Activation of M3-AChR and IP3/Ca2+/PKC signaling pathways by pilocarpine increases glycine-induced currents in ventral horn neurons of the spinal cord

Our study aimed to determine the effects of pilocarpine and the mechanisms involving muscarinic acetylcholine receptors (mAChRs) on glycine receptors (GlyRs) in neurons of the spinal cord ventral horn. An enzymatic digestion combined with acute mechanical separation was applied to isolate neurons from the spinal cord ventral horn. Patch-clamp recording was then used to investigate the outcomes of pilocarpine. Our results indicate that pilocarpine increased the glycine currents in a concentration-dependent manner, which was blocked by the M3- AChR selective antagonists 4-DAMP and J104129. Pilocarpine also enhanced the glycine currents in nominally Ca2+-free extracellular solution. Conversely, the enhancement of glycine currents by pilocarpine disappeared when intracellular Ca2+ was chelated by BAPTA. Heparin and Xe-C, which are IP3 receptor antagonists, also totally abolished the pilocarpine effect. Furthermore, Bis-IV, a PKC inhibitor, eliminated the pilocarpine effect. Additionally, PMA, a PKC activator, mimicked the pilocarpine effect. These results indicate that pilocarpine may increase the glycine currents by activating the M3-AChRs and IP3/Ca2+/PKC pathways.