This competing renewal application is based on the hypothesis that gut epithelial barrier function is deranged by cellular acidosis. In the currently funded research, the investigator has shown that ileal mucosal acidosis in pigs increases mucosal permeability in vivo, and that exposure of mucosal cell (CaCo-2BBe) monolayers to acid also induce an increase in epithelial permeability in vitro. The investigator's studies have also shown that acidosis promotes lipid peroxidation, increases intracellular Fe2+, and partly depletes cellular ATP in the CaCo-2BBe enterocytes. These studies have demonstrated also that inhibition of calpain and phospholipases ameliorated acid induced mucosal hyperpermeability. These studies collectively support the concept that intracellular calcium related derangements may be important in the pathogenesis of acid induced mucosal barrier dysfunction. The present proposal will extend the studies of CaCo-2BBe cells and monocytes by: 1) measuring intracellular [H+], 2) assessing the effect of acidosis and ATP depletion on epithelial permeability to water soluble macromolecule as well as to particulate matter such as LPS and E.coli bacteria. In addition the proposal will focus on cytoskeletal integrity and its relationship to acidosis induced mucosal cell permeability dysfunction in CaCo-2BBe monolayers. Studies will evaluate also the effect of acidosis or moderate ATP depletion in the enterocytes on [Ca2+]i, and the effects of A23187, BAPTA, and various modifiers of phospholipases calpains and PKC on the acid and/or low ATP level induced mucosal permeability dysfunction. all of the aforementioned studies are to be carrier in the CaCo-2BBe cell line in vitro. Finally, experiments will ascertain the various determinants of ileal mucosal permeability dysfunction in the in vivo studies in a rat model of hemorrhagic injury. These studies will employ in vivo methodology to determine both translocation of LPS and permeability of macromolecules across the ileal epithelium.
Showing the most recent 10 out of 30 publications
