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炎症性腸疾患における腸管神経の機能変化に対するグリア細胞の関与

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Please use this identifier to cite or link to this item:https://doi.org/10.14943/doctoral.k9046

Title: 炎症性腸疾患における腸管神経の機能変化に対するグリア細胞の関与
Other Titles: Involvement of enteric glial cells in functional changes of enteric neurons in inflammatory bowel disease : Study using in vitro model system
Authors: 村上, 真津香1 Browse this author
Authors(alt): Murakami, Matsuka1
Issue Date: 25-Mar-2009
Publisher: Hokkaido University
Abstract: To identify the action of BK in the enteric nervous system (ENS), the effects of bradykinin (BK) on the enteric neuron and glial cells were examined by focusing on the interaction between them, using primary culture of myenteric plexus cells from the rat intestine. Furthermore, the impacts of lipopolysaccharide (LPS) or inflammatory cytokines, elevated in serum of inflammatory bowel disease (IBD) patients, on the interaction were investigated. 1. The immunocytochemical analysis using anti-protein gene product (PGP9.5) and anti-S100 antibodies showed that cells in primary culture of myenteric plexus were composed of neurons (30%) and enteric glial cells (68%). The processes of these neurons connected with other neurons and formed neural networks. 2. In both primary cultured myenteric plexus and whole mount preparation of myenteric plexus, less than 10% of neurons showed an immunoreactive (IR) positive to calbindin, a marker of intrinsic primary afferent neuron. BK increased [Ca2+]i to the same extent in both calbindin IR-positive or -negative neurons. 3. BK evoked [Ca2+]i rise in neurons in a dose-dependent manner. The number of neurons responded to BK were increased by increasing concentrations of BK. 4. Both B1 and B2 receptor mRNAs were detected in cultured myenteric plexus cells. The neural [Ca2+]i increase by BK was abolished by a B2 receptor antagonist HOE140, but not affected by a B1 receptor antagonist Lys-des-Ag9-HOE140. A B1 receptor agonist des-Arg9-BK failed to cause a [Ca2+]i response. 5. Double immunostaining using antibodies against B2 receptors together with PGP9.5 or S100 indicated that B2 receptors were expressed in both enteric neurons and glial cells. 6. The neural [Ca2+]i response to BK was attenuated by removal of external Ca2+ or by Cd2+, a blocking agent of voltage dependent Ca2+ channels. In the neurons depleted of Ca2+ from internal stores by thapsigargin, [Ca2+] response to BK became smaller. 7. Indomethacin, a non-selective cyclooxygenase (COX) inhibitor, significantly suppressed the neural [Ca2+]i response to BK. Pretreatment with prostaglandin E2 (PGE2), but not PGI2, significantly potentiated the [Ca2+]i response to BK. Neither PGE2 nor PGI2 affected the resting [Ca2+]i level. An EP1 receptor antagonist SC19220 suppressed the neural response to BK. Unlike PGE2, an EP3 receptor agonist sulprostone did not potentiate the neural response to BK. 8. The release of PGE2 from cultured myenteric plexus cells was increased by BK in a dose-dependent manner, which was inhibited by indomethacin. 9. The [Ca2+]i response to BK in neurons cultured at the high density was significantly larger than that at the low density. The response to BK in neurons was greatly diminished by reducing the number of enteric glial cells in culture. Indomethacin inhibited the response to BK in neurons cultured at the high density, but not the low density. Under the culture conditions of lowering the number of enteric glial cells, indomethacin failed to inhibit the neural response to BK. 10. In cultured myenteric neurons, BK evoked a slow and sustained depolarization and action potential discharges superimposed on it in some neurons. BK-induced depolarization and action potentials were suppressed by indomethacin. The current intensity required for evoking action potentials was decreased by BK, without changing membrane potential threshold of evoking action potentials. 