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Mechanisms of opioid signal transduction in SH-SY5Y neural cells.

机译：SH-SY5Y神经细胞中阿片类药物信号转导的机制。

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摘要

The mechanisms of opioid receptor coupling through G protein to adenylate cyclase (AC) during acute and long-term receptor occupancy by ligand were investigated in human neuroblastoma SH-SY5Y cells. In intact cells, the {dollar}mu{dollar} agonist DAMGO (Tyr-D-Ala-Gly-(Me)Phe-Gly-ol) bound to two sites of which less than 10% were of high-affinity and pertussis toxin (PTX) sensitive. In isolated membranes, DAMGO bound to a single population of sites with a K{dollar}sb{lcub}rm D{rcub}{dollar} similar to the high-affinity K{dollar}sb{lcub}rm D{rcub}{dollar} in cells and the addition of GTP reduced the B{dollar}sb{lcub}rm max{rcub}{dollar} by 87%. In intact cells and isolated membranes, the kinetic constants for opioid stimulation of low-K{dollar}sb{lcub}rm m{rcub}{dollar} GTPase and inhibition of AC were similar to the high affinity K{dollar}sb{lcub}rm D{rcub}{dollar}, indicating analogous efficiency of signal transduction in both preparations with a large reserve of uncoupled receptors whose population in cells is regulated by endogenous GTP.; Inhibition of AC in SH-SY5Y membranes by DAMGO was blocked 50-60% by either of two antibodies to G{dollar}alpha{dollar}o, while antibodies to G{dollar}alpha{dollar}i{dollar}sb{lcub}1,2{rcub}{dollar} or G{dollar}alpha{dollar}i{dollar}sb3{dollar} had marginal effects. In contrast, inhibition by the {dollar}delta{dollar} agonist DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen-OH) was most sensitive to G{dollar}alpha{dollar}i{dollar}sb{lcub}1,2{rcub}{dollar} antibodies. Similar results were observed with DAMGO in membranes from rat brain stratium. Treatment of SH-SY5Y cells for 24 hrs with DAMGO reduced G{dollar}alpha{dollar}o levels by 26% without altering, G{dollar}alpha{dollar}i{dollar}sb{lcub}1,2{rcub}{dollar}, G{dollar}alpha{dollar}i{dollar}sb3{dollar} or G{dollar}alpha{dollar}s content. Addition of purified Go to membranes from PTX-treated cells restored DAMGO inhibition of AC by 70%, while recovery of DPDPE effects required much higher concentrations. These results describe a novel role for Go, preferential G protein coupling of {dollar}mu{dollar} and {dollar}delta{dollar} receptors, and reduced levels of Go during opioid tolerance.; Long-term treatment of the cells with agonists which were potent in acutely inhibiting AC decreased the B{dollar}sb{lcub}rm max{rcub}{dollar} of antagonist binding by 80-95%, increased the K{dollar}sb{lcub}rm s{rcub}{dollar} for GTPase stimulation 10- to 14-fold, and the K{dollar}sb{lcub}rm i{rcub}{dollar} for AC inhibition 2- to 3-fold. These parameters were only marginally affected by agonists of lower acute potency, regardless of their I{dollar}sb{lcub}rm max{rcub}{dollar}. DAMGO, a potent agonist, retained