庆大霉素对急性分离小鼠耳蜗螺旋神经节神经元细胞电生理特性的影响
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摘要
目的:探讨庆大霉素对急性分离小鼠耳蜗螺旋神经节神经元(SGN)细胞电生理特性的影响及其意义。方法:急性酶解分离小鼠耳蜗SGN细胞,并利用间接免疫荧光细胞化学染色法对其进行鉴定。应用全细胞电压钳技术对急性分离小鼠SGN胞膜上的钾、钠离子通道电生理活动进行记录和分析,并研究庆大霉素对钾、钠离子通道电流的峰值的影响和与细胞外液中庆大霉素浓度的关系,以及庆大霉素洗脱后电流的恢复情况。
结果:在荧光显微镜下呈亮绿色的细胞为SGN细胞。在SGN细胞膜上记录到对氯化四乙胺(TEA-C1)、4-氨基吡啶(4-AP)敏感的外向延迟整流钾电流和A型钾电流,以及对河豚毒素(TTX)敏感的内向钠电流,离子通道具有电压依赖性。庆大霉素能抑制电压依赖性钾通道,但不能抑制电压依赖性钠通道,其钾通道抑制作用与细胞外液中庆大霉素的浓度呈剂量依赖性,庆大霉素洗脱后电流恢复不完全。结论:急性酶解分离的小鼠耳蜗SGN细胞膜上主要具有延迟整流钾通道、A型钾通道和钠通道,其电生理特性为听觉传导机制的研究提供了重要的参考,并从电生理的角度解释了庆大霉素通过抑制螺旋神经节神经元细胞的钾离子通道而产生耳毒性的机制。
Objective: To explore the influence of gentamicin on acutely dissociated murine coclear spiral ganglion neurons' electrophysiological properties and its significance.Method: Murine coclear spiral ganglion neurons ( SGN) were dissociated acutely using trypsin. Indirect immunocytochemical identification was performed and cells were observed under fluorescent microscope. Potassium and sodium channel currents of acutely trypsin-dissociated spiral ganglion neurons were recorded and analyzed, using whole-cell variation of the voltage clamp technique. We studied gentamicin's influence on the peak currents of potassium and sodium channels on cell membranes of spiral ganglion neurons, and the relationship to gentamicin's concentration in extracellular fluid, and the currents' recovery after gentamicin being washed out.Results: The light-green cells under fluorescent microscope were spiral ganglion neurons. In the membranes of spiral ganglion neurons, we recorded
tetraethylammonium chloride (TEA-C1) -sensitive and 4-aminopyridine (4-AP) -sensitive outside delayed rectifier potassium currents and A-type potassium currents, and tetrodotoxin (TTX) -sensitive inside sodium currents. Ion channels' activities had the character of voltage-dependence. Gentamicin could inhibit voltage-dependent potassium channels, but it couldn't inhibit voltage-dependent sodium channels. Gentamicin's inhibitation on potassium currents had dose-dependence on gentamicin's concemtration in extracellular fluid and the currents recoverd incompletely after gentamicin being washed out.Conclusion: The electrophysiological properties of acutely dissociated SGN were the important reference to the research of the mechanics of auditory propagation. There were mainly delayed rectifier potassium channel, A-type potassium channel and sodium channel on cell membranes of acutely dissociated spiral ganglion neurons. And this research explained the ototoxic mechanism of gentamicin through its action on keeping from SGN' potassium channels from the electrophysiological aspect.
