Soroka C J, Chew C S, Hanzel D K, Smolka A, Modlin I M, Goldenring J R
Department of Surgery, Yale University School of Medicine, New Haven, CT 06510-8062.
Eur J Cell Biol. 1993 Feb;60(1):76-87.
Primary cultures of rabbit gastric parietal cells respond to various gastric secretagogues as evidenced by morphological alterations and [14C]aminopyrine uptake. The availability of cultures of > 95% purity has allowed us to utilize immunofluorescence and confocal microscopy to observe the direct effect of histamine upon the distribution of membrane and cytoskeletal proteins in parietal cells. Cells cultured for 3 days were incubated for 45 min with or without 10(-4) M histamine, washed, and fixed with 3% paraformaldehyde. Immunofluorescence was performed with antibodies against H+/K(+)-ATPase, Na+/K(+)-ATPase, ezrin, and beta-tubulin, as well as with Bodipy-phallacidin. Anti-H+/K(+)-ATPase antibody stained resting cells in a vesicular cytoplasmic pattern. Stimulation with histamine resulted in the development of a well-defined linear pattern, outlining the expanded secretory canaliculi. The Na+/K(+)-ATPase was restricted to predominantly the lateral surface in both the resting and stimulated cells, suggesting that the cultured parietal cells retain membrane polarity. Ezrin was visualized outlining the intracellular canaliculi in the resting state, and surrounding the large secretory canaliculi in the stimulated cell. Phallacidin labeling of F-actin localized to an area tightly surrounding the intracellular canaliculi in the resting cell, and was comparable with the staining observed with ezrin. In the stimulated cells this fluorescence pattern became more diffuse and surrounded the expanded secretory surface. In both the resting and stimulated cells, antibodies to beta-tubulin revealed a microtubular pattern located predominantly in the basal portion of the cell. These results demonstrate that the cells are capable of translocating the H+/K(+)-ATPase-containing tubulovesicles to a secretory surface, and that they exhibit organization and maintenance of basolateral and canalicular membrane domains. Furthermore, these studies demonstrate the directed movement of membrane and cytoskeletal proteins upon stimulation of the cultured parietal cells.
兔胃壁细胞的原代培养物对各种胃促分泌素产生反应，形态学改变和[14C]氨基比林摄取可证明这一点。纯度大于95%的培养物使我们能够利用免疫荧光和共聚焦显微镜观察组胺对壁细胞中膜蛋白和细胞骨架蛋白分布的直接影响。将培养3天的细胞在有或无10(-4) M组胺的情况下孵育45分钟，洗涤后用3%多聚甲醛固定。用抗H+/K(+)-ATP酶、Na+/K(+)-ATP酶、埃兹蛋白和β-微管蛋白的抗体以及Bodipy-鬼笔环肽进行免疫荧光检测。抗H+/K(+)-ATP酶抗体以囊泡状细胞质模式对静息细胞进行染色。用组胺刺激导致形成明确的线性模式，勾勒出扩张的分泌小管。Na+/K(+)-ATP酶在静息和刺激细胞中主要局限于侧面，这表明培养的壁细胞保持膜极性。埃兹蛋白在静息状态下勾勒出细胞内小管，在刺激细胞中围绕大的分泌小管。鬼笔环肽对F-肌动蛋白的标记定位于静息细胞中紧密围绕细胞内小管的区域，与用埃兹蛋白观察到的染色相当。在刺激细胞中，这种荧光模式变得更加弥散并围绕扩张的分泌表面。在静息和刺激细胞中，抗β-微管蛋白抗体均显示出主要位于细胞基部的微管模式。这些结果表明，细胞能够将含H+/K(+)-ATP酶的微管小泡转运至分泌表面，并且它们表现出基底外侧膜和小管膜结构域的组织和维持。此外，这些研究证明了培养的壁细胞受到刺激时膜蛋白和细胞骨架蛋白的定向运动。
