Wang Jun Ming, Brinton Roberta Diaz
Department of Pharmacology and Pharmaceutical Sciences and Program in Neuroscience, University of Southern California, Los Angeles, CA 90089, USA.
BMC Neurosci. 2008 Dec 3;9 Suppl 2(Suppl 2):S11. doi: 10.1186/1471-2202-9-S2-S11.
Factors that regulate intracellular calcium concentration are known to play a critical role in brain function and neural development, including neural plasticity and neurogenesis. We previously demonstrated that the neurosteroid allopregnanolone (APalpha; 5alpha-pregnan-3alpha-ol-20-one) promotes neural progenitor proliferation in vitro in cultures of rodent hippocampal and human cortical neural progenitors, and in vivo in triple transgenic Alzheimer's disease mice dentate gyrus. We also found that APalpha-induced proliferation of neural progenitors is abolished by a calcium channel blocker, nifedipine, indicating a calcium dependent mechanism for the proliferation.
In the present study, we investigated the effect of APalpha on the regulation of intracellular calcium concentration in E18 rat hippocampal neurons using ratiometric Fura2-AM imaging.
Results indicate that APalpha rapidly increased intracellular calcium concentration in a dose-dependent and developmentally regulated manner, with an EC50 of 110 +/- 15 nM and a maximal response occurring at three days in vitro. The stereoisomers 3beta-hydroxy-5alpha-hydroxy-pregnan-20-one, and 3beta-hydroxy-5beta-hydroxy-pregnan-20-one, as well as progesterone, were without significant effect. APalpha-induced intracellular calcium concentration increase was not observed in calcium depleted medium and was blocked in the presence of the broad spectrum calcium channel blocker La3+, or the L-type calcium channel blocker nifedipine. Furthermore, the GABAA receptor blockers bicuculline and picrotoxin abolished APalpha-induced intracellular calcium concentration rise.
Collectively, these data indicate that APalpha promotes a rapid, dose-dependent, stereo-specific, and developmentally regulated increase of intracellular calcium concentration in rat embryonic hippocampal neurons via a mechanism that requires both the GABAA receptor and L-type calcium channel. These data suggest that APalpha-induced intracellular calcium concentration increase serves as the initiation mechanism whereby APalpha promotes neurogenesis.
已知调节细胞内钙浓度的因素在脑功能和神经发育中起关键作用，包括神经可塑性和神经发生。我们之前证明，神经甾体别孕烯醇酮（APα；5α-孕烷-3α-醇-20-酮）在体外可促进啮齿动物海马体和人类皮质神经祖细胞培养物中的神经祖细胞增殖，在体内可促进三重转基因阿尔茨海默病小鼠齿状回中的神经祖细胞增殖。我们还发现，钙通道阻滞剂硝苯地平可消除APα诱导的神经祖细胞增殖，表明增殖存在钙依赖性机制。
在本研究中，我们使用比率式Fura2-AM成像技术研究了APα对E18大鼠海马神经元细胞内钙浓度调节的影响。
结果表明，APα以剂量依赖性和发育调节的方式迅速增加细胞内钙浓度，EC50为110±15 nM，最大反应出现在体外培养三天时。立体异构体3β-羟基-5α-羟基-孕烷-20-酮、3β-羟基-5β-羟基-孕烷-20-酮以及孕酮均无显著影响。在缺钙培养基中未观察到APα诱导细胞内钙浓度升高，在存在广谱钙通道阻滞剂La3+或L型钙通道阻滞剂硝苯地平时，该升高被阻断。此外，GABAA受体阻滞剂荷包牡丹碱和印防己毒素消除了APα诱导的细胞内钙浓度升高。
总体而言，这些数据表明，APα通过一种需要GABAA受体和L型钙通道的机制，促进大鼠胚胎海马神经元细胞内钙浓度迅速、剂量依赖性、立体特异性和发育调节性增加。这些数据表明，APα诱导的细胞内钙浓度升高是APα促进神经发生的起始机制。