结果:在荧光显微镜下呈亮绿色的细胞为SGN细胞。在SGN细胞膜上记录到对氯化四乙胺(TEA-C1)、4-氨基吡啶(4-AP)敏感的外向延迟整流钾电流和A型钾电流,以及对河豚毒素(TTX)敏感的内向钠电流,离子通道具有电压依赖性。庆大霉素能抑制电压依赖性钾通道,但不能抑制电压依赖性钠通道,其钾通道抑制作用与细胞外液中庆大霉素的浓度呈剂量依赖性,庆大霉素洗脱后电流恢复不完全。结论:急性酶解分离的小鼠耳蜗SGN细胞膜上主要具有延迟整流钾通道、A型钾通道和钠通道,其电生理特性为听觉传导机制的研究提供了重要的参考,并从电生理的角度解释了庆大霉素通过抑制螺旋神经节神经元细胞的钾离子通道而产生耳毒性的机制。
Objective: To explore the influence of gentamicin on acutely dissociated murine coclear spiral ganglion neurons' electrophysiological properties and its significance.Method: Murine coclear spiral ganglion neurons ( SGN) were dissociated acutely using trypsin. Indirect immunocytochemical identification was performed and cells were observed under fluorescent microscope. Potassium and sodium channel currents of acutely trypsin-dissociated spiral ganglion neurons were recorded and analyzed, using whole-cell variation of the voltage clamp technique. We studied gentamicin's influence on the peak currents of potassium and sodium channels on cell membranes of spiral ganglion neurons, and the relationship to gentamicin's concentration in extracellular fluid, and the currents' recovery after gentamicin being washed out.Results: The light-green cells under fluorescent microscope were spiral ganglion neurons. In the membranes of spiral ganglion neurons, we recorded
tetraethylammonium chloride (TEA-C1) -sensitive and 4-aminopyridine (4-AP) -sensitive outside delayed rectifier potassium currents and A-type potassium currents, and tetrodotoxin (TTX) -sensitive inside sodium currents. Ion channels' activities had the character of voltage-dependence. Gentamicin could inhibit voltage-dependent potassium channels, but it couldn't inhibit voltage-dependent sodium channels. Gentamicin's inhibitation on potassium currents had dose-dependence on gentamicin's concemtration in extracellular fluid and the currents recoverd incompletely after gentamicin being washed out.Conclusion: The electrophysiological properties of acutely dissociated SGN were the important reference to the research of the mechanics of auditory propagation. There were mainly delayed rectifier potassium channel, A-type potassium channel and sodium channel on cell membranes of acutely dissociated spiral ganglion neurons. And this research explained the ototoxic mechanism of gentamicin through its action on keeping from SGN' potassium channels from the electrophysiological aspect.
引文
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4. Adamson CL, Reid MA, Mo ZL, et al. Firing features and potassium channel content of murine spiral ganglion neurons vary with cochlear location. J Comp Neurol. 2002, 447(4): 331-350.
5. Mo ZL, Davis RL. Endogenous Firing Patterns of Murine Spiral Ganglion Neurons. J Neurophysiol. 1997, 77: 1294-1305.
6. Jagger D J, Housley GD. A-Type Potassium Currents Dominate Repolarisation of Neonatal Rat Primary Auditory Neurons in Situ. Neuroscience. 2002, 109: 169-182.
7. Morizono T. Ototopical agents: ototoxicity in animal studies. Ann Otol Rhino Laryngol. 1988, 97(suppl 131): 28-30.
8. Wright CG, Meyerhoff WL. Ototoxocity of otic drops applied to the middle ear in the chinchilla. Am J Otolaryngol. 1984, 5: 166-170.
9. Hutchin T, Higashi K, Stoneking M, et al. A molecular basis for human hypersensitivity to aminoglycoside antibiotics. Nucleic Acids Res. 1993, 21: 4174-4179.
10. Sha SH, Schacht J. Formation of reactive oxygen species following bioactivation of gentamicin. Free Radical Biol. Med. 1999, 26: 341.
11. Raffray M, Cohen GM. Apoptosis and necrosis in toxicology: a continuum or distinct modes of cell death? Pharmacol Ther. 1997, 75: 153-177.
12.陈贤明,王锦玲,屈玲.庆大霉素及其代谢产物对外毛细胞内游离钙浓度的影响.听力学与言语疾病杂志.2001,9(2):89-91.
13. Hinojosa R, Nelson EG, Lerner SA, et al. Aminoglycoside ototoxicity: a human temporal bone study. Laryngoscope. 2001, 111: 1797-1805.
14. Moore EJ, Hall DB, Narahashi T. Sodium and potassium currents of type Ⅰ spiral ganglion cells from rat. Acta Otolaryngol. 1996, 116(4): 552-560.
15. Chen C. Hyperpolarization-activated current (I_h) in primary auditory neurons. Hear Res. 1997, 110(1-2): 179-190.
16. Ingram SL, Williams JT. Modulation of hyperpolarization-activated current (I_h) by cyclic nuclaotides in guinea pig primary afferent neurons. J Physiol. (Lond) 1996, 492: 97-106.
17. Kuijpers W, Tonnaer ELG, Peter TA, et al. Expression of intermediat filament proteins in the mature inner ear of the rat and guinea pig. Hear Res. 1991, 52(1): 133-140.
18. Darnell J. Molecular cell biology. New York: Scientific American Books Inc. 1995: 640-644, 944-952,
19.孙伟,丁大连,金晓杰,等.小鼠耳蜗螺旋神经节细胞电压依赖性离子通道的研究.中华耳鼻咽喉科杂志.2001,36(3):178-182.
20. Garcia-Diaz JF. Development of a fast transient potassium current in chick cochlear ganglion neurons. Hear Res. 1999, 135(1-2): 124-134.
21. Hoffman DA, Magee JC, Colbert CM, et al. K~+ channel regulation of signal propagation in dendrites of hippocampal pyramidal neurons. Nature. 1997, 387: 869-875.
