Inappropriately regulated inflammation of the gastrointestinal tract is common and debilitating, but poorly understood. This proposal focuses on neurogenic mechanisms of intestinal inflammation that induce plasma extravasation and leukocyte infiltration from post- capillary venules. Hypothesis: (1) Intestinal inflammation in initiated by release of the peptide substance P from sensory nerves and generation of bradykinin in plasma and tissues, and their interaction with specific receptors on endothelial cells. (2) Inflammation is terminated by cell-surface proteases that degrade substance P and bradykinin and by desensitization and down-regulation of receptors. (3) Pharmacological blockade or genetic deletion of these receptors or these proteases results in inappropriately initiated or terminated intestinal inflammation. These hypothesis will be tested in mice, due to preliminary results, and the availability of genetic models where receptors and enzymes have been deleted. Inflammation will be assessed by (1) quantifying and localizing extravasation of plasma proteins using Evans blue and Monastral blue, respectively; (2) assessing gap formation between endothelial cells by microscopy: (3) quantifying and localizing neutrophil infiltration using myeloperoxidase.
Specific Aim 1 will examine the involvement of substance P and bradykinin in initiation of inflammation. The time course and duration of effects of exogenous and endogenous peptides will be determined. The receptors mediating these effects will be identified using specific antagonists and genetic deletion, and localized using antibodies.
Specific Aim 2 will examine the role of cell-surface proteases and receptor desensitization in terminating inflammation. The role of neutral endopeptidase and angiotensin converting enzyme in terminating inflammation will be determined using specific inhibitors and genetic deletion. Desensitization of receptors will be examined by delineating the pathway of receptor endocytosis and recycling in endothelial cells.
Specific Aim 3 will evaluate if defects in initiation and termination mechanisms result in an inappropriate inflammatory response in well defined models of intestinal inflammation that have a known neurogenic component. Inflammation will be evaluated in mice in which the key receptors or cell-surface proteases are genetically deleted or are pharmacologically inhibited. These experiments will determine the mechanisms of initiation and termination of intestinal inflammation, evaluate if abnormalities in these mechanisms lead to an inappropriately regulated response, and provide new insights into the mechanisms and possible treatment for intestinal inflammation.
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