Audesirk G, Audesirk T, Ferguson C, Lomme M, Shugarts D, Rosack J, Caracciolo P, Gisi T, Nichols P
Biology Department, University of Colorado, Denver 80204.
Brain Res Dev Brain Res. 1990 Aug 1;55(1):109-20. doi: 10.1016/0165-3806(90)90111-b.
The intracellular free Ca2+ concentration, [Ca2+]i, plays an important role in regulating neurite growth in cultured neurons. Insofar as [Ca2+]i is partly a function of Ca2+ influx through voltage-sensitive calcium channels (VSCC), Ca2+ entry through VSCC should influence neurite growth. Vertebrate neurons may possess several types of VSCC. The most frequently described VSCC types are usually designated L, T and N. In most preparations, these VSCC types respond differently to certain pharmacological agents, including Cd2+, Ni2+, the dihydropyridines nifedipine and BAY K8644, and the aminoglycoside antibiotics. We used these agents to study the role of Ca2+ influx in regulating neurite initiation and length in cultures of chick embryo brain neurons and N1E-115 mouse neuroblastoma cells. In chick neurons, nifedipine and Cd2+ (less than 50 microM), which have been reported to inhibit L-type channels, reduced neurite initiation, but not mean neurite length. Ni2+ (less than 100 microM), reported to inhibit T-type channels, had no effect on either initiation or length. Low concentrations of most aminoglycosides (less than 300 microM), reported to inhibit N-type channels, had no effect on neurite initiation, but high concentrations of streptomycin (great than 300 microM), reported to inhibit both L- and N-type channels, reduced neurite initiation. BAY K8644, which enhances current flow through L-type channels, had no effect except at high concentration (50 microM), which inhibited initiation. N1E-115 neuroblastoma cells have been reported to contain L-type and T-type channels, but thus far no channel similar to the N-type has been described. In cultured N1E-115 cells, nifedipine (5 microM), Cd2+ (5 microM), and streptomycin (200 microM) reduced neurite initiation, while nickel (50 microM) and neomycin (100 microM) did not affect initiation. None of these agents altered neurite length. In N1E-115 cells, whole-cell voltage clamp recordings showed that nifedipine and Cd2+ inhibited L-type channels but not T-type channels, while Ni2+ inhibited T-type channels but not L-type channels. Streptomycin slightly inhibited L-type channels but enhanced current flow through T-type channels. Neomycin slightly inhibited both channel types. These data indicated that neurite initiation in these two cell types may be modulated by Ca2+ influx through L-type channels, but not T- or N-type channels. Neurite length was not significantly influenced by any of the agents tested, suggesting that Ca2+ influx through VSCC may not affect neurite elongation.
细胞内游离钙离子浓度[Ca2+]i在调节培养神经元的神经突生长中起重要作用。由于[Ca2+]i部分是通过电压敏感性钙通道（VSCC）流入钙离子的函数，通过VSCC的钙离子内流应会影响神经突生长。脊椎动物神经元可能拥有几种类型的VSCC。最常描述的VSCC类型通常被指定为L型、T型和N型。在大多数标本中，这些VSCC类型对某些药理试剂反应不同，包括Cd2+、Ni2+、二氢吡啶类硝苯地平和BAY K8644，以及氨基糖苷类抗生素。我们使用这些试剂研究钙离子内流在调节鸡胚脑神经元和N1E - 115小鼠神经母细胞瘤细胞培养物中神经突起始和长度方面的作用。在鸡神经元中，据报道可抑制L型通道的硝苯地平和Cd2+（小于50 microM）减少了神经突起始，但不影响神经突平均长度。据报道可抑制T型通道的Ni2+（小于100 microM）对起始或长度均无影响。据报道可抑制N型通道的大多数氨基糖苷类低浓度（小于300 microM）对神经突起始无影响，但高浓度链霉素（大于300 microM）据报道可抑制L型和N型通道，减少了神经突起始。增强通过L型通道电流的BAY K8644除高浓度（50 microM）时抑制起始外无其他影响。据报道N1E - 115神经母细胞瘤细胞含有L型和T型通道，但迄今为止尚未描述类似于N型的通道。在培养的N1E - 115细胞中，硝苯地平（5 microM）、Cd2+（5 microM）和链霉素（200 microM）减少了神经突起始，而镍（50 microM）和新霉素（100 microM）不影响起始。这些试剂均未改变神经突长度。在N1E - 115细胞中，全细胞电压钳记录显示硝苯地平和Cd2+抑制L型通道但不抑制T型通道，而Ni2+抑制T型通道但不抑制L型通道。链霉素轻微抑制L型通道但增强通过T型通道的电流。新霉素轻微抑制两种通道类型。这些数据表明这两种细胞类型中的神经突起始可能受通过L型通道的钙离子内流调节，而不是T型或N型通道。所测试的任何试剂均未显著影响神经突长度，这表明通过VSCC的钙离子内流可能不影响神经突伸长。
