Pharmacologic Characterization of Intrinsic Mechanisms Controlling Tone and Relaxation of Porcine Lower Esophageal Sphincter

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Abstract
The neurotransmitters mediating relaxation of lower esophageal sphincter (LES) were studied using circular LES strips from adult pigs in organ baths. LES relaxation by sodium nitroprusside (1 nM–3 μM), vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP; 1 nM–1 μM), ATP (10 μM–30 mM), and tricarbonyldichlororuthenum dimer (1 μM–1 mM) was unaffected by tetrodotoxin (1 μM) or l-NG-nitroarginine methyl ester (l-NAME; 100 μM). Calcitonin gene-related peptide (CGRP; 1 nM–1 μM) did not affect LES tone. ATP relaxation was blocked by 1 μM apamin and the P2Y1 antagonist MRS 2179 (N6-methyl 2′-deoxyadenosine 3′,5′-bisphosphate; 10 μM). Apamin inhibited PACAP relaxation. VIP and PACAP relaxation was blocked by 10 U/ml α-chymotrypsin. l-NAME (–62.52 ± 13.13%) and 1H-[1,2,4]oxadiazole-[4,3-α]quinoxalin-1-one (ODQ; 10 μM, –67.67 ± 6.80%) similarly inhibited electrical LES relaxation, and apamin blocked non-nitrergic relaxation. Nicotine relaxation (100 μM) was inhibited by l-NAME (–60.37 ± 10.8%) and ODQ (–41.90 ± 7.89%), and apamin also blocked non-nitrergic relaxation. Non-nitrergic and apamin-sensitive LES relaxation by electrical stimulation or nicotine was strongly inhibited by MRS 2179, slightly inhibited by α-chymotrypsin and the P2X1,2,3 receptor antagonist NF 279 (8,8¢-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt; 10 μM), and unaffected by tin protoporphyrin IX (100 μM). Porcine LES relaxation after stimulation of intrinsic inhibitory motor neurons is mediated by two main neuromuscular pathways: nitric oxide through guanylate cyclase signaling and apamin-insensitive mechanisms and by non-nitrergic apamin-sensitive neurotransmission mainly mediated by ATP, ADP, or a related purine acting on P2Y1 receptors and a minor contribution of purinergic P2X1,2,3 receptors and PACAP. Nitrergic and purinergic co-transmitters show parallel effects of similar magnitude without major interplay. Our study shows no role for CGRP and only a minor one for VIP and carbon monoxide in porcine LES relaxation.