Cholinergic neurotransmission in the stomach is mediated via muscarinic M ₃ receptors expressed by interstitial cells of Cajal and not directly by smooth muscle

Enteric motor neurotransmission regulates motor functions throughout the gastrointestinal (GI) tract. Acetylcholine (ACh), the predominant neurotransmitter released from enteric excitatory motor neurons, targets muscarinic receptors (M ₂ and M ₃ ) expressed by post-junctional cells. Previous studies, using global knockouts of M ₂ and/or M ₃ receptors, concluded that expression of both muscarinic receptor subtypes by smooth muscle cells (SMCs) is necessary for cholinergic responses. Other studies suggested that cholinergic neurotransmission is transduced primarily by interstitial cells of Cajal (ICC) that are in close association with motor neurons and electrically coupled to SMCs. We examined responses of gastric fundus muscles from mice with conditional knockouts of M ₃ receptors in SMCs or ICC to cholinergic neurotransmission. Electrical and mechanical responses were normal in mice with knockdown of M ₃ in SMCs, but greatly depressed in mice with M ₃ knockdown in ICC. Knowing that SMCs also express muscarinic receptors (primarily M ₂ ), we increased the duration of nerve stimulation and blocked acetylcholinesterase (AChE) to attempt to elicit SMC responses. Large contractions developed after inhibition of AChE, and these responses were blocked by the M ₂ antagonist AF-DX 116, but not by an antagonist of ANO1 channels, a signature conductance that mediates cholinergic responses in ICC. Our experiments demonstrate that cholinergic neurotransmission activates excitatory responses in proximal stomach primarily by ACh binding to M ₃ receptors expressed by ICC. Responses are activated in SMCs, via M ₂ receptors, perhaps only during extreme levels of nerve stimulation or when AChE is inhibited, allowing ACh to overflow from neural-ICC junctions to SMCs.