Expression of aquaporin-4 water channels in the digestive tract of the guinea pig

Expression of the aquaporin-4 (AQP4) water channel was systematically studied in the digestive tract of the guinea pig using Western blot and immunofluorescence techniques. The results showed that AQP4 was expressed widely in different segments of the guinea pig digestive tract. AQP4-immunoreactivity was confined to parietal cells in the stomach, and absorptive and glandular epithelial cells of small and large intestine. AQP4 protein was also expressed by enteric glial cells of submucosal and myenteric ganglia and primary nerve trunks. AQP4 was expressed by both type I and type II enteric gliocytes, but not by type III or type IV enteric gliocytes, indicating that enteric gliocytes have a heterogeneous distribution in the gut wall. In addition, different patterns of AQP4 expression in the enteric nervous system of human, guinea pig, rat and mouse colon mucosa were identified: in rat and mouse AQP4 was localised to a small subpopulation of neurons; in the guinea pig AQP4 was localised to enteric glial cells; and in the human colon mucosa, AQP4 was also detected mainly in the glial cells. It has been speculated that AQP4 may be involved in water transport in the gastrointestinal tract. Its role in enteric neurons and glia is unknown, but, by analogy with the brain, AQP4 may be involved in the formation and resolution of edema.     

Control of peripheral glial cell proliferation: enteric neurons exert an inhibitory influence on Schwann cell and enteric glial cell DNA synthesis in culture

Neuronal membranes from rat dorsal root ganglia provide a mitogenic signal to cultured Schwann cells and it has been suggested this is an important factor in regulating Schwann cell numbers during development. In this study, the influence of enteric neurons on the DNA synthesis of both Schwann cells and enteric glia has been investigated as well as the effect of axonal membrane fractions (axolemma) on enteric glia. The proliferation rate of rat Schwann cells and enteric glia was assessed in culture using [3H]thymidine uptake and autoradiography in combination with immunolabelling to identify cell types. When purified rat Schwann cells were co-cultured with guinea pig enteric neurons, their DNA synthesis rate was reduced compared with control cultures of pure Schwann cells or Schwann cells not close to neurites or neuronal cell bodies. Nevertheless, in accordance with previous findings that sensory neurons stimulate Schwann cell division, these Schwann cells increased their DNA synthesis rate when in contact with neurites from purified guinea pig or adult rat dorsal root ganglion neurons and on exposure to bovine axolemmal fractions. The enteric neurons also suppressed the DNA synthesis of enteric glia in co-cultures of purified enteric neurons and enteric glia, while bovine axolemma stimulated their DNA synthesis. These results indicate that a mitotic inhibitory signal is associated with enteric neurons and can exert its effect on both Schwann cells and enteric glia, and that enteric glia, like Schwann cells, are stimulated to divide by axolemmal fractions. It thus seems possible that during development glial cell numbers in the peripheral nervous system may be controlled by both positive and negative regulators of cell growth.     

Role of enteric glia in intestinal physiology: effects of the gliotoxin fluorocitrate on motor and secretory function

The role of enteric glia in gastrointestinal physiology remains largely unexplored. We examined the actions of the gliotoxin fluorocitrate (FC) on intestinal motility, secretion, and inflammation after assessing its efficacy and specificity in vitro. FC (100 microM) caused a significant decrease in the phosphorylation of the glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diaz-4-yl)amino]-2-deoxyglucose in enteric glial cultures and a reduction in glial uptake of the fluorescent dipeptide Ala-Lys-7-amino-4-methylcoumarin-3-acetic acid in both the ileum and colon. Dipeptide uptake by resident murine macrophages or guinea pig myenteric neurons was unaffected by FC. Incubation of isolated guinea pig ileal segments with FC caused a specific and significant increase in glial expression of the phosphorylated form of ERK-1/2. Disruption of enteric glial function with FC in mice reduced small intestinal motility in vitro, including a significant decrease in basal tone and the amplitude of contractility in response to electrical field stimulation. Mice treated with 10 or 20 micromol/kg FC twice daily for 7 days demonstrated a concentration-dependent decrease in small intestinal transit. In contrast, no changes in colonic transit or ion transport in vitro were observed. There were no changes in glial or neuronal morphology, any signs of inflammation in the FC-treated mice, or any change in the number of myenteric nitric oxide synthase-expressing neurons. We conclude that FC treatment causes enteric glial dysfunction, without causing intestinal inflammation. Our data suggest that enteric glia are involved in the modulation of enteric neural circuits underlying the regulation of intestinal motility.     

