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

确定被μ阿片受体激活的G蛋白α亚基的分子特性，并评估其寡脱氧核苷酸（AS-ODN）、核酶或DNAzymes。设计了针对不同G蛋白α亚基（Gi1 ~ 3α、Goα和Gsα） mRNA的硫代ODN，研究了各ODN注入大鼠脑导水管周围灰质（PAG）后G蛋白α亚基mRNA或蛋白水平的变化。每个AS-ODN（1.7 nmol）在PAG中微注射3次，间隔48小时。末次注射24小时后，用溴酚蓝对注射区域进行精确取样，分别用RT-PCR和Western blot检测G蛋白α亚基mRNA或蛋白水平。靶向Gi1α的AS-ODN不仅抑制了Gi1α的表达，还抑制了Goα的表达。所选目标位点与Goα具有12个碱基同源性。部分同源序列缺失的短AS-ODN只抑制Gi1α而不抑制Goα的水平，这种短AS-ODN针对的是Gi1α或Goα抑制的吗啡诱导镇痛。为了提高特异性，我们设计了核酶和DNAzymes。微量注射核酶并没有抑制G蛋白α亚基mRNA的水平，这可能是由于核酶在大脑中的不稳定性。经两端o -甲基修饰的DNAzymes可抑制脑内G蛋白α亚基mRNA的表达。修饰后的DNAzymes靶向Gi1α或Goα抑制吗啡诱导的镇痛。综上所述，这些结果表明吗啡诱导的镇痛部分是通过Gi1α和Goα介导的。

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.