大鼠前庭内侧核胆碱能受体和γ-氨基丁酸受体介导的钙离子荧光显像及信息转导机制研究
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摘要
第一部分乙酰胆碱对大鼠前庭内侧核钙离子荧光强度的影响
目的:探讨大鼠前庭内侧核胆碱能受体介导的细胞内游离钙离子水平的变化。方法:应用激光共聚焦显微镜离子显像技术对前庭内侧核离体脑片中被荧光颜料Fluo-3AM标记的细胞内游离钙离子浓度的变化进行实时监测。结果:大鼠前庭内侧核细胞对乙酰胆碱敏感,10-5~10-2 M乙酰胆碱可以浓度依赖性的引起细胞内游离钙离子浓度的增高,其中1mM是最大药效浓度。胆碱能M受体拮抗剂阿托品(1μM)可以使1mM乙酰胆碱引起的前庭内侧核神经元钙离子荧光的增强幅度降低55%,神经元胆碱能N受体的拮抗剂美加明(10μM)可以使1mM乙酰胆碱引起的前庭内侧核神经元钙离子荧光的增强幅度降低49%(n=5)。两种拮抗剂之间对前庭内侧核钙离子荧光的影响没有显著性差异(P>0.05)。结论:前庭内侧核胆碱能受体可以介导细胞内游离钙离子浓度的升高,呈浓度依赖性,并且,毒蕈碱受体和烟碱受体均参与了此效应。
第二部分大鼠前庭内侧核毒蕈碱受体介导的钙离子荧光显像研究
目的:探讨大鼠前庭内侧核中毒蕈碱受体对细胞内游离钙离子浓度的调控,及其调控机制。方法:应用激光共聚焦显微镜离子显像技术技术对前庭内侧核离体脑片中被荧光颜料Fluo-3AM标记的细胞内游离钙离子浓度的变化进行实时监测。结果: 5~100μM的传统的毒蕈碱受体的激动剂Muscarine chloride可以浓度依赖性的引起细胞内游离钙离子浓度的增高,50μM是最大药效浓度。该效应可以被thapsigargin明显抑制(P<0.01),而在无钙脑脊液或存在EGTA的情况下,该效应无明显变化(P>0.05)。并且,Muscarine chloride引起的细胞内游离钙离子浓度增高的效应可以被毒蕈碱m1和m3受体的特异性拮抗剂pirenzepine和4-DAMP明显抑制(P<0.05),而毒蕈碱m2和m4受体的特异性拮抗剂methoctramine和tropicamide无明显抑制作用(P>0.05)。结论:毒蕈碱受体通过前庭内侧核细胞内钙库对钙离子的释放介导细胞内游离钙离子浓度的升高。并且,此效应是通过毒蕈碱m1和m3亚型受体来实现的。
第三部分大鼠前庭内侧核烟碱受体介导的钙离子荧光显像研究
目的:探讨大鼠前庭内侧核中烟碱受体对细胞内游离钙离子浓度的调控,及其调控机制。方法:应用激光共聚焦显微镜离子显像技术对大鼠前庭内侧核离体脑片中被荧光颜料Fluo-3AM标记的细胞内游离钙离子浓度的变化进行实时监测。结果:10～500μM的传统的烟碱受体的激动剂nicotine可以浓度依赖性的引起细胞内游离钙离子浓度的增高,100μM是最大药效浓度。该效应可以被EGTA和nifedipine明显抑制(P<0.01),在无钙人工脑脊液中,该效应亦可被明显抑制(P<0.01)。但是,mibefradil及thapsigargin对nicotine引起的细胞内游离钙离子浓度增高的效应均无明显抑制作用(P>0.05)。此外,烟碱α4β2亚型受体的特异性拮抗剂Dihydro-β-erythroidine对nicotine引起的效应有明显的抑制作用(P<0.01) ,而烟碱α7亚型受体的拮抗剂methyllycaconitine则无明显效果(P>0.05)。结论:烟碱受体可以介导前庭内侧核细胞内钙离子水平呈浓度依赖性的升高,是通过细胞外钙离子内流来实现的,至少有L型电压门控钙离子通道的开放参与了此过程,并且,该效应是通过烟碱α4β2亚型受体实现的,而非烟碱α7亚型受体。
第四部分大鼠前庭内侧核γ-氨基丁酸受体介导的钙离子荧光显像研究
目的:探讨大鼠前庭内侧核中γ-氨基丁酸受体介导的细胞内游离钙离子浓度变化,以及对胆碱能受体效应的影响,并探讨其调控机制。方法:应用激光共聚焦显微镜离子显像技术对大鼠前庭内侧核离体脑片中被荧光颜料Fluo-3AM标记的细胞内游离钙离子浓度的变化进行实时监测。结果: 5μM、20μM、100μM、500μM GABA对前庭内侧核钙离子荧光强度没有显著性影响(P>0.05),对毒蕈碱受体激动剂muscarine chloride引起的前庭内侧核钙离子荧光强度变化也没有显著影响(P>0.05),而对100μM nicotine引起的前庭内侧核钙离子荧光强度的增强有明显的抑制作用(P<0.05),呈浓度依赖性,其中,100μM GABA可以达到最大抑制效应。GABAB受体选择性激动剂baclofen对50μM muscarine chloride和100μM nicotine引起的钙离子荧光增强均无明显抑制作用(P>0.05)。结论: GABA为抑制性神经递质,对前庭内侧核细胞内钙离子浓度没有直接的影响,但是可以对烟碱受体引起的细胞外钙离子的内流有抑制作用,并且该效应是通过GABA能A型受体来实现,机制可能是阻断电压门控钙离子通道的开放。
PART 1: An Increase in Intracelluar Free Calcium Ions by Cholinergic Receptors in Rat Medial Vestibular Nucleus
