This 5-year renewal application is directed at characterizing a newly discovered high affinity kappa opioid binding site expressed on the murine thymoma cell line R1.1. Membranes prepared from this cell line bound the kappa-selective alkaloid [3H]U69,593 will high affinity (Kd = 0.23 + 0.018 nM) and specificity. Opioids known to inhibit kappa opioid binding to brain membranes were effective at inhibiting the binding of both [3H]U69,593 and (-)[3H]bremazocine to R1.1 cell membranes. The dynorphin peptides inhibited binding with Ki values of less than 0.5 nM. Neither a mu- nor a delta- opioid binding site was detected on membranes from R1.1 cells. The subtype of kappa binding site on R1.1 cells will be determined by further characterizing the opioid binding properties of this cell line. Sodium inhibited agonist binding, and in the presence of sodium, GTP further attenuated (-)[3H]bremazocine binding to R1.1 membranes, suggesting that this kappa opioid binding site may be coupled to a G protein. Studies will be directed at determining if kappa agonists alter cyclic AMP production. As an indicator of coupling to a G protein, the ability of opioids to stimulate low Km GTPase activity will be examined in R1.1 cell membranes. The specificity of any agonistic effect on either adenylyl cyclase or low Km GTPase activity will be determined by testing whether the kappa-selective antagonist, nor-BNI, can block an agonistic effect and whether mu- and delta-selective opioids can produce an effect. The ability of kappa agonists to down-regulation, and antagonists to upregulate, this binding site will be investigated by culturing R1.1 cells in the presence of kappa-selective agonists and antagonists. The Kd and Bmax values for kappa opioid binding to membranes prepared from control and treated cells will be compared. If this kappa opioid binding site is coupled to adenylyl cyclase, desensitization of this second messenger system will be investigated concomitantly with changes in the affinity and number of kappa opioid binding sites. Murine thymocytes and T-cell populations, isolated based on their ability to recognize dynorphin, will be studied to determine if a population of cells expressing a kappa opioid binding site, similar to the one observed on the R1.1 thymoma, can be detected and characterized. The experiments described in this proposal will result in an understanding of the high affinity binding sites and second messenger systems by which kappa opioids can influence immunocompetence.
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