Comparison of effects of a selective 5-HT reuptake inhibitor versus a 5-HT4 receptor agonist on in vivo neurogenesis at the rectal anastomosis in rats

It was recently reported that activation of enteric neural 5-HT(4) receptors (SR4) promotes reconstruction of enteric neural circuit injury in distal gut of guinea pigs and that this reconstruction involves neural stem cells. We aimed to explore a novel approach using a selective serotonin reuptake inhibitor (SSRI), which increases endogenous 5-HT, to repair enteric nerve fiber injury in the rat distal gut. Enteric nerve fiber injury was performed by rectal transection and subsequent end-to-end one-layer anastomosis. The SSRI fluvoxamine maleate (100 μmol/l) was applied locally at the anastomotic site to compare with the 5-HT(4) agonist mosapride citrate (100 μmol/l) (applied for patent) applied locally and orally. Unlike mosapride, fluvoxamine failed to promote the regeneration of the nerve fiber tract across the anastomosis. Furthermore, fluvoxamine did not generate anti-distal-less homeobox 2 (DLX2)- and anti-SR4-positive cells (neural stem cells) and/or anti-neurofilament (NF)-positive cells (neural cells) in newly formed granulation tissue at the anastomosis, whereas these cell types were observed in mosapride-treated preparations. In contrast to its effects in guinea pigs, mosapride generated 5-bromo-2'-deoxyuridine (BrdU)-positive neural cells in ganglia sites 3 mm oral and anal from the anastomosis 2 wk after nerve fiber injury. All actions of mosapride were observed after local and or oral applications. These findings indicate that local SSRI treatment does not induce in vivo nerve fiber tract growth across the anastomosis in the rat distal gut. Mosapride induces nerve fiber tract growth across the anastomosis, mediated through enteric neural stem cells possibly from neural crest-derived stem cells or mesenchymal stem cells in the bone marrow.     

Physiological analysis of delta opioid receptors in the guinea pig myenteric plexus

Ilea taken from guinea pigs that had been chronically exposed to morphine exhibit a greater tolerance to morphine and normorphine than to the opioid peptides D-ala2-D-leu5-enkephalin (DADLE) or D-met2-pro5-enkephalinamide (DMPE). This differential tolerance strongly implies the existence of at least two different types of opioid receptor in the guinea pig myenteric plexus or two different mechanisms of interaction between opioids and their receptor complex. Since DADLE is considered to be the prototypic ligand for the delta receptor, the above results imply the presence of delta receptors in the guinea pig myenteric plexus and furthermore, that this subtype of opioid receptor is associated with the modulation of release of enteric acetylcholine.     

Enteric motor neurons form synaptic-like junctions with interstitial cells of Cajal in the canine gastric antrum

Morphological studies have shown synaptic-like structures between enteric nerve terminals and interstitial cells of Cajal (ICC) in mouse and guinea pig gastrointestinal tracts. Functional studies of mice lacking certain classes of ICC have also suggested that ICC mediate enteric motor neurotransmission. We have performed morphological experiments to determine the relationship between enteric nerves and ICC in the canine gastric antrum with the hypothesis that conservation of morphological features may indicate similar functional roles for ICC in mice and thicker-walled gastrointestinal organs of larger mammals. Four classes of ICC were identified based on anatomical location within the tunica muscularis. ICC in the myenteric plexus region (IC-MY) formed a network of cells that were interconnected to each other and to smooth muscle cells by gap junctions. Intramuscular interstitial cells (IC-IM) were found in muscle bundles of the circular and longitudinal layers. ICC were located along septa (IC-SEP) that separated the circular muscle into bundles and were also located along the submucosal surface of the circular muscle layer (IC-SM). Immunohistochemistry revealed close physical associations between excitatory and inhibitory nerve fibers and ICC. These contacts were synaptic-like with pre- and postjunctional electron-dense regions. Synaptic-like contacts between enteric neurons and smooth muscle cells were never observed. Innervated ICC formed gap junctions with neighboring smooth muscle cells. These data show that ICC in the canine stomach are innervated by enteric neurons and express similar structural features to innervated ICC in the murine GI tract. This morphology implies similar functional roles for ICC in this species.     

