A major objective of this 3-year competitive renewal proposal is to isolate a subpopulation of mouse lymphocytes that express an opioid receptor. Previous studies from this laboratory have shown that the mouse R1.1 thymoma cell line expresses a kappa1 opioid receptor that is negatively coupled to adenylyl cyclase through a pertussis toxin- sensitive G protein, suggesting that certain cells of the immune system can express opioid receptors. Two approaches will be used to isolate a subpopulation of mouse lymphocytes that express opioid receptors. One approach involves conjugating a 14beta-bromoacetamido derivative of naltrexone to magnetic beads, containing a thiol group. Mouse thymocytes, splenocytes, and enriched T-cell populations will be incubated with the opioid-conjugated beads, followed by washing. Cells, containing an opioid receptor, will bind to the beads, and with the use of magnet, these cells will be separated from cells that lack opioid receptors. The affinity ligand recognizes me, delta, and kappa opioid receptors, and should be useful in isolating cells that contain any type of opioid receptor. The second approach involves the use of a high affinity kappa-selective fluorescent opioid to label the cells, followed by an amplification procedure using phycoerythrin. Both of these procedures have been shown to selectively label the R1.1. and derivative thymoma cell lines, suggesting that these approaches will be successful in isolating a murine lymphocyte subpopulation that expresses opioid receptors. Phenotypic markers will be used to characterize the purified cells and the receptors properties will be determined by binding and second messenger assays. Two commercially available cell lines, derived from the R1.1 thymoma, express a larger number of kappa opioid receptors than the parent R1.1 cells. The potency of -GTP to inhibition agonist binding and the maximal inhibition of adenylyl cyclase activity varies among the three cell lines. Using specific antibodies, the G proteins that are coupled to the kappa opioid receptor in the three cell lines will b e determined. Opioid stimulation of low Km GTPase activity will b e used as a measure of kappa opioid receptor coupling to G proteins. Studies will also determine the correlation between the number of spare receptors and desensitization/downregulation of the kappa opioid receptor. By determining the types of immune cells that express opioid receptors and the factors involved in the coupling of the lymphocytic kappa opioid receptor to adenylyl cyclase, the proposed studies will result in a better understanding of the mechanisms involved in the alteration of immune function by narcotics.
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