Harris H W, Wade J B, Handler J S
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1988 Mar;85(6):1942-6. doi: 10.1073/pnas.85.6.1942.
Antidiuretic hormone (ADH) increases the water permeability of the toad urinary bladder. The increase occurs in the apical plasma membrane of granular cells that line the urinary surface of the bladder and is produced by the insertion of water permeability units that have been identified by freeze-fracture electron microscopy as intramembrane particle aggregates. Under water-impermeable conditions, particle aggregates reside in intracellular vesicles called "aggrephores." In response to ADH, the aggrephores fuse with the apical plasma membrane and render it water permeable. When ADH is removed, intramembrane particle aggregates and aggrephores are retrieved from the apical membrane, and it returns to a water-impermeable state. To identify proteins involved in the water permeability response, we used lactoperoxidase/glucose oxidase to 125I-label external apical membrane proteins to compare control and ADH-treated bladders. Several polypeptides were consistently labeled in ADH-treated bladders and not in paired controls. After demonstrating that lactoperoxidase behaves as a fluid-phase marker and is sequestered in aggrephore-like vesicles when ADH is withdrawn, we used the technique of Mellman et al. [Mellman, I.S., Steinman, R. M., Unkeless, J. C. & Cohn, Z. A. (1980) J. Cell Biol. 86, 712-722] to label proteins endocytosed when water permeability declines after ADH is withdrawn to test whether the membrane proteins labeled in ADH-treated bladders behaved like particle aggregates. The internalized membranes contained polypeptides of the same molecular weights (55,000, 17,000-14,000, and 7,000) as those labeled on the apical surface of ADH-treated but not control bladders. These polypeptides are evidently involved in the ADH-stimulated water permeability response and may be components of particle aggregates.
抗利尿激素（ADH）可增加蟾蜍膀胱的水通透性。这种增加发生在膀胱尿表面的颗粒细胞顶端质膜上，是由水通透性单位的插入所导致的，这些水通透性单位经冷冻蚀刻电子显微镜鉴定为膜内颗粒聚集体。在水不通透的条件下，颗粒聚集体存在于称为“聚集小泡”的细胞内小泡中。在ADH的作用下，聚集小泡与顶端质膜融合，使其具有水通透性。当去除ADH后，膜内颗粒聚集体和聚集小泡从顶端膜回收，顶端膜恢复到水不通透状态。为了鉴定参与水通透性反应的蛋白质，我们使用乳过氧化物酶/葡萄糖氧化酶对顶端膜外部蛋白质进行125I标记，以比较对照膀胱和经ADH处理的膀胱。在经ADH处理的膀胱中，有几种多肽持续被标记，而在配对的对照膀胱中则没有。在证明乳过氧化物酶表现为液相标记物且在去除ADH时被隔离在类似聚集小泡的小泡中之后，我们使用Mellman等人[Mellman, I.S., Steinman, R. M., Unkeless, J. C. & Cohn, Z. A. (1980) J. Cell Biol. 86, 712 - 722]的技术，在去除ADH后水通透性下降时标记内吞的蛋白质，以测试在经ADH处理的膀胱中标记的膜蛋白是否表现得像颗粒聚集体。内化的膜含有与在经ADH处理但未经对照处理的膀胱顶端表面标记的多肽分子量相同（55,000、17,000 - 14,000和7,000）的多肽。这些多肽显然参与了ADH刺激的水通透性反应，可能是颗粒聚集体的组成成分。
