HEPATOCYTE:KUPFFER CELL INTERACTIONS IN SURGICAL SEPSIS

University Of Pittsburgh At Pittsburgh, Pittsburgh PA

Investigators

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Abstract

This grant has been devoted to the regulation of the inducible isoform of nitric oxide synthase in trauma and sepsis. The paradigm has emerged that NO as a product of iNOS is diffusible and toxic. In fact, NO can be either protective of toxic within the same organ depending on the setting. In the liver, the investigators hypothesize that its dominant cytoprotective action is due, in part, to tight regulation on the subcellular localization of iNOS such that NO produced by iNOS interacts with specific molecular targets. They also propose that the subcellular localization of iNOS will be determined by specific molecular interactions of iNOS with other proteins and organelles in the cell. In support of this hypothesis, their preliminary data show tight interactions of iNOS with the proteins calmodulin and caspase-3, and selected subcellular structures, including the subcortical cytoskeleton and peroxisomes. Under AIM 1, they propose to define the factors that regulate the subcellular localization of the iNOS molecule during its induction and expression within the cell. This will be accomplished using a combination of fluorescent microscopy, immunoelectron microscopy, and green fluorescent protein-tagged iNOS in living cells. They plan to test the hypothesis that iNOS-calmodulin interaction regulates subcellular localization and that iNOS localization to peroxisomes favors the conversion of NO to reaction products that can carry out nitration and nitrosation. Under AIM 2, the molecular interactions between the caspase family of enzymes and iNOS will be defined. The applicants' preliminary data show that iNOS-caspase interaction results in the proteolytic cleavage of iNOS. It is likely that the iNOS fragments produce toxic oxygen radicals. Conversely, this group has previously shown that NO from iNOS can inhibit caspase activity. Therefore, they propose the iNOS cleavage by caspase is a mechanism to degrade iNOS. To investigate the mechanisms, iNOS will be co-localized with caspase in cells and mutate the putative binding/ cleavage sites in iNOS. They also plan to determine the consequences of iNOS cleavage, to quantitate oxygen radical production, and to determine the effect on cell viability. Completion of these aims will significantly advance our understanding of how the function of iNOS is regulated in the cell and, more importantly, how iNOS can carry out seemingly opposing actions.