@phdthesis{oai:sucra.repo.nii.ac.jp:00017932,
 author = {AMRITA, DUDANI},
 month = {},
 note = {xx, 97 p., Background and Aim: Motilin and ghrelin are released in the interdigestive state to initiate and regulate phase III contractions of migrating motor complex (MMC). The MMC is responsible for emptying the stomach during the interdigestive period to prepare for the next meal. Gastric phase III contractions of the MMC originate in the stomach and propagate downward in the alimentary canal. Previously, I found that motilin and ghrelin synergistically induced gastric contractions both in vitro and in vivo. Motilin-induced contractions are regulated by a ghrelin-mediated GABAergic pathway. Therefore, I hypothesized that some regions of the stomach are more responsive to motilin and ghrelin and propagate strong contractions. The present study determined active responsive sites for motilin- and ghrelin-induced contractions in the stomach and elucidated mechanisms underlying the induction of these contractions.

Methods: The stomachs of Suncus murinus or Asian house shrew, a small insectivorous mammal, were dissected, and the fundus, proximal corpus, distal corpus, and antrum were isolated to examine the effect of motilin- and/or ghrelin-induced contractions by using an organ bath system. The stomach segments were pretreated with tetrodotoxin, atropine, bicuculline, phaclofen, adenosine, and dopamine to determine the involvement of neural pathways. Quantitative PCR (qPCR) was performed to measure the mRNA expression of the motilin receptor GPR38. Distribution of ghrelin-immunopositive cells and mRNA expression of the GHSR in the different segments of the suncus stomach were examined by performing immunohistochemical analysis and RT-PCR, respectively.

Results: Results of this in vitro study showed that treatment with 10-10 M motilin induced contractions only in the proximal corpus. In contrast, treatment with 10-9 M motilin induced strong contractions in the other segments of the suncus stomach. Motilin-induced contractions in each dissected stomach segment were inhibited by tetrodotoxin and atropine pretreatment, suggesting that these contractions were mediated by a cholinergic neural pathway in the myenteric plexus. Treatment with ghrelin (10-11-10-7 M) in the presence of low-dose motilin (10-10 M) induced gastric contractions in a dose-dependent manner in the fundus and proximal corpus but not in the distal corpus and antrum. In addition, pretreatment with ghrelin antagonist D-Lys3-GHRP6 blocked motilin-induced contractions in all the stomach segments. In contrast, treatment with GABA antagonists reversed this blockade in all the stomach segments. Treatment with adenosine A2A receptor and dopamine D2 receptor agonists also reversed ghrelin antagonist-induced inhibition of motilin-induced contractions. The mRNA expression of motilin receptor, GPR38 was highest in the proximal corpus and was the lowest in the antrum. The mRNA expression of GPR38 varied, with low expression in the mucosal layer and higher expression in the muscle layer. The mRNA expression of the ghrelin receptor, GHSR was detected in all the stomach segments. Density of ghrelin-immunopositive cells was significantly higher in the fundus and proximal corpus than in the other stomach segments.

