中华大蟾蜍卵母细胞质膜存在钙依赖性的氯离子通道
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摘要
目的:研究中华大蟾蜍卵母细胞内源性电压门控型离子通道的成份及其生理特性。
方法:采用双微电极电压钳法。
结果:(1)发现卵母细胞去极化至-30mv及更偏正时,有一持续的电压依赖性的外向电流出现。该电流被最大抑制剂量的钾离子通道拮抗剂TEA(10mmol/L)和4-AP(10mmol/L)协同作用时,只能被抑制到最大电流幅度的23.4±2.4%;
(2)用无氯Ringer氏液与钾离子通道拮抗剂(TEA10mmol/L和4-AP10mmol/L)协同作用时,可将此电流基本抑制,仅剩2.2±0.2%。用氯离子通道拮抗剂NPPB(30μmol/L)与钾离子通道拮抗剂(TEA10mmol/L和4-AP10mmol/L)协同作用时,该电流也可被基本抑制,只剩2.1±0.2%;
(3)用无钙Ringer氏液与钾离子通道拮抗剂(TEA10mmol/L和4-AP10mmol/L)协同作用时,可将此电流基本抑制,仅剩3.2±0.3%。用钙离子通道拮抗剂维拉帕米(40μmol/L),与钾离子通道拮抗剂(TEA10mmol/L和4-AP10mmol/L)协同作用时,该电流也可被基本抑制,只剩3.1±0.3%。
结论:中华大蟾蜍卵母细胞质膜上除有钾离子通道之外,还存在钙依赖性的氯离子通道.
Objective: To study membrane properties of the fully-grown oocytes from toad, Bufo bufo gargarizans.
Method: Two-microelectrode voltage-clamp(TEA)method was used.
Results: (1)It was found that a sustained outward current was elicited by membrane depolarization to -30mv or more positive value. Potassium channel blockers(TEA10mmol/L and 4-AP10mmol/L) partly reduced the majority of outward current to 23.4±2.4%.(2)Bath application of Cl—-free Ringer’s solution and potassium channel blockers(TEA10mmol/L and 4-AP10mmol/L) could almost completely block the outward current, only remaining 2.2±0.2% .Bath application of chloride channel blocke(rNPPB30μmol/L)and potassium channel blockers(TEA10mmol/L and 4-AP10mmol/L) could almost completely block the outward current, only remaining 2.1±0.2%.Bath application of Ca2+-free Ringer’s solution and potassium channel blockers(TEA10mmol/L and 4-AP10mmol/L) could almost completely block the outward current, only remaining 3.2±0.3%.(3) Bath application of calcium channel blockers(Verapamil40μmol/L)and potassium channel blockers(TEA10mmol/L and 4-AP10mmol/L) could almost completely block the outward current, only remain 3.1±0.3%.
Conclusion: It was firstly concluded that plasma membrane of Bufo oocyte exist calcium-dependent chloride channel, except potassium channel.
方法:采用双微电极电压钳法。
结果:(1)发现卵母细胞去极化至-30mv及更偏正时,有一持续的电压依赖性的外向电流出现。该电流被最大抑制剂量的钾离子通道拮抗剂TEA(10mmol/L)和4-AP(10mmol/L)协同作用时,只能被抑制到最大电流幅度的23.4±2.4%;
(2)用无氯Ringer氏液与钾离子通道拮抗剂(TEA10mmol/L和4-AP10mmol/L)协同作用时,可将此电流基本抑制,仅剩2.2±0.2%。用氯离子通道拮抗剂NPPB(30μmol/L)与钾离子通道拮抗剂(TEA10mmol/L和4-AP10mmol/L)协同作用时,该电流也可被基本抑制,只剩2.1±0.2%;
(3)用无钙Ringer氏液与钾离子通道拮抗剂(TEA10mmol/L和4-AP10mmol/L)协同作用时,可将此电流基本抑制,仅剩3.2±0.3%。用钙离子通道拮抗剂维拉帕米(40μmol/L),与钾离子通道拮抗剂(TEA10mmol/L和4-AP10mmol/L)协同作用时,该电流也可被基本抑制,只剩3.1±0.3%。
结论:中华大蟾蜍卵母细胞质膜上除有钾离子通道之外,还存在钙依赖性的氯离子通道.
