Alternative Pathways of Acetylcholine Release in the Colon: Role of High‐Affinity Choline Transporters

ABSTRACT Background Cholinergic neuromuscular transmission is central to gastrointestinal (GI) motility and is traditionally attributed to calcium‐dependent, vesicular acetylcholine (ACh) release from enteric neurons. However, non‐quantal, calcium‐independent mechanisms—possibly involving transporter‐mediated ACh efflux—may also contribute to cholinergic signaling. Aim To investigate both classical and alternative mechanisms of ACh release in the colon, focusing on the potential role of non‐vesicular, transporter‐dependent pathways in modulating smooth muscle contractility. Methods Experiments were performed on full‐thickness and epithelium‐depleted rat colonic muscle strips. Neostigmine, a reversible acetylcholinesterase inhibitor, was used to enhance cholinergic mechanisms. A panel of pharmacological agents—including tetrodotoxin (TTX selective blocker of Na + channels), ω‐conotoxin GVIA (Ca 2+ N ‐type channel blocker), Hemicholinium (choline transporter inhibitor), corticosterone (OCTs inhibitor), and hexamethonium (nicotinic receptor antagonist)—was applied to differentiate neural, non‐neural, and transporter‐mediated contributions to ACh release. Key Results Neostigmine‐induced contractions were preserved in epithelium‐depleted strips, following neural blockade with TTX and ω‐conotoxin GVIA. Hemicholinium concentration‐dependently attenuated these contractions, suggesting involvement of high‐affinity choline transporters operating in reverse mode. In contrast, corticosterone and hexamethonium had negligible effects, arguing against substantial roles for OCTs and nicotinic transmission. Conclusions and Inferences These findings support the existence of a non‐vesicular, transporter‐dependent cholinergic signaling mechanism in the colon. This alternative pathway may contribute to the regulation of colonic motility and represents a novel target in GI motility modulation.