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Preferential stimulation of a neural secretory pathway in the rat colon by acetylcholine
Mammalian colonic mucosa contains cholinergic neurones whose cell bodies are located in the submucosal plexus. Stimulation of such neurones is thought to release both acetylcholine (ACh) and a non-cholinergic neurotransmitter at the neuro-epithelial junction to evoke an increase in basal short-circuit current (Isc) in mucosal preparations (Kuwahara et al, 1987; Przyborski & Levin ,1997). Despite such observations and the demonstration of M3 muscarinic receptors located on epithelial cells low concentrations of ACh were shown to increase Isc in rat colon by a tetrodotoxin-sensitive mechanism. A direct effect on epithelial cells could be demonstrated but only by increasing the concentration of ACh in the presence of tetrodotoxin (O’Malley et al, 1995). The aim of the present study was to investigate the mechanism by which low concentrations of ACh stimulate neural pathways in rat colonic mucosa in preference to a direct action on epithelial cells. Muscle-stripped preparations of colon from male Wistar rats (250-350g) were mounted in Ussing chambers for recording of transepithelial Isc. Values quoted are mean +/- SEM µA.cm-2, statistical analysis used the Mann-Whitney U test for unpaired data. Serosal application of ACh (1-1000µM, n=4) or bethanechol (100-3000µM, n=8)gave concentration dependent increases in Isc with EC50 values and maximum responses of 14+/-1µM and 188+/-16µA.cm-2 and 216+/-45µM and 155+/-23µA.cm-2 respectively. Responses to submaximal concentrations of ACh (10µM) or bethanechol (300µM) were then obtained before and after administration of antagonist drugs or control vehicle (H20). Administration of antagonist drugs was never restricted to a particular region of the colon. In the presence of tetrodotoxin (1µM) hexamethonium (100µM) and hyoscine (0.1µM) responses to ACh were reduced by 90% (167+/-23 to 17+/-6 µA.cm-2, n=8, P<0.05), 64% (123+/-8 to 44+/-9 µA.cm-2, n=8, P<0.05) and 100% (75+/-30 to 0+/-0 µA.cm-2, n=3, P<0.05) respectively. Tetrodotoxin (1µM) and hexamethonium (100µM) reduced responses to bethanechol (300µM) by 78% (89+/-20 to 20+/-8 µA.cm-2, n=3, P<0.05) and 60% (153+/-23 to 61+/-11 µA.cm-2, n =7, P<0.05) respectively. Concentration-response curves were obtained to bethanechol in the absence (EC50 216+/-45µM, n=8) and presence (EC50 1050+/-180µM, n=6) of pirenzepine (0.1µM) giving an apparent pKb of 7.6. Secretory responses to ACh involve activation of both muscarinic and nicotinic receptors. As nicotinic receptors are not found on epithelial cells (Przyborski & Levin, 1997) inhibition of bethanechol by hexamethonium suggests that interneurones and post-synaptic neuroeffector motor neurones are activated by the agonist. In conclusion the neurally mediated responses to both ACh and bethanechol probably result from an action on M1 muscarinic receptors located on interneurones. These release endogenous ACh which stimulates nicotinic receptors on post-synaptic neuroeffector motor neurones .The non-cholinergic neurotransmitter released at neuroepithelial junctions is unknown although in rat ileal mucosa it appears to be vasoactive intestinal peptide (Banks et al, 2005).
Banks MR et al. (2005) Br J Pharmacol. 144:994-1001. |
