaesthesia and neuroprotection.Trend in Pharmacological Sciences.2004,25:601-608.
2.Amanda J.Patel and Eric Honore.Properties and modulation of mammalian 2P domain K+channels.Trend in Neurosciences.2001,24,339-346.
3.Medhurst,A.D.et al.Distribution analysis of human two pore domain potassium channels in tissues of the central nervous systerm and periphery.Brain Research and Molecular Brain Research.86,101-114.
4.Hervieu,G.J.et al.Distribution and expression of TREK-1,a two-pore-domain potassium channel,in the adult rat CNS.Neuroscience.103:899-919.
5.Edmund M.Talley et al.CNS Distribution of Members of the two-pore-domain(KCNK)Potassium Channel Family.J.Neuroscience.2001,21:7491-7505.
6.Patel,A.J.and Honore,E.Properties and modulation of mammalian 2P domain K~+channels.Trends Neurosci.1999.24:422-426.
7.Maingret,F et al.Lysophospholipids open the two P domain mechano-gated K~+ channels TREK-1 and TRAAK.J.Biol.Chem.2000,275:10128-10133.
8.Koh,S.D.etal.TREK-1 regulation by nitric oxide and cGMP-dependent protein kinase.J.Biol.Chem 2001,276,44338-44346.
9.Patel,A.J.et al.A mammalian two pore domain mechano-gated S-like K~+ channel.EMBO J.1998,17:4283-4290.
10.Maingret,F.et al.TRAAK is a mammalian neuronal mechanogated K~+ channel.J.Biol.Chem.1999,274:1381-1387.
11.Patel,A.J.et al.Lipid and mechano-gated 2P domain K~+ channels.Curr.Opin.Cell Biol.2001,13:422-428.
12.Maingret,F.et al.TREK-1 is a heat-activated background K~+ channel.EMBO J.2000,19:2483-2491.
13 Maingret,F.et al.Mechano- or acid stimulation,two interactive modes of activation of the TREK-1 potassium channel.J.Biol.Chem.1999,274:26691-26696.
14 Honore',E.et al.An intracellular proton sensor commands lipid- and mechano-gating of the KC channel TREK-1.EMBO J.2002,21:2968-2976.
15 Lesage,F.et al.Human TREK2,a 2P domain mechanosensitive K~+ channel with multiple regulations by polyunsaturated fatty acids,lysophospholipids,and Gs,Gi,and Gq protein-coupled receptors.J.Biol.Chem.2000,275:28398-28405.
16.Kim,Y.et al.Synergistic interaction and the role of C-terminus in the activation of TRAAK K~+ channels by pressure,free fatty acids and alkali.Pflugers Arch.2001,442:64-72.
17.Kim,Y.et al.Localization of TREK-2 K~+ channel domains that regulate channel kinetics and sensitivity to pressure,fatty acids and phi.Pflugers Arch.2001,442:952-960.
18.Fink,M.et al.A neuronal two P domain K~+ channel activated by arachidonic acid and polyunsaturated fatty acid.EMBO J.1998,17:3297-3308.
19.Maingret,E et al.Molecular basis of the voltage-dependent gating of TREK-1,a mechano-sensitive K~+ channel.Biochem.Biophys.Res.Commun.2002,292:339-346.
20.Duprat,F.et al.The neuroprotective agent riluzole activates the two P domain K~+ channels TREK-1 and TRAAK.Mol.Pharmacol.2000,57:906-912.
21.Meadows,H.et al.The neuroprotective agent sipatrigine(BW619C89) potently inhibits the human tandem pore-domain K~+ channels TREK-1 and TRAAK.Brain Res.2001,892:94-101.
23.Maingret,E et al.Lysophospholipids open the two P domain mechano-gated K~+ channels TREK-1 and TRAAK.J.Biol.Chem.2000,275:10128-10133.
24.Enyeart,J.J.et al.An ACTH- and ATP-regulated background K~+ channel in adrenocortical cells is TREK-1.J.Biol.Chem.2002,277:49186-49199.
25.Punke,M.A.et al.Inhibition of human TREK-1 channels by bupivacaine.Anesth.Analg.2003,96:1665-1673.
26.Bockenhauer,D.et al.KCNK2:reversible conversion of a hippocampal potassium leak into a voltage-dependent channel.Nat.Neurosci.2001,4:486-491.
27.Koh,S.D.et al.TREK-1 regulation by nitric oxide and cGMP-dependent protein kinase.J. Biol.Chem.2001,276:44338-44346.
