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Molecular characterization and distribution of motilin and motilin receptor in the Japanese medaka Oryzias latipes

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Abstract

Motilin (MLN) is a peptide hormone originally isolated from the mucosa of the porcine intestine. Its orthologs have been identified in various vertebrates. Although MLN regulates gastrointestinal motility in tetrapods from amphibians to mammals, recent studies indicate that MLN is not involved in the regulation of isolated intestinal motility in zebrafish, at least in vitro. To determine the unknown function of MLN in teleosts, we examined the expression of MLN and the MLN receptor (MLNR) at the cellular level in Japanese medaka (Oryzias latipes). Quantitative PCR revealed that mln mRNA was limitedly expressed in the gut, whereas mlnr mRNA was not detected in the gut but was expressed in the brain and kidney. By in situ hybridization and immunohistochemistry, mlnr mRNA was detected in the dopaminergic neurons of the area postrema in the brain and the noradrenaline-producing cells in the interrenal gland of the kidney. Furthermore, we observed efferent projections of mlnr-expressing dopaminergic neurons in the lobus vagi (XL) and nucleus motorius nervi vagi (NXm) of the medulla oblongata by establishing a transgenic medaka expressing the enhanced green fluorescence protein driven by the mlnr promoter. The expression of dopamine receptor mRNAs in the XL and cholinergic neurons in NXm was confirmed by in situ hybridization. These results indicate novel sites of MLN activity other than the gastrointestinal tract. MLN may exert central and peripheral actions through the regulation of catecholamine release in medaka.

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Data Availability

The data supporting the findings of present study are available from the corresponding author (MA) upon reasonable request.

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Acknowledgements

We thank Ms. Sayaka Sunada of the laboratory in the University of Toyama for technical assistance. We also thank Enago (www.enago.jp) for English-language editing.

Funding

This study was partly supported by JSPS KAKENHI Grants (Numbers 20K15834 to MA, 20K06717 to NK, and 22H02659 to HK). Japan Society for the Promotion of Science, 20K15834, Morio Azuma, 20K06717, Norifumi Konno, NK,Hiroyuki Kaiya, 22H02659, Hiroyuki Kaiya.

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Authors and Affiliations

  1. Division of Molecular Pharmacology, Department of Pharmacology, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi, Japan

    Morio Azuma & Taka-aki Koshimizu

  2. Department of Biological Science, Graduate School of Science and Engineering, University of Toyama, Gofuku, Toyama, 3190, Japan

    Norifumi Konno & Hiroyuki Kaiya

  3. Area of Regulatory Biology, Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimookubo, Saitama, Japan

    Ichiro Sakata

  4. Division of Drug Discovery, Grandsoul Research Institute for Immunology, Inc. 8-1 Utano-Matsui, Uda, Nara, Japan

    Hiroyuki Kaiya

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  1. Morio Azuma
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  4. Taka-aki Koshimizu
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Contributions

MA coordinated the project, wrote the manuscript, and performed in situ hybridization and immunohistochemistry; NK performed phylogenetically analysis, quantitative PCR, and establishment MLNR-EGFP transgenic medaka; IS supervised and validated the project; TK supervised and validated the project; HK performed measurement of intracellular Ca2+ mobilization. All authors read, edited, and approved the final manuscript.

Corresponding author

Correspondence to Morio Azuma.

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Ethics approval

All animal experiments were performed after receiving approval from the Institutional Animal Experiment Committee of Jichi Medical University (Approval number: 18023–01) and the University of Toyama (Approval number: G2019SCI-3, G2024SCI-03).

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Not applicable.

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Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Figure 1. Sequence and phylogenetic tree of motilin (MLN) precursors. 

The amino acid sequences obtained from GenBank and a genome browser for vertebrate genomes (Ensemble) were edited using Biological Sequence Alignment Editor version 7.2 (BioEdit software) and the ClustalW algorithm (DNA Data Bank of Japan). Molecular phylogenetic relationships were analyzed using the maximum-likelihood method with MEGA11 software (Tamura et al., 2021). To estimate the reliability of the trees, bootstrap** of the data (1000 replicates) was performed. a Nucleotide sequences and deduced amino acid sequences of the MLN precursor identified in the Japanese medaka Oryzias latipes. The predicted signal peptide is underlined. The mature peptide is indicated with a gray background. The dibasic cleavage sites are indicated in the box. b The phylogenetic tree was constructed from the alignment of amino acid sequences of MLN precursors by MEGA11 using the maximum-likelihood method. Data were resampled with 1,000 bootstrap replicates. The accession numbers of the sequences used in this analysis are shown in Table S2. (TIF 26782 KB)

Supplementary Figure 2. Multiple alignment of deduced amino acid sequences of motilin receptor (MLNR) from human, chicken, clawed frog, zebrafish, and medaka. 

