CAJ | 학술논문

应用全细胞膜片钳技术探讨β-淀粉肽(1-40)(β-amyloid peptide1-40,Aβ1-40)对新鲜分离的大鼠海马CA1区锥体神经元高电压依赖性钙通道电流(high voltage-activated calcium channel current,IHVA)的作用并观察银杏内酯B(ginkgolideB,GB)对该作用的影响.利用细胞外灌流或者电极内液给药的方法,比较加药前后电流幅度的变化以判断药物是否发挥作用.细胞外给予老化处理的Aβ1-40可以浓度依赖性地增强IHVA的幅度,Aβ1-40的浓度为0.01～30 μmol/L时可分别使IHVA幅度增加(5.43±3.01)%(n=8,P＞0.05)、(10.49±4.13)%(n=11,P＞0.05)、(40.69±8.01)%(n=16,P＜0.01)、(58.32±4.85)%(n=12,P＜0.01)和(75.45±5.81)%(n=6,P＜0.01);新鲜配制的Aβ1-40对IHVA几乎没有影响(n=5,P＞0.05).L-型钙通道阻断剂nifedipine可以抵消Aβ1-40对IHVA的增强作用.Aβ1-40(1.0μmol/L)对IHVA的增强作用可以被cAMP的类似物8-Br-cAMP和腺苷酸环化酶(adenylyl cyclase,AC)的激动剂forskolin增强[分别为(66.19±5.74)%,P＜0.05和(73.21±6.90)%,P＜0.05],被蛋白激酶A(protein kinaseA,PKA)的抑制剂H-89减弱[(20.08±2.18)%,P＜0.05].GB可有效地减弱Aβ1-40对IHVA的增强作用.以上结果表明Aβ1-40可通过AC-cAMP-PKA增强IHVA引起胞内钙超载,这可能是其产生神经毒性作用的机制之一.GB可通过抑制Aβ1-40引起的异常钙离子内流对神经元起一定保护作用.
응용전세포막편겸기술탐토β-정분태(1-40)(β-amyloid peptide1-40,Aβ1-40)대신선분리적대서해마CA1구추체신경원고전압의뢰성개통도전류(high voltage-activated calcium channel current,IHVA)적작용병관찰은행내지B(ginkgolideB,GB)대해작용적영향.이용세포외관류혹자전겁내액급약적방법,비교가약전후전류폭도적변화이판단약물시부발휘작용.세포외급여노화처리적Aβ1-40가이농도의뢰성지증강IHVA적폭도,Aβ1-40적농도위0.01～30 μmol/L시가분별사IHVA폭도증가(5.43±3.01)%(n=8,P＞0.05)、(10.49±4.13)%(n=11,P＞0.05)、(40.69±8.01)%(n=16,P＜0.01)、(58.32±4.85)%(n=12,P＜0.01)화(75.45±5.81)%(n=6,P＜0.01);신선배제적Aβ1-40대IHVA궤호몰유영향(n=5,P＞0.05).L-형개통도조단제nifedipine가이저소Aβ1-40대IHVA적증강작용.Aβ1-40(1.0μmol/L)대IHVA적증강작용가이피cAMP적유사물8-Br-cAMP화선감산배화매(adenylyl cyclase,AC)적격동제forskolin증강[분별위(66.19±5.74)%,P＜0.05화(73.21±6.90)%,P＜0.05],피단백격매A(protein kinaseA,PKA)적억제제H-89감약[(20.08±2.18)%,P＜0.05].GB가유효지감약Aβ1-40대IHVA적증강작용.이상결과표명Aβ1-40가통과AC-cAMP-PKA증강IHVA인기포내개초재,저가능시기산생신경독성작용적궤제지일.GB가통과억제Aβ1-40인기적이상개리자내류대신경원기일정보호작용.
Whole-cell patch clamp recording was used to investigate the action of β-amyloid peptide1-40 (Aβ1-40) on high voltageactivated calcium channel current (IHVA) in acutely isolated hippocampal CA1 pyramidal neurons in rats and observe its modulation by ginkgolide B (GB). Drug was applied by extracellular bath or adding in the pipette solution, and its effect was determined by comparing the amplitude of IHVA before and after the drug application. Bath application of aggregated Aβ1-40 at concentrations of 0.01 ～30 μmol/L increased the amplitude of IHVA in a dose-dependent manner by (5.43+3.01)% (n=8, P＞0.05), (10.49+4.13)% (n=11, P＞0.05), (40.69+8.01)% (n=16, P＜0.01), (58.32+4.85)% (n=12, P＜0.01), and (75.45+5.81)% (n=6, P＜0.01), respectively, but had no effect on the I-V curve of IHVA; fresh Aβ1-40 almost had no effect on IHVA (n=5, P＞0.05). L-type calcium channel antagonist nifedipine abolished the increase of IHVA by Aβ1-40. The increase of IHVA by Aβ1-40 (1.0 μmol/L) was enhanced to (66.19+5.74)% (P＜0.05) by 8-Br-cAMP (membrane permeable analogue of cAMP) and to (73.21 +6.90)% (P＜0.05) by forskolin, an adenylyl cyclase (AC) agonist, and reduced to (20.08+2.18)% (P＜0.05) by H-89, cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) antagonist. GB effectively inhibited the increase of IHVA by Aβ1-40. The results indicate that Aβ1-40 leads to an intracellular calcium overload by increasing IHVA via AC-cAMP-PKA. This may be one of the mechanisms for its neurotoxicity. GB can prevent neurons from neurotoxicity by inhibiting abnormal calcium influx caused by Aβ1-40.