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S Fukusumi,
Y Habata,
H Yoshida,
N Iijima, Y Kawamata,
M Hosoya,
R Fujii,
S Hinuma,
C Kitada,
Y Shintani,
M Suenaga,
H Onda,
O Nishimura,
M Tanaka,
Y Ibata,
M Fujino
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ABSTRACT: We have recently identified RFamide-related peptide (RFRP) gene that would encode three peptides (i.e., RFRP-1, -2, and -3) in human and bovine, and demonstrated that synthetic RFRP-1 and -3 act as specific agonists for a G protein-coupled receptor OT7T022. However, molecular characteristics and tissue distribution of endogenous RFRPs have not been determined yet. In this study, we prepared a monoclonal antibody for the C-terminal portion of rat RFRP-1. As this antibody could recognize a consensus sequence among the C-terminal portions of rat, human, and bovine RFRP-1, we purified endogenous RFRP-1 from bovine hypothalamus on the basis of immunoreactivity to the antibody. The purified bovine endogenous RFRP-1 was found to have 35-amino-acid length that corresponds to 37-amino-acid length in human and rat. We subsequently constructed a sandwich enzyme immunoassay using the monoclonal antibody and a polyclonal antibody for the N-terminal portion of rat RFRP-1, and analyzed the tissue distribution of endogenous RFRP-1 in rats. Significant levels of RFRP-1 were detected only in the central nervous system, and the highest concentration of RFRP-1 was detected in the hypothalamus. RFRP-1-positive nerve cells were detected in the rat hypothalamus by immunohistochemical analyses using the monoclonal antibody. In culture, RFRP-1 lowered cAMP production in Chinese hamster ovary cells expressing OT7T022 and it was abolished by pre-treatment with pertussis toxin, suggesting that OT7T022 couples G(i)/G(o) in the signal transduction pathway.
Biochimica et Biophysica Acta 10/2001; 1540(3):221-32. · 4.66 Impact Factor
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Y Kawamata,
Y Habata,
S Fukusumi,
M Hosoya,
R Fujii,
S Hinuma,
N Nishizawa,
C Kitada,
H Onda,
O Nishimura,
M Fujino
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ABSTRACT: We analyzed the tissue distribution of apelin mRNA in rats by a quantitative reverse transcription-polymerase chain reaction and that of immunoreactive apelin (ir-apelin) by an enzyme immunoassay (EIA) using a monoclonal antibody. The expression levels of apelin mRNA and ir-apelin seemed to be consistent among tissues: they were highly expressed in the lung and mammary gland. By the combination of gel filtration and EIA, we found that the molecular forms of apelin differ among respective tissues: apelin molecules with sizes close to apelin-36 (long forms) were major components in the lung, testis, and uterus, but both long and short (whose sizes were close to [<Glu(65)]apelin-13) forms were detected in the mammary gland. In Scatchard analyses, the radioiodinated apelin-36 analogue bound to the receptor, APJ, with high affinity. In competitive binding assays, apelin-36 and apelin-19 far more efficiently inhibited the binding of the labeled apelin-36 analogue with APJ than [<Glu(65)]apelin-13. In analyses for the dissociation of apelin from APJ, unlabeled apelin-36 replaced more rapidly the labeled apelin-36 analogue bound with APJ than [<Glu(65)]apelin-13. Our results demonstrate that the long and short forms of apelin differently interact with APJ.
Biochimica et Biophysica Acta 05/2001; 1538(2-3):162-71. · 4.66 Impact Factor
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S Hinuma,
Y Shintani,
S Fukusumi,
N Iijima,
Y Matsumoto,
M Hosoya,
R Fujii,
T Watanabe,
K Kikuchi,
Y Terao, [......], Y Kawamata,
Y Habata,
M Asada,
C Kitada,
T Kurokawa,
H Onda,
O Nishimura,
M Tanaka,
Y Ibata,
M Fujino
[show abstract]
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ABSTRACT: Only a few RFamide peptides have been identified in mammals, although they have been abundantly found in invertebrates. Here we report the identification of a human gene that encodes at least three RFamide-related peptides, hRFRP-1-3. Cells transfected with a seven-transmembrane-domain receptor, OT7T022, specifically respond to synthetic hRFRP-1 and hRFRP-3 but not to hRFRP-2. RFRP and OT7T022 mRNAs are expressed in particular regions of the rat hypothalamus, and intracerebroventricular administration of hRFRP-1 increases prolactin secretion in rats. Our results indicate that a variety of RFamide-related peptides may exist and function in mammals.
