INTESTINAL PERFUSION AND PERMEABILITY IN SEPSIS

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DESCRIPTION: (Adapted from the applicant's abstract). The long- term goal of this application is to elucidate the fundamental mechanisms responsible for intestinal barrier dysfunction in states associated with acute tissue hypoxia and/or inflammation. One unifying hypothesis is that derangements in cellular energy metabolism cause or contribute to alterations in epithelial barrier function in critical illness. Aim I is to study cytokine mediated repression of hypoxia-inducible factor-1 (HIF-1)-dependent adaptive epithelial responses to hypoxia during sepsis. HIF-1 is a transcription factor that regulates the expression of a number of genes associated with adaptive cellular responses to hypoxia. In preliminary studies, they demonstrated that HIF-1 DNA-binding activity is increased when cultured hepatocytes and enterocytes are incubated with a mixture of IFN-gamma and TNF under normoxia. However, these cytokines fail to induce the expression of a HIF-1-dependent luciferase reporter gene. More recently, these cytokines inhibit HIF-1-dependent reporter activity during hypoxia. The applicants hypothesize that (1) HIF-1 DNA-binding activity will increase in the liver and intestinal mucosa of septic animals; (2) adaptive cellular responses to hypoxia will be impaired in cells or tissues that have been exposed to a pro-inflammatory milieu (such as occurs in sepsis); (3) signaling initiated by IFN-gamma and TNF suppresses HIF-1-induced gene expression by blocking the recruitment of CBP/p300 to hypoxia-inducible promoters. Aim II is to evaluate one potential way that an increase in cytosolic ionized calcium concentration, [Ca2+] i, could act to increase intestinal epithelial paracellular permeability. They previously showed that epithelial hyperpermeability caused by ATP depletion is dependent upon the resultant increase in [Ca2+] i. The mechanism(s) whereby increases in [Ca2+] i promote hyperpermeability are unknown. In Aim II they will conduct experiments to test the hypothesis that elevation of [Ca2+] i leads to activation of a calcium-dependent enzyme, myosin light chain kinase (MLCK), and thereby increases phosphorylation of the 20-kDa cytoskeletal protein, myosin light chain (MLC20), resulting in cytoskeletal contraction and epithelial hyperpermeability on that basis. Aim III is to investigate the effect of cytokines, hypoxia, or metabolic inhibition on the polarized basolateral-to-apical transport of complex carbohydrates and other hydrophilic compounds across the intestinal epithelium. These studies are prompted by preliminary data they have obtained, which indicate that a wide variety of compounds, including dextrans and various anionic dyes, are transported across rat colonic mucosa in the serosa-to-mucosa direction via a process that apparently is energy-dependent. Based on these findings, we hypothesize that that impaired barrier function in sepsis (or other forms of acute illness) may not just reflect increased passive permeation in the apical-to basolateral direction, but also decreased active pumping (scavenging) in the opposite direction.