A new approach for high‐pressure freezing of primary culture cells: the fine structure and stimulation‐associated transformation of cultured rabbit gastric parietal cells

• Published in: Journal of microscopy (Oxford) Vol. 208; no. 3; pp. 158 - 166
• Main Authors: Sawaguchi, A., Mcdonald, K. L., Karvar, S., Forte, J. G.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.12.2002
• Subjects: Animals; Cells, Cultured; Cimetidine - pharmacology; Electron microscopy; Freeze Substitution; gastric parietal cell; H(+)-K(+)-Exchanging ATPase - metabolism; high‐pressure freezing; Histamine - pharmacology; Immunohistochemistry; Microscopy, Electron - methods; Parietal Cells, Gastric - drug effects; Parietal Cells, Gastric - ultrastructure; primary culture; Rabbits; transformation
• ISSN: 0022-2720; 1365-2818
• DOI: 10.1046/j.1365-2818.2002.01085.x

Summary A newly designated procedure for high‐pressure freezing of primary culture cells provided excellent ultrastructure of rabbit gastric parietal cells. The isolated parietal cells were cultivated on Matrigel‐coated aluminium plates for conventional subsequential cryoimmobilization by high‐pressure freezing. The ultrastructure of different organelles (Golgi apparatus, mitochondria, multivesicular bodies, etc.) was well preserved compared to conventional chemical fixation. In detail, actin filaments were clearly shown within the microvilli and the subapical cytoplasm. Another striking finding on the cytoskeleton system is the abundance of microtubules among the tubulovesicles. Interestingly, some microtubules appeared to be associating with tubulovesicles. A large number of electron‐dense coated pits and vesicles were observed around the apical membrane vacuoles in cimetidine‐treated resting parietal cells, consistent with an active membrane uptake in the resting state. Immunogold labelling of H+/K+‐ATPase was seen on the tubulovesicular membranes. When stimulated with histamine, the cultured parietal cells undergo morphological transformation, resulting in great expansion of apical membrane vacuoles. Immunogold labelling of H+/K+‐ATPase was present not only on the microvilli of expanded apical plasma membrane vacuoles but also in the electron‐dense coated pits. The present findings provide a clue to vesicular membrane trafficking in cultured gastric parietal cells, and assure the utility of the new procedure for high‐pressure freezing of primary culture cells.