Nature Cell Biology 11/2000; 2(10):703-8. · 19.49 Impact Factor
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M Hosoya,
T Moriya, Y Kawamata,
S Ohkubo,
R Fujii,
H Matsui,
Y Shintani,
S Fukusumi,
Y Habata,
S Hinuma,
H Onda,
O Nishimura,
M Fujino
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ABSTRACT: Neuromedin U is a bioactive peptide isolated originally from the porcine spinal cord. We recently identified neuromedin U as the cognate ligand for the orphan G protein-coupled receptor FM-3. In this study, we isolated cDNA coding for a novel G protein-coupled receptor, TGR-1, which was highly homologous with FM-3. We found that neuromedin U specifically and clearly elevated the extracellular acidification rates, arachidonic acid metabolite release, and intracellular Ca(2+) mobilization in Chinese hamster ovary cells expressing TGR-1. Radiolabeled neuromedin U specifically bound with high affinity to membrane fractions prepared from these cells. These results show that TGR-1, like FM-3, is a specific and functional receptor for neuromedin U. We analyzed TGR-1 mRNA tissue distribution in rats using quantitative reverse transcription-polymerase chain reaction and found it to considerably differ from that of FM-3 mRNA. TGR-1 mRNA was primarily expressed in the uterus, suggesting that TGR-1 mediates the contractile activity of neuromedin U in this tissue. The identification of specific and functional receptor subtypes for neuromedin U will facilitate the study of their physiological roles and the search for their specific agonists and antagonists.
Journal of Biological Chemistry 10/2000; 275(38):29528-32. · 4.77 Impact Factor
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[show abstract]
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ABSTRACT: Neuromedin U is a bioactive peptide first isolated from porcine spinal cord. In this paper, we demonstrate that neuromedin U is the cognate ligand for the orphan G protein-coupled receptor, FM-3, isolated originally as a homologue of neurotensin and growth hormone secretogogue receptors. Neuromedin U induced specific and evident elevation of extracellular acidification rates, arachidonic acid metabolite release, and intracellular Ca(2+) mobilization in Chinese hamster ovary cells expressing human FM-3. In addition, radiolabeled neuromedin U specifically bound to membrane fractions prepared from these cells with high affinity. We subsequently analyzed the tissue distribution of neuromedin U and FM-3 mRNAs in rats using quantitative reverse transcription-polymerase chain reaction. Neuromedin U mRNA was highly expressed in the gastrointestinal tract, and the highest expression was detected in the pituitary gland. On the other hand, FM-3 mRNA was highly expressed in the small intestine and lung, suggesting that neuromedin U plays important roles in these tissues. The identification of a specific and functional receptor for neuromedin U will facilitate studies on their physiological roles and the search for receptor agonists and antagonists.
Journal of Biological Chemistry 08/2000; 275(28):21068-74. · 4.77 Impact Factor
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ABSTRACT: We validated the effect of prolactin-releasing peptide (PrRP) on prolactin (PRL) secretion from rat anterior pituitary cells in in vitro culture. We found that culture conditions considerably influenced the response of the anterior pituitary cells to PrRP. Longer culture term (4 d) was required to obtain better responses of the anterior pituitary cells to PrRP in comparison to thyrotropin-releasing hormone (TRH). Under the culture conditions employed here, PrRP was comparable to TRH in the potency promoting PRL secretion, and the action of PrRP was very specific for PRL secretion. The susceptibility of the anterior pituitary cells to PrRP varied in female rats depending on the process of reproduction: the cells prepared from lactating rats were the most sensitive to PrRP compared with those from random-cycle and pregnant rats. Because the expression levels of PrRP receptor mRNA in the pituitary varied during the reproductive process, we speculated that the susceptibility of the anterior pituitary cells would reflect cellular changes including the expression level of PrRP receptors. In addition, treatment with estrogen in vivo enhanced the susceptibility of the cultured anterior pituitary cells in male rats. Our results indicate that the susceptibility of the rat anterior pituitary cells to PrRP is regulated by physiological mechanisms.
