This competitive renewal proposal is directed at determining which cells from the mouse immune system express 6, g and K opioid receptors, and whether activation of these cells alters the amount of receptor expressed and the distribution of the multiple opioid receptors among the different lymphocyte populations. By using fluorescent opioids and flow cytometry, we have developed a novel indirect fluorescent method for detecting about: opioid receptors. This method is more sensitive than radioreceptor binding methodology. Double-labeling experiments have shown the highest level of about receptor expression on mouse thymocytes and macrophages, and on human microglial cells. The studies proposed in this application focus on the detection of 6 and/.t opioid receptors. Based on functional studies, the first hypothesis to be tested is that mouse thymocytes and CD4+ and CD8+ T cells express ;5 opioid receptors. To detect 6 receptors, two approaches will be compared. FITC-labeled derivatives of the 6-selective ligand, naltrindole, will be used in a manner similar to the labeling of the receptor with the FITC-conjugated K ligand. The second approach will use specific antibodies directed against the 6 receptor. The second hypothesis to be tested is that mouse peritoneal and T cells express the/,t opioid receptor. The g receptor will be labeled with either a FITC-labeled derivative of fentanyl or morphine, and/or antibodies directed against the la opioid receptor. Flow cytometry will be used to detect the labeled receptors. For both 6 and g receptors, the goal is to develop an optimal method for detecting the receptor, and then to use this method to study receptor expression. The third hypothesis is that activation of lymphocytes will increase the expression of opioid receptors since levels of receptor mRNA have been shown to increase with cell activation. Collectively, these studies will use an innovative approach and technique to determine which cells from the immune express about:, 6, and about opioid receptors and if receptor expression is altered with activation of the cells. These studies will lay the foundation for determining the cellular mechanisms by which opioids alter immune function and immunological responses to viral infection, including AIDS.
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