Functional interactions between the SK2 channel and the nicotinic acetylcholine receptor in enteric neurons of the guinea pig ileum

The neurotransmitter acetylcholine (ACh) plays a critical role in gastrointestinal function. The role of the small conductance Ca²⁺-activated K⁺ (SK) channel in ACh release was examined using myenteric plexus preparations of guinea pig ileum. Apamin, an inhibitor of the SK channel, significantly enhanced nicotine-induced ACh release, but neither electrical field stimulation- nor 5-hydroxytryptamine-induced ACh release, suggesting that SK channels might be selectively involved in the regulation of nicotine-induced ACh release. Therefore, we investigated the distribution of SK2 and SK3 subunits and the interaction between SK2 channels and nicotinic ACh receptors (nAChRs) in the guinea pig ileum. The immunoreactivity of SK2 subunits was located in enteric neuronal cells. Furthermore, SK2-immunoreactive cells stained with an antibody for choline acetyltransferase, a marker for cholinergic neurons, and with an antibody for the α3/5 subunits of nAChR. In contrast, immunoreactivity of SK3 subunits was not found in enteric neurons. A co-immunoprecipitation assay with Triton X-100-soluble membrane fractions prepared from the ileum revealed an association of the SK2 subunit with the α3/5 subunits of nAChR. These results suggest that SK2 channels negatively regulate the excitation of enteric neurons via functional interactions with nAChRs.     

Local shwartzman phenomenon in animals with a complete absence and a reduction of the normal microflora]

Macro- and microscopic changes were studied in case of reproduction of the local Schwartzmann phenomenon in animals with various extent of bioisolation. It was revealed that the Schwartzmann phenomenon was positive in the usual animals and negative in the germ-free guinea pigs and in the animals with reduced enteric microbial flora given sterile diet. In the absence of microscopic changes in the germ-free animals there was revealed in their skin a neutrophilic-mononuclear infiltration of the derma, dilatation of the vessels, thrombosis of individual vessels. Apart from the same changes in guinea pigs with a reduced microbial flora, there were found focal extravasation and thrombosis of a somewhat greater number of vessels. There was no marked thromboses of small branches of the vessels, extensive hemorrhages or necroses which usually accompanied positive Schwartzmann phenomenon.     

Immunohistochemical localization of microtubule-associated proteins in the nervous system of the small intestine of guinea pig

Summary Layers containing Auerbach's and Meissner's plexuses were dissected from the small intestine of guinea pig and immunostained with affinity-purified antibodies against brain-specific microtubule-associated proteins (MAPs): MAP1, MAP2 and tau and a MAP with a molecular weight of 190000 dalton purified from bovine adrenal cortex (190-kDa MAP). MAP1 antibody stained the network of nerve fibers and the cell bodies of enteric neurons in both Auerbach's and Meissner's plexuses. Staining with anti-tau antibody gave the same results. Antibody against MAP2 stained neuronal cell bodies and short thin processes extending from them. Interganglionic strands composed mainly of long processes were unstained. Anti-190-kDa MAP antibody stained both the neuronal cell bodies and bundles of nerve fibers. However, the staining was less intense than that with anti-MAP1 and tau antibodies. Differentiation in the structure of the cytoskeleton probably exists in the neuronal processes of the enteric neurons as is shown in the dendrites and axons in some neurons of the central nervous system. Thus, enteric neurons possess axon-like processes containing MAP1, tau and probably lower amounts of 190-kDa MAP. Cell bodies and dendrite-like structures of these neurons contain MAP2 in addition to MAP1, tau and 190-kDa MAP.     

