Hack N, Hidaka H, Wakefield M J, Balázs R
Netherlands Institute for Brain Research, Amsterdam.
Neuroscience. 1993 Nov;57(1):9-20. doi: 10.1016/0306-4522(93)90108-r.
Cerebellar granule cells in culture develop survival requirements which can be met either by chronic membrane depolarization (25 mM K+) or by stimulation of ionotropic excitatory amino acid receptors. We observed previously that this trophic effect is mediated via Ca2+ influx, either through dihydropyridine-sensitive, voltage-dependent calcium channels (activated directly by high K+ or indirectly by kainate) or through N-methyl-D-aspartate receptor-linked ion channels. Steps after Ca2+ entry in the transduction cascade mediating the survival-supporting effect of high K+ and excitatory amino acids have now been examined. Using protein kinase inhibitors (H-7, polymixin B and gangliosides), and modulating protein kinase C activity by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, we obtained evidence against the involvement of protein kinase C and cyclic nucleotide-dependent protein kinases in the transduction cascade. On the other hand, calmidazolium (employed as a calmodulin inhibitor) counteracted the trophic effect of elevated K+ with high potency (IC50 0.3 microM), which exceeded by approximately 10-fold the potency for the blockade by the drug of voltage-sensitive calcium channels. The potency of calmidazolium in interfering with the N-methyl-D-aspartate rescue of cells was also much higher in comparison with the inhibition of 45Ca2+ influx through N-methyl-D-aspartate receptor-linked channels. Our results indicated that after calmodulin the next step in the trophic effects involves Ca2+/calmodulin-dependent protein kinase II activity. KN-62, a fairly specific antagonist of this enzyme, compromised elevated K+ or excitatory amino acid-supported cell survival with high potency (IC50 2.5 microM). In the relevant concentration range, KN-62 had little or no effect on Ca2+ entry through either voltage- or N-methyl-D-aspartate receptor-gated channels. Combining information on the toxic action of glutamate in "mature" granule cells with the trophic effect of either excitatory amino acids or high K+ treatment on "young" cells, we conclude that after the initial steps involving calcium in both cases the respective transduction pathways diverge. The toxic action of glutamate seems to be mediated through protein kinase C [Favaron et al. (1990) Proc. natn. Acad. Sci. U.S.A. 87, 1983-1987 whereas a Ca2+/calmodulin-dependent protein kinase, which can be inhibited by KN-62 (but is resistant to gangliosides and to inhibitors whose potency is higher for protein kinase C than for Ca2+ calmodulin-dependent protein kinases, such as H-7 and polymixin B), is involved critically in the trophic effect.
培养的小脑颗粒细胞形成了生存需求，这可以通过慢性膜去极化（25 mM K+）或通过刺激离子型兴奋性氨基酸受体来满足。我们之前观察到，这种营养作用是通过Ca2+内流介导的，要么通过对二氢吡啶敏感的电压依赖性钙通道（直接由高K+激活或间接由海藻酸激活），要么通过与N-甲基-D-天冬氨酸受体相连的离子通道。现在已经研究了Ca2+进入介导高K+和兴奋性氨基酸的生存支持作用的转导级联反应后的步骤。使用蛋白激酶抑制剂（H-7、多粘菌素B和神经节苷脂），并用佛波酯12-O-十四酰佛波醇-13-乙酸酯处理来调节蛋白激酶C的活性，我们获得了证据，表明蛋白激酶C和环核苷酸依赖性蛋白激酶不参与转导级联反应。另一方面，钙调蛋白拮抗剂（用作钙调蛋白抑制剂）以高效力（IC50 0.3 microM）抵消了升高的K+的营养作用，其效力比该药物阻断电压敏感性钙通道的效力高约10倍。与抑制通过与N-甲基-D-天冬氨酸受体相连的通道的45Ca2+内流相比，钙调蛋白拮抗剂干扰细胞的N-甲基-D-天冬氨酸挽救作用的效力也高得多。我们的结果表明，在钙调蛋白之后，营养作用的下一步涉及Ca2+/钙调蛋白依赖性蛋白激酶II的活性。KN-62是该酶的一种相当特异的拮抗剂，以高效力（IC50 2.5 microM）损害升高的K+或兴奋性氨基酸支持的细胞存活。在相关浓度范围内，KN-62对通过电压门控或N-甲基-D-天冬氨酸受体门控通道的Ca2+内流几乎没有影响。将关于谷氨酸在“成熟”颗粒细胞中的毒性作用的信息与兴奋性氨基酸或高K+处理对“年轻”细胞的营养作用相结合，我们得出结论，在这两种情况下涉及钙的初始步骤之后，各自的转导途径发生分歧。谷氨酸的毒性作用似乎是通过蛋白激酶C介导的[法瓦龙等人（1990年）《美国国家科学院院刊》8
