Schnitzer J E, Oh P, Pinney E, Allard J
Department of Pathology, Harvard Medical School, Beth Israel Hospital, Boston, Massachusetts 02215.
J Cell Biol. 1994 Dec;127(5):1217-32. doi: 10.1083/jcb.127.5.1217.
Caveolae or noncoated plasmalemmal vesicles found in a variety of cells have been implicated in a number of important cellular functions including endocytosis, transcytosis, and potocytosis. Their function in transport across endothelium has been especially controversial, at least in part because there has not been any way to selectively inhibit this putative pathway. We now show that the ability of sterol binding agents such as filipin to disassemble endothelial noncoated but not coated plasmalemmal vesicles selectively inhibits caveolae-mediated intracellular and transcellular transport of select macromolecules in endothelium. Filipin significantly reduces the transcellular transport of insulin and albumin across cultured endothelial cell monolayers. Rat lung microvascular permeability to albumin in situ is significantly decreased after filipin perfusion. Conversely, paracellular transport of the small solute inulin is not inhibited in vitro or in situ. In addition, we show that caveolae mediate the scavenger endocytosis of conformationally modified albumins for delivery to endosomes and lysosomes for degradation. This intracellular transport is inhibited by filipin both in vitro and in situ. Other sterol binding agents including nystatin and digitonin also inhibit this degradative process. Conversely, the endocytosis and degradation of activated alpha 2-macroglobulin, a known ligand of the clathrin-dependent pathway, is not affected. Interestingly, filipin appears to inhibit insulin uptake by endothelium for transcytosis, a caveolae-mediated process, but not endocytosis for degradation, apparently mediated by the clathrin-coated pathway. Such selective inhibition of caveolae not only provides critical evidence for the role of caveolae in the intracellular and transcellular transport of select macromolecules in endothelium but also may be useful for distinguishing transport mediated by coated versus noncoated vesicles.
在多种细胞中发现的小窝或无包被质膜囊泡与许多重要的细胞功能有关，包括内吞作用、转胞吞作用和胞饮作用。它们在内皮细胞跨膜运输中的功能一直存在特别大的争议，至少部分原因是一直没有办法选择性地抑制这条假定的途径。我们现在表明，诸如制霉菌素之类的固醇结合剂能够选择性地分解内皮细胞的无包被而非有包被的质膜囊泡，从而抑制小窝介导的特定大分子在内皮细胞中的细胞内运输和跨细胞运输。制霉菌素显著降低胰岛素和白蛋白跨培养的内皮细胞单层的跨细胞运输。制霉菌素灌注后，大鼠肺微血管对白蛋白的原位通透性显著降低。相反，小分子溶质菊粉的细胞旁运输在体外或原位均未受到抑制。此外，我们表明小窝介导构象修饰白蛋白的清道夫内吞作用，以便将其递送至内体和溶酶体进行降解。这种细胞内运输在体外和原位均受到制霉菌素的抑制。包括制霉菌素和洋地黄皂苷在内的其他固醇结合剂也抑制这一降解过程。相反，网格蛋白依赖性途径的已知配体——活化的α2-巨球蛋白的内吞作用和降解不受影响。有趣的是，制霉菌素似乎抑制内皮细胞摄取胰岛素用于转胞吞作用（这是一个小窝介导的过程），但不抑制用于降解的内吞作用（显然由网格蛋白包被途径介导）。对小窝的这种选择性抑制不仅为小窝在内皮细胞中特定大分子的细胞内和跨细胞运输中的作用提供了关键证据，而且可能有助于区分由包被囊泡与无包被囊泡介导的运输。