Effects of animal or plant protein diets on cecal fermentation in guinea pigs (Cavia porcellus), rats (Rattus norvegicus) and chicks (Gallus gallus domesticus)

Monogastric herbivores such as the guinea pig depend on energy supply from enteric fermentation as short-chain fatty acids (SCFA) corresponding to 30-40% of their maintenance energy requirements. They evolved specific digestive system to adapt their indigenous microflora to plant polysaccharides fermentation. No information has been available about the adaptability of microbial fermentation in hindgut of the monogastric herbivorous to an animal protein diet. We investigated if the guinea pig can fully retrieve energy of an animal protein diet by hindgut fermentation compared with a plant protein diet. For comparison, we also studied two omnivores. End products of in vitro cecal fermentation (SCFA, ammonia and gases) were measured to judge how well an animal protein diet could be fermented. The animal protein diet resulted in the less intensive fermentation with increased feed intake and volume of cecal contents than the plant protein diet only in guinea pigs. This may be due to a limited capacity of the hindgut microflora to adapt to the substrate rich in animal protein. We also found that chick cecal contents produced methane at higher emission rate than ruminants.     

Clinical salmonellosis in a guinea pig colony caused by a new Salmonella serotype, Salmonella ochiogu

During a severe outbreak of clinical salmonellosis in an experimental guinea pig colony, a new strain of Salmonella was isolated and identified. The new serotype, with the antigenic structure 1,3,19 : Z38 : e, n, Z15, for which the name Salmonella ochiogu has been suggested, caused both enteric and systemic infection in the animal colony. During the outbreak a total of 127 animals died (26.9%). All ages of animal were affected. Treatment with oral tetracycline was successful when combined with strict hygienic measures.     

A new possibility for repairing the anal dysfunction by promoting regeneration of the reflex pathways in the enteric nervous system

Moderate rectal distension elicits recto-rectal reflex contractions and simultaneous recto-internal anal sphincter reflex relaxations that together comprise the defecation reflex. Both reflexes are controlled by 1) pelvic nerves, 2) lumbar colonic nerves, and 3) enteric nervous system. The aim of the present study was to explore a novel approach to repairing the defecation reflex dysfunction by using the plasticity of enteric nervous pathways. Experiments were performed in anesthetized guinea pigs with ethyl carbamate. The rectum 30 mm oral from the anal verge was transected without damage to extrinsic nerves, and subsequent end-to-end one-layer anastomosis was performed. Recovery of the defecation reflex and associated reflex pathways were evaluated. Eight weeks after sectioning of intrinsic reflex nerve pathways in the rectum, the defecation reflex recovered to the control level, accompanied with regeneration of reflex pathways. The 5-HT(4)-receptor agonist mosapride (0.5 and 1.0 mg/kg) significantly (P < 0.01) enhanced the recovered defecation reflex 8 wk after surgery. Two weeks after local treatment with brain-derived neurotrophic factor (BDNF: 10(-6) g/ml) at the rectal anastomotic site, the recto-internal anal sphincter reflex relaxations recovered and some bundles of fine nerve fibers were shown to interconnect the oral and anal ends of the myenteric plexus. These results suggested a possibility for repairing the anal dysfunction by promoting regeneration of the reflex pathways in the enteric nervous system with local application of BDNF.     

