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

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.

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.

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 (Ca²⁺ 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.

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.

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.