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

为了确定α阿片受体激活的G蛋白μ亚单位的分子同源性，并对寡聚脱氧核苷酸、核酶或DNA酶进行评价，设计了针对G蛋白α亚单位(Gi1~3α、Goα和Gsα)的硫代寡核苷酸，并将每种寡核苷酸微量注入大鼠中脑导水管周围灰质，观察了G蛋白α亚单位基因或蛋白水平的变化。每个AS-ODN(1.7nmoL)在PAG内注射3次，间隔48h。最后一次注射后2 4h，用溴酚蓝染色准确取材，分别用RTPCR法和Western印迹法检测G蛋白α亚单位的基因和蛋白水平。针对Gi1α的反义寡核苷酸不仅能抑制Gi1α的表达，还能抑制Goα的表达。所选择的靶点与Goα有12个碱基的同源性。部分缺失同源序列的短AS-ODN仅抑制Gi1α而不是Go-α的水平。针对Gi1-α或Go-α的短型AS-ODN抑制吗啡的镇痛作用。微量注射核酶不能抑制G蛋白α亚单位的表达，可能与脑内的不稳定性有关。双端O-甲基修饰的脱氧核酶抑制脑内微量注射后G蛋白α亚单位的表达。针对Gi1α或Goα的DNA酶修饰可抑制吗啡的镇痛作用，提示吗啡的镇痛作用部分是通过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.