研究报告：缓激肽上调豚鼠肠道黏膜下神经丛环氧合酶2的表达

目的缓激肽和缓激肽B2受体在肠神经系统中起重要作用。缓激肽通常参与肠道的炎症反应和神经保护,这种作用取决于缓激肽诱导前列腺素的形成。环氧合酶1 (COX1)和环氧合酶2 (COX2)催化花生四烯酸转化为前列腺素。本研究旨在探讨缓激肽刺激对豚鼠肠神经前列腺素E2 (p GE2)释放和COX2表达的影响及信号机制。方法本文通过免疫荧光检测肠神经细胞中COX2与神经细胞标志物Anti-Hu和ch AT的表达;采用PCR及蛋白质印迹(Western blot)检测不同条件下缓激肽刺激对COX2表达的影响;使用缓激肽B1受体的选择性拮抗剂Leu-8和B2受体的选择性拮抗剂HOE-140,研究缓激肽影响COX2表达的信号机制;利用COX2选择性拮抗剂NS398和COX1拮抗剂FR12207,观察COX2在缓激肽诱导p EG2释放的作用。结果 COX2与神经细胞标志物Anti-Hu和ch AT在肠神经细胞上共同表达,缓激肽可通过B2受体诱导肠神经细胞COX2的表达。缓激肽刺激引起的肠神经细胞p GE2的释放与COX2表达升高密切相关。结论缓激肽通过B2R影响肠道黏膜下神经丛COX2的表达,肠道缓激肽...     

Morphine Decreases Enteric Neuron Excitability via Inhibition of Sodium Channels

Gastrointestinal peristalsis is significantly dependent on the enteric nervous system. Constipation due to reduced peristalsis is a major side-effect of morphine, which limits the chronic usefulness of this excellent pain reliever in man. The ionic basis for the inhibition of enteric neuron excitability by morphine is not well characterized as previous studies have mainly utilized microelectrode recordings from whole mount myenteric plexus preparations in guinea pigs. Here we have developed a Swiss-Webster mouse myenteric neuron culture and examined their electrophysiological properties by patch-clamp techniques and determined the mechanism for morphine-induced decrease in neuronal excitability. Isolated neurons in culture were confirmed by immunostaining with pan-neuronal marker, β-III tubulin and two populations were identified by calbindin and calretinin staining. Distinct neuronal populations were further identified based on the presence and absence of an afterhyperpolarization (AHP). Cells with AHP expressed greater density of sodium currents. Morphine (3 µM) significantly reduced the amplitude of the action potential, increased the threshold for spike generation but did not alter the resting membrane potential. The decrease in excitability resulted from inhibition of sodium currents. In the presence of morphine, the steady-state voltage dependence of Na channels was shifted to the left with almost 50% of channels unavailable for activation from hyperpolarized potentials. During prolonged exposure to morphine (two hours), action potentials recovered, indicative of the development of tolerance in single enteric neurons. These results demonstrate the feasibility of isolating mouse myenteric neurons and establish sodium channel inhibition as a mechanism for morphine-induced decrease in neuronal excitability.     

Interleukin-1beta activates specific populations of enteric neurons and enteric glia in the guinea pig ileum and colon

Fos expression was used to assess whether the proinflammatory cytokine interleukin-1beta (IL-1beta) activated specific, chemically coded neuronal populations in isolated preparations of guinea pig ileum and colon. Whether the effects of IL-1beta were mediated through a prostaglandin pathway and whether IL-1beta induced the expression of cyclooxygenase (COX)-2 was also examined. Single- and double-labeling immunohistochemistry was used after treatment of isolated tissues with IL-1beta (0.1-10 ng/ml). IL-1beta induced Fos expression in enteric neurons and also in enteric glia in the ileum and colon. For enteric neurons, activation was concentration-dependent and sensitive to indomethacin, in both the myenteric and submucosal plexuses in both regions of the gut. The maximum proportion of activated neurons differed between the ileal (approximately 15%) and colonic (approximately 42%) myenteric and ileal (approximately 60%) and colonic (approximately 75%) submucosal plexuses. The majority of neurons activated in the myenteric plexus of the ileum expressed nitric oxide synthase (NOS) or enkephalin immunoreactivity. In the colon, activated myenteric neurons expressed NOS. In the submucosal plexus of both regions of the gut, the majority of activated neurons were vasoactive intestinal polypeptide (VIP) immunoreactive. After treatment with IL-1beta, COX-2 immunoreactivity was detected in the wall of the gut in both neurons and nonneuronal cells. In conclusion, we have found that the proinflammatory cytokine IL-1beta specifically activates certain neurochemically defined neural pathways and that these changes may lead to disturbances in motility observed in the inflamed bowel.     

Blastocystis hominis: pathogenic potential in human patients and in gnotobiotes.

