In the gastrointestinal tract, tachykinins are peptide neurotransmitters in nerve circuits that regulate intestinal motility, secretion, and vascular functions. Tachykinins also contribute to transmission from spinal afferents that innervate the gastrointestinal tract and have roles in the responses of the intestine to inflammation. Tachykinins coexist with acetylcholine, the primary transmitter of excitatory neurons innervating the muscle, and act as a co-neurotransmitter of excitatory neurons. Excitatory transmission is mediated through NK1 receptors (primarily on interstitial cells of Cajal) and NK2 receptors on the muscle. Tachykinins participate in slow excitatory transmission at neuro-neuronal synapses, through NK1 and NK3 receptors, in both ascending and descending pathways affecting motility. Activation of receptors (NK1 and NK2) on the epithelium causes fluid secretion. Tachykinin receptors on immune cells are activated during inflammation of the gut. Finally, tachykinins are released from the central terminals of gastrointestinal afferent neurons in the spinal cord, particularly in nociceptive pathways.
1 Isometric contractile responses to carbachol were studied in ileal longitudinal smooth muscle strips from wild-type mice and mice genetically lacking M 2 or M 3 muscarinic receptors, in order to characterize the mechanisms involved in M 2 and M 3 receptor-mediated contractile responses. 2 Single applications of carbachol (0.1-100 mM) produced concentration-dependent contractions in preparations from M 2 -knockout (KO) and M 3 -KO mice, mediated via M 3 and M 2 receptors, respectively, as judged by the sensitivity of contractile responses to blockade by the M 2 -preferring antagonist methoctramine (300 nM) or the M 3 -preferring antagonist 4-DAMP (30 nM). 3 The M 2 -mediated contractions were mimicked in shape by submaximal stimulation with high K þ concentrations (up to 35 mM), almost abolished by voltage-dependent Ca 2 þ channel (VDCC) antagonists or depolarization with 140 mM K þ medium, and greatly reduced by pertussis toxin (PTX) treatment. 4 The M 3 -mediated contractions were only partially inhibited by VDCC antagonists or 140 mM K þ -depolarization medium, and remained unaffected by PTX treatment. The contractions observed during high K þ depolarization consisted of different components, either sensitive or insensitive to extracellular Ca 2 þ . 5 The carbachol contractions observed with wild-type preparations consisted of PTX-sensitive and -insensitive components. The PTX-sensitive component was functionally significant only at low carbachol concentrations. 6 The results suggest that the M 2 receptor, through PTX-sensitive mechanisms, induces ileal contractions that depend on voltage-dependent Ca 2 þ entry, especially associated with action potential discharge, and that the M 3 receptor, through PTX-insensitive mechanisms, induces contractions that depend on voltage-dependent and -independent Ca 2 þ entry and intracellular Ca 2 þ release. In intact tissues coexpressing M 2 and M 3 receptors, M 2 receptor activity appears functionally relevant only when fractional receptor occupation is relatively small.
We investigated the responses of morphologically identified myenteric neurons of the guinea-pig ileum to inflammation that was induced by the intraluminal injection of trinitrobenzene sulphonate, 6 or 7 days previously. Electrophysiological properties were examined with intracellular microelectrodes using in vitro preparations from the inflamed or control ileum. The neurons were injected with marker dyes during recording and later they were recovered for morphological examination. A proportion of neurons with Dogiel type I morphology, 45% (32/71), from the inflamed ileum had a changed phenotype. These neurons exhibited an action potential with a tetrodotoxin-resistant component, and a prolonged after-hyperpolarizing potential followed the action potential. Of the other 39 Dogiel type I neurons, no changes were observed in 36 and 3 had increased excitability. The afterhyperpolarizing potential (AHP) in Dogiel type I neurons was blocked by the intermediate conductance, Ca 2+ -dependent K + channel blocker TRAM-34. Neurons which showed these phenotypic changes had anally directed axonal projections. Neither a tetrodotoxin-resistant action potential nor an AHP was seen in Dogiel type I neurons from control preparations. Dogiel type II neurons retained their distinguishing AH phenotype, including an inflection on the falling phase of the action potential, an AHP and, in over 90% of neurons, an absence of fast excitatory transmission. However, they became hyperexcitable and exhibited anodal break action potentials, which, unlike control Dogiel type II neurons, were not all blocked by the h current (I h ) antagonist Cs + . It is concluded that inflammation selectively affects different classes of myenteric neurons and causes specific changes in their electrophysiological properties.
1 In guinea-pig ileal longitudinal muscle, muscarinic partial agonists, 4-(N-[3-chlorophenyl]-carbomoyloxy)-2-butynyl-trimethylammonium (McN-A343) and pilocarpine, each produced parallel increases in tension and cytosolic Ca 2+ concentration ([Ca 2+ ]c) with a higher EC 50 than that of the full agonist carbachol. The maximum response of [Ca 2+ ]c or tension was not much different among the three agonists. The Ca 2+ channel blocker nicardipine markedly inhibited the effects of all three agonists 2 The contractile response to any agonist was antagonized in a competitive manner by M 2 receptor selective antagonists (N,benzodiazepine-6-one), and the apparent order of M 2 antagonist sensitivity was McN-A3434pilo-carpine4carbachol. M 3 receptor selective antagonists, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide and darifenacin, both severely depressed the maximum response for McN-A343, while darifenacin had a similar action in the case of pilocarpine. Both M 3 antagonists behaved in a competitive manner in the case of the carbachol response. -releasing action of pilocarpine was very weak compared with that of carbachol. All three agonists were capable of increasing Ca 2+ sensitivity of the contractile proteins. 4 McN-A343 rarely produced membrane depolarization, but always accelerated electrical spike discharge. Pilocarpine effect was more often accompanied by membrane depolarization, as was usually seen using carbachol. 5 The results suggest that muscarinic agonist-evoked contractions result primarily from the integration of Ca 2+ entry associated with the increased spike discharge and myofilaments Ca 2+ sensitization, and that Ca 2+ store release may contribute to the contraction indirectly via potentiation of the electrical membrane responses. They may also support the idea that an interaction of M 2 and M 3 receptors plays a crucial role in mediating the contraction response.
