M2-receptor subtype does not mediate muscarine-induced increases in [Ca2+](i) in nociceptive neurons of rat dorsal root ganglia

Multiple muscarinic receptor subtypes are present on sensory neurons that m ay be involved in the modulation of nociception. In this study we focused o n the presence of the muscarinic receptor subtypes, M2 and M3 (M2R, M3R), i n adult rat lumbar dorsal root ganglia (DRG) at the functional ([Ca2+](i) m easurement), transcriptional (RT- PCR), and translational level (immunohist ochemistry). After 1 day in culture exposure of dissociated medium-sized ne urons (20-35 mm diam) to muscarine was followed by rises in [Ca2+](i) in 76 % of the neurons. The [Ca2+](i) increase was absent after removal of extrac ellular calcium and did not desensitize after repetitive application of the agonist. This rise in [Ca2+](i) may be explained by the expression of M3R, which can induce release of calcium from internal stores via inositoltrisp hospate. Indeed the effect was antagonized by the muscarinic receptor antag onist atropine as well as by the M3R antagonist, 4-diphenylacetoxy-N-(2 chl oroethyl)-piperidine hydrochloride (4-DAMP). The pharmacological identifica tion of M3R was corroborated by RT- PCR of total RNA and single-cell RT- PC R, which revealed the presence of mRNA for M3R in lumbar DRG and in single sensory neurons. In addition, RT- PCR also revealed the expression of M2R, which did not seem to contribute to the calcium changes since it was not pr evented by the M2 receptor antagonist, gallamine. Immunohistochemistry demo nstrated the presence of M2R and M3R in medium-sized lumbar DRG neurons tha t also coexpressed binding sites for the lectin I-B4, a marker for mainly c utaneous nociceptors. The occurrence of muscarinic receptors in putative no ciceptive I-B4-positive neurons suggests the involvement of these acetylcho line receptors in the modulation of processing of nociceptive stimuli.