Conclusions: These results suggest that each gastric segment shows a different response toward motilin and/or ghrelin. The fundus and proximal corpus, including the cardia, are the most sensitive and responsive to motilin- and/or ghrelin-induced synergistic gastric contractions, suggesting that the proximal part of the stomach along with fundus is the first contractile site for MMC onset. In addition, present results indicate that adenosine(via A2A receptor) and dopamine (via D2 receptor) play vital roles in regulating motilin- and ghrelin-induced gastric contractions., Declaration　I
Thesis dedicated to　II
Acknowledgement　III
Table of contents　VI
List of figures　XI
List of tables　XIII
List of appendix　XIV
Abbreviations　XV
Abstract　XVIII
Chapter 1: General Introduction and Objectives　1
1.1. Research background　1
1.1.1. The migrating motor complex (MMC) of the gastrointestinal (GI) motility　1
1.1.2. Motilin and its regulatory mechanism　2
1.1.3. Ghrelin and GI motility　3
1.1.4. Similar properties of motilin and ghrelin　4
1.1.5. Asian house shrew (Suncus murinus)　5
1.2. Hypothesis and objectives　7
Chapter 2: The proximal gastric corpus is the most responsive site of motilin-induced contractions in the stomach of the Asian house shrew in vitro　8
2.1. Introduction　8
2.1.1. Motilin as an important regulator of MMC　8
2.1.2. Advantages of suncus for studying gastrointestinal physiology including gastric motility　9
2.2. Materials and Methods　11
2.2.1. Ethical Approval　11
2.2.2. Animals　11
2.2.3. Drugs used　11
2.2.4. Preparation of S. murinus isolated stomach　12
2.2.5. Gastric contractility study　13
2.2.6. GPR38 mRNA expression　14
2.2.7. Statistical analysis　15
2.3. Results　16
2.3.1. Spontaneous contractile pattern in the different segments of isolated stomach　16
2.3.2. Responses to motilin in different segments of stomach　16
2.3.3. The cholinergic pathway of the motilin-induced contraction　18
2.3.4. The myenteric plexus pathway of the motilin-induced contraction　18
2.3.5. GPR38 mRNA expression in the stomach　18
2.4. Discussion　20
2.4.1. Sensitivity towards motilin is region-specific　20
2.4.2. Physiological correlation of MMC and motilin　21
2.4.3. Regulatory mechanism of motilin-induced gastric contraction　21
2.4.4. Motilin receptor GPR38 mRNA expression　22
2.5. Summary
24 Chapter 3: Synergistic effect of motilin and ghrelin induces different responses in different segments of the stomach of Suncus murinus in vitro　25
3.1. Introduction　25
3.1.1. Regulatory mechanism underlying motilin- and ghrelin-induced gastric contractions　25
3.2.2 Adenosine and dopamine as neurotransmitters and neuromodulators　26
3.2. Materials and Methods　28
3.2.1. Ethical approval　28
3.2.2. Animals　28
3.2.3. RT-PCR　29
3.2.4. Tissue preparation for morphological analysis　29
3.2.5. Immunohistochemical analysis　30
3.2.6. Morphometric analysis　31
3.2.7. Preparation of the isolated S. murinus stomach　31
3.2.8. Analysis of in vitro gastric contractions　32
3.2.9. Drugs used　33
3.2.10. Statistical analysis　33
3.3. Results　35
3.3.1. Synergistic effect of motilin and ghrelin on the different segments of the suncus stomach　35
3.3.2. Motilin and ghrelin synergistically induce gastric contractions through a cholinergic neural pathway　35
3.3.3. GHS-R mRNA expression in the different segments of the suncus stomach　35
3.3.4. Localization of ghrelin-ip cells in the suncus stomach　36
3.3.5. Effect of D-Lys3-GHRP6 and GABA receptor antagonists on motilin-induced gastric contractions in vitro　36
3.3.6. Effect of non-selective adenosine and dopamine receptor agonists on motilin-induced gastric contractions　37
3.3.7. Effect of adenosine A2A receptor agonist and dopamine D2 receptor agonist on motilin-induced gastric contractions　37
3.4. Discussion　39
3.4.1. Role of ghrelin in the MMC　39
3.4.2. Involvement of the GABAergic pathway in motilin-induced gastric contractions　40
3.4.3. Adenosine and dopamine are the key molecules for mediating motilin-induced gastric contractions　41
4. Summary and Conclusion　43
5. Figures, Tables and Appendix　45
6. References　78, 主指導教員 : 坂井貴文, text, application/pdf},
 school = {埼玉大学},
 title = {Motilin- and ghrelin-induced gastric contractions in different parts of Suncus stomach in vitro},
 year = {2017},
 yomi = {アムリタ, ドゥダニ}
}