The late afterhyperpolarizing potential (AHP) that follows the action potential in intrinsic primary afferent neurons of the gastrointestinal tract has a profound influence on their firing patterns. There has been uncertainty about the identity of the channels that carry the late AHP current, especially in guinea pigs, where the majority of the physiological studies have been made. In the present work, the late AHP was recorded with intracellular microelectrodes from myenteric neurons in the guinea pig small intestine. mRNA was extracted from the ganglia to determine the identity of the guinea pig intermediate conductance potassium (I(K)) channel gene transcript. The late AHP was inhibited by two blockers of I(K) channels, TRAM34 (0.1-1 microM) and clotrimazole (10 microM), and was enhanced by the potentiator of the opening of these channels, DC-EBIO (100 nM). Action potential characteristics were unchanged by TRAM34 or DC-EBIO. The full sequence of the gene transcript and the deduced amino acid sequence were determined from extracts including myenteric ganglia and from bladder urothelium, which is a rich source of I(K) channel mRNA. This showed that the guinea pig sequence has a high degree of homology with other mammalian sequences but that the guinea pig channel lacks a phosphorylation site that was thought to be critical for channel regulation. It is concluded that the channels that carry the current of the late afterhyperpolarizing potential in guinea pig enteric neurons are I(K) channels.
Background and purpose: The functional roles of M 2 and M 3 muscarinic receptors in neurogenic cholinergic contractions in gastrointestinal tracts remain to be elucidated. To address this issue, we studied cholinergic nerve-induced contractions in the ileum using mutant mice lacking M 2 or M 3 receptor subtypes. Experimental approach: Contractile responses to transmural electrical (TE) stimulation were isometrically recorded in ileal segments from M 2 -knockout (KO), M 3 -KO, M 2 /M 3 -double KO, and wild-type mice. Key results: TE stimulation at 2-50 Hz frequency-dependently evoked a fast, brief contraction followed by a slower, longer one in wild-type, M 2 -KO or M 3 -KO mouse preparations. Tetrodotoxin blocked both the initial and later contractions, while atropine only inhibited the initial contractions. The initial cholinergic contractions were significantly greater in wild-type than M 2 -KO or M 3 -KO mice; the respective mean amplitudes at 50 Hz were 91, 74 and 68 % of 70mM K þ -induced contraction. Pretreatment with pertussis toxin blocked the cholinergic contractions in M 3 -KO but not in M 2 -KO mice. Cholinergic contractions also remained in wild-type preparations, but their sizes were reduced by 20-30 % at 10-50 Hz. In M 2 /M 3 -double KO mice, TE stimulation evoked only slow, noncholinergic contractions, which were significantly greater in sizes than in any of the other three mouse strains. Conclusion and Implications: These results demonstrate that M 2 and M 3 receptors participate in mediating cholinergic contractions in mouse ileum with the latter receptors assuming a greater role. Our data also suggest that the lack of both M 2 and M 3 receptors causes upregulation of noncholinergic excitatory innervation of the gut smooth muscle.
AnimalsMale Syrian hamsters (110-130 g) were maintained in temperature-controlled rooms and the experiments were performed in accordance with the Gifu University Animal Care and Use Committee and Japanese Department of Agriculture guidelines. Tissue preparationAnimals were anaesthetized with sodium pentobarbital (60 mg kg _1 , I.P.). Following exsanguination through the axillary arteries, the whole oesophagus of the hamster was dissected out and excised from the larynx to the level of the diaphragm, together Oesophageal peristalsis is controlled by vagal motor neurones, and intrinsic neurones have been identified in the striated muscle oesophagus. However, the effect(s) of intrinsic neurones on vagally mediated contractions of oesophageal striated muscles has not been defined. The present study was designed to investigate the role of intrinsic neurones on vagally evoked contractions of oesophageal striated muscles, using hamster oesophageal strips maintained in an organ bath. Stimulation (30 ms, 20 V) of the vagus nerve trunk produced twitch contractions. Piperine inhibited vagally evoked contractions, while capsaicin and N G -nitro-L-arginine methyl ester (L-NAME) abolished the inhibitory effect of piperine. The effect of L-NAME was reversed by subsequent addition of L-arginine, but not by D-arginine. L-NAME did not have any effect on the vagally mediated contractions and presumed 3 H-ACh release. NONOate, a nitric oxide donor, and dibutyryl cyclic GMP inhibited twitch contractions. Inhibition of vagally evoked contractions by piperine and NONOate was fully reversed by ODQ, an inhibitor of guanylate cyclase. Immunohistochemical staining showed immunoreactivity for nitric oxide synthase (NOS) in nerve cell bodies and fibres in the myenteric plexus and the presence of choline acetyltransferase and NOS in the motor endplates. Only a few NOS-immunoreactive portions in the myenteric plexus showed vanilloid receptor 1 (VR1) immunoreactivity. Our results suggest that there is a local neural reflex that involves capsaicin-sensitive neurones, nitrergic myenteric neurones and vagal motor neurones.
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