Transient, mechanically induced disruptions of cell plasma membranes are now known to occur in cells of gut, skin and muscle. These cell membrane wounds occur not only after an experimentally induced mechanical injury but also during the normal functioning of these tissues, suggesting that they have biological as well as pathological significance. However, as a newly discovered cellular phenomenon, cell membrane wounding remains almost entirely uncharacterized at all basic levels: histological, cellular and molecular. Two basic histological questions - where and at what frequency in stomach and colon does cell wounding occur - that could not be answered in earlier exploratory studies that utilized whole-mounts of excised gut will now be addressed by use of frozen sections of stomach and colon; two recently developed probes for cell wounding, fixable fluorescein dextran and bovine serum albumin; and a newly developed, computer-assisted method for quantitative analysis of cell wounding in frozen sections. In vitro model systems for wounding cell membranes will be used to answer three fundamental questions about the cell biological mechanisms mediating resealing of cell membrane wounds. Flow cytofluorometry will be used to measure how long survivable membrane wounds remain open. Secondly, we will determine quantitatively whether, as in amoeba and other non-mammalian cells, extracellular Ca++ is necessary for mammalian cell survival of plasma membrane wounding. Third, using in vitro models, we propose to test the hypothesis that the actin and/or tubulin based cytoskeleton is involved in membrane resealing. We will apply to mammalian cells wounded at their plasma membranes the fluorescence analogue cytochemical and electron microscopic techniques that had been successfully used to study the role of actin in wound resealing in amoeba; use immunofluorescence and immunoelectron microscopy to visualize the actin- and tubulin-based cytoskeleton during resealing, and use cytoskeletal disrupting agents in functional tests of the requirement of actin and tubulin for membrane resealing. Finally, we propose initial tests of the hypothesis that basic fibroblast growth factor (bFGF) release through gut epithelial cell membranes mediates mucosal repair after injury and/or the cell renewal occurring in undisturbed gut. Bioassay, biochemical and immunochemical approaches previously developed for studying release of BFGF through endothelial membranes as well as previously developed models of mucosal injury will be used in combination to test this last hypothesis.
| Paimela, H; Goddard, P J; Carter, K et al. (1993) Restitution of frog gastric mucosa in vitro: effect of basic fibroblast growth factor. Gastroenterology 104:1337-45 |
