Yu O, Chuang D M
Section on Molecular Neurobiology, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892-1272, USA.
J Neurochem. 1997 Jan;68(1):68-77. doi: 10.1046/j.1471-4159.1997.68010068.x.
Long-term survival of cultured rat cerebellar granule neurons requires depolarizing concentrations of potassium (high potassium; 25 mM KCl). A high-potassium culturing condition has been reported to increase the intracellular calcium concentration ([Ca2+]i) and the expression of brain-derived neurotrophic factor (BDNF), which in turn induces the expression of neurotrophin-3 (NT-3) in these neurons. We therefore examined the neurotrophic effect of these two neurotrophins in low-potassium (5 mM) cultures and their neuroprotective capabilities against sodium nitroprusside-induced neurotoxicity in both low- and high-potassium cultures. Neuronal survival and neurotrophic effects were monitored by [3H]ouabain binding and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. In low-potassium cultures, the neurotrophic effect of BDNF approached that found in high-potassium cultures but was much more robust than that of NT-3. In contrast, undifferentiated neurons cultured in high-potassium medium were much less responsive to BDNF and not responsive at all to NT-3. Induction of nitroprusside neurotoxicity occurred more readily in low- than in high-potassium cultures. BDNF, NT-3, and a high potassium concentration, alone or in combination, were unable to protect neurons treated with nitroprusside at 50 or 100 microM. However, the neurotoxicity of a lower dose of nitroprusside (10 microM) was reversed by the combined actions of these two neurotrophins in low-potassium cultures and by BDNF alone in high-potassium cultures. Because nitroprusside neurotoxicity is less robust in high-potassium cultures, high-potassium-induced BDNF expression and subsequent NT-3 expression may participate in its neuroprotection and neurotrophism in these cultures. Also, we found that toxic doses of nitroprusside antagonized KCl- and NMDA-induced rises in [Ca2+]i, suggesting that this effect is related to nitroprusside-induced neurotoxicity.
培养的大鼠小脑颗粒神经元的长期存活需要去极化浓度的钾（高钾；25 mM KCl）。据报道，高钾培养条件会增加细胞内钙浓度（[Ca2+]i）和脑源性神经营养因子（BDNF）的表达，进而诱导这些神经元中神经营养因子-3（NT-3）的表达。因此，我们研究了这两种神经营养因子在低钾（5 mM）培养物中的神经营养作用，以及它们在低钾和高钾培养物中对硝普钠诱导的神经毒性的神经保护能力。通过[3H]哇巴因结合和3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐试验监测神经元存活和神经营养作用。在低钾培养物中，BDNF的神经营养作用接近在高钾培养物中发现的作用，但比NT-3的作用更强。相反，在高钾培养基中培养的未分化神经元对BDNF的反应性低得多，对NT-3完全无反应。硝普钠神经毒性在低钾培养物中比在高钾培养物中更容易诱导。BDNF、NT-3和高钾浓度单独或联合使用，均无法保护用50或100 microM硝普钠处理的神经元。然而，较低剂量的硝普钠（10 microM）的神经毒性在低钾培养物中被这两种神经营养因子的联合作用逆转，在高钾培养物中被单独的BDNF逆转。由于硝普钠神经毒性在高钾培养物中不那么强烈，高钾诱导的BDNF表达以及随后的NT-3表达可能参与了这些培养物中的神经保护和神经营养作用。此外，我们发现硝普钠的毒性剂量拮抗KCl和NMDA诱导的[Ca2+]i升高，表明这种效应与硝普钠诱导的神经毒性有关。
