OPIOID MODULATION OF IMMUNOCOMPETENCE

这份为期5年的续订申请旨在描述一种新的 发现小鼠表达高亲和力kappa阿片结合位点 胸腺瘤细胞系R1.1。用该细胞系制备的膜结合了 Kappa选择性生物碱U69,593具有很高的亲和力(Kd=0.23+0.018 NM)和特异性。已知阿片类药物抑制kappa阿片类药物与 脑膜能有效地抑制两者的结合。 [~3H]U69,593和(-)[~H]Bremazocine对R1.1细胞膜的作用。强啡肽 多肽抑制与Ki值小于0.5 nM的结合。既不是A 在R1.1膜上检测到MU-NOR-阿片结合位点 细胞。将确定R1.1细胞上kappa结合位点的亚型 通过进一步鉴定该细胞系的阿片结合特性。 钠抑制激动剂结合，在钠存在的情况下，GTP 进一步减弱(-)[~H]Bremazocine与R1.1膜的结合，提示 该kappa阿片结合位点可能与G蛋白偶联。研究 将针对确定kappa激动剂是否改变环状AMP 制作。作为与G蛋白偶联的指标， 将在R1.1细胞中检测刺激低KM GTP酶活性的阿片类药物 膜。任何激动剂作用于任何一种腺苷的特异性 环化酶或低KM GTP酶活性将通过测试是否 Kappa选择性拮抗剂，Nor-BNI，可以阻断激动剂效应和 Mu-和Delta-选择性阿片类药物是否能产生效果。一种能力 Kappa激动剂下调，拮抗剂上调，这 将通过培养R1.1细胞来研究结合部位 Kappa选择性激动剂和拮抗剂。的Kd值和Bmax值 Kappa阿片类药物与对照细胞和处理细胞膜的结合 将会被比较。如果该kappa阿片结合位点与腺苷偶联 环化酶，这个第二信使系统的脱敏 伴随着kappa亲和力和数量的变化而进行研究 阿片类药物结合部位。分离的小鼠胸腺细胞和T细胞群 根据它们识别强啡肽的能力，将进行研究以确定 如果一群表达kappa阿片结合位点的细胞，类似于 在R1.1胸腺瘤上观察到的一种，可以被检测和表征。 本提案中描述的实验将导致一种理解 高亲和力结合位点和第二信使系统 Kappa阿片类药物会影响免疫活性。

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.