Apical vacuole formation by gastric parietal cells in primary culture: effect of low extracellular Ca2+

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.