Endocrine 07/2000; 12(3):215-21. · 1.42 Impact Factor
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M Hosoya, Y Kawamata,
S Fukusumi,
R Fujii,
Y Habata,
S Hinuma,
C Kitada,
S Honda,
T Kurokawa,
H Onda,
O Nishimura,
M Fujino
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ABSTRACT: We have recently identified apelin as the endogenous ligand for human APJ. In rats, the highest expression of APJ mRNA was detected in the lung, suggesting that APJ and its ligand play an important role in the pulmonary system. When apelin-36 and its pyroglutamylated C-terminal peptide, [<Glu(65)]apelin-13, were compared in microphysiometric analyses, the elevation of extracellular acidification induced in cells expressing APJ by [<Glu(65)]apelin-13 was transient, whereas that by apelin-36 was sustained. These responses were almost completely inhibited by a specific inhibitor for G(i) or that for Na(+)/H(+) exchanger. (125)I -Labeled [<Glu(65)]apelin-13 analogue specifically bound to APJ with a high affinity, and [<Glu(65)]apelin-13 was more potent than apelin-36 in competitive inhibition assays. Because pretreatment with apelin-36 but not [<Glu(65)]apelin-13 drastically reduced the binding of the labeled apelin to APJ, the different patterns of acidification induced by these two peptides appeared to reflect their dissociation rather than association with APJ. Apelin elicited the migration of APJ-expressing cells, and [<Glu(65)]apelin-13 was more potent than apelin-36 in this activity. Heterogeneous molecular forms of apelin corresponding to apelin-36 and [<Glu(65)]apelin-13 were produced in bovine colostrum. Apelin-36 and [<Glu(65)]apelin-13 might have different functions in vivo and in vitro.
Journal of Biological Chemistry 07/2000; 275(28):21061-7. · 4.77 Impact Factor
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Y Habata,
R Fujii,
M Hosoya,
S Fukusumi, Y Kawamata,
S Hinuma,
C Kitada,
N Nishizawa,
S Murosaki,
T Kurokawa,
H Onda,
K Tatemoto,
M Fujino
[show abstract]
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ABSTRACT: By using a strategy that we have developed to search for the ligands of orphan seven-transmembrane-domain receptors [S. Hinuma et al., Nature 393 (1998) 272-276], we have recently identified a natural ligand, apelin, for the orphan 7TMR, APJ [K. Tatemoto et al., Biochem. Biophys. Res. Commun. 251 (1998) 471-476]. In this paper, we isolated rat and mouse apelin cDNAs, and analyzed the tissue distribution of apelin mRNA in rats. Although apelin mRNA was widely detected in a variety of tissues, the highest expression of apelin mRNA was detected in the mammary gland of pregnant rats. In the mammary gland, biologically active apelin and its mRNA considerably increased during pregnancy and lactation, and reached a maximal level around parturition. Moreover, a large amount of apelin (14-93 pmol/ml) was found to be secreted in the bovine colostrum, and it was still detectable even in commercial bovine milk. Since apelin partially suppressed cytokine production by mouse spleen cells in response to T cell receptor/CD3 cross-linking, the oral intake of apelin in the colostrum and milk might modulate immune responses in neonates.
Biochimica et Biophysica Acta 11/1999; 1452(1):25-35. · 4.66 Impact Factor
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R Fujii,
S Fukusumi,
M Hosoya, Y Kawamata,
Y Habata,
S Hinuma,
M Sekiguchi,
C Kitada,
T Kurokawa,
O Nishimura,
H Onda,
Y Sumino,
M Fujino
[show abstract]
[hide abstract]
ABSTRACT: Prolactin-releasing peptide (PrRP) is a novel bioactive peptide, originally isolated from bovine hypothalamus by utilizing an orphan seven-transmembrane-domain receptor expressed in the human pituitary gland. In this paper, we analyzed the tissue distribution of rat and human PrRP and their receptor mRNAs by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Northern blotting. In RT-PCR analysis, rat PrRP receptor mRNA was detected in the central nervous system, and the highest expression was detected in the pituitary gland. In addition, in situ hybridization revealed that rat PrRP receptor mRNA was highly expressed in the anterior lobe of the pituitary. On the other hand, rat PrRP mRNA was most abundantly expressed in the medulla oblongata, while significant levels of expression were widely detected in other tissues. In Northern blot analyses, human PrRP receptor mRNA was detected only in the pituitary gland among tissues examined. Human PrRP mRNA was detected in the medulla oblongata and in the pancreas. In contrast to the pattern of mRNA expression, the highest content of bioactive PrRP was found in the hypothalamus rather than the medulla oblongata in the rat brain, indicating that PrRP mRNA does not always parallel with mature PrRP in tissue distribution. The wide distribution of PrRP and its receptor suggests that they have various functions not only in the pituitary gland but also in the other tissues.