Germfree guinea pigs were inoculated orally, in some experiments, and intracecally, in others, with Blastocystis hominis and the enteric flora from symptomatic patients. Other germfree guinea pigs received the parasite from axenic culture and still others from monoxenic culture with Proteus vulgaris. Fourteen of 43 animals inoculated orally with B. hominis and patient's enteric flora developed B. hominis infections and those with particularly heavy infections developed watery diarrhea of more than 1 week's duration immediately prior to sacrifice. Similar results were obtained from intracecal inoculations in that 13 of 28 animals developed infections and those with the greatest numbers of B. hominis had watery diarrhea for more than 1 week prior to sacrifice. Gross pathologic changes in these animals were mostly unremarkable, with only a slight hyperemia observed in several of the symptomatic animals. Microscopic examination, however, revealed frequent penetration of intestinal epithelium by B. hominis and the parasites in significant numbers were observed within the epithelium. There was a slight increase in cellularity in the lamina propria but parasites were not observed therein and their presence in the epithelium did not provoke an inflammatory response. Only one of eight animals inoculated from monoxenic cultures developed B. hominis infection (asymptomatic), and infections were not produced in animals inoculated from axenic culture. As a result of our observations of diarrhea in patients with particularly heavy infections with B. hominis together with the demonstration of similar symptoms in animals heavily infected with this parasite, we believe B. hominis may occasionally be related causally to the production of such symptoms by a mechanism not completely understandable.     

Endothelin-Stimulated Capacitative Calcium Entry in Enteric Glial Cells: Synergistic Effects of Protein Kinase C Activity and Nitric Oxide

Abstract: Depletion of intracellular calcium stores by agonist stimulation is coupled to calcium influx across the plasma membrane, a process termed capacitative calcium entry. Capacitative calcium entry was examined in cultured guinea pig enteric glial cells exposed to endothelin 3. Endothelin 3 (10 n M ) caused mobilization of intracellular calcium stores followed by influx of extracellular calcium. This capacitative calcium influx was inhibited by Ni²⁺ (89 ± 2%) and by La³⁺ (78 ± 2%) but was not affected by L-, N-, or P-type calcium channel blockers. Chelerythrine, a specific antagonist of protein kinase C, dose-dependently inhibited capacitative calcium entry. The nitric oxide synthase inhibitor N ^G-nitro- l -arginine decreased calcium influx in a dose-dependent manner. The combination of chelerythrine and N ^G-nitro- l -arginine produced synergistic inhibitory effects. Capacitative calcium entry occurs in enteric glial cells via lanthanum-inhibitable channels through a process regulated by protein kinase C and nitric oxide.     

Sensory peptide neurotransmitters mediating mucosal and distension evoked neural vasodilator reflexes in guinea pig ileum

The aim was to determine the role CGRP and/or tachykinins released from sensory neural mechanisms in enteric neural vasodilator pathways. These pathways project through the myenteric plexus to submucosal vasodilator neurons. Submucosal arterioles were exposed in the distal portion of an in vitro combined submucosal-myenteric guinea pig ileal preparation, and dilation was monitored with videomicroscopy. Vasodilator neural reflexes were activated by gently stroking the mucosa with a fine brush or by distending a balloon placed beneath the flat-sheet preparation in the proximal portion. Dilations evoked by mucosal stroking were inhibited 64% by the CGRP 8-37 and 37% by NK3 (SR 142801) antagonists. When the two antagonists were combined with hexamethonium, only a small vasodilation persisted. Balloon distension-evoked vasodilations were inhibited by NK3 antagonists (66%) but were not altered by CGRP 8-37. In preparations in which myenteric descending interneurons were directly activated by electrical stimulation, combined application of CGRP 8-37 and the NK antagonists had no effect. Stimulation of capsaicin sensitive nerves in the myenteric plexus did not activate these vasodilator reflexes. These findings suggest that mucosal-activated reflexes result from the release of CGRP and tachykinins from enteric sensory neurons. Distension-evoked responses were significantly blocked by NK3 antagonists, suggesting that stretch activation of myenteric sensory neurons release tachykinins that activate NK3 receptors on myenteric vasodilator pathways.