| The enteric nervous system (ENS) is an independent nervous system which is located in the wall of gastrointestinal tract. It controls and coordinates motility, blood flow, secretion, water and electrolyte metabolism of gastrointestinal tract to meet the digestion needs of the individual. According to the distribution of enteric neurons in the wall of gastrointestinal tract, ENS was divided into two major parts: the myenteric plexus between the longitudinal and circular muscles and the submucosal plexuses associated with the mucosal epithelium. In the gastrointestinal tract, the neural reflex activity need to rely on the complex interactions between a number of neural active molecules and their receptors. Opioids are distributed in the gastrointestinal tract and modulate their activity as neural active molecules. If the opioid peptides were used for a long time in vivo,it can induc abnormal activity of ENS, leading to gastrointestinal disorders. Opioid receptors are widely distributed in the gastrointestinal tract as the messenger throgh which opioid peptides can modulate the gastrointestinal motolity. In the gastrointestinal tract, the distributions of MORs are more abundant than other receptors. Endomorphin-1 (EM1) and endomorphin-2 (EM2), the two endogenous ligands for MOR, selectively bind to the MOR with high affinity. EM specifically activate MOR and induce its endocytosis in enteric neurons. The pharmacological and physiological results showed that EM can modulate the stomach and small intestine motility via MOR, but their distribution and modulatory mechanism in the gastrointestinal tract are unclear. In this study, we studied the distribution pattern and neurochemical phenotype in the myenteric and submucosal plexus of rat colon, and further investigated the possible regulatory mechanisms of EM2 on the colonic motolity.Methods and results:1. The distribution and neurochemical phenotype of EM2-containg neurons in the rat colon.The distribution pattern and neurochemical phenotype of EM2-containning neurons in the rat colon were examined by whole-mount preparations and immunohistochemistry.(1) The distribution pattern and neurochemical phenotype of EM2-containg neurons in the myenteric plexus.EM2–immunoreactive (IR) neurons were found in myenteric plexus. EM2-IR neurons had an oval cell body, with a few thick dendrites protruding from the soma, and a long axonal process that could extend for up to 100μm or more. EM2-IR neuronal cell bodies were 35.5±10.8μm (length) by 17.8±4.6μm (width). There were 10.55±4.2 EM2-IR neuronal cells per ganglion. EM2-immunoreactivities were located in 57±4.1 % of neurons stained with neuron-specific enolase (NSE). In the myenteric plexus, 53±4.6%, 55±4.5%, 37±4.3%, 49±4.2%, 26±4.5% and 26±2.8% of EM2- IR neurons were also positive for choline acetyl transferase (ChAT), calretinin (CR), 5-hydroxytryptamine (5-HT), nitric oxide synthetase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP), respectively. In addition, EM2 was detected in ChAT-, 5-HT- and SP-IR neurons for 74±4.7%, 76±3.2% and 88±2.5%, and all CR-, VIP- and NOS-IR neurons co-located with EM2-IR. EM2 and calcitonin gene-related peptide (CGRP) were not co-localized in neuronal cell body but CGRP-IR nerve fibers ran among EM2-IR neurons and surrounded them. Triple-stained results showed that EM2 and ChAT with VIP or NOS were co-located in neurons of myenteric plexus.( 2 ) The distribution and neurochemical phenotype of EM2-containg neurons in the submucosal plexus.In the submucosal plexus, about 6±4.2 EM2-IR neurons aggregated into a ganglion, and the distribution of ganglia is quite dispersed. EM2-immunoreactivities were co-localized with 68±1.5 % of neurons stained with NSE. In submucosal plexus, 91±2.6 %, 44±4.7%, 36±2.4% and 44±2.5% of EM2-IR neurons were also positive for ChAT, NOS, SP and VIP, respectively. In addition, EM2 was detected in ChAT-, SP-, VIP-IR neurons for 53±2.8%, 75±3.4% and 73.3±2.6%, and all NOS-IR neurons co-localized with EM2-IR. EM2 and CGRP were not co-located in neurons but CGRP-IR nerve fibers ran among EM2-IR neurons and surrounded them. Triple-stained results showed that EM2 with ChAT and VIP were colacated in submucosal plexus. The present results indicate that EM2-containning neurons exist in the myenteric and submucosal plexus of the rat colon, and EM2 are co-expressed with neurochemically distinct populations of myenteric and submucosal neurons, which play the roles of motor neurons or interneurons.2. Effect of EM2 on the motility of the rat colon in vitroIn this study, the effects of EM2 were evaluated on colonic motility in vitro using electrical field stimulation (EFS), and investigated the motor neuromechanism with various MOR antagonists and neural blocking agents.(1) Direct effects of EM2 on the rat colon.Carbachol (10–6 M) and 5-HT (10–6 M) caused contractions of the colonic segments. EM2 (10–6 to 10–9 M) alone did not affect carbachol and 5-HT pre-stimulated colonic segments or the unstimulated colonic segments.(2) Effects of EM2 on the electrically induced colon.The electrically stimulated colonic segments produced a twitch-like contraction response during stimulation (4.38±0.13g, control). EM2 dose-dependently (10–9 to 10–6 M) reduced the contractile response to EFS. At 10–6 M, EM2 elicited a maximally inhibitory effect (62±7.1% of control). Naloxone (10–5 M) orβ-funaltrexamin (β-FNA, 10–6 M) blocked the inhibitory responses of EM2 ( 98.8±13.4% and 97.7±12.1% of control ).(3) Effects of various antagonists on the electrically induced colon.Tetrodotoxin (TTX, 10–4 M) abolished the contractile responses to field stimulation. Atropine (10-6 M) inhibited the response of the electrically induced colonic contraction (53.2±7.1% of control). EM2 still inhibited contraction after blockade by atropine (24.3±7.8% of control), regardless of the application sequence. Similarly, 3-tropanylindole-3-carboxylate methiodide (ICS, 10-6 M), inhibited field stimulation (74.9±8.9% of control), and EM2 potentiated this effect (35.4±7.8% of control). EM2 produced a small inhibition after NG-Nitro-L-arginine Methyl Ether, Hydrochloride (L-NAME, 10–4 M) pre-incubation (86.7±6.5% of control), but L-NAME induced a small rebound contraction with EM2 incubation (86.7±8.7% of control). Similarly, EM2 also produced a small inhibition after vasoactive intestinal peptide fragment 6-28 (VIP6-28,10-6 M) pre-incubation (82.9±5.8% of control ).The present results suggest EM2 mediates the motor response of rat colon via enteric motor neurons by inducing enteric neurons to release inhibitory neurotransmitters under electrical stimulus.In summary, EM2-IR neurons exist in the myenteric plexus of the rat colon and EM2 co-existed with many neurochemical maekers in distinct populations of myenteric and submucosal neurons. Under electrical stimulus, EM2 mediates the motor response of rat colon via enteric motor neurons, which naturally express the specific opioid receptor, MOR. EM2 can principally induce enteric neurons to release inhibitory neurotransmitters, but not to inhibit the release of ACh or 5-HT.
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