Shu-ichi Kojima

Contractile response to a cannabimimetic eicosanoid, 2-arachidonoylglycerol, of longitudinal smooth muscle from the guinea-pig distal colon in vitro

The effect of 2-arachidonoylglycerol, a cannabimimetic eicosanoid, was studied on mucosa-free longitudinal muscle strips isolated from the guinea-pig distal colon. In the presence of indomethacin (3 microM) and N(G)-nitro-L-arginine (100 microM), 2-arachidonoylglycerol (10 nM-10 microM) produced concentration-dependent and tetrodotoxin (1 microM)-sensitive contractions of the longitudinal muscle strips. The contractions were markedly attenuated in the presence of atropine (0.2 microM), and partially by hexamethonium (100 microM) pretreatment. The response to 2-arachidonoylglycerol was mimicked with N-arachidonoylethanolamine (anandamide, 0.1-30 microM), another cannabimimetic eicosanoid, but the cannabinoid CB(1)/CB(2) receptor agonist, R-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3,-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2) (0.1-10 microM), and the vanilloid receptor agonist, (all Z)-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide (AM 404) (10-30 microM), were without effect. The cannabinoid CB(1) receptor antagonist, N-piperidino-5-(4-chlorophenyl)-l-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-caroxamide (SR141716A) (1 microM), the cannabinoid CB(2) receptor antagonist, [N-[1S]-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-l-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) (1 microM), and the vanilloid receptor antagonist, capsazepine (10 microM), did not shift the concentration-response curve for 2-arachidonoylglycerol to the right. The contractile action of 2-arachidonoylglycerol was also partially attenuated in the presence of nordihydroguaiaretic acid (10 microM), a lipoxygenase inhibitor. These results indicate that 2-arachidonoylglycerol produces contraction of longitudinal muscle of the guinea-pig distal colon via mainly stimulation of myenteric cholinergic neurones, and that neither cannabinoid CB(1)/CB(2) receptors nor vanilloid receptors contributed to the response. The present results suggest the possibility that lipoxygenase metabolites may also contribute, at least in part, to the contractile action of 2-arachidonoylglycerol.

Investigation into the 5-hydroxytryptamine-induced relaxation of the circular smooth muscle of guinea-pig stomach fundus

The 5-HT receptor that mediates relaxation of circular muscle strips of the guinea-pig stomach fundus under resting tone was investigated. Concentration-dependent relaxation was obtained in the presence of atropine (0.2 microM) with 5-hydroxytryptamine (5-HT) (apparent mean pEC50 value, 5.27), 5-carboxamidotryptamine (7.35), 5-methoxytryptamine (4.98) and 5-methyltryptamine (4.58). 1-(m-Trifluoromethyl-phenyl)piperazine and 8-hydroxy-2-(di-n- propylamino)tetralin acted as partial agonists while 2-methyl-5-hydroxytryptamine, alpha-methyl-5-hydroxytryptamine, sumatriptan, metoclopramide and cisapride had little or no effect on the guinea-pig stomach fundus. The concentration-response curve for 5-HT was not affected by tetrodotoxin (0.3 microM), guanethidine (5 microM) or indomethacin (2 microM), suggesting that the relaxation is non-neuronal in origin and is independent of the release of catecholamines or prostanoids. The non-selective 5-HT receptor antagonist, metitepine (0.03-0.1 microM), the 5-HT1C/5-HT2 receptor antagonists, mianserin (0.3-1 microM), pizotifen (0.3-1 microM), ketanserin (3-10 microM), and the 5-HT1A/5-HT2 receptor antagonist, spiperone (3 microM), shifted the concentration-response curves for 5-HT to the right. A 5-HT3 receptor antagonist, ICS205-930 (1 microM), propranolol (1 microM) and phentolamine (1 microM) failed to block the 5-HT-induced relaxation. In conclusion, the results found with agonists and antagonists are compatible with the view that a 5-HT1-like receptor is involved in 5-HT-induced direct relaxation of circular muscle of guinea-pig stomach fundus.

