Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA.
Am J Physiol Cell Physiol. 2012 Dec 15;303(12):C1301-11. doi: 10.1152/ajpcell.00244.2012. Epub 2012 Oct 24.
In primary culture, the gastric parietal cell's deeply invaginated apical membrane, seen in microscopy by phalloidin binding to F-actin (concentrated in microvilli and a subapical web), is engulfed into the cell, separated from the basolateral membrane (which then becomes the complete plasma membrane), and converted, from a lacy interconnected system of canaliculi, into several separate vacuoles. In this study, vacuolar morphology was achieved by 71% of parietal cells 8 h after typical collagenase digestion of rabbit gastric mucosa, but the tight-junctional protein zonula occludens-1 (ZO-1) was completely delocalized after ∼2 h, when cells were ready for culturing. Use of low-Ca(2+) medium (4 mM EGTA) to release cells quickly from gastric glands yielded parietal cells in which ZO-1 was seen in a small spot or ring, a localization quickly lost if these cells were then cultured in normal Ca(2+) but remaining up to 20 h if they were cultured in low Ca(2+). The cells in low Ca(2+) mostly retained, at 20 h, an intermediate morphology of many bulbous canalicular expansions ("prevacuoles"), seemingly with narrow interconnections. Histamine stimulation of 20-h cells with intermediate morphology caused colocalization of proton-pumping H-K-ATPase with canaliculi and prevacuoles but little swelling of those structures, consistent with a remaining apical pore through which secreted acid could escape. Apparent canalicular interconnections, lack of stimulated swelling, and lingering ZO-1 staining indicate inhibition of membrane fission processes that separate apical from basolateral membrane and vacuoles from each other, suggesting an important role for extracellular Ca(2+) in these, and possibly other, endocytotic processes.
在原代培养中，胃壁细胞的顶面膜深深内陷，在显微镜下可以看到鬼笔环肽结合 F-肌动蛋白（集中在微绒毛和亚顶网上），被细胞吞噬，与基底外侧膜分离（然后成为完整的质膜），并从花边状相互连接的小管系统转变为几个单独的空泡。在这项研究中，在典型的兔胃黏膜胶原酶消化 8 小时后，71%的壁细胞达到空泡形态，但当细胞准备培养时，紧密连接蛋白 ZO-1 完全定位。使用低钙（4 mM EGTA）培养基快速释放细胞从胃腺中获得壁细胞，其中 ZO-1 可见于一个小点或环，当这些细胞在正常钙中培养时，这种定位很快丢失，但如果在低钙中培养，最多可持续 20 小时。在低钙中的细胞大多在 20 小时时保持许多球泡状小管扩张的中间形态（“前空泡”），似乎具有狭窄的连接。组胺刺激具有中间形态的 20 小时细胞会导致质子泵 H-K-ATP 酶与小管和前空泡共定位，但这些结构的肿胀很少，这与分泌酸可以通过的剩余顶端孔一致。明显的小管连接、缺乏刺激肿胀和持续的 ZO-1 染色表明，膜分裂过程受到抑制，这些过程将顶面膜与基底外侧膜和空泡彼此分离，这表明细胞外 Ca(2+) 在这些过程中（可能还有其他内吞过程）起着重要作用。