28.Miller,P.et al.Acute hypoxia occludes hTREK-1 modulation:re-evaluation of the potential role of tandem P domain K~+ channels in central neuroprotection.J.Physiol.2003,548:31-37.
29.Patel,A.J.et al.Inhalational anaesthetics activate two-poredomain background K~+channels.Nat.Neurosci.1999,2:422-426.
30.Harinath,S.and Sikdar,S.K.Trichloroethanol enhances the activity of recombinant human TREK-1 and TRAAK channels.Neuropharmacology.2004,46:750-760.
31.Winegar,B.D.et al.Volatile general anesthetics produce hyperpolarization of Aplysia neurons by activation of a discrete population of baseline potassium channels.Anesthesiology.1996,85:889-900.
32 Franks,N.E et al.How does xenon produce anaesthesia? Nature 1998,396,324.
33.Jevtovic-Todorovic,V.et al.Nitrous oxide(laughing gas) is an NMDA antagonist,neuroprotectant and neurotoxin.Nat.Med.1998,4:460-463.
34.Gruss,M.et al.Two:pore-domain K~+ channels are a novel target for the anesthetic gases xenon,nitrous oxide,and cyclopropane.Mol.Pharmacol.2004,65:443-452.
35.Xing,Y.et al.The use of the potassium channel activator riluzole to test whether potassium channels mediate the capacity of isoflurane to produce immobility.Anesth.Analg.2003,97:1020-1024.
36.Mantz,J.et al.Anesthetic properties of riluzole(54274 RP),a new inhibitor of glutamate neurotransmission.Anesthesiology.1992,76:844-848.
37.Heurteaux,C.et al.TREK-l,a K~+ channel involved in neuroprotection and general anesthesia.EMBO J.2004,23:2684-2695.
38.Franks,N.R and Lieb,W.R.Molecular and cellular mechanisms of general anaesthesia.Nature.1994,367:607-614.
39.Patel,A.J.and Honore',E.Anesthetic-sensitive 2P domain K~+ channels.Anesthesiology.2001,95:1013-1025
40.Gray,A.T.et al.TOK1 is a volatile anesthetic stimulated K~+ channel.Anesthesiology.1998,88:1076-1084.
41.Franks, N.P. and Lieb, W.R. Volatile general anaesthetics activate a novel neuronal K~+ current. Nature 1988, 333: 662-664.
42.Gray, A.T. et al. Volatile anesthetics activate the human tandem pore domain baseline K~+ channel KCNK5.Anesthesiology. 2000, 92: 1722-1730.
43.Talley, E.M. and Bayliss, D.A. Modulation of TASK-1 (Kcnk3) and TASK-3 (Kcnk9) potassium channels: volatile anesthetics and neurotransmitters share a molecular site of action. J. Biol. Chem. 2002, 277: 17733-17742
44. Sirois, J.E. et al. The TASK-1 two-pore domain K~+ channel is a molecular substrate for neuronal effects of inhalational anesthetics. J. Neurosci. 2000, 20: 6347-6354.
45. Meadows, H.J. and Randall, A.D. Functional characterization of human TASK-3, anacid-sensitive two-pore domain potassium channel. Neuropharmacology. 2001, 40: 551—559.
46. Gerstin, K.M. et al. Mutation of KCNK5 or Kir3.2 potassium channels in mice does not change minimum alveolar anesthetic concentration. Anesth. Analg. 2003, 96: 1345-1349.
47. Lauritzen, 1. et al. Poly-unsaturated fatty acids are potent neuroprotectors. EMBO J. 2000,19: 1784-1793.
48. Lang-Lazdunski, L. et al. Linolenic acid prevents neuronal cell death and paraplegia after transient spinal cord ischemia in rats. J. Vasc. Surg. 2003, 38: 564-575.
49. Blondeau, N. et al. Polyunsaturated fatty acids induce ischemic and epileptic tolerance.Neuroscience. 2002, 109: 231-241.
50. Blondeau, N. et al. A potent protective role of lysophospholipids against global cerebral ischemia and glutamate excitotoxicity in neuronal cultures. J. Cereb. Blood Flow Metab. 2002,22: 821-834.
51. Chemin, J. et al. Mechanisms underlying excitatory effects of group I metabotropic glutamate receptors via inhibition of 2P domain K~+ channels. EMBO J. 2003, 22: 5403-5411.