Rapid communicationThe role of the G protein γ2 subunit in opioid antinociception in mice
Introduction
The synthetic cyclic derivative of the endogenous opioid agonist, enkephalin, c[D-Pen2,D-Pen5]enkephalin (DPDPE), has been shown to produce supraspinal analgesia in mice. The analgesia is antagonized by naltrexone, indicating the involvement of opioid receptors. Previous reports have established the role of Gi/o proteins in DPDPE-mediated analgesia, as the supraspinal analgesia induced by DPDPE was blocked by pertussis toxin (Sanchez-Blazquez and Garzon, 1992) and by antisense phosphorothioate oligodeoxynucleotides to the α subunits of Gi/o proteins (Standifer et al., 1996). However, the molecular mechanisms mediating opioid analgesia have not been established unequivocally.
Agonist binding to opioid receptors has been shown to lead to the inhibition of cAMP formation, the activation of inwardly rectifying K+ channels (GIRKs), the inhibition of voltage-sensitive Ca2+ channels, the stimulation of phospholipase Cβ isoenzymes, and the activation of the mitogen-activated protein (MAP) kinase pathway (Quock et al., 1999). The inhibition of cAMP formation by opioid receptors is thought to be mediated directly by the activated α subunit of the Gi/o proteins. However, recent evidence indicates the involvement of Gβγ subunits of the heterotrimeric G proteins in the regulation of the GIRK and voltage-sensitive Ca2+ channels, certain adenylyl cyclases, the pertussis-toxin-sensitive regulation of PLCβ, and the activation of the MAP kinase pathway (Clapham and Neer, 1997).
Genes encoding 20 different α-, six β- and 12 γ subunits of the G proteins have been identified in mammalian systems (Hamm, 1998). The rules determining the specificity of the interaction between receptors and G protein subunits or between effectors and G protein subunits are not known. In intact cells using antisense oligodeoxynucleotides (Kleuss et al., 1993) or ribozymes (Wang et al., 1997), both the receptor–βγ subunit and βγ subunit–effector interactions were shown to be highly specific.
The amino acid sequences of the β subunits are highly homologous, while the sequences of the γ subunits are more divergent (27–75% homology). Consequently, the specificity of receptor–βγ subunit interactions is thought to be conferred by the γ subunit of the complex. The γ2 subunit is the most abundant G protein (-subunit in brain (Betty et al., 1998). Therefore, we selected the Gγ2 subunit to test the role of the βγ subunit regulated second messenger pathway(s) in the production of analgesia in mouse brain.
In this report, we investigated the involvement of the Gγ2 subunit in antinociception mediated by DPDPE in mice after intracerebroventricular (i.c.v.) treatment with antisense-, mismatch- or missense phosphorothioate oligodeoxynucleotides to the Gγ2 subunit. We found that the treatment with antisense phosphorothioate oligodeoxynucleotide against the Gγ2 subunit produced a significant reduction in antinociception mediated by DPDPE as compared to vehicle-, mismatch phosphorothioate oligodeoxynucleotide- and missense phosphorothioate oligodeoxynucleotide treatments.
Section snippets
Materials and methods
We measured antinociception 24 h after the last oligodeoxynucleotide administration using the 55°C warm water tail-flick test. Male ICR mice were used for this study. All compounds were injected by the i.c.v. route into mice lightly anesthetized with ether. An initial latency time was determined before the mice were included in the study. Mice not responding within 5 s were excluded from further experiments. A phosphorothioate-modified antisense oligodeoxynucleotide, corresponding to
Results and discussion
DPDPE produced 84.0±9.0% antinociception in vehicle-treated mice. In contrast, DPDPE produced 24.4±7.4% antinociception in mice treated with antisense phosphorothioate oligodeoxynucleotide to the Gγ2 subunit. However, mice treated with either mismatch- or missense phosphorothioate oligodeoxynucleotide exhibited 65.1±10.3% and 76.4±23.6% antinociception, respectively (Fig. 1). The differences between antisense phosphorothioate oligodeoxynucleotide-treated mice and vehicle-, mismatch
Acknowledgements
This research was supported by a grant from the National Institute of Drug Abuse (DA 06284).
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