Investigation of nicotine-induced relaxation of circular smooth muscle of the guinea-pig gastric fundus

A possible mechanism for the nicotine-induced relaxation of circular muscle strips of the guinea-pig gastric fundus was investigated. In the presence of atropine (0.2 microM), nicotine produced concentration-dependent relaxation with a maximum effect at 100 microM (mean pEC50 value, 4.60). The maximum relaxation due to nicotine was greatly reduced by pretreatment with tetrodotoxin (0.3 microM) or hexamethonium (10 microM), but not with metitepine (0.3 microM). Combined pretreatment with timolol (0.3 microM) and phentolamine (0.3 microM) or chemical sympathectomy by 6-hydroxydopamine pretreatment partially inhibited the nicotine-induced relaxation. alpha-Chymotrypsin (2 u/ml) which abolished the equivalent relaxation induced by vasoactive intestinal polypeptide (VIP) had no effect on nicotine-induced relaxation. NG-Nitro-L-arginine (L-NNA) and NG-Nitro-L-arginine methyl ester (L-NAME) caused a concentration-dependent inhibition of the nicotine-induced relaxation (98% inhibition at 10 microM of L-NNA), but had no effect on sodium nitroprusside- or noradrenaline-induced relaxation. The inhibitory effect of L-NNA or L-NAME was reversed completely by L-arginine (3 mM), but not by D-arginine (3 mM). From these results, we concluded that nicotine-induced relaxation of the guinea-pig gastric fundus is mediated largely by the release of nitric oxide or a related substance and partially by the release of noradrenaline. Possible contributions of 5-hydroxytryptamine or VIP to the nicotine-induced relaxation appear to be negligible.

An enhancing effect of 5-hydroxytryptamine on electrically evoked atropine-resistant contraction of guinea-pig proximal colon.

1 In the presence of atropine (0.2 μm) and indomethacin (2 μm), the effects of 5‐hydroxytryptamine (5‐HT) have been studied on electrically‐evoked, neurogenic contractions of the guinea‐pig proximal colon in vitro. 2 5‐HT, at higher concentrations than 1 nm, caused an increase in electrically (1 Hz, 0.3 ms, 160 mA)‐evoked, atropine‐resistant contractions in a concentration‐dependent manner and at 30 nm produced a maximal effect (pEC50 value of 8.20 ± 0.11, n = 6). The enhancing effects of 5‐HT on the electrically evoked contractions were mimicked by α‐methyl‐5‐HT (pEC50 value of 6.59 ± 0.05, n = 6). 3 Both hexamethonium (100 μm) and spantide (10 μm), selective antagonists for nicotinic and tachykinin receptors respectively, significantly reduced the enhancement of the electrically evoked contractions by 5‐HT (30 nm). 4 DAU 6285 (3 μm), a 5‐HT4 receptor antagonist, abolished the enhancing action of 5‐HT (30 nm), but metitepine (0.03 μm), a 5‐HT1/5‐HT2 receptor antagonist, ketanserin (0.01 μm), a 5‐HT2 receptor antagonist, and ondansetron (1 μm), a 5‐HT3 receptor antagonist, had no effect on the enhancement. The enhancing effects of a‐methyl‐5‐HT (1 μm) were also abolished by DAU 6285 (3 μm). 5 Both 5‐HT (30 nm) and α‐methyl‐5‐HT (1 μm) had no effect on contractions to exogenous substance P (0.15–0.3 nm). 6 These results indicate that in the guinea‐pig proximal colon, 5‐HT produced an enhancement of atropine‐resistant neurogenic contraction induced by electrical field stimulation through pre‐junctional mechanisms and that the enhancement is mediated by the stimulation of 5‐HT4 receptors located on intramural preganglionic cholinergic neurones and tachykininergic neurones.

Facilitation by endogenous acetylcholine and nitric oxide of luminal serotonin release from the guinea-pig colon

The present study was designed to determine the influence of endogenous acetylcholine and nitric oxide (NO) on spontaneous luminal serotonin (5-hydroxytryptamine, 5-HT) release in the luminally perfused isolated guinea-pig proximal colon in vitro. 5-HT was determined by high-performance liquid chromatography with electro-chemical detection. The luminal outflow of 5-HT was significantly reduced by atropine (0.2 microM), hexamethonium (100 microM), the NO synthase inhibitor NG-nitro-L-arginine (L-NNA, 10 microM) and the NO-trapping agent 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO, 30 microM). Addition of excess L-arginine (300 microM) reversed the inhibitory effect of L-NNA on the 5-HT outflow. Physostigmine (1 microM) caused a great increase (atropine-sensitive) in 5-HT outflow. The enhancing action of physostigmine on 5-HT outflow was partially inhibited by L-NNA (100 microM) or carboxy-PTIO (30 microM), but was unaffected by the muscarinic M1 receptor antagonist pirenzepine (0.2 microM) or a muscarinic M3 receptor antagonist 4-diphenyl-acetoxy-N-methyl-piperidine methiodide (0.2 microM). These results suggest that 5-HT release from luminally perfused proximal colon of the guinea pig is stimulated via a NO pathway and cholinergic pathways which utilize muscarinic synapses and nicotinic synapses. Further, an intrinsic cholinergic-NO link appears to play a role in the stimulation of luminal 5-HT release, which may reflect the release of 5-HT from entero-chromaffin cells.