Objective: To Study the modulations of intracellular free Ca2+ concentrations by cholinergic receptors in rat medial vestibular nucleus. Methods: The fluorescence intensity of medial vestibular nucleus in Fluo-3 AM loaded acute brainstem slices was detected by applying intracellular free Ca2+ measurement technique via confocal laser scanning microscope. Results: Acetylcholine was effective at increasing the fluorescence intensity of medial vestibular nucleus in a concentration dependent manner at a concentration range of 10-5~10-2 M, and the increase by acetylcholine was saturable with increasing concentrations of up to 1mM. The increase of intracelluar free calcium ions levels by 1mM acetylcholine could be inhibited by atropine and mecamylamine, and there is little difference between the inhibition between atropine and mecamylamine (P>0.05). Conclusion: The data provide the evidence that cholinergic receptors induce the increase of intracellular free Ca2+ levels, in a manner related to both muscarinic receptors and nicotinic receptors in rat medial vestibular nucleus.
PART 2: An Increase in Intracelluar Free Calcium Ions by Muscarinic Receptors in Rat Medial Vestibular Nucleus
Objective: To Study the modulations of intracellular free Ca2+ concentrations by muscarinic receptors in rat medial vestibular nucleus, and the mechanisms of the modulations. Methods: The fluorescence intensity of medial vestibular nucleus in Fluo-3 AM loaded acute brainstem slices was detected by applying intracellular free Ca2+ measurement technique via confocal laser scanning microscope. Results: Muscarine chloride (a general muscarinic cholinergic agonist) induced a marked increase of fluorescence intensity in medial vestibular nucleus in a concentration dependent fashion at a concentration range of 5~100μM, and the increase by muscarine chloride was saturable with increasing concentrations of up to 50μM. The enhancement of fluorescence intensity by muscarine chloride was significantly reduced by thapsigargin (P<0.01), rather than Ca2+ free artifical cerebrospinal fluid (ACSF) and EGTA (P>0.05). And the increase of fluorescence intensity was also significantly inhibited by pirenzepine and 4-DAMP (P<0.05), rather than methoctramine and tropicamide (P>0.05). Conclusion: The data provide the evidence that muscarinic receptors induce the increase of intracellular free Ca2+ levels through the Ca2+ release of intracellular Ca2+ stores rather than influx of extracellular Ca2+, in a manner related to m1 and m3 subtypes rather than m2 and m4 subtypes of muscarinic receptors in rat medial vestibular nucleus.