its ability to induce tolerance even under sub-maximal signal, produced by lower concentrations or by PTX pretreatment. Alkylation of 50% of the receptors by {dollar}beta{dollar}-chlornaltrexamine did not significantly alter the rank order of opioids based on their maximum acute effect. The results show that receptor down-regulation and effector desensitization correlate with the acute potency, not the maximum effect of an opioid in interacting with the effector.; Among differentiating agents investigated, phorbol ester increased the B{dollar}sb{lcub}rm max{rcub}{dollar} of {dollar}mu{dollar}, but not {dollar}delta{dollar} opioid binding, while not affecting receptor coupling to AC. These results indicate potential regulation of opioid signal transduction by specific differentiating agents.
机译：在人类神经母细胞瘤SH-SY5Y细胞中研究了配体对急性和长期受体的阿片样物质受体通过G蛋白偶联至腺苷酸环化酶（AC）的机制。在完整细胞中，{dol} mu {dollar}激动剂DAMGO（Tyr-D-Ala-Gly-（Me）Phe-Gly-ol）结合到两个位点，其中不到10％是高亲和力和百日咳毒素（PTX）敏感。在孤立的膜中，DAMGO与一个高亲和力的K {dollar} sb {lcub} rm D {rcub} {}结合到单个种群的K {dollar} sb {lcub} rm D {rcub} {美元}和GTP的添加将B {dollar} sb {lcub} rm max {rcub} {dollar}降低了87％。在完整的细胞和分离的膜中，阿片样物质刺激低K {dolb} sb {lcub} rm m {rcub} {dollar} GTP酶的动力学常数和AC抑制作用与高亲和力K {dollar} sb {lcub相似} rm D {rcub} {dollar}，表示两种制剂中信号转导的相似效率，这些制剂具有大量未偶联的受体，这些受体的细胞数量受内源性GTP调节。 DAMGO对SH-SY5Y膜中AC的抑制作用被两种抗G {dollar} alpha {dollar} o抗体中的任一种阻断50-60％，而抗G {dollar} alpha {dollar} i {dollar} sb {lcub抗体} 1,2 {rcub} {dollar}或G {dollar} alpha {dollar} i {dollar} sb3 {dollar}的影响很小。相比之下，{dollar}δ{dollar}激动剂DPDPE（Tyr-D-Pen-Gly-Phe-D-Pen-OH）的抑制作用对G {dollar} alpha {dollar} i {dollar} sb { lcub} 1,2 {rcub} {dollar}抗体。用DAMGO在大鼠脑层的膜中观察到了相似的结果。 DAMGO处理SH-SY5Y细胞24小时可将G {dollar} alpha {dollar} o水平降低26％，而不会改变G {dollar} alpha {dollar} i {dollar} sb {lcub} 1,2 {rcub} {dollar}，G {dollar} alpha {dollar} i {dollar} sb3 {dollar}或G {dollar} alpha {dollar}的内容。从PTX处理过的细胞的膜中加入纯化的Go可以使DAMGO对AC的抑制作用恢复70％，而DPDPE效果的恢复需要更高的浓度。这些结果描述了Go的新作用，{μm}和{delta}美元受体的优先G蛋白偶联，以及在阿片样物质耐受期间降低的Go水平。用能强烈抑制AC的激动剂长期处理细胞，可使拮抗剂结合的B {dollar} sb {lcub} rm max {rcub} {dollar}降低80-95％，K {dollar} sb升高对于GTPa​​se刺激，{scub} rm s {rcub} {dollar}为10到14倍，而对AC抑制的K {dolub} sb {lcub} rm i {rcub} {dollar}为2到3倍。这些参数仅受较低急性效力的激动剂的影响，而与Imax（rcub} rm max {rcub} {dollar}无关）。 DAMGO是一种强效激动剂，即使在较低浓度或PTX预处理产生的亚最大信号下，也保持了诱导耐受的能力。 {dol} beta {dollar} -chlornaltrexamine使50％的受体烷基化，但基于其最大的急性作用，并未显着改变阿片类药物的等级顺序。结果表明，受体的下调和效应子的脱敏与急性效力有关，而不是阿片样物质与效应子相互作用的最大作用。在所研究的区分剂中，佛波酯增加了{mu}的B {dolb} sb {lcub} rm max {rcub} {dollar}，但没有增加{dollar} delta {dollar}阿片样物质的结合，同时不影响受体耦合到交流。这些结果表明特定分化剂对阿片样物质信号传导的潜在调节。

著录项

  • 作者

    Carter, Bruce Douglas.;

  • 作者单位

    University of Michigan.;

  • 授予单位 University of Michigan.;
  • 学科 Biology Neuroscience.; Health Sciences Pharmacology.; Biology Cell.
  • 学位 Ph.D.
  • 年度 1992
  • 页码 139 p.
  • 总页数 139
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 神经科学;药理学;细胞生物学;
  • 关键词

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