Objective: To study membrane properties of the fully-grown oocytes from toad, Bufo bufo gargarizans.
Method: Two-microelectrode voltage-clamp(TEA)method was used.
Results: (1)It was found that a sustained outward current was elicited by membrane depolarization to -30mv or more positive value. Potassium channel blockers(TEA10mmol/L and 4-AP10mmol/L) partly reduced the majority of outward current to 23.4±2.4%.(2)Bath application of Cl—-free Ringer’s solution and potassium channel blockers(TEA10mmol/L and 4-AP10mmol/L) could almost completely block the outward current, only remaining 2.2±0.2% .Bath application of chloride channel blocke(rNPPB30μmol/L)and potassium channel blockers(TEA10mmol/L and 4-AP10mmol/L) could almost completely block the outward current, only remaining 2.1±0.2%.Bath application of Ca2+-free Ringer’s solution and potassium channel blockers(TEA10mmol/L and 4-AP10mmol/L) could almost completely block the outward current, only remaining 3.2±0.3%.(3) Bath application of calcium channel blockers(Verapamil40μmol/L)and potassium channel blockers(TEA10mmol/L and 4-AP10mmol/L) could almost completely block the outward current, only remain 3.1±0.3%.
Conclusion: It was firstly concluded that plasma membrane of Bufo oocyte exist calcium-dependent chloride channel, except potassium channel.
引文
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16.姚永、顾全保、朱辉.中华大蟾蜍卵母细胞质膜的外向整流型钾离子通道.生理学报,1992,44:461-469.
17.顾全保、朱辉、姚永.大鼠脑神经递质受体和通道在中华大蟾蜍卵母细胞的表达.第一届中科院神经科学学术会议论文摘要,1990,p28.
18.Barish,ME. A transient calcium-dependent chloride current in the immature Xenopus oocytes. J. Physiol.(Lond).1983,342:309-325.
19.Boton R. Dascal N. Gillo B. and Lass Y.Tow calcium-actived chloride conductances in Xenopus laevis oocytes permeabilized with the ionophore A23187,J. Physical,(Long.)1989,408,511-534.
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25.王幽兰.长年控制蟾蜍生殖期的研究.动物学报,1954, 6: 81-84.
26.朱冼、王幽兰.长年高温养育对蟾蜍卵球发育和成熟的影响.Scientia Sinica,1963,12:1165-1168.
27.赵剑星、朱光、徐国江、王幽兰.用实验条件改变离体高温卵巢卵对孕酮的应答能力.实验生物学报,1988,21:169-177.
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29.Cavarra MS, Kotisias BA. Chloride channel blockers activate an endogenous cationic current in oocytes of Bufo arenarum. J.Neuroethology,2004,190(7):531-537.
30.Rafael V, Luc B, Luisa L, Marcela C. Bufo marinus oocytes as a model for ion channel protein expression and functional characterization for electrophysiological studies. Cellular Physiology and Biochemistry, 2004, 14:197-202.
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2. Dascal N. The use of Xenopus oocytes for the study of ion channeis. Ibid, 1987,22(4):317-387.
3. 田辉凯,李学军.爪蟾卵母细胞表达体系在功能基因组学研究中的应用.国外 医学分子生物学分册,2002,24(4):193-196.
4 .Dick DA, Fry DJ. Sodium fluxes in single amphibian oocytes: further studies and a new model. J. Physiol,1975,247(1):91-116.
5. Barnard EA, Miledi R, Sumikawa K. Translation of exogenous messenger RNA coding for nicotinic acetylcholine receptors produces functional receptors in Xenopus oocytes.Proc.R.Soc.Lond,1982,B215:241-246.