11. BK evoked dose-dependent increases of [Ca2+]i in enteric glial cells, which were hardly diminished by the removal of external Ca2+. In glial cells depleted Ca2+ from internal stores by thapsigargin, BK failed to evoke [Ca2+]i increase. BK-induced glial [Ca2+]i increase was abolished by a phospholipase C inhibitor U73122. 12. [Ca2+]i responses to BK in enteric neurons and glial cells were potentiated by treatment with LPS or IL-1β, but not with TNF-α Similar potentiation was induced by LPS or IL-1β in the pure culture of glial cells. These augmenting effects of LPS on responses to BK were suppressed by pretreatment with an interleukin-1 receptor antagonist IL-1ra. 13. In cultured myenteric plexus cells, the augmenting effects of LPS and IL-1β on neural [Ca2+]i responses to BK were suppressed by reducing the number of enteric glial cells in culture. 14. LPS increased IL-1β secretion either from cultured myenteric plexus cells containing neurons and glial cells or from pure culture of glial cells. 15. IL-1β augmented BK-induced [Ca2+]i increases in both enteric neurons and glial cells in exposure time-dependent manner. In untreated neurons and glial cells, BK-induced [Ca2+]i increase was not affected by a B1 receptor antagonist des-Arg9-HOE140, but abolished by a B2 receptor antagonist HOE140. After cultured with IL-1β, the augmented [Ca2+]i response to BK in both cell types were partly inhibited by the B1 receptor antagonist. The B2 receptor antagonist completely abolished the responses to BK in neurons but not in enteric glial cells. 16. In glial cells cultured without IL-1β, two different B1 receptor agonists, Lys-des-Arg9-BK and BK fragment 1-8, evoked tiny changes in [Ca2+]i. After culture with IL-1β, substantial [Ca2+]i increase occurred with both B1 receptor agonists in enteric glial cells but not neurons. 17. IL-1β potentiated the expression level of mRNA of B1 receptor but not B2 receptor in cultured myenteric plexus cells. The signal intensity of B1 receptor IR increased in enteric glial cells immunoreactive to S100. 18. The EP1 receptor antagonist SC19220 suppressed the IL-1β-enhancing neural response to BK. The amount of PGE2 release by BK from myenteric plexus cells was increased by IL-1β. The B1 receptor agonist produced a significant release of PGE2 from IL-1β-treated myenteric plexus cells but not from untreated cells. 19. Under the conditions of depletion of Ca2+ from internal stores by thapsigargin and the removal of external Ca2+, BK failed to evoke PGE2 release from cultured myenteric plexus cells. A23187, a Ca2+ ionophore, significantly increased PGE2 release, which was suppressed by a non-selective phospholipase A2 inhibitor aristolochic acid. 20. After cultured with IL-1β, either aristolichic acid or indomethacin suppressed the neural [Ca2+]i response to BK and BK-induced PGE2 release from myenteric plexus cells. A selective COX-2 inhibitor, nimesulide, significantly, but not completely, abolished the augmented [Ca2+]i responses to BK. This drug inhibited partly the BK-induced PGE2 release from myenteric plexus cells cultured with IL-1β but not without IL-1β. 21. IL-1β increased the intensity of COX-2 IR in all positive to S100 IR-positive, but not PGP9.5 IR-positive cells. These results shows that BK causes a [Ca2+]i increase and depolarization in rat myenteric neurons through the activation of B2 receptors, which was partly associated with PGE2 released from glial cells in response to BK. Furthermore, these results also indicate that LPS up-regulates B1 receptors and COX-2 in enteric glial cells via secretion of IL-1β in an autocrine fashion. The enhancement of PGE2 production results in the alteration of cross-talk of the neuron-glial interaction. Such functional changes of enteric glial cells may cause dysfunction in the gut of IBD patients.
Conffering University: 北海道大学
Degree Report Number: 甲第9046号
Degree Level: 博士
Degree Discipline: 獣医学
Type: theses (doctoral)
URI: http://hdl.handle.net/2115/38246
Appears in Collections:学位論文 (Theses) > 博士 (獣医学)

Submitter: 村上 真津香

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