Evaluation of Muscarinic Agonist-Induced Analgesia in Muscarinic Acetylcholine Receptor Knockout Mice

Abstract

Centrally active muscarinic agonists display pronounced analgesic effects. Identification of the specific muscarinic acetylcholine receptor (mAChR) subtype(s) mediating this activity is of considerable therapeutic interest. To examine the roles of the M 2 and M 4 receptor subtypes, the two G i /G o -coupled mAChRs, in mediating agonist-dependent antinociception, we generated a mutant mouse line deficient in both M 2 and M 4 mAChRs [M 2 /M 4 double-knockout (KO) mice]. In wild-type mice, systemic, intrathecal, or intracerebroventricular administration of centrally active muscarinic agonists resulted in robust analgesic effects, indicating that muscarinic analgesia can be mediated by both spinal and supraspinal mechanisms. Strikingly, muscarinic agonist-induced antinociception was totally abolished in M 2 /M 4 double-KO mice, independent of the route of application. The nonselective muscarinic agonist oxotremorine showed reduced analgesic potency in M 2 receptor single-KO mice, but retained full analgesic activity in M 4 receptor single-KO mice. In contrast, two novel muscarinic agonists chemically derived from epibatidine, CMI-936 and CMI-1145, displayed reduced analgesic activity in both M 2 and M 4 receptor single-KO mice, independent of the route of application. Radioligand binding studies indicated that the two CMI compounds, in contrast to oxotremorine, showed Ͼ6-fold higher affinity for M 4 than for M 2 receptors, providing a molecular basis for the observed differences in agonist activity profiles. These data provide unambiguous evidence that muscarinic analgesia is exclusively mediated by a combination of M 2 and M 4 mAChRs at both spinal and supraspinal sites. These findings should be of considerable relevance for the development of receptor subtype-selective muscarinic agonists as novel analgesic drugs.

Alterations in Behavioral Responses to a Cholinergic Agonist in Post-Pubertal Rats with Neonatal Ventral Hippocampal Lesions: Relationship to Changes in Muscarinic Receptor Levels

“…M 2 receptors were shown to play a predominant role in the hypothermic effect of oxotremorine (Bymaster et al, 2003;Gomeza et al, 1999) with an additional contribution of the M 3 subtype (Bymaster et al, 2003). Finally, the analgesic effect of oxotremorine was reported to be predominantly modulated by M 2 receptors with a small contribution of the M 4 subtype (Gomeza et al, 1999;Duttaroy et al, 2002). Further studies will be required to establish which muscarinic receptor subtype(s) is mostly involved in oxotremorine-hyper-responsiveness in NVH lesioned rats.…”

Knockdown of the type 2 and 3 inositol 1,4,5-trisphosphate receptors suppresses muscarinic antinociception in mice

“…Other reports suggest a role of M 2 and M 4 muscarinic receptors in the induction of supraspinal antinociception using muscarinic receptor knockout mice (Duttaroy et al, 2002). The M 2 -M 4 receptors are preferentially coupled via G i proteins to inhibit adenylate cyclase (Caulfield and Birdsall 1998).…”

Modulation of pain transmission by G-protein-coupled receptors

“…Strong M 2 immunoreactivity is located in laminae I-III of the rat and mouse spinal cord (Duttaroy et al, 2002;Li et al, 2002). Using mAChR subtype knockout mice, it has been shown that the M 2 subtype is abundant in the superficial laminae of the dorsal horn, where the M 4 subtype is expressed only at a low level (Duttaroy et al, 2002;Chen et al, 2005a (Chen et al, 2005a).…”

“…In this regard, blockade of mAChRs with atropine in the spinal cord causes a large decrease in the nociceptive threshold in rats (Zhuo and Gebhart, 1991). Intrathecal administration of mAChR agonists or acetylcholinesterase inhibitors produces a potent analgesic effect in rats, mice, and humans (Naguib and Yaksh, 1994;Hood et al, 1997;Ellis et al, 1999;Duttaroy et al, 2002;Chen and Pan, 2003c), and this analgesic effect is blocked by atropine (Naguib and Yaksh, 1994). The mAChRs also mediate some of the antinociceptive effects of opioids (Chen and Pan, 2001) and can enhance the analgesic effect of systemic opioids in humans (Hood et al, 1997).…”

“…But recently, the use of mAChR subtype knockout mice has clearly shown that the M 2 and M 4 subtypes are mainly responsible Duttaroy et al, 2002). Also, activation of the M 2 subtype present on cutaneous nociceptors can suppress the transduction of nociceptive information (Bernardini et al, 2002).…”