title: Fast freeze-fixation/freeze-substitution reveals the secretory membranes of the gastric parietal cell as a network of helically coiled tubule. A new model for parietal cell transformation.
abstract: The parietal cell of the gastric mucosa undergoes rapid morphological transformation when it is stimulated to produce hydrochloric acid. In chemically fixed cells, this process is seen as a reduction in number of cytoplasmic 'tubulovesicles' as the apical surface of the cell progressively invaginates to increase the secretory surface area. It is widely believed that the tubulovesicles represent stored secretory membrane in the cytoplasm of the unstimulated cell, which is incorporated into the apical membrane upon stimulation, because they share H+,K+-ATPase activity with the apical membrane. However, fusion of tubulovesicles with the apical membrane concomitant with parietal cell activation has never been convincingly demonstrated. We have used fast freeze-fixation and freeze-substitution to study stages of morphological transformation in these cells. Tubulovesicles were not seen in the cytoplasm of any of our cryoprepared cells. Instead, the cytoplasm of the unstimulated cell contained numerous and densely packed helical coils of tubule, each having an axial core of cytoplasm. The helical coils were linked together by connecting tubules, lengths of relatively straight tubule. Lengths of straight connecting tubule also extended from coils lying adjacent to the apical and canalicular surfaces and ended at the apical and canaliculus membranes. Immunogold labelling with alpha- and beta-subunit-specific antibodies showed that the gastric H+,K+-ATPase was localized to the membranes of this tubular system, which therefore represented the configuration of the secretory membrane in the cytoplasm of the unstimulated parietal cell. Stimulation of the cells with histamine and isobutylmethylxanthine lead to modification of the tubular membrane system, correlated with progressive invagination of the apical membrane. The volume of the tubule lumen increased and, as this occurred, the tight spiral twist of the helical coils was lost, indicating that tubule distension was accounted for by partial unwinding. This exposed the cores of cytoplasm in the axes of the coils as rod-shaped elements of a three-dimensional reticulum, resembling a series of microvilli in random thin sections. Conversely, treatment with the H2 antagonist cimetidine caused severe contraction of the tubular membrane system and intracellular canaliculi. Our results indicate that tubulovesicles are an artifact of chemical fixation; consequently, they cannot have a role in parietal cell transformation. From our findings we propose an alternative model for morphological transformation in the parietal cell. This model predicts cytoskeleton-mediated control over expansion and contraction of the tubular membrane network revealed by cryopreparation. The model is compatible with the localization of cytoskeletal components in these cells.
authors.affiliation: Department of Pathology and Immunology, Monash University Medical School, Prahran, Victoria, Australia.
authors.forename: J M
authors.initials: JM
authors.lastname: Pettitt
authors.forename: D C
authors.initials: DC
authors.lastname: Humphris
authors.forename: S P
authors.initials: SP
authors.lastname: Barrett
authors.forename: B H
authors.initials: BH
authors.lastname: Toh
authors.forename: I R
authors.initials: IR
authors.lastname: van Driel
authors.forename: P A
authors.initials: PA
authors.lastname: Gleeson
mesh_terms: D000818:Animals; D002927:Cimetidine / Q000494:pharmacology; D003593:Cytoplasm / Q000648:ultrastructure; D017110:Freeze Substitution; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism; D006632:Histamine / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D008871:Microvilli / Q000648:ultrastructure; D008954:Models, Biological; D010295:Parietal Cells, Gastric / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D017494:Proton Pumps / Q000378:metabolism
chemical_list: D017494:Proton Pumps; D002927:Cimetidine; D006632:Histamine; D017506:H(+)-K(+)-Exchanging ATPase