Role of M2, M3, and M4 muscarinic receptor subtypes in the spinal cholinergic control of nociception revealed using siRNA in rats.
Muscarinic acetylcholine receptors (mAChRs) are involved in the control of nociception in the spinal cord. The M(2), M(3), and M(4) mAChR subtypes are present in the spinal dorsal horn. However, the role of the individual subtypes in the anti-nociceptive effect produced by mAChR agonists is uncertain. Here, we determined the contribution of M(2), M(3), and M(4) subtypes to spinal muscarinic analgesia by using small-interference RNA (siRNA) targeting specific mAChR subtypes in rats. The neuronal uptake and distribution of a chitosan-siRNA conjugated fluorescent dye in the spinal cord and dorsal root ganglion were confirmed after intrathecal injection. The control and gene-specific siRNA-chitosan complexes were injected intrathecally for three consecutive days. Quantitative reverse-transcription polymerase chain reaction analysis showed that treatment with siRNA targeting M(2), M(3), or M(4) subtype produced a large reduction in the corresponding mRNA levels in the dorsal root ganglion and dorsal spinal cord. Also, the protein levels of the mAChR subtypes in the spinal cord were significantly down-regulated by siRNA treatment, as determined by the immunoprecipitation and receptor-binding assay. Treatment with the M(2)-siRNA caused a large reduction in the inhibitory effect of muscarine on the nociceptive withdrawal threshold. Furthermore, M(4) knockdown at the spinal level significantly reduced the anti-nociceptive effect of muscarine. However, the anti-nociceptive effect of muscarine was not significantly changed by the M(3)-specific siRNA. Our study suggests that chitosan nanoparticles can be used for efficient delivery of siRNA into the neuronal tissues in vivo. Our findings also provide important functional evidence that M(2) and M(4), but not M(3), contribute to nociceptive regulation by mAChRs at the spinal level.
Department of Anesthesiology and Perioperative Medicine, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.
You-Qing
YQ
Cai
Shao-Rui
SR
Chen
Hee-Dong
HD
Han
Anil K
AK
Sood
Gabriel
G
Lopez-Berestein
Hui-Lin
HL
Pan
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D001672:Biocompatible Materials / Q000494:pharmacology; D048271:Chitosan / Q000008:administration & dosage; D005727:Ganglia, Spinal / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D006930:Hyperalgesia / Q000188:drug therapy / Q000378:metabolism*; D007278:Injections, Spinal; D008297:Male; D009116:Muscarine / Q000494:pharmacology; D018721:Muscarinic Agonists / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D043588:Receptor, Muscarinic M4; D011976:Receptors, Muscarinic / Q000235:genetics / Q000502:physiology*; D013116:Spinal Cord / Q000166:cytology / Q000502:physiology*
D001672:Biocompatible Materials; D018721:Muscarinic Agonists; D034741:RNA, Small Interfering; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D043588:Receptor, Muscarinic M4; D011976:Receptors, Muscarinic; D009116:Muscarine; D048271:Chitosan; D000109:Acetylcholine