Regulatory Peptides 09/1999; 83(1):1-10. · 2.11 Impact Factor
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ABSTRACT: We have previously reported a hypothalamic peptide that shows specific prolactin (PRL)-releasing activity in vitro, named prolactin-releasing peptide (PrRP). However, its activity in vivo has not yet been shown. In this study, we examined whether PrRP could induce specific PRL release in vivo using normal cycling female and male rats. Intravenous injection of PrRP31 increased plasma PRL levels in rats in a dose-dependent manner. PrRP31 (50 nmol/kg i.v.) significantly (P < 0.05) stimulated plasma PRL levels within 25 min after injection in rats in proestrus, estrus, and metestrus. A higher dose of PrRP31 (500 nmol/kg i.v.) was necessary for a significant increase in plasma PRL levels in male rats. These results clearly indicate that female rats, especially at proestrus, are more sensitive to PrRP-induced PRL secretion than male rats. The effect of PrRP on PRL release is affected considerably by the estrous cycle and sex, which suggests that PrRP sensitivity is controlled by the endogenous hormonal milieu, such as estrogen levels. PrRP31 did not affect other pituitary hormone secretions. The results indicate that PrRP shows specific PRL-releasing activity in vivo as well as in vitro and suggest that it plays an important role in the regulation of PRL release under certain physiological conditions.
Biochemical and Biophysical Research Communications 07/1999; 259(2):321-4. · 2.48 Impact Factor
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K Tatemoto,
M Hosoya,
Y Habata,
R Fujii,
T Kakegawa,
M X Zou, Y Kawamata,
S Fukusumi,
S Hinuma,
C Kitada,
T Kurokawa,
H Onda,
M Fujino
[show abstract]
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ABSTRACT: In the search for an endogenous ligand of the orphan G protein-coupled receptor APJ, the presence of the ligand in various tissue extracts was examined by measuring the increase in extracellular acidification rate of the cells expressing the APJ receptor as a specific signal induced by the interaction of the receptor and ligand. By monitoring this activity, we isolated an APJ receptor ligand, designated apelin, from bovine stomach extracts. The structures of bovine and human apelin preproproteins were deduced from the sequences of the corresponding cDNAs. The preproproteins consisted of 77 amino acid residues, and the apelin sequence was encoded in the C-terminal regions. Synthetic peptides derived from the C-terminal amino acid sequence of bovine preproapelin were capable of specifically promoting the acidification rate in the cells expressing the APJ receptor in a range from 10(-7) to 10(-10) M, indicating that apelin is an endogenous ligand for the APJ receptor.
Biochemical and Biophysical Research Communications 11/1998; 251(2):471-6. · 2.48 Impact Factor
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S Hinuma,
Y Habata,
R Fujii, Y Kawamata,
M Hosoya,
S Fukusumi,
C Kitada,
Y Masuo,
T Asano,
H Matsumoto,
M Sekiguchi,
T Kurokawa,
O Nishimura,
H Onda,
M Fujino
[show abstract]
[hide abstract]
ABSTRACT: Hypothalamic peptide hormones regulate the secretion of most of the anterior pituitary hormones, that is, growth hormone, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone and adrenocorticotropin. These peptides do not regulate the secretion of prolactin, at least in a specific manner, however. The peptides act through specific receptors, which are referred to as seven-transmembrane-domain receptors or G-protein-coupled receptors. Although prolactin is important in pregnancy and lactation in mammals, and is involved in the development of the mammary glands and the promotion of milk synthesis, a specific prolactin-releasing hormone has remained unknown. Here we identify a potent candidate for such a hormone. We first proposed that there may still be unknown peptide hormone factors that control pituitary function through seven-transmembrane-domain receptors. We isolated the complementary DNA encoding an 'orphan' receptor (that is, one for which the ligand is unknown). This receptor, hGR3, is specifically expressed in the human pituitary. We then searched for the hGR3 ligand in the hypothalamus and identified a new peptide, which shares no sequence similarity with known peptides and proteins, as an endogenous ligand. We show that this ligand is a potent prolactin-releasing factor for rat anterior pituitary cells; we have therefore named this peptide prolactin-releasing peptide.
Nature 06/1998; 393(6682):272-6. · 36.28 Impact Factor
