CELLULAR UPTAKE OF THYROID HORMONE BY HUMAN HEPATOCYTES

人肝细胞对甲状腺激素的细胞摄取

• 批准号: 3463405
• 负责人: DAVID H SARNE
• 金额: $ 9.3万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3463405
• 关键词: adenosine triphosphate; albumins; amiodarone; free fatty acids; furosemide; hormone binding protein; hormone receptor; human tissue; hyperthyroidism; hypothyroidism; liver cells; liver metabolism; membrane permeability; neoplastic cell culture for noncancer research; peptide hormone metabolism; pinocytosis; propranolol; thyroid hormone binding protein; thyroid hormones; thyroxine; triiodothyronine

The entry of thyroid hormone (TH) into cells is a critical, but poorly understood process. Alterations of TH entry into cells may contribute to changes in thyroid hormone metabolism during fasting, in severely ill patients (NTI), in hypothyroidism, and as a consequence of drugs. Altered entry of TH into cells has been described in patients with abnormal TH serum binding proteins and as a primary inherited defect producing generalized resistance to TH. The long term goal of these studies is to delineate the mechanisms and regulation of the entry of thyroid hormone into human cells and to evaluate its contributions to clinical problems. Cultured human hepatocytes, Hep G2, will be utilized as they retain many properties of normal human liver cells and allow experimental conditions to be controlled. Incubation of these cells with radioiodine labeled T3 and T4 with unlabeled TH and inactive metabolites will establish if human liver cells have saturable uptake systems for TH that are functional at physiologic concentrations, and specific for active TH. Incubation with varying concentrations of free T3 and T4 and purified albumin, thyroxine binding prealbumin (TBPA), and thyroxine binding globulin (TBG) will indicate if these proteins facilitate uptake of TH. Incubation of cells with radioiodine labeled albumin, TBPA, and TBG will determine whether these serum proteins are specifically bound to human liver cells. Uptake of tracer TH from serum of subjects with abnormal serum TH binding proteins will evaluate whether these proteins alter the cellular uptake. Incubation of TH with furosemide and free fatty acids will determine whether these postulated inhibitors of TH binding to serum proteins in NTI also interfere with cellular entry of TH. Treatment of Hep G2 cells with drugs which block cellular ATP generation will indicate whether the uptake of thyroid hormone into human cells is energy dependent. Pre-treatment with drugs which inhibit endocytosis will indicate whether this process is important in the uptake of TH by human cells. Membrane preparations obtained from the Hep G2 cells will be combined with varying concentrations of labeled and unlabeled T3 and T4 to detect specific membrane receptors. Incubation of labeled TH with ipodate, amiodarone and propranolol will evaluate if they alter cellular entry of TH by competing for uptake, inhibiting deiodination, or decreasing cellular ATP content. The effect of long term treatment of Hep G2 cells with hypothyroid serum and serum with excess TH will establish whether thyroid hormone status may alter the entry of TH into human cells.

甲状腺激素(TH)进入细胞是一个关键，但 对流程知之甚少。进入单元格的更改可以 与甲状腺激素代谢的变化有关 禁食，在重症患者(NTI)，在甲状腺功能减退，并作为 毒品的后果。TH进入细胞的改变已经被 TH血清结合蛋白异常患者的描述 作为一种主要的遗传缺陷，产生了普遍的 对TH的抵抗。这些研究的长期目标是 描述甲状腺进入的机制和调节 荷尔蒙注入人体细胞并评估其对 临床问题。培养的人肝细胞，Hep G2，将被 因为它们保留了正常人类肝脏的许多特性而被利用 细胞，并允许控制实验条件。 用放射性碘标记的T3和T4与这些细胞孵育 未标记的TH和非活性代谢物将确定人类 肝细胞对TH有饱和的摄取系统， 在生理浓度下起作用，并对活动具有特异性 那就是。用不同浓度的游离T_3、T_4和 纯化白蛋白、甲状腺激素结合前白蛋白(TBPA)和 甲状腺激素结合球蛋白(TBG)将指示这些蛋白质是否 促进对TH的吸收。用放射性碘孵育细胞 标记的白蛋白、总胆红素和总胆红素将决定这些 血清蛋白与人体肝细胞特异性结合。 血清异常者对示踪剂TH的摄取 TH结合蛋白将评估这些蛋白是否会改变 细胞摄取。TH与速尿和游离脂肪的孵育 酸性物质将决定这些假定的 NTI中与血清蛋白的结合也干扰细胞进入 那就是。阻断细胞生长的药物对Hep G2细胞的作用 ATP的生成将指示甲状腺的摄取 荷尔蒙进入人体细胞是能量依赖的。前处理 抑制内吞作用的药物将表明这是否 过程在人体细胞对TH的摄取中起着重要作用。 从Hep G2细胞获得的膜制剂将是 结合不同浓度的标记和未标记的T3 和T4检测特异性膜受体。孵化 用异丙酚、胺碘酮和普萘洛尔标记TH将评估 如果它们通过竞争摄取来改变TH的细胞进入， 抑制脱碘，或降低细胞内的ATP含量。这个 Hep G2细胞长期治疗对甲状腺功能减退的影响 血清和血清中TH含量过高将确定甲状腺 荷尔蒙状态可能会改变TH进入人体细胞的方式。