Kozak A, Yavin E
Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel.
J Mol Neurosci. 1992;3(4):203-12. doi: 10.1007/BF03380140.
Rat PC12 pheochromocytoma cells loaded with the fluorescent Ca2+ dye fluo-3 or indo-1 and scanned fluorimetrically on a cell sorter apparatus showed a rapid cell density-dependent increase in free cytosolic calcium concentration [Ca2+]i when maintained in suspension cultures. Cell adhesion, measured under a defined set of conditions, was low when cells were seeded at 1.5 x 10(4) cells/ml but reached maximal levels after addition of A23187 calcium ionophore. A six to sevenfold increase in cell density mimicked the effect of the ionophore. Densities above 2 x 10(6) cells/ml caused a decrease in cell adhesion, which was further reduced by the addition of A23187. BAPTA, AM (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and nifedipine (10 microM each), partially inhibited cell attachment (34% and 44% reduction), but at 0.25 microM and 1 microM, respectively, they enhanced attachment (46% and 67% increase). The data suggest that a certain permissive level of [Ca2+]i, attained by either increasing cell density or by the presence of a calcium ionophore, is sufficient for maximal cell adhesion. Above the permissive level, manipulation of [Ca2+]i either by altering cell density or by the addition of calcium blocking agents in high concentrations results in a significant reduction in cell adhesion. Based on these observations, we were able to isolate a biochemically and morphologically distinct cell population. The variant, designated PC12ds (density selected), differed substantially from the original cells. Most notable was a relatively lower content of free [Ca2+]i in the PC12ds cells, as independently assayed by using fluo-3 and indo-1 dyes. In addition, the variant cells exhibited a significantly diminished rate of 45Ca2+ uptake, most likely due to less efficient L-type voltage-dependent calcium (VDC) channels. Addition of several calcium channels agonists and antagonists suggested that PC12ds cells contained relatively more N-type VDC channels, possibly indicating a shift to a neuronal phenotype.
用荧光钙染料fluo - 3或indo - 1加载的大鼠嗜铬细胞瘤PC12细胞，在细胞分选仪上进行荧光扫描，结果显示，当维持悬浮培养时，游离胞质钙浓度[Ca2+]i会随着细胞密度的增加而迅速升高。在一组特定条件下进行测量，当以1.5×10(4)个细胞/毫升的密度接种细胞时，细胞黏附率较低，但在添加A23187钙离子载体后达到最高水平。细胞密度增加6到7倍可模拟离子载体的作用。细胞密度高于2×10(6)个细胞/毫升会导致细胞黏附率下降，添加A23187后进一步降低。BAPTA-AM（1,2-双（邻氨基苯氧基）乙烷-N,N,N',N'-四乙酸）和硝苯地平（各10微摩尔）部分抑制细胞附着（分别降低34%和44%），但在0.25微摩尔和1微摩尔时，它们分别增强了附着（分别增加46%和67%）。数据表明，通过增加细胞密度或存在钙离子载体达到的一定允许水平的[Ca2+]i，足以实现最大细胞黏附。超过允许水平，通过改变细胞密度或添加高浓度钙阻断剂来操纵[Ca2+]i会导致细胞黏附显著降低。基于这些观察结果，我们能够分离出一个在生化和形态上不同的细胞群体。该变体称为PC12ds（密度选择），与原始细胞有很大差异。最显著的是，使用fluo - 3和indo - 1染料独立测定时，PC12ds细胞中游离[Ca2+]i的含量相对较低。此外，变体细胞的45Ca2+摄取率显著降低，最可能的原因是L型电压依赖性钙（VDC）通道效率较低。添加几种钙通道激动剂和拮抗剂表明，PC12ds细胞含有相对较多的N型VDC通道，这可能表明向神经元表型转变。
