ZINC METABOLISM IN ISOLATED LIVER CELLS

我们的目标是研究锌的调控机制 和铜的代谢。 这是使用primary， 大鼠肝实质细胞的单层培养物。 研究 表明糖皮质激素增加细胞内积累 锌的再分布和金属硫蛋白的表达增强 (MT)基因 胰高血糖素增强这一过程。 铜的积累是 受到肾上腺素的刺激 糖皮质激素增加铜蓝蛋白（Cp） 合成和分泌，而肾上腺素增加细胞的流出， 铜作为血浆铜蓝蛋白。 我们现在可以看到 更详细的机制，并将它们放在功能上下文中。 在 下一阶段，将进一步评估铜和锌吸收动力学 通过总细胞滞留，快速积累/快速 交换细胞池和外排交换。 这些参数将 在来自胎鼠、肾上腺切除的年轻成年大鼠、老年大鼠和成年大鼠的细胞中进行了比较。 成年大鼠和链脲佐菌素诱导的糖尿病大鼠。 的影响 地塞米松、肾上腺素、胰高血糖素和加压素以及它们的组合 这些以及LEM和白细胞介素-1对细胞铜和锌摄取的影响 铜蓝蛋白的合成-分泌和MT的合成-降解 考察 放线菌素D抑制蛋白质合成， 放线菌酮和蛋白分泌与衣霉素和亮抑酶肽将是 用于评估与MT和Cp相关的机制。 肝细胞毒素， 最初溴苯和叔丁基过氧化氢和Fe诱导脂质 将评价过氧化作用对摄取动力学、激素 反应性和细胞金属硫蛋白含量和合成， 血浆铜蓝蛋白合成和分泌，细胞内谷胱甘肽（GSH和 GSSG）、脂质过氧化、GSH过氧化物酶和GSH还原酶、H202 生产，葡萄糖生产和3 H-脯氨酸掺入胶原蛋白。 MT mRNA和Cp mRNA水平的变化将通过32 P-cDNA测量。 杂交和北方印迹。 细胞会变得缺铜 或通过改变培养基中的Cu和Zn含量来降低锌缺乏。 后 评估缺陷的标准得到满足， 缺乏激素刺激的细胞和缺乏细胞的能力， 处理细胞毒素的方法将被测试 铜、锌的保护作用 补充，硒，α-胆固醇，13-顺-维甲酸和特定激素 例如将评价地塞米松。

Our goal is to investigate the mechanisms involved in regulation of zinc and copper metabolism by the liver. This is approached using primary, monolayer cultures of rat liver parenchymal cells. Research has demonstrated that glucocorticoids increase the intracellular accumulation and redistribution of zinc and enhanced expression of the metallothionein (MT) gene. Glucagon augments this process. Copper accumulation is stimulated by epinephrine. Glucocorticoids increase ceruloplasmin (Cp) synthesis and secretion while epinephrine increases the efflux of cellular copper as ceruloplasmin. We are now in a position to look at the mechanisms in more detail and place them within a functional context. In the next phase, copper and zinc uptake kinetics will be further evaluated via total cellular retention, retention of the fast accumulated/rapidly exchanged cellular pool and efflux exchange. These parameters will be compared in cells from fetal rats, adrenalectomized young adult rats, old adult rats and streptozotocin-induced diabetic rats. The influences of dexamethasone, epinephrine, glucagon and vasopressin and combinations of these as well as LEM and interleukin-1 on cellular copper and zinc uptake and ceruloplasmin synthesis-secretion and MT synthesis-degradation will be examined. Inhibitions of protein synthesis with actinomycin D and cycloheximide and protein secretion with tunicamycin and leupeptin will be used to evaluate the mechanisms relating to MT and Cp. Liver cell toxins, initially bromobenzene and t-butyl hydroperoxide and Fe-induced lipid peroxidation will be evaluated for an effect on uptake kinetics, hormonal responsiveness and cellular metallothionein content and synthesis, ceruloplasmin synthesis and secretion, intracellular glutathione (GSH and GSSG), lipid peroxidation, GSH peroxidase and GSH reductase, H202 production, glucose production and 3H-proline incorporation into collagen. Changes in MT mRNA and Cp mRNA levels will be measured by 32P-cDNA hybridization and northern blotting. Cells will be made copper deficient or zinc deficient by altering the Cu and Zn content of the media. After criteria to evaluate deficiency have been satisfied, the response of deficient cells to hormonal stimulation and the ability of defient cells to handle the cell toxins will be tested. The protective effect of Cu and Zn repletion, Se, Alpha-tocophenol, 13-cis-retinoic acid and specific hormones e.g. dexamethasone will be evaluated.