Lockerbie R O, Autillo-Touati A, Araud D, Seite R, Chneiweiss H, Glowinski J, Prochiantz A
Chaire de Neuropharmacologie, INSERM U 114, College de France, Paris.
Neuroscience. 1989;28(2):443-54. doi: 10.1016/0306-4522(89)90191-7.
We have recently shown that isolated neuronal growth cones from developing rat forebrain possess an appreciable activity of adenylate cyclase, producing cyclic adenosine monophosphate, which can be stimulated by various neurotransmitter receptor agonists and by forskolin [Lockerbie R. O., Hervé D., Blanc G., Tassin J. P. and Glowinski J. (1988) Devl Brain Res. 38, 19-25]. In the present study, we have investigated the effect of cyclic adenosine monophosphate in an in vitro adhesion assay established between [3H]GABA-labelled isolated growth cones and a Simian virus-40 transformed astrocytic cell line from embryonic mouse striatum. Adhesion of the isolated growth cones onto the astrocytic clone increased steadily up to about 45 min before it began to level off at ca 16-18% of total [3H]GABA-labelled isolated growth cones added. Adhesion of the isolated growth cones onto the astrocytic clone was much superior to that seen on polyornithine and, in particular, on non-treated tissue culture wells. Adhesion "at plateau" was independent of both temperature and extracellular Ca2+ and was markedly reduced (ca 50%) by trypsin pre-treatment of the isolated growth cones. Pre-treatment of the isolated growth cones with either forskolin or lipophilic analogues of cyclic adenosine monophosphate attenuated adhesion in a time- and concentration-dependent manner. Approximately 30% reduction in adhesion to the astrocytic clone "at plateau" was observed after a 15 min pre-treatment of the isolated growth cones with forskolin at 10(-4) M or cyclic adenosine monophosphate analogues at 10(-3) M. A cyclic guanosine monophosphate analogue was without effect on adhesion of isolated growth cones. Scanning electron microscope analysis showed that isolated growth cones pre-treated with either cyclic adenosine monophosphate analogues or forskolin had a simpler morphology when attached to the astrocytic clone than isolated growth cones under control conditions. Pre-treatment of the isolated growth cones with low concentrations of cyclic adenosine monophosphate analogues increased protein kinase activity, measured using an exogenous histone phosphate acceptor, to a level which could not be further stimulated by cyclic adenosine monophosphate. Pre-treatment with a cyclic guanosine monophosphate analogue produced the same effect but only at much higher concentrations than those required for cyclic adenosine monophosphate analogues.
我们最近发现，从发育中的大鼠前脑分离出的神经元生长锥具有相当可观的腺苷酸环化酶活性，可产生环磷酸腺苷，各种神经递质受体激动剂和福斯可林均可刺激该酶活性[洛克比·R·O、埃尔韦·D、布兰克·G、塔辛·J·P和格洛温斯基·J（1988年）《发育脑研究》38卷，19 - 25页]。在本研究中，我们在[³H]γ-氨基丁酸标记的分离生长锥与来自胚胎小鼠纹状体的猿猴病毒40转化星形胶质细胞系之间建立的体外黏附试验中，研究了环磷酸腺苷的作用。分离的生长锥与星形胶质细胞克隆的黏附在约45分钟前稳步增加，然后开始趋于平稳，达到所添加的[³H]γ-氨基丁酸标记的分离生长锥总数的约16 - 18%。分离的生长锥与星形胶质细胞克隆的黏附远优于在聚鸟氨酸上的黏附，特别是在未处理的组织培养孔上的黏附。“平稳期”的黏附与温度和细胞外钙离子均无关，并且通过对分离的生长锥进行胰蛋白酶预处理可使其显著降低（约50%）。用福斯可林或环磷酸腺苷的亲脂性类似物对分离的生长锥进行预处理，会以时间和浓度依赖的方式减弱黏附。在用10⁻⁴M福斯可林或10⁻³M环磷酸腺苷类似物对分离的生长锥进行15分钟预处理后，观察到与星形胶质细胞克隆“平稳期”的黏附减少了约30%。环磷酸鸟苷类似物对分离的生长锥的黏附没有影响。扫描电子显微镜分析表明，用环磷酸腺苷类似物或福斯可林预处理的分离生长锥在附着于星形胶质细胞克隆时，其形态比对照条件下的分离生长锥更简单。用低浓度的环磷酸腺苷类似物对分离的生长锥进行预处理，使用外源性组蛋白磷酸受体测量，可使蛋白激酶活性增加到环磷酸腺苷无法进一步刺激的水平。用环磷酸鸟苷类似物进行预处理也产生了相同的效果，但所需浓度远高于环磷酸腺苷类似物所需的浓度。
