OPIATE RECEPTOR MEDIATED EFFECTS OF STRESS ON IMMUNITY

β-内啡肽是一种内源性阿片肽，由 脑下垂体对应激源的反应和合成 脾单个核细胞亚群。尽管它的外围设备 靶点定义不明确，受体的存在对 参与宿主防御的细胞上的β-内啡肽提供了一个可能的位置 对β-内啡肽的作用。事实上，有几个组织报告说 丝裂原诱导的混合淋巴样细胞增殖受到影响 以耐纳洛酮的方式被β-内啡肽抑制。阐明机制 由此纳洛酮耐药受体和/或阿片受体(OR)介导 这些对T淋巴细胞细胞周期的影响将需要 相对均一的正常细胞群。我们计划将CD4+ 与来自不同小鼠的CD8+T细胞(经FAC分选获得99%以上纯度)的对比 菌株，以表征(I)药理和(Ii) 阿片类药物对细胞信号转导通路的影响 交联型T细胞抗原受体的增殖反应 抗CD3-epsilon的(TCR)/CD3复合体。β-内啡肽的作用 关于抗CD3诱导的增殖，将测量以下方面：细胞 倍增时间，细胞按细胞周期时相分布，~3H- 胸腺嘧啶核苷掺入和IL-2分泌。依赖于浓度的 β-内啡肽的作用将与其他OR特异性激动剂进行比较 例如DAGO(Mu)、DPDP-脑啡肽和D-Ala2，Glu4-Deltorphin (delta1vs.delta2)和U50,488(Kappa)；特定的OR拮抗剂也将 被评估。对药物影响的研究背后的假设 介导T细胞的早期信号转导通路上的阿片类药物 增殖是阿片类药物刺激cAMP的合成，这是 作为一种直接的、抗增殖的信号在高度 纯化的正常CD4+和CD8+T细胞。实验将测量 阿片类药物对~3H-cAMP形成和肌醇1，4，5的影响 三磷酸(IP3)和Ras-MAPK(丝裂原活化蛋白激酶) 小路。这两条介导TCR诱导的增殖的途径都是 被营地限制住了。因此，阿片类药物与Forsklin对TCR的影响。 磷脂酶cGamma1(PLCGamma1)的依赖刺激及其结果 将对IP3的形成进行比较。自酪氨酸磷酸化以来 激活PLC-Gamma1，测量其磷酸化程度 提供活动指数。同样，阿片剂与阿司可林的作用 对抗CD3诱导的RAS-MAPK活性进行检测。金额的多少 激活的、过度磷酸化的MAPK可以被量化，因为激活 在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法上，MAPK的活性降低。 此外，MAPK，ERK2的生物活性可以通过定量测定 添加底物的磷酸化，髓鞘碱性蛋白。激活 IL-2高亲和力的IL-2受体复合体是疾病进展所必需的 从G1期进入S期(DNA复制)；这些后来的事件也是 参与TCR介导的增殖。因为我们知道cAMP能抑制 IL-2受体亚单位的表达，阿片类药物与。 Forsklin对IL-2α和β受体亚单位及mRNAs表达的影响 将会被确定。总之，这些调查人员将澄清 阿片类药物对TCR诱导增殖的调控机制 确定对早期和后期信号通路的影响。

Beta-Endorphin is an endogenous opioid peptide, secreted from the pituitary gland in response to stressors and synthesized by subpopulations of splenic mononuclear cells. Although its peripheral targets are not well defined, the existence of receptors selective for Beta-endorphin on cells involved in host defense provides a likely site for the action of Beta-endorphin. Indeed, several groups have reported that mitogen-induced proliferation of mixed lymphoid cells is affected by Beta-endorphin in a naloxone-resistant manner. Elucidating mechanisms whereby a naloxone-resistant receptor and/or opiate receptor (OR) mediate these effects on the cell cycle of T lymphocytes will require a relatively homogenous population of normal cells. We plan to compare CD4+ vs. CD8+ T-cells (more than 99% pure by FAC sorting) from different mouse strains, in order to characterize (i) the pharmacology and (ii) the effects of opioids on signal transduction pathways mediating cell proliferation in response to cross-linking the T-cell antigen receptor (TCR)/CD3 complex with anti-CD3-epsilon. The effects of Beta-endorphin on anti-CD3-induced proliferation will be measured with respect to: cell doubling time, distribution of cells by phase of the cell cycle, 3H- thymidine incorporation and IL-2 secretion. The concentration-dependent effects of Beta-endorphin will be compared to other OR-specific agonists such as DAGO (mu), DPDP-enkephalin and D-ala2,Glu4-deltorphin (delta1vs.delta2) and U50,488 (kappa); specific OR antagonists will also be evaluated. The hypothesis underlying studies of the effects of opiates on early signal transduction pathways mediating T cell proliferation is that opiates stimulate the synthesis of cAMP, which is well established as a direct, anti-proliferative signal in highly purified normal CD4+ and CD8+ T cells. Experiments will measure the effects of opiates on 3H-cAMP formation, and on the inositol 1,4,5 triphosphate (IP3) and Ras-MAPK (mitogen activated protein kinases) pathways. Both of these pathways mediating TCR-induced proliferation are inhibited by cAMP. Thus, effects of opiates vs forskolin on the TCR- dependent stimulation of phospholipase cgamma1 (PLCgamma1) and consequent formation of IP3 will be compared. Since tyrosine phosphorylation activates PLC-gamma1, measuring the extent of its phosphorylation will provide an activity index. Similarly, effects of opiates vs. forskolin on anti-CD3-induced Ras-MAPK activation can be measured. The amount of activated, hyperphosphorylated MAPK can be quantified, since activation of MAPK retards its mobility on SDS-polyacrylamide gel electrophoresis. Also, bioactivity of the MAPK, ERK2, can be assayed by quantifying the phosphorylation of added substrate, myelin basic protein. Activation of high affinity IL-2 receptor complexes by IL-2 is required for progression from G1 into S phase (DNA duplication); these later events are also involved in TCR-mediated proliferation. Since cAMP is known to inhibit expression of IL-2 receptor subunits, the effects of opiates vs. forskolin on expression IL-2 alpha and Beta receptor subunits and mRNAs will be determined. Together, these investigators will clarify mechanisms underlying opiate modulation of TCR-induced proliferation by identifying effects on both early and later signaling pathways.