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

## TL;DR

The study shows that acetylcholine can be released in the colon through a non-vesicular pathway involving high-affinity choline transporters, which could influence gut motility.

## Contribution

The paper identifies a novel, non-vesicular mechanism of acetylcholine release in the colon mediated by high-affinity choline transporters.

## Key findings

- Neostigmine-induced contractions persisted even after neural blockade, indicating a non-vesicular release mechanism.
- Hemicholinium reduced these contractions, implicating high-affinity choline transporters in the process.
- This pathway may represent a new target for modulating gastrointestinal motility.

## Abstract

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.

Neostigmine‐induced contractions in rat colon persist despite neural blockade, indicating a non‐vesicular mechanism of acetylcholine release.Hemicholinium significantly inhibits these contractions, implicating high‐affinity choline transporters operating in reverse as a key mediator of this alternative cholinergic pathway.High‐affinity choline transporters could provide a novel pathway for acetylcholine release originating from neuronal sources.

Neostigmine‐induced contractions in rat colon persist despite neural blockade, indicating a non‐vesicular mechanism of acetylcholine release.

Hemicholinium significantly inhibits these contractions, implicating high‐affinity choline transporters operating in reverse as a key mediator of this alternative cholinergic pathway.

High‐affinity choline transporters could provide a novel pathway for acetylcholine release originating from neuronal sources.

Mechanisms of Acetylcholine release, including typical and atypical release under neuronal blockade. Left panel illustrates the classical (quantal) cholinergic transmission and the right panel the mechanism described in the present study. High‐Affinity Choline Transporter (HCT), Acetylcholine (ACh), Acetylcholinesterase (ACE), and Tetrodotoxin (TTX).