Bernier-Valentin F, Kostrouch Z, Rabilloud R, Rousset B
Institut National de la Santé et de la Recherche Médicale, U 197, Faculté de Médecine Alexis Carrel, Lyon, France.
Endocrinology. 1991 Oct;129(4):2194-201. doi: 10.1210/endo-129-4-2194.
We have designed a new experimental system based on in vitro reconstituted thyroid follicles (RTF) to study the relative implication of macropinocytosis and micropinocytosis processes in the internalization of thyroglobulin (Tg). Thyrocytes cultured in the presence of TSH reorganize in histiotypic and functional follicles. Tg, which accumulates into the newly formed intrafollicular lumen (IL), was pulse labeled with [125I]iodide. Basal or TSH-activated Tg internalization, i.e. transfer from IL to cells, was assessed by measuring [125I]Tg in the cells and the IL; the IL fraction was collected after selective opening of lumina by a short treatment of RTF in a calcium-free medium. We used the ratio between cellular and IL labeled Tg contents as an endocytic index. TSH caused a very rapid increase in the cellular uptake of labeled Tg; the endocytic index increased by a factor of 4-8. The TSH effect was maximum after 15-20 min. TSH had no effect when the chase-incubation was performed at 4 C, but exhibited the same stimulatory action in terms of both time course and amplitude of action at 20 and 37 C. The macropinocytosis-related cellular structures, the pseudopods, were never observed in RTF maintained at 20 C; they were rare at 37 C and only found after 30 min of TSH treatment. At 20 as well as 37 C, the action of TSH on Tg endocytosis was concentration dependent in the range of 0.05-10 mU/ml. A fraction of Tg internalized by thyrocytes was found in coated vesicles. The labeled Tg content of purified coated vesicles varied with the temperature of the chase-incubation and was increased in TSH-treated RTF. Taken together, these data show that endocytosis of Tg by thyroid follicular cells in resting or moderately activated states does not proceed via the pseudopod formation-dependent mechanism, also termed macropinocytosis. Tg internalization would be related to what is referred as micropinocytosis and would involve a coated vesicle-dependent endocytic pathway.
我们设计了一种基于体外重建甲状腺滤泡（RTF）的新实验系统，以研究巨吞饮作用和微吞饮作用过程在甲状腺球蛋白（Tg）内化中的相对影响。在促甲状腺激素（TSH）存在下培养的甲状腺细胞重组形成组织型和功能性滤泡。积聚在新形成的滤泡内腔（IL）中的Tg用[125I]碘化物进行脉冲标记。通过测量细胞和IL中的[125I]Tg来评估基础状态或TSH激活状态下的Tg内化，即从IL向细胞的转移；在无钙培养基中对RTF进行短时间处理使内腔选择性开放后，收集IL部分。我们将细胞和IL中标记的Tg含量之比用作内吞指数。TSH导致标记的Tg细胞摄取非常迅速地增加；内吞指数增加了4至8倍。TSH作用在15至20分钟后达到最大值。当在4℃进行追踪孵育时，TSH没有作用，但在20℃和37℃时，在时间进程和作用幅度方面都表现出相同的刺激作用。在20℃下维持的RTF中从未观察到与巨吞饮作用相关的细胞结构——伪足；它们在37℃时很少见，并且仅在TSH处理30分钟后才出现。在20℃和37℃时，TSH对Tg内吞作用的影响在0.05 - 10 mU/ml范围内呈浓度依赖性。甲状腺细胞内化的一部分Tg存在于被膜小泡中。纯化的被膜小泡中标记的Tg含量随追踪孵育温度而变化，并且在TSH处理的RTF中增加。综上所述，这些数据表明，处于静止或适度激活状态的甲状腺滤泡细胞对Tg的内吞作用不是通过依赖伪足形成的机制（也称为巨吞饮作用）进行的。Tg内化可能与所谓的微吞饮作用有关，并且涉及依赖被膜小泡的内吞途径。