Investigation into the 5‐hydroxytryptamine‐induced atropine‐resistant neurogenic contraction of guinea‐pig proximal colon

1 The aim of this study was to characterize the receptors mediating the atropine‐resistant neurogenic contraction to 5‐hydroxytryptamine (5‐HT) in the longitudinal muscle of the guinea‐pig proximal colon and to determine the type of tachykinin receptors involved in the contractile response to 5‐HT by the use of selective antagonists. 2 In the presence of atropine (0.3 μm), guanethidine (5 μm), hexamethonium (100 μm), ketanserin (0.1 μm) and indomethacin (3 μm), 5‐HT (0.01–3 μm) produced concentration‐dependent neurogenic contractions of colonic strips and at 0.3 μm produced a maximal effect (pEC50 = 7.39±0.09, n = 18). The 5‐HT4 receptor stimulant, 5‐methoxytryptamine (5‐MeOT, 0.03–10 μm) also produced neurogenic contractions with similar maximum effect to those of 5‐HT (pEC50 = 6.89±0.16). 3 The 5‐HT4 receptor antagonist, DAU 6285 (3 μm) shifted the concentration‐response curves to both 5‐HT and 5‐MeOT to the right without significant depression of the maximum, but the 5‐HT1/5‐HT2 receptor antagonist, metitepine (0.1 μm) and the 5‐HT3 receptor antagonist, ondansetron (0.3 μm) had no effect on the control curves to 5‐HT and 5‐MeOT. 4 The selective NK1 receptor antagonist, FK 888 (1 μm) markedly attenuated the contractions to 5‐HT and 5‐MeOT. In contrast, the selective NK2 receptor antagonist, SR 48968 (10 nM) and the selective NK3 receptor antagonist, SR 142801 (10 nM) had no effect on the contractions to 5‐HT and 5‐MeOT. 5 These results indicate that the 5‐HT‐induced atropine‐resistant neurogenic contraction of guinea‐pig proximal colon is due to activation of 5‐HT4 receptors, presumably located on excitatory motor neurones, innervating the longitudinal muscle. The contraction evoked by activation of the 5‐HT4 receptors is mediated primarily via NK1 receptors but not NK2 or NK3, suggesting that the 5‐HT4 receptor‐mediated contraction is evoked indirectly via tachykinin release from tachykinin‐releasing excitatory neurones.

Histamine responses mediated via H1 and H2−receptors in the isolated portal vein of the dog

The effects of histamine were studied on the isolated circular muscle strip and longitudinal muscle strip of the dog portal vein. Histamine‐induced contractions of the circular muscle were inhibited by H1‐receptor antagonist pyrilamine but increased by H2‐receptor antagonist ranitidine. When the tissues were contracted with PGF2α in the presence of pyrilamine, histamine produced the relaxation of the circular muscle but not longitudinal muscle. The relaxation of the circular muscle was inhibited by ranitidine in a concentration dependent manner, the pA2 value for ranitidine being 6·97 (6·55–7·39). It is concluded that the response of the circular muscle to histamine is the sum of two components, H1‐receptor mediated contraction and H2‐receptor mediated relaxation.