PART 3: An Increase in Intracelluar Free Calcium Ions by Nicotinic Receptors in Rat Medial Vestibular Nucleus
Objective: To Study the modulations of intracellular free Ca2+ concentrations by nicotinic receptors in rat medial vestibular nucleus, and the mechanisms of the modulations. Methods: The fluorescence intensity of medial vestibular nucleus in Fluo-3 AM loaded acute brainstem slices was detected by applying intracellular free Ca2+ measurement technique via confocal laser scanning microscope. Results: Fluorescence intensity of medial vestibular nucleus was markedly increased by nicotine (a general nicotinic cholinergic agonist) in a dose dependent manner at a concentration range of 10 to 500μM. and the increase by nicotine was saturable with increasing concentrations of up to 100μM. The enhancement of fluorescence intensity by nicotine was significantly reduced by EGTA, nifedipine and in Ca2+ free ASCF (P<0.01), but not in the presence of mibefradil or thapsigargin (P>0.05). The increase was also significantly reduced by Dihydro-β-erythroidine (P<0.01), rather than methyllycaconitine (P>0.05). Conclusion: The results suggest that nicotine increase intracellular free Ca2+ concentrations via the influx of extracellular Ca2+ at least across L-type voltage-gated Ca2+ channels rather than Ca2+ release from intracellular stores, in a manner related to theα4β2 subtype of nicotinic receptors, but notα7 subtype in rat medial vestibular nucleus.
PART 4: A Change in Intracelluar Free Calcium Ions by GABAergic Receptors in Rat Medial Vestibular Nucleus
Objective: To Study the modulations of intracellular free Ca2+ concentrations by GABAergic receptors in rat medial vestibular nucleus, and the mechanisms of the modulations. Methods: The fluorescence intensity of medial vestibular nucleus in Fluo-3 AM loaded acute brainstem slices was detected by applying intracellular free Ca2+ measurement technique via confocal laser scanning microscope. Results: 5~500μM GABA could not induce change of the intracellular free Ca2+ concentrations (P>0.05), or inhibit the increase of the intracellular free Ca2+ concentrations by 50μM muscarine chloride (P>0.05). GABA induced the inhibition of increase by 100μM nicotine in a dose dependent manner at a concentration range of 5 to 500μM (P<0.05), and the inhibition by GABA was saturable with increasing concentrations of up to 100μM. 1μM~100μM baclofen (a selective GABAB agonist) could not change the increase in intracellular free Ca2+ levels by 50μM muscarine chloride or 100μM nicotine (P>0.05). Conclusion: GABAergic receptors induce the inhibition of the increase in intracellular free Ca2+ concentrations by nicotinic receptors, in a manner related to GABAA subtype, though blocking L-type voltage-gated Ca2+ channels possibly in rat medial vestibular nucleus.
