This is an application for three years of support for a collaboration between the U.S. Principal Investigator and Dr. Hirzir Hurtel of the Department of Physiology of Marmara University School of Medicine in Istanbul, Turkey. The purpose of the proposed work is to determine the role of endothelin-1 in producing the mucosal and microvascular damage that occurs as a result of ischemia and reperfusion of the circulation of the small intestine. It is known that reperfusion of the ischemic intestine results in an acute inflammatory-like response including neutrophil infiltration, increased microvascular permeability, the formation of reactive oxygen metabolites, and intestinal mucosal barrier failure. Endothelin-1, which is a 21-amino acid peptide made in vascular endothelial cells, is a potent vasoconstrictor, modulator of neutrophils, and enhancer of superoxide production by neutrophils. Although endothelin-1 has been implicated in the pathogenesis of various organ disorders, its possible role in inducing intestinal damage following an ischemic-reperfusion episode has not been characterized. Therefore, the goal of the proposed research is to test the hypothesis that endothelin-1 contributes to the disturbances in mucosal microcirculatory function resulting from an episode of intestinal ischemia and reperfusion. There are three specific aims for the proposed work. The first is to determine whether endothelin-1 can cause intestinal neutrophil infiltration and mucosal damage, when infused into the abdominal aorta of the rat, and whether this response is associated with the production of reactive oxygen metabolites and increased permeability of mucosal and/or capillary membranes. Neutrophil infiltration will be assessed by monitoring myeloperoxidase activity in intestinal tissue samples, the production of oxygen metabolites will be examined by measuring lipid, protein and sulfhydryl oxidations, microvascular permeability will be measured by the penetration of plasma protein-bound Evan's blue dye into intestinal tissue, and mucosal permeability will be estimated using the blood to intestinal lumen clearance of 51Cr-EDTA. To determine whether putative permeability changes produced by endothelin-1 are mediated by polymorphonuclear leukocytes, anti-neutrophil serum, monoclonal antibodies against leukocyte or endothelial cell adhesion molecules, and receptor antagonists against LTB4 or PAF will be administered to some animals prior to the infusion of endothelin-1. The second specific aim is to determine whether levels of endothelin-1 change in the intestine after an ischemic-reperfusion episode, and whether endothelin-1 antagonists can protect against the mucosal and permeability changes that occur in response to ischemia-reperfusion. The third specific aim will be to determine whether endothelin-1 antagonists can protect against the neutrophil infiltration and oxidant production that results from ischemia and reperfusion.
