GROWTH AND DEVELOPMENT OF DOPAMINERGIC NEURONS

胚胎中脑多巴胺能神经元的培养 大鼠在完全限定的Neurobasal/B27补充的无血清培养基中生长 介质 当暴露于增加的细胞外K+浓度时， 这些培养物主要通过NMDA受体被破坏。 因为在体内 细胞暴露于高浓度的细胞外K+， 缺血，缺氧，或癫痫发作，我们研究了相关的机制 在K+诱导的神经毒性，并比较他们参与 NMDA诱导的神经毒性。 暴露于高K+后的损伤 水平被MK-801（2.5 μ M）阻断，表明NMDA 受体。 CNQX不能保护神经元免受高K+诱导的损伤。 这种损伤在无Ca ~（2+）的培养基中不发生，Ca ~（2+）/钙调素 拮抗剂W-5在150-200 μ M时完全保护神经元免受 损害 如果KN-62，钙调蛋白依赖性蛋白激酶抑制剂 II以1 μ M存在时，约60-70%的神经元存活，但如果在 此外，环孢菌素A，一种朊蛋白磷酸酶的抑制剂， 钙调神经磷酸酶以1 μ M存在于培养基中，仅30-40%的神经元 生存 由于这些发现涉及Ca 2+和Ca 2 +/钙调素依赖性， 在高K+诱导的神经毒性的机制，我们进一步研究 可能与谷氨酸诱导的毒性有关。 W-5（50和 100 μ M）使K+诱导的谷氨酸释放减少30- 50%。在200 uM W-5 延迟谷氨酸释放，但谷氨酸水平最终达到那些 在没有W5的情况下。 KN-62减少谷氨酸释放30-50% 在0.2和5 μ M时为100%，在1 μ M时为100%。 述钙调磷酸酶抑制剂 环孢菌素A单独仅轻微释放谷氨酸，但与 KN-62延迟谷氨酸的释放，并将其最终水平降低到40%。 与KN-62相比 似乎谷氨酸盐释放是 由进入细胞的Ca 2+激活钙调蛋白触发。 的 对钙调蛋白激活的最早反应是钙调神经磷酸酶催化的 蛋白质去磷酸化 最终细胞外的谷氨酸 浓度是由两个活动的平衡来调节的 钙调蛋白依赖性酶 II和钙调神经磷酸酶。

Cultures of dopaminergic neurons from the mesencephalon of embryonic rat were grown in fully defined Neurobasal/B27 supplemented serum-free medium. When exposed to increased extracellular K+ concentrations, these cultures are damaged mainly via NMDA-receptors. Because in vivo cells are exposed to high concentrations of extracellular K+ during ischemia, anoxia, or seizures, we investigated the mechanisms involved in K+-induced neurotoxicity and compared them to those involved in NMDA-induced neurotoxicity. Damage following exposure to high K+ levels is blocked by MK-801 (2.5uM), indicating a role for NMDA receptors. CNQX does not protect neurons from high K+-induced damage. This damage does not occur in Ca2+-free medium, and the Ca2+/calmodulin antagonist W-5 at 150-200 uM completely protects neurons from the damage. If KN-62, an inhibitor of calmodulin-dependent Protein Kinase II is present at 1 muM, about 60-70% of neurons survive, but if, in addition, cyclosporin A, an inhibitor of the pritein phosphatase, calcineurin, is present in medium at 1 muM, only 30-40% of the neurons survive. Because these findings implicated Ca2+ and Ca2+/calmodulin dependent mechanisms in the high K+-induced neurotoxicity, we examined further their possible relationship glutamate-induced toxicity. W-5 (50 and 100muM) reduced K+-induced glutamate release by 30-50%. At 200 uM W-5 delayed glutamate release, but glutamate levels eventually reached those seen in the absence of W5. KN-62 reduced glutamate release by 30-50% at 0.2 and 5 uM and 100% at 1 uM . The calcineurin inhibitor cyclosporin A alone only slightly glutamate release, but together with KN-62 delayed glutamate release and reduced its final level to 40% of that seen with KN-62 alone. It appears that the lutamate release is triggered by activation of calmodulin by Ca2+ entering the cells. The earliest response to calmodulin activation is calcineurin-catalyzed protein dephosphorylation. Eventually the extracellular glutamate concentration is regulated by the balance of the activities of the two calmodulin-dependent enzymes; Ca2+/ calmodulin- dependent protein kinase II and calcineurin.