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

Abstract

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.

Gastric Acid Secretion from Parietal Cells Is Mediated by a Ca2+ Efflux Channel in the Tubulovesicle

“…In cultured parietal cells, upon secretagogue stimulation, apical canalicular membranes are engulfed into the cell to form multiple actin-wrapped vacuoles known as vacuolar apical compartments (VACs), which remain separate from the basolateral membrane and free TVs in the cytosol (Nakada et al, 2012). Hence, total VAC membrane area provides a quantitative measurement of TV exocytosis (Nakada et al, 2012).…”

“…Hence, total VAC membrane area provides a quantitative measurement of TV exocytosis (Nakada et al, 2012). In resting WT cells, small (diameter < 2 μm) VACs were observed occasionally with a total surface area < 20 μm 2 (Fig.…”

“…5A). Native VAC membranes are comprised of both apical membranes and TV membranes (Nakada et al, 2012) (Fig. 5A).…”

Computer modeling of gastric parietal cell: significance of canalicular space, gland lumen, and variable canalicular [K+]

“…Examination of many of our micrographs of adult rabbit gastric mucosae revealed ϳ200 gastric glands per linear cm, thus ϳ40,000 glands/cm 2 . Diameter of a single gland lumen, modeled as a simple cylindrical tube, was approximated as 3 m (35,48,49), resulting in a total ϳ0.003 cm 2 of cross-sectional luminal area through which parietal cell secretion would flow to exit 1 cm 2 of flattened mucosa. A gland luminal length of 300 m was chosen for distance from mucosal bath to an average parietal cell (16).…”

“…The very beginning of SC is the only point at which V t is driven to zero solely by the applied current. With the use of ⌬V t and ⌬I from the model to calculate resting R t , 35 (43).…”