Hydropathy analysis was performed using the Kyte–Doolittle algorithm to predict seven putative hydrophobic transmembrane regions characteristic of GPCRs. Computer analysis of the presence of glycosylation sites was performed using NetNGlyco1.0 software (https://services.healthtech.dtu.dk/services/NetNGlyc-1.0/). The deduced amino acid sequences were aligned using the ClustalW algorithm [asterisks, identical amino acid residues; open box, predicted transmembrane regions; black box, putative N-glycosylation sites; gray character, putative phosphorylation sites; hash, ERY motif; black triangle, NPxxY motif (where x represents any amino acid)]. The accession numbers of the sequences used in this analysis are shown in Table S2. (TIF 22060 KB)

Supplementary Figure 3. Phylogenetic analysis of the motilin receptor (MLNR) and ghrelin receptor (GHSR) in vertebrates.

 The phylogenetic tree was constructed using the maximum-likelihood method using MEGA11 software (Tamura et al., 2021). Data were resampled with 1,000 bootstrap replicates. The accession numbers of the sequences are shown in Table S2. (TIF 25002 KB)

Supplementary Figure 4. Construction of a vector for establishing MLNR transgenic medaka.

The DNA fragment of the 5′-upstream region of the medaka mlnr gene (amplified the 4,040 bp) and the ISpBSIISK+EGFP vector (Ogino et al., 2006) with medaka heat-shock protein 70 core promoter (hsp) were ligated using in-Fusion technology (TaKaRa Bio, Shiga, Japan) to generate the IS-medaka mlnr+EGFP plasmid. The injection solution with linear plasmid was injected into the cytoplasm of one-cell stage medaka embryos. (TIF 21209 KB)

Supplementary Figure 5. Expression of catecholamine-synthesizing enzymes in the medaka area postrema and interrenal gland.

 In situ hybridization was performed with a fluorescein-labeled antisense cRNA probe for catecholamine-synthesizing enzyme (a, f tyrosine hydroxylase 1, th1; b, g tyrosine hydroxylase 2, th2; c, h dopa decarboxylase, ddc;d, i dopamine beta-hydroxylase, dbh; e, j phenylethanolamine N-methyltransferase, pnmt) mRNAs to determine the types of catecholamines produced in the medaka area postrema and interrenal gland. Dopamine and noradrenaline were produced in the area postrema by the expression of th1, ddc, and dbh mRNAs. Noradrenaline and adrenaline were produced in the interrenal gland by the expression of th1,ddc, dbh, and pnmt mRNAs. Asterisks show the vein of the kidney. In situ hybridization with 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP,blue). Bars, 10 μm (a–e), 100 μm (f–j). (TIF 19121 KB)

Supplementary Figure 6. Expression of dopamine receptors in projection areas of MLNR-expressing neurons in the medulla oblongata. 

In situ hybridization was performed with a DIG-labeled antisense cRNA probe for the D1-types (d1a,d1b, d5a, d5b, d6a) and the D2-types (d2a, d3,d4a, d4b, d3, d9) of the medaka dopamine receptor in the lobus vagi (XL) and nucleus motorius nervi vagi (NXm). The dopamine receptors of d1b and d2a were qualitatively major types in these areas compared with d5a, d3, and d8. The expression of d1a,d5b, d6a, d4a, d4b, and d9 mRNAs was not detected in these areas. In situ hybridization with NBT/BCIP (blue). Bars, 100 μm. (TIF 20428 KB)

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Azuma, M., Konno, N., Sakata, I. et al. Molecular characterization and distribution of motilin and motilin receptor in the Japanese medaka Oryzias latipes. Cell Tissue Res (2024). https://doi.org/10.1007/s00441-024-03896-5

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