目的:探讨大鼠前庭内侧核胆碱能受体介导的细胞内游离钙离子水平的变化。方法:应用激光共聚焦显微镜离子显像技术对前庭内侧核离体脑片中被荧光颜料Fluo-3AM标记的细胞内游离钙离子浓度的变化进行实时监测。结果:大鼠前庭内侧核细胞对乙酰胆碱敏感,10-5~10-2 M乙酰胆碱可以浓度依赖性的引起细胞内游离钙离子浓度的增高,其中1mM是最大药效浓度。胆碱能M受体拮抗剂阿托品(1μM)可以使1mM乙酰胆碱引起的前庭内侧核神经元钙离子荧光的增强幅度降低55%,神经元胆碱能N受体的拮抗剂美加明(10μM)可以使1mM乙酰胆碱引起的前庭内侧核神经元钙离子荧光的增强幅度降低49%(n=5)。两种拮抗剂之间对前庭内侧核钙离子荧光的影响没有显著性差异(P>0.05)。结论:前庭内侧核胆碱能受体可以介导细胞内游离钙离子浓度的升高,呈浓度依赖性,并且,毒蕈碱受体和烟碱受体均参与了此效应。
第二部分大鼠前庭内侧核毒蕈碱受体介导的钙离子荧光显像研究
目的:探讨大鼠前庭内侧核中毒蕈碱受体对细胞内游离钙离子浓度的调控,及其调控机制。方法:应用激光共聚焦显微镜离子显像技术技术对前庭内侧核离体脑片中被荧光颜料Fluo-3AM标记的细胞内游离钙离子浓度的变化进行实时监测。结果: 5~100μM的传统的毒蕈碱受体的激动剂Muscarine chloride可以浓度依赖性的引起细胞内游离钙离子浓度的增高,50μM是最大药效浓度。该效应可以被thapsigargin明显抑制(P<0.01),而在无钙脑脊液或存在EGTA的情况下,该效应无明显变化(P>0.05)。并且,Muscarine chloride引起的细胞内游离钙离子浓度增高的效应可以被毒蕈碱m1和m3受体的特异性拮抗剂pirenzepine和4-DAMP明显抑制(P<0.05),而毒蕈碱m2和m4受体的特异性拮抗剂methoctramine和tropicamide无明显抑制作用(P>0.05)。结论:毒蕈碱受体通过前庭内侧核细胞内钙库对钙离子的释放介导细胞内游离钙离子浓度的升高。并且,此效应是通过毒蕈碱m1和m3亚型受体来实现的。
第三部分大鼠前庭内侧核烟碱受体介导的钙离子荧光显像研究
目的:探讨大鼠前庭内侧核中烟碱受体对细胞内游离钙离子浓度的调控,及其调控机制。方法:应用激光共聚焦显微镜离子显像技术对大鼠前庭内侧核离体脑片中被荧光颜料Fluo-3AM标记的细胞内游离钙离子浓度的变化进行实时监测。结果:10～500μM的传统的烟碱受体的激动剂nicotine可以浓度依赖性的引起细胞内游离钙离子浓度的增高,100μM是最大药效浓度。该效应可以被EGTA和nifedipine明显抑制(P<0.01),在无钙人工脑脊液中,该效应亦可被明显抑制(P<0.01)。但是,mibefradil及thapsigargin对nicotine引起的细胞内游离钙离子浓度增高的效应均无明显抑制作用(P>0.05)。此外,烟碱α4β2亚型受体的特异性拮抗剂Dihydro-β-erythroidine对nicotine引起的效应有明显的抑制作用(P<0.01) ,而烟碱α7亚型受体的拮抗剂methyllycaconitine则无明显效果(P>0.05)。结论:烟碱受体可以介导前庭内侧核细胞内钙离子水平呈浓度依赖性的升高,是通过细胞外钙离子内流来实现的,至少有L型电压门控钙离子通道的开放参与了此过程,并且,该效应是通过烟碱α4β2亚型受体实现的,而非烟碱α7亚型受体。
第四部分大鼠前庭内侧核γ-氨基丁酸受体介导的钙离子荧光显像研究
目的:探讨大鼠前庭内侧核中γ-氨基丁酸受体介导的细胞内游离钙离子浓度变化,以及对胆碱能受体效应的影响,并探讨其调控机制。方法:应用激光共聚焦显微镜离子显像技术对大鼠前庭内侧核离体脑片中被荧光颜料Fluo-3AM标记的细胞内游离钙离子浓度的变化进行实时监测。结果: 5μM、20μM、100μM、500μM GABA对前庭内侧核钙离子荧光强度没有显著性影响(P>0.05),对毒蕈碱受体激动剂muscarine chloride引起的前庭内侧核钙离子荧光强度变化也没有显著影响(P>0.05),而对100μM nicotine引起的前庭内侧核钙离子荧光强度的增强有明显的抑制作用(P<0.05),呈浓度依赖性,其中,100μM GABA可以达到最大抑制效应。GABAB受体选择性激动剂baclofen对50μM muscarine chloride和100μM nicotine引起的钙离子荧光增强均无明显抑制作用(P>0.05)。结论: GABA为抑制性神经递质,对前庭内侧核细胞内钙离子浓度没有直接的影响,但是可以对烟碱受体引起的细胞外钙离子的内流有抑制作用,并且该效应是通过GABA能A型受体来实现,机制可能是阻断电压门控钙离子通道的开放。
PART 1: An Increase in Intracelluar Free Calcium Ions by Cholinergic Receptors in Rat Medial Vestibular Nucleus
Objective: To Study the modulations of intracellular free Ca2+ concentrations by cholinergic receptors in rat medial vestibular nucleus. Methods: The fluorescence intensity of medial vestibular nucleus in Fluo-3 AM loaded acute brainstem slices was detected by applying intracellular free Ca2+ measurement technique via confocal laser scanning microscope. Results: Acetylcholine was effective at increasing the fluorescence intensity of medial vestibular nucleus in a concentration dependent manner at a concentration range of 10-5~10-2 M, and the increase by acetylcholine was saturable with increasing concentrations of up to 1mM. The increase of intracelluar free calcium ions levels by 1mM acetylcholine could be inhibited by atropine and mecamylamine, and there is little difference between the inhibition between atropine and mecamylamine (P>0.05). Conclusion: The data provide the evidence that cholinergic receptors induce the increase of intracellular free Ca2+ levels, in a manner related to both muscarinic receptors and nicotinic receptors in rat medial vestibular nucleus.