22.刘新民,徐韬园,张克义,等.实用临床治疗药典.沈阳:辽宁科学技术出版社,2003.685-686.
23. Wanamaker HH, Gruenwald L, Damm KJ, et al. Dose-related vestibular and cochlear effects of transtympanic gentamicin. American Journal of Otology. 1998, 19: 170-179.
24. Perez R, Freeman S, Sohmer H, et al. Vestibular and coclear ototoxicity of topical antiseptics assessed by evoked potentials. Laryngoscope. 2000, 110: 1522-1527.
2. Arnold W, Wang JB, Linnenkohl S. Anatomical observations in the spiral ganglion of human newborns (author's transl). Arch Otorhinolaryngol. 1980, 228(2): 69-84.
3. Lin X, Chen S. Endogenously generated spontaneous spiking activities recorded from postnatal spiral ganglion neurons in vitro. Brain Res Dev Brain Res. 2000, 119(2): 297-305.
4. Adamson CL, Reid MA, Mo ZL, et al. Firing features and potassium channel content of murine spiral ganglion neurons vary with cochlear location. J Comp Neurol. 2002, 447(4): 331-350.
5. Mo ZL, Davis RL. Endogenous Firing Patterns of Murine Spiral Ganglion Neurons. J Neurophysiol. 1997, 77: 1294-1305.
6. Jagger D J, Housley GD. A-Type Potassium Currents Dominate Repolarisation of Neonatal Rat Primary Auditory Neurons in Situ. Neuroscience. 2002, 109: 169-182.
7. Morizono T. Ototopical agents: ototoxicity in animal studies. Ann Otol Rhino Laryngol. 1988, 97(suppl 131): 28-30.
8. Wright CG, Meyerhoff WL. Ototoxocity of otic drops applied to the middle ear in the chinchilla. Am J Otolaryngol. 1984, 5: 166-170.
9. Hutchin T, Higashi K, Stoneking M, et al. A molecular basis for human hypersensitivity to aminoglycoside antibiotics. Nucleic Acids Res. 1993, 21: 4174-4179.
10. Sha SH, Schacht J. Formation of reactive oxygen species following bioactivation of gentamicin. Free Radical Biol. Med. 1999, 26: 341.
11. Raffray M, Cohen GM. Apoptosis and necrosis in toxicology: a continuum or distinct modes of cell death? Pharmacol Ther. 1997, 75: 153-177.
12.陈贤明,王锦玲,屈玲.庆大霉素及其代谢产物对外毛细胞内游离钙浓度的影响.听力学与言语疾病杂志.2001,9(2):89-91.
13. Hinojosa R, Nelson EG, Lerner SA, et al. Aminoglycoside ototoxicity: a human temporal bone study. Laryngoscope. 2001, 111: 1797-1805.
14. Moore EJ, Hall DB, Narahashi T. Sodium and potassium currents of type Ⅰ spiral ganglion cells from rat. Acta Otolaryngol. 1996, 116(4): 552-560.
15. Chen C. Hyperpolarization-activated current (I_h) in primary auditory neurons. Hear Res. 1997, 110(1-2): 179-190.
16. Ingram SL, Williams JT. Modulation of hyperpolarization-activated current (I_h) by cyclic nuclaotides in guinea pig primary afferent neurons. J Physiol. (Lond) 1996, 492: 97-106.
17. Kuijpers W, Tonnaer ELG, Peter TA, et al. Expression of intermediat filament proteins in the mature inner ear of the rat and guinea pig. Hear Res. 1991, 52(1): 133-140.
18. Darnell J. Molecular cell biology. New York: Scientific American Books Inc. 1995: 640-644, 944-952,
19.孙伟,丁大连,金晓杰,等.小鼠耳蜗螺旋神经节细胞电压依赖性离子通道的研究.中华耳鼻咽喉科杂志.2001,36(3):178-182.
20. Garcia-Diaz JF. Development of a fast transient potassium current in chick cochlear ganglion neurons. Hear Res. 1999, 135(1-2): 124-134.
21. Hoffman DA, Magee JC, Colbert CM, et al. K~+ channel regulation of signal propagation in dendrites of hippocampal pyramidal neurons. Nature. 1997, 387: 869-875.
22.刘新民,徐韬园,张克义,等.实用临床治疗药典.沈阳:辽宁科学技术出版社,2003.685-686.
23. Wanamaker HH, Gruenwald L, Damm KJ, et al. Dose-related vestibular and cochlear effects of transtympanic gentamicin. American Journal of Otology. 1998, 19: 170-179.
24. Perez R, Freeman S, Sohmer H, et al. Vestibular and coclear ototoxicity of topical antiseptics assessed by evoked potentials. Laryngoscope. 2000, 110: 1522-1527.