Hidalgo J, García A, Oliva A M, Giralt M, Gasull T, González B, Milnerowicz H, Wood A, Bremner I
Departamento de Biología Celular y Fisiología, Universidad Autónoma de Barcelona, Bellaterra, Spain.
Chem Biol Interact. 1994 Dec;93(3):197-219. doi: 10.1016/0009-2797(94)90020-5.
The knowledge of brain metallothionein (MT) regulation and especially of MT presence in specific cell types is scarce. Therefore, the effect of several well-known MT inducers, measured by radioimmunoassays using antibodies that cross-react with MT-I and MT-II or specific for MT-I and which do not cross-react with human growth inhibitory factor (GIF or MT-III), has been studied in primary cultures of neurons or astrocytes obtained from rat cerebrum. MT-I levels in glial cells were about ten times higher than those in neuronal cells (538 +/- 194 vs. 49 +/- 16 pg MT-I/micrograms protein, mean +/- S.D. from three separate cell preparations). Increasing the concentration of Zn in the bovine serum albumin (BSA)-containing culture medium up to 50 microM significantly increased MT-I levels by up to 3.5-fold in neurons and 2.5-fold in astrocytes. In contrast, Cu up to 50 microM increased MT-I levels in a saturable manner in both neurons (up to 5-fold) and astrocytes (up to 1.5-fold), the maximum effect occurring at 5 microM Cu. In general, the combination of Zn and Cu further increased MT-I levels. The effect of the metals on MT-I appeared to reflect metal uptake, since MT-I induction was less marked when the BSA concentration in the medium was increased from 2 to 10 mg/ml. Dexamethasone increased MT-I levels in both neurons and astrocytes in vitro in a concentration-dependent manner. Endotoxin, IL-1 and IL-6 did not have a significant effect on glial MT levels at the concentrations studied. The administration of dexamethasone to rats increased MT-I levels in non-frontal cortex, cerebellum, pons+medulla, midbrain and hippocampus, but not in hypothalamus, frontal cortex and striatum. Endotoxin increased liver but not brain MT-I levels. Immunocytochemical studies in adult rat brain preparations with a polyclonal antibody that cross-reacts with MT-I and MT-II indicated that immunostaining was always nuclear in glial cells, whereas in neurons it was nuclear in the cerebral cortex, hippocampus and the granular layer of the cerebellum, and nuclear plus cytoplasmic in Purkinje cells in the cerebellum, hypothalamic nuclei and gigantocellular reticular nucleus in the brain stem. Meninges, choroidal plexus, ependymal and endothelial cells were also MT-immunoreactive.
关于脑金属硫蛋白（MT）调节的知识，尤其是特定细胞类型中MT的存在情况，目前还很匮乏。因此，我们使用与MT-I和MT-II交叉反应或对MT-I特异且不与人生长抑制因子（GIF或MT-III）交叉反应的抗体，通过放射免疫测定法研究了几种著名的MT诱导剂对从大鼠大脑获得的原代神经元或星形胶质细胞培养物的影响。神经胶质细胞中的MT-I水平比神经元细胞中的高约10倍（538±194对49±16 pg MT-I/μg蛋白质，来自三个独立细胞制剂的平均值±标准差）。将含牛血清白蛋白（BSA）的培养基中锌的浓度提高到50 μM，可使神经元中的MT-I水平显著提高多达3.5倍，星形胶质细胞中提高2.5倍。相比之下，高达50 μM的铜在神经元（高达5倍）和星形胶质细胞（高达1.5倍）中均以饱和方式提高MT-I水平，最大效应出现在5 μM铜时。一般来说，锌和铜的组合进一步提高了MT-I水平。金属对MT-I的影响似乎反映了金属摄取，因为当培养基中BSA浓度从2 mg/ml增加到10 mg/ml时，MT-I诱导作用不太明显。地塞米松在体外以浓度依赖性方式提高了神经元和星形胶质细胞中的MT-I水平。在所研究的浓度下，内毒素、白细胞介素-1和白细胞介素-6对神经胶质细胞MT水平没有显著影响。给大鼠注射地塞米松可提高非额叶皮质、小脑、脑桥+延髓、中脑和海马中的MT-I水平，但在下丘脑、额叶皮质和纹状体中则没有。内毒素提高了肝脏而非大脑中的MT-I水平。用与MT-I和MT-II交叉反应的多克隆抗体对成年大鼠脑制剂进行免疫细胞化学研究表明，神经胶质细胞中的免疫染色始终在细胞核中，而在神经元中，大脑皮质、海马和小脑颗粒层的免疫染色在细胞核中，小脑中的浦肯野细胞、下丘脑核和脑干中的巨细胞网状核的免疫染色在细胞核加细胞质中。脑膜、脉络丛、室管膜和内皮细胞也呈MT免疫反应阳性。
