Cholinergic neurons and muscarinic receptors regulate anion secretion in pig distal jejunum

The neurotransmitter acetylcholine is thought to modulate epithelial ion transport in the mammalian small intestine. In this study, the non-selective acetylcholine agonist, carbachol, produced rapid changes in short-circuit current (Isc), an electrical measure of active anion secretion, across isolated sheets of the distal jejunal mucosa-submucosa from swine. The potency of carbachol in elevating Isc was decreased 26-fold in the presence of 10 nM atropine, but remained unchanged by 1-100 μM hexamethonium or 0.1 μM tetrodotoxin. The acetylcholine antagonists produced little or no decrease in spontaneous Isc, whereas tetrodotoxin decreased Isc by 54 μA/cm². [³H]Quinuclidinyl benzilate (QNB), a muscarinic acetylcholine receptor blocker, bound to a single species of sites within the mucosa-submucosa with a K_D = 38 pM and B_max = 94 fmol/mg protein. Selective muscarinic acetylcholine receptor blockers competed with [³H]QNB for this site with a rank order of affinity indicative of an interaction with a M₃-muscarinic acetylcholine receptor. Specific [³H]QNB binding sites were autoradiographically localized in the jejunal wall to the epithelium, submucosa and muscularis propria. Transmural electrical stimulation (3-600 pulses/10 s, 0.5 ms, 60 V) of the mucosa-submucosa produced increases in Isc which were dependent upon the number of impulses delivered and did not undergo tachyphylaxis upon repeated stimulation. Responses to electrical transmural stimulation were inhibited by atropine and hexamethonium, as well as the respective neuronal Na⁺ and Ca²⁺ channel blockers tetrodotoxin and ω-conotoxin GVIA, suggesting that electrical transmural stimulation depolarizes submucosal cholinergic neurons which terminate on ion-transporting epithelial cells. Thus acetylcholine, released upon electrical depolarization of intrinsic submucosal neurons, appears to stimulate anion secretion through interactions with M₃-muscarinic acetylcholine receptors in the mucosa of the porcine distal jejunum.