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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,INVA)的作用并观察银杏内酯B(ginkgolide B,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(1．0 μmol/L)对IHVA的增强作垌可以被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 kinase A,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。
Whole cell patch clamp technique was used to investigate the high voltage-dependent calcium channel currents of pyramidal neurons in hippocampal CA1 region of freshly isolated rat hippocampus induced by β-amyloid peptide 1-40 (Aβ1-40) The effect of voltage-activated calcium channel current (INVA) on the effect of ginkgolide B (GB) was observed. Using extracellular perfusion or intra-electrode dosing methods, the changes in current amplitude before and after dosing were compared to determine whether the drug worked. Extracellular administration of Aβ1-40 with aging treatment can increase the amplitude of IHVA in a concentration-dependent manner. Aβ1-40 concentration of 0.01-30 μmol/L can increase IHVA amplitude (5.43±3.01)%, respectively. (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); Freshly formulated A[beta]l-40 had little effect on IHVA (n=5, P>0.05). The L-type calcium channel blocker nifedipine can counteract the enhancement of IHVA by Aβ1-40. The enhancement of IHVA by Aβ1-40 (1.0 μmol/L) can be enhanced by cAMP analog 8-Br-cAMP and adenylyl cyclase (AC) agonist forskolin [respectively ( 66.19±5.74)%, P<0.05 and (73.21±6.90)%, P<0.05], inhibitor of protein kinase A (PKA) H- 89 decreased [(20.08±2.18)%, P<0.05]. GB can effectively attenuate the enhancement of IHVA by Aβ1-40. The above results indicate that Aβ1-40 can increase intracellular calcium overload induced by IHVA through AC-cAMP-PKA, which may be one of the mechanisms of its neurotoxicity. GB can protect neurons by inhibiting the abnormal calcium influx induced by Aβ1-40.