Analysis of mechanisms regulation ion transport activities of Na pump isoforms in neurons.

在培养的神经元中存在三种Na泵同种型α1、α2和α3同种型，并且同种型的离子转运活性受到差异调节。在此，我们发现细胞外K^+对α2/α3（一种神经元型钠泵）亚型的抑制是差异调节的关键机制。在基础条件下，生理浓度的K^+仅抑制成熟神经元中的α2/α3亚型。抑制消失后，谷氨酸兴奋的神经元和孵育与钙调素拮抗剂或钙调素激酶II的抑制剂。与成熟的神经元相比，在未成熟的神经元中抑制作用较小，其中亚型活性不受差异调节。此外，当神经元中ATP浓度降低时，抑制减弱，差异调节变得越来越突出。我们还表明，星形胶质细胞衍生因子指导小鼠ES细胞分化为神经元。在星形胶质细胞条件培养基中培养 ...更多信息 在自由漂浮条件下，在4天内，未分化的ES细胞集落产生具有同心层状结构的漂浮球。具体地说，球体具有神经干细胞的外围、分裂的ES细胞的核心和中间层。在ACM中的粘性基质上培养球体促进神经发生。附着的球体在外周产生含有神经元的细胞簇，并且许多具有神经突的神经元在5天内从簇迁移。基因表达分析和电生理学证实了细胞的神经元特性。神经元表达神经元型Na泵，α2和α3亚型。免疫荧光分析显示，许多NF^+神经元和神经突起也呈酪氨酸羟化酶阳性。相反，既不形成星形胶质细胞也不形成少突胶质细胞。该通路为神经发生机制提供了新的见解，也为神经干细胞和神经元的产生提供了简单的程序。少

Three Na pump isoforms α1, α2 and α3 isoforms, are present in cultured neurons and ion transport activities of the isoforms are differentially regulated. Here we show that the inhibition of α2/α3, a neuron type Na pump, isoform by extracellular K^+ is a key mechanism of the differential regulation. Only the α2/α3 isoform is inhibited by physiological concentrations of K^+ under basal conditions in mature neurons. The inhibition was vanished after glutamate excitation of the neurons and by incubation with a calmodulin antagonist or an inhibitor of CaM kinase II. In contrast to mature neurons the inhibition is small in immature neurons, in which the isoform activities are not regulated differentially. Additionally, when ATP concentration in the neurons becomes low, the inhibition diminishes and the differential regulation becomes increasingly prominent. We also show that astrocyte-derived factors instruct mouse ES cells to differentiate into neurons. Cultured in astrocyte-conditioned med … More ium (ACM) under free-floating conditions, within 4 days, colonies of undifferentiated ES cells give rise to floating spheres with a concentric stratiform structure. Specifically, the spheres have a periphery of neural stem cells, a core of dividing ES cells and an intermediate layer. Culturing the spheres on an adhesive substrate in ACM promotes neurogenesis. The attached spheres give rise to clusters of cells containing neurons at the periphery, and many neurons with neurites migrate from the clusters within 5 days. Gene expression analyses and electrophysiology confirm the neuronal properties of the cells. The neurons express a neuron type Na pump, the α2 and α3 isoforms. Immunofluorescence analyses show that many NF^+ neurons and neurites are also positive for tyrosine hydroxylase. In contrast, neither astrocytes nor oligodendrocytes are formed. This pathway provides a new insight into mechanisms of neurogenesis and also provides a simple procedure for generation of neural stem cells and neurons. Less