OPIATE RECEPTOR MEDIATED EFFECTS OF STRESS ON IMMUNITY

β-内啡肽是一种内源性阿片肽，由 脑下垂体对压力的反应， 脾单核细胞亚群。 虽然其外围 目标没有很好地定义，受体的存在选择性 参与宿主防御的细胞上的β-内啡肽提供了一个可能的位点， β-内啡肽的作用。 事实上，一些团体报告说， 有丝分裂原诱导的混合淋巴细胞增殖受到影响， 通过抗纳洛酮的方式被β-内啡肽抑制。 阐明机制 由此纳洛酮抗性受体和/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 相对同质的正常细胞群。我们计划比较CD 4 + vs.来自不同小鼠的CD 8 + T细胞（通过FAC分选纯度超过99%） 菌株，以表征（i）药理学和（ii） 阿片类药物对细胞信号转导通路的影响 响应于交联T细胞抗原受体的增殖 (TCR)/CD 3复合物与抗CD 3-CD 4。 β-内啡肽的作用 将测量抗CD 3诱导的增殖的以下方面： 倍增时间、细胞周期各阶段的细胞分布、3 H- 胸苷掺入和IL-2分泌。 的浓度依赖 β-内啡肽的作用将与其他OR特异性激动剂进行比较 如DAGO（mu）、DPDP-脑啡肽和D-Ala 2、Glu 4-deltorphin （δ 1对δ 2）和U 50，488（κ）;特异性OR拮抗剂也将 被评价。 研究的基本假设是 阿片类药物对T细胞介导的早期信号转导通路的影响 阿片类药物刺激cAMP的合成， 作为一种直接的抗增殖信号， 纯化的正常CD 4+和CD 8 + T细胞。 实验将测量 阿片类药物对3 H-cAMP形成和肌醇1，4，5的影响 三磷酸（IP 3）和Ras-MAPK（丝裂原活化蛋白激酶） 途径。 这两种介导TCR诱导的增殖的途径都是 被cAMP抑制。 因此，阿片类药物与毛喉素对TCR- 磷脂酶c γ 1（PLC γ 1）的依赖性刺激， 将比较IP 3的形成。 由于酪氨酸磷酸化 激活PLC-γ 1，测量其磷酸化程度将 提供活动指数。 同样，阿片类药物与毛喉素的影响 可以测量抗CD 3诱导的Ras-MAPK活化。 的量 活化的、过度磷酸化的MAPK可以定量，因为活化 在SDS-聚丙烯酰胺凝胶电泳上，MAPK的活化使其迁移率降低。 此外，MAPK，ERK 2的生物活性可以通过定量MAPK，ERK 2的活性来测定。 添加的底物髓鞘碱性蛋白的磷酸化。 激活 IL-2与IL-2受体形成高亲和力复合物是疾病进展所必需的 从G1期进入S期（DNA复制）;这些后期事件也是 参与TCR介导的增殖。 由于已知cAMP抑制 IL-2受体亚单位的表达，阿片类药物对IL-2受体亚单位表达的影响。 毛喉素对IL-2 α和β受体亚单位和mRNA表达的影响 将被确定。 这些调查人员将一起澄清 阿片调节TCR诱导的增殖的机制 识别对早期和晚期信号通路的影响。

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.