Differential blockade of opioid analgesia by gene-specific therapeutics directed against various G protein α subnits

针对各种 G 蛋白 α 亚基的基因特异性疗法差异性阻断阿片类镇痛

• 批准号: 11670103
• 负责人: KOBAYASHI Hiroyuki
• 金额: $ 2.3万
• 依托单位: Tokai University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670103/
• 关键词: μ-opioid receptor; G protein α subunits; antisense oligodeoxynucleotide; morphine-induced analgesia; ribozyme; DNAzyme; RT-PCR; ミューオピオイド受容体; in situ hybridization; アンチセンスオリゴヌクレオチド

To define the molecular identity of the G protein α subunits activated by the μ opioid receptor and assess the oligodeoxynucleotides (AS-ODN), ribozymes, or DNAzymes.As phosphorothioate ODNs directed against various G protein α subunits (Gi1〜3α, Goα, and Gsα) mRNA were designed, and the changes in G protein α subunit mRNA or protein levels in the periaqueductal gray (PAG) of rat brain were investigated after the microinjection of each ODN into PAG. Each AS-ODN(1.7 nmol) was microinjected into PAG three times at the interval of 48 hours. Twenty four hours after the last injection, the region injected was stained by bromophenol blue for sampling precisely, and the G protein α subunit mRNA or protein levels were determined by RT-PCR or Western blot, respectively. AS-ODN directed against Gi1α suppressed the level of not only Gi1α but also Goα. The target site selected shared 12-bases sequence homology with Goα. Short AS-ODN, of which the homologous sequence was partly deleted, suppressed the level of only Gi1α not Goα This short-type AS-ODNs directed against Gi1α or Goα suppressed morphine-induced analgesia.To improve the specificity, we designed ribozymes and DNAzymes. Microinjection of ribozymes did not suppress the levels of G protein α subunits mRNA probably due to the instability in the brain. Some DNAzymes modified with O-methy1 group at both ends suppressed the levels of G protein α subunits mRNA after microinjection into brain. The modified DNAzymes directed against Gi1α or Goα suppressed morphine-induced analgesia.Together, these results suggest that morphine-induced analgesia is partly mediated through Gi1α and Goα.

为了确定μ阿片受体激活的G蛋白α亚基的分子身份并评估寡脱氧核苷酸（AS-ODN）、核酶或脱氧核糖核酸酶。设计了针对各种G蛋白α亚基（Gi1〜3α，Goα和Gsα）mRNA的硫代磷酸酯ODN，并研究了G蛋白α亚基的变化 将每种 ODN 显微注射到 PAG 中后，研究了大鼠大脑导水管周围灰质 (PAG) 中的 mRNA 或蛋白质水平。将每个AS-ODN(1.7nmol)显微注射到PAG中3次，间隔48小时。最后一次注射后24小时，将注射区域用溴酚蓝染色精确取样，分别采用RT-PCR或Western blot测定G蛋白α亚基mRNA或蛋白水平。针对 Gi1α 的 AS-ODN 不仅抑制 Gi1α 的水平，还抑制 Goα 的水平。选择的目标位点与Goα共享12个碱基序列同源性。短AS-ODN，其同源序列被部分删除，仅抑制Gi1α而不是Goα的水平。这种针对Gi1α或Goα的短型AS-ODN抑制吗啡诱导的镇痛。为了提高特异性，我们设计了核酶和DNA酶。核酶的显微注射并没有抑制 G 蛋白 α 亚基 mRNA 的水平，这可能是由于大脑中的不稳定性。一些两端带有O-甲基1基团修饰的DNA酶在显微注射到大脑后抑制了G蛋白α亚基mRNA的水平。针对 Gi1α 或 Goα 的修饰 DNAzyme 抑制吗啡诱导的镇痛。总而言之，这些结果表明吗啡诱导的镇痛部分是通过 Gi1α 和 Goα 介导的。

项目成果

Yoshikawa M, et al.: "Time course of changes in mu-opioid receptor mRNA levels in the periaqueductal gray of rat brain by a single or repeated injections of antisense oligodeoxynuclcotides"Jpn. J. Pharmacol. 81. 209-215 (1999)

Yoshikawa M等人：“通过单次或重复注射反义寡脱氧核苷酸，大鼠大脑导水管周围灰质中μ-阿片受体mRNA水平变化的时间过程”Jpn。

Y.Yoshikawa., et al.: "Time course of changes in mu-opioid receptor mRNA levels"Jpn. J. Pharmacol.. 81. 209-215 (1999)

Y.Yoshikawa.等人：“μ-阿片受体mRNA水平变化的时间过程”Jpn。

Oka T, et al.: "Study of μ,∫, and k opioid receptors."In "Basic and clinical aspects of opioids". 172-175 (2000)

Oka T 等人：“μ、∫ 和 k 阿片受体的研究”。《阿片类药物的基本和临床方面》172-175 (2000)。

岡哲雄, 他: "受容体mRNAに対するアンチセンスオリゴデオキシヌクレオチドの特異的及び非特異的作用"医学のあゆみ(別冊7回膜貫通型受容体研究の新展開号). 106-109 (2001)

Tetsuo Oka 等人：“反义寡脱氧核苷酸对受体 mRNA 的特异性和非特异性作用”医学史（7 次跨膜受体研究新进展的单独问题）106-109 (2001)。

Oka T, et al.: "Effects of antisense oligodeoxynucleotides against opioid receptors on the receptor mRNA contents."Life Sci.. 68. 2221-2225 (2001)

Oka T 等人：“针对阿片受体的反义寡脱氧核苷酸对受体 mRNA 含量的影响。”Life Sci.. 68. 2221-2225 (2001)