Intestinal ischemia and reperfusion is a major problem associated with a high morbidity and mortality following trauma and hemorrhagic shock. Apoptosis is the major mode of cell death following reperfusion. The cytoskeleton damage precedes the apoptotic final microscopic features. Calcium plays a central role in apoptosis. Therefore, we studied whether verapamil could preserve the function of the cytoskeleton in an in vitro intestinal model following hypoxia-reoxygenation (H/R). Our goal was to assess mainly the cytoskeleton functions, which includes IgA transport and the cell monolayer barrier integrity.METHODS
Confluent HT29 intestinal monolayers grown in a two-chamber cell culture system were held under hypoxic (5% CO2) conditions for 90 minutes followed by normoxia (21% O2) (H/R). Cell subsets were exposed to lipopolysaccharide (10 μg/mL) before H/R. Verapamil (8 μM) was added to HT29 cell subsets after H/R treatment. Dimeric IgA was added to the basal compartment, and apical media were sampled at intervals to quantitate IgA transcytosis using enzyme-linked immunosorbent assay. HT29 cells held under normoxic conditions served as controls. HT29 permeability to FD4 was assessed at the end of each experiment. In a separate experiment, HT29 cells were stained for F actin using rhodamine-labeled phalloidin.RESULTS
Intestinal monolayer permeability was increased following treatment with H/R and/or lipopolysaccharide. Verapamil treatment prevented increased permeability in HT29 cells and led to an increase in IgA transport. Disruption of actin microfilaments was demonstrated following H/R insult but was abrogated by the addition of verapamil following H/R insult.CONCLUSION
Reperfusion can lead to both physical and immune derangement of epithelial cell barrier function. Verapamil may be important in preserving gut barrier function. Additional studies including in vivo confirmation in animal shock models are needed to validate these findings.