6. Koltchine VV, Anantharam V, Bayley H, Alternative splicing of the NMDARI subunit affeits by calcium[J]. Moleculer Brain Res, 1996, 39(1-2):99-108.
7. Lane CD. The fate of genes, messengers, and proteins introduced into Xenopus oocytes. Current Topics of Dev. Biol.1983, 18:89-116.
8. Soreq,H. The biosynthesis of biologically active proteins in mRNA-microinjected Xenopus oocytes. CRC Critical Reviews in Biochemistry, 1985,18(3):199-238.
9. Hollman M, O’Shea-Greenfield A, Rogers SW, Heinemann S. Cloning by functional expression of a member of the glutamate receptor family.Nature,1989,342:643-648.
10.Dumont JW. Oogenesis in Xenopus laevis (Daudin),Stage of oocyte development in laboratory maintained animals[J].J Morphol, 1972, 136:153-180.
11.顾全保、朱辉、李巍.红藻氨酸和 GABA 受体在中华大蟾蜍卵母细胞的表达.生理学报,1992,44(5):470-477.
12.朱幸、朱辉、包永德.鸡视网膜谷氨酸受体和 GABA 受体在两栖类卵母细胞中的表达.生理学报,1994,46:417-426.
13.朱辉、朱幸、包永德.鲫鱼脑 mRNA 在中华大蟾蜍卵母细胞中的表达。第一届全国神经科学学术会议论文摘要汇编。生理通讯增刊号,1992,3:61-62.
14.朱辉、朱幸、包永德.鲫鱼脑氨基酸类神经递质受体在两栖类卵母细胞中的表达。生理学报,1995,47(1):1-10.
15.张晓东、臧益民、谢安.用于离子通道基因表达的爪蟾卵母细胞的分离和培养.心脏杂志,2001,13:120-122.
16.姚永、顾全保、朱辉.中华大蟾蜍卵母细胞质膜的外向整流型钾离子通道.生理学报,1992,44:461-469.
17.顾全保、朱辉、姚永.大鼠脑神经递质受体和通道在中华大蟾蜍卵母细胞的表达.第一届中科院神经科学学术会议论文摘要,1990,p28.
18.Barish,ME. A transient calcium-dependent chloride current in the immature Xenopus oocytes. J. Physiol.(Lond).1983,342:309-325.
19.Boton R. Dascal N. Gillo B. and Lass Y.Tow calcium-actived chloride conductances in Xenopus laevis oocytes permeabilized with the ionophore A23187,J. Physical,(Long.)1989,408,511-534.
20.Dascal,N. The use of Xenopus oocytes for the study of ion channels. Ibid,1987,22(4):317-387.
21.Parker I, Ivorra I. A slowly inactivating potassium current in native oocytes of Xenopus laevis.Proc.R.Soc.Lond.1990,B238:369-381.
22.Descal D. Snutch TP. Lucbbert H. Davidson N. and Lester HA. Expression and modulation of voltage-gated calcium channels after RNA injection in Xenopus oocytes. Science,1986,231:1147-1150.
23.Baud C. Kado R. Introduction and disappearance of excitability in the oocytes of Xenopus laevis: a voltage-clamp study. J. Physiol, (Lond.) 1981,356:275-289.
24.Tchou S, Wang YL. La succession d’ovogenese et l’impossibilite de maturation ovulaire chez le milieu a haute temperature pendant toute une annee. Scientia Scienca, 1963, 12(8):1165-1168.
25.王幽兰.长年控制蟾蜍生殖期的研究.动物学报,1954, 6: 81-84.
26.朱冼、王幽兰.长年高温养育对蟾蜍卵球发育和成熟的影响.Scientia Sinica,1963,12:1165-1168.
27.赵剑星、朱光、徐国江、王幽兰.用实验条件改变离体高温卵巢卵对孕酮的应答能力.实验生物学报,1988,21:169-177.
28.Dick DA, Fry DJ. Sodium fluxes in single amphibian oocytes:further studies and a new model. J Physiol, 1997,247(1):91-116.
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