KW-5092, a novel gastrokinetic agent, facilitates luminal serotonin release from the guinea-pig colon

The present study was designed to determine the influence of KW-5092 ((1-[2-[[[5-(piperidinomethyl)-2-furanyl]methyl]amino]ethyl]-2- imidazolidinylidene) propanedinitrile fumarate), a novel gastroprokinetic agent on intraluminal serotonin (5-hydroxytryptamine, 5-HT) release which reflects the release of 5-HT from enterochromaffin cells, using the luminally perfused isolated guinea-pig proximal colon in vitro. 5-HT was determined by high-performance liquid chromatography with electrochemical detection. KW-5092 (1-10 microM) concentration-dependently caused an increase in the luminal 5-HT outflow. In the presence of atropine (0.2 microM) or tetrodotoxin (0.3 microM), the stimulatory action of KW-5092 (10 microM) was inhibited by 94% and 74%, respectively. These results suggest that KW-5092 stimulates intraluminal 5-HT release from luminally perfused proximal colon of the guinea-pig via the stimulation of cholinergic neurons. Because 5-HT is recognized as an important messenger substance in the control of intestinal motility, this stimulatory effect could be considered as an indirect action of KW-5092 that may contribute to its prokinetic effects.

Calcitonin gene‐related peptide (CGRP)‐enhanced non‐adrenergic non‐cholinergic contraction of guinea‐pig proximal colon

1 We have investigated the effect of calcitonin gene‐related peptide (CGRP) on non‐adrenergic, non‐cholinergic (NANC) excitatory transmission to the longitudinal muscle of the guinea‐pig proximal colon. 2 In the presence of atropine (0.3μm), guanethidine (5 μm), hexamethonium (100 μm) and indomethacin (3 μm), electrical field stimulation (EFS, 1 Hz, 0.3 ms for 10 s) produced tetrodotoxin‐(300 nM)‐sensitive contractions which were reduced by the combined administration of FK 888 (10 μm) and MEN 10,376 (0.3 μm), to block tachykinin NKi and NK2 receptors, respectively. Thus, the EFS‐induced NANC contractions are a tachykinin‐mediated response. 3 CGRP, at concentrations higher than 0.1 nM, caused an increase in the electrically‐evoked, NANC contractions in a concentration‐dependent manner and at 10 nM produced a maximal effect (pEC50 = 9.20±0.17, n = 6). 45 ‐Hydroxytryptamine (5‐HT, 1–100 nM) also caused an increase in the EFS‐induced NANC contractions in a concentration‐dependent manner and at 30 nM produced a maximal effect (pEC50 = 8.06 ±0.09, n = 4), but calcitonin (10–100 nM) failed to enhance the EFS‐induced NANC responses. Moreover, a 5‐HT4 receptor antagonist, DAU 6285 (3 μm) abolished the enhancing action of 5‐HT (30 nM). 5 The combined administration of FK 888 (10 μm) plus MEN 10,376 (0.3 μm) abolished the enhancement of EFS‐induced NANC contractions by CGRP (10 nM), but DAU 6285 (3 μm) had no effect on the enhancement. 6 Human CGRP8_37 (1 μm), a CGRP1 receptor antagonist had no effect on the submaximal enhancement of the electrically‐evoked, NANC contractions by CGRP (1 nM). 7 CGRP (30 nM) had no effect on contractions evoked by exogenous substance P (0.3‐1 nM). 8 These results indicate that in the guinea‐pig proximal colon, CGRP produced an enhancement of NANC contraction induced by EFS through prejunctional mechanisms and that the enhancement is mediated by the stimulation of non‐CGRP1 receptors located on intramural tachykininergic neurones. Further, the possible contribution of 5‐HT to the enhancing effect of CGRP appeared to be negligible.

An Endogenous Tachykinergic NK2/NK3 Receptor Cascade System Controlling the Release of Serotonin from Colonic Mucosa

5-Hydroxytryptamine (5-HT) released from colonic mucosal enterochromaffin (EC) cells is a major signaling molecule, which participates in the pathophysiological regulation of colonic functions in gut disorder including irritable bowel syndrome (IBS), but the endogenous modulator system for the 5-HT release is not yet well elucidated. Our in vitro studies in guinea-pig colon have indicated that the cascade pathway of neuronal tachykinergic NK3 receptors and NK2 receptors on peptide YY (PYY)-containing endocrine L cells represents an endogenous modulator system for 5-HT release from EC cells and that melatonin, endogenous tachykinins and PYY play important roles in modulation of the release of 5-HT from EC cells via the endogenous NK2/NK3 receptor cascade system. This review aims at examining the potential role of the endogenous tachykinergic NK2/NK3 receptor cascade system controlling the release of 5-HT from EC cells, with special attention being paid to the pathophysiology of gut disorders including IBS.