The role of arginine in the function of macrophages has gained importance with the discovery of the inducible nitric oxide synthase pathway. Nitric oxide produced from arginine through this pathway plays key roles in host defenses. Arginases are a family of enzymes that can also be expressed in immune cells, providing an alternative pathway for the metabolism of arginine. Arginases produce L-ornithine, the precursor of polyamines and proline, substances essential for cellular proliferation and repair. Arginases can also compete with nitric oxide synthase for L- arginine, therefore limiting the production of nitric oxide in some cases. Our laboratory has recently described increased arginase I isoenzyme expression in macrophages after trauma, and that this increase in activity can be blocked with beta-adrenergic blockers. We have also shown that arginase activity can be increased in vitro in normal and transformed macrophages with the use of catecholamines. The present research proposal has as its main specific aim to determine the role played by catecholamines in the metabolism of arginine in the macrophage.
This aim will answer the following questions: 1) What type of arginase isoenzyme is upregulated by catecholamines? 2) What is the interaction between catecholamines and other cytokines known to induce arginase (such as IL-4 and IL-10) in the expression of arginase? 3) What are the effects of catecholamines on the expression of other arginine metabolizing enzymes? Carefully designed experiments using a macrophage cell line, RAW 264.7, will be used to answer these questions. Expression of arginine-metabolizing enzyme activity will be measured by functional assays, while protein and mRNA expression will be evaluated with the use of Western blotting, RT-PCR and Northern blotting. Because altered arginine metabolism in immune cells in trauma and surgery affects cellular immunity, the completion of the present proposal will provide key evidence for the role of catecholamines in the modulation of the immune system. Post-injury infection resulting from immune dysfunction is the principal cause of mortality in the intensive care unit. Therefore, understanding the mechanisms of immune dysfunction after injury has great clinical significance.
