Mogensen H S, Hack N, Balázs R, Jørgensen O S
Department of Pharmacology, University of Copenhagen, Denmark.
Int J Dev Neurosci. 1994 Aug;12(5):451-60. doi: 10.1016/0736-5748(94)90029-9.
The effects of K(+)-induced membrane depolarization were studied on the survival and biochemical parameters in mouse and rat cerebellar granule cells grown in micro-well cultures. Cell numbers were determined by estimating DNA content using the Hoechst 33258 fluorochrome binding assay. DNA from degenerated cells was removed by prior DNAase treatment. These DNA estimates of cell numbers were comparable with values obtained by direct counting of fluorescein diacetate-stained viable cells. In agreement with previous studies, the survival of rat granule cells was promoted by increasing the concentration of K+ in the medium from 5 to 25 mM throughout a 7-day culture period. In contrast, mouse granule cells survived in culture containing 'low' K+ (5 or 10 mM), as well as in the presence of 'high' K+ (25 mM). On the other hand, several biochemical parameters in mouse granule cells were markedly increased by cultivation in 'high' as compared with 'low' K(+)-containing media, demonstrated by increased fluorescein diacetate esterase activity, enhanced rate of NADPH-dependent tetrazolium reduction, augmented 2-deoxy-D-glucose accumulation and increased N-methyl-D-aspartate-evoked 45Ca2+ influx. It was concluded that although cultivation in 'high' K+ promotes biochemical differentiation in mouse cerebellar granule cells, these cells differ from their rat counterparts in that they do not develop a survival requirement for K(+)-induced membrane depolarization.
研究了钾离子诱导的膜去极化对微孔培养的小鼠和大鼠小脑颗粒细胞存活及生化参数的影响。通过使用Hoechst 33258荧光染料结合测定法估计DNA含量来确定细胞数量。通过预先的DNA酶处理去除退化细胞的DNA。这些细胞数量的DNA估计值与通过直接计数荧光素二乙酸酯染色的活细胞获得的值相当。与先前的研究一致，在整个7天的培养期内，将培养基中钾离子浓度从5 mM增加到25 mM可促进大鼠颗粒细胞的存活。相比之下，小鼠颗粒细胞在含有“低”钾（5或10 mM）的培养基中以及在“高”钾（25 mM）存在的情况下均能存活。另一方面，与含“低”钾的培养基相比，在含“高”钾的培养基中培养可使小鼠颗粒细胞的几个生化参数显著增加，表现为荧光素二乙酸酯酶活性增加、NADPH依赖性四氮唑还原速率增强、2-脱氧-D-葡萄糖积累增加以及N-甲基-D-天冬氨酸诱发的45Ca2+内流增加。得出的结论是，尽管在“高”钾条件下培养可促进小鼠小脑颗粒细胞的生化分化，但这些细胞与大鼠颗粒细胞不同，它们对钾离子诱导的膜去极化没有生存需求。