PART 2: An Increase in Intracelluar Free Calcium Ions by Muscarinic Receptors in Rat Medial Vestibular Nucleus
Objective: To Study the modulations of intracellular free Ca2+ concentrations by muscarinic receptors in rat medial vestibular nucleus, and the mechanisms of the modulations. Methods: The fluorescence intensity of medial vestibular nucleus in Fluo-3 AM loaded acute brainstem slices was detected by applying intracellular free Ca2+ measurement technique via confocal laser scanning microscope. Results: Muscarine chloride (a general muscarinic cholinergic agonist) induced a marked increase of fluorescence intensity in medial vestibular nucleus in a concentration dependent fashion at a concentration range of 5~100μM, and the increase by muscarine chloride was saturable with increasing concentrations of up to 50μM. The enhancement of fluorescence intensity by muscarine chloride was significantly reduced by thapsigargin (P<0.01), rather than Ca2+ free artifical cerebrospinal fluid (ACSF) and EGTA (P>0.05). And the increase of fluorescence intensity was also significantly inhibited by pirenzepine and 4-DAMP (P<0.05), rather than methoctramine and tropicamide (P>0.05). Conclusion: The data provide the evidence that muscarinic receptors induce the increase of intracellular free Ca2+ levels through the Ca2+ release of intracellular Ca2+ stores rather than influx of extracellular Ca2+, in a manner related to m1 and m3 subtypes rather than m2 and m4 subtypes of muscarinic receptors in rat medial vestibular nucleus.
PART 3: An Increase in Intracelluar Free Calcium Ions by Nicotinic Receptors in Rat Medial Vestibular Nucleus
Objective: To Study the modulations of intracellular free Ca2+ concentrations by nicotinic receptors in rat medial vestibular nucleus, and the mechanisms of the modulations. Methods: The fluorescence intensity of medial vestibular nucleus in Fluo-3 AM loaded acute brainstem slices was detected by applying intracellular free Ca2+ measurement technique via confocal laser scanning microscope. Results: Fluorescence intensity of medial vestibular nucleus was markedly increased by nicotine (a general nicotinic cholinergic agonist) in a dose dependent manner at a concentration range of 10 to 500μM. and the increase by nicotine was saturable with increasing concentrations of up to 100μM. The enhancement of fluorescence intensity by nicotine was significantly reduced by EGTA, nifedipine and in Ca2+ free ASCF (P<0.01), but not in the presence of mibefradil or thapsigargin (P>0.05). The increase was also significantly reduced by Dihydro-β-erythroidine (P<0.01), rather than methyllycaconitine (P>0.05). Conclusion: The results suggest that nicotine increase intracellular free Ca2+ concentrations via the influx of extracellular Ca2+ at least across L-type voltage-gated Ca2+ channels rather than Ca2+ release from intracellular stores, in a manner related to theα4β2 subtype of nicotinic receptors, but notα7 subtype in rat medial vestibular nucleus.
PART 4: A Change in Intracelluar Free Calcium Ions by GABAergic Receptors in Rat Medial Vestibular Nucleus
Objective: To Study the modulations of intracellular free Ca2+ concentrations by GABAergic receptors in rat medial vestibular nucleus, and the mechanisms of the modulations. Methods: The fluorescence intensity of medial vestibular nucleus in Fluo-3 AM loaded acute brainstem slices was detected by applying intracellular free Ca2+ measurement technique via confocal laser scanning microscope. Results: 5~500μM GABA could not induce change of the intracellular free Ca2+ concentrations (P>0.05), or inhibit the increase of the intracellular free Ca2+ concentrations by 50μM muscarine chloride (P>0.05). GABA induced the inhibition of increase by 100μM nicotine in a dose dependent manner at a concentration range of 5 to 500μM (P<0.05), and the inhibition by GABA was saturable with increasing concentrations of up to 100μM. 1μM~100μM baclofen (a selective GABAB agonist) could not change the increase in intracellular free Ca2+ levels by 50μM muscarine chloride or 100μM nicotine (P>0.05). Conclusion: GABAergic receptors induce the inhibition of the increase in intracellular free Ca2+ concentrations by nicotinic receptors, in a manner related to GABAA subtype, though blocking L-type voltage-gated Ca2+ channels possibly in rat medial vestibular nucleus.
引文
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