論文 - 北村 和雄
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Mitsuda Y., Takimoto A., Kamitani S., Kitamura K., Sakata T., Mitsushima K.
Protein Expression and Purification 25 ( 3 ) 448 - 455 2002年11月
掲載種別：研究論文（学術雑誌） 出版者・発行元：Protein Expression and Purification
Human adrenomedullin (hAM) is a 52-amino-acid regulatory peptide containing a six-membered ring structure and an amidated C-terminus, features that are essential for its biological activity. Here, we describe a simple and effective protocol for producing large quantities of highly pure, functional recombinant hAM. A peptide precursor (hAM-Gly) was expressed in Escherichia coli as a fusion protein with thioredoxin and collected as inclusion bodies. The fusion protein was then digested with BLase, a glutamate-specific endopeptidase, to prepare hAM-Gly. The essential ring structure formed spontaneously, while the terminal amide was generated by conversion of the added glycine residue using peptidylglycine α-amidating enzyme. The low solubility of hAM-Gly enabled the use of a selective precipitation/extraction method to generate a product that was 80-90% pure, which was sufficient to proceed with the α-amidating enzyme reaction. The resultant hAM was then purified further by column chromatography. The final yield was about 82 mg/L of bacterial culture, and the purity, determined by reverse phase HPLC, was >99:5%. The recombinant hAM was biologically active, eliciting concentration-dependent increases in cAMP in CHO-K1 cells expressing a specific hAM receptor and hypotensive responses when intravenously injected into rats. This new approach to the synthesis of hAM is simpler and more cost-effective for large-scale production than chemical synthesis. It therefore represents a new powerful tool that has the potential to facilitate analysis of the structure and function of hAM, as well as the development of new therapeutic protocols for the treatment of ailments such as hypertension. © 2002 Elsevier Science (USA). All rights reserved.
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Kato J., Kitamura K., Uemura T., Kuwasako K., Kita T., Kangawa K., Eto T.
Hypertension Research 25 ( 6 ) 887 - 892 2002年11月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Hypertension Research
Adrenomedullin (AM) and atrial and brain natriuretic peptides (ANP and BNP) exert vasodilator and natriuretic actions and are thought to share roles in counteracting the progression of hypertension or heart failure as circulating or locally-acting hormones. However, little data is available with regard to their roles in subjects who have no apparent cardiovascular diseases. The present study was carried out to identify the factors that affect plasma levels of AM, ANP and BNP in the general population. We measured the plasma levels of AM, ANP and BNP in 184 local residents who had a scheduled regular health checkup, and compared the findings with those for other clinical parameters. Univariate analyses showed that the plasma levels of AM, ANP and BNP were significantly correlated with age. The plasma levels of ANP and BNP were also significantly correlated with systolic blood pressure (SBP) and with pulse pressure (PP), an indicator of the stiffness of the great vessels. Multivariate analyses conducted using a stepwise method revealed that age was a significant, independent variable for the plasma levels of AM, ANP and BNP. In addition, PP was a significant factor for the plasma levels of ANP and BNP, while the plasma AM was significantly associated with body mass index (BMI). Thus, the plasma levels of AM, ANP and BNP all increased in association with aging, and those of ANP and BNP increased in association with PP, suggesting possible relationships between the plasma levels and age-related changes in the cardiovascular system.
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Nagoshi Y., Kuwasako K., Ito K., Uemura T., Kato J., Kitamura K., Eto T.
European Journal of Pharmacology 450 ( 3 ) 237 - 243 2002年8月
掲載種別：研究論文（学術雑誌） 出版者・発行元：European Journal of Pharmacology
The receptor activity-modifying protein (RAMP)/calcitonin receptor-like (CRL) receptor heterodimer is thought to function as a receptor for either a calcitonin gene-related peptide (CGRP) (CRL receptor/RAMP1) or adrenomedullin (CRL receptor/RAMP2 or -3), depending on the RAMP isoform present. We examined the receptor specificity of adrenomedullin-induced increases in cAMP in human embryonic kidney (HEK)293 cells coexpressing human CRL receptor and human RAMP1 or RAMP2. In cells expressing CRL receptor/RAMP1, adrenomedulin-induced increases in cAMP were comparable to those induced by α-CGRP, and the CGRP receptor antagonist α-CGRP-(8-37), but not the adrenomedullin receptor antagonist adrenomedullin-(22-52), blocked the adrenomedullin-evoked responses. Cells expressing CRL receptor/RAMP2 responded more selectively to adrenomedullin; in this case, the effect was blocked by adrenomedullin-(22-52) but not by α-CGRP-(8-37). Real-time quantitative polymerase chain reaction confirmed that cotransfection of CRL receptor and RAMP1 had no effect on the endogenous expression of RAMP2. Thus, CRL receptor/RAMP1 likely functions as an adrenomedullin receptor as well as a CGRP receptor, which may explain why many of the actions of adrenomedullin are potently antagonized by α-CGRP-(8-37). © 2002 Elsevier Science B.V. All rights reserved.
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Ishiyama Y., Kitamura K., Eto T.
Biochemical and Biophysical Research Communications 293 ( 2 ) 741 - 746 2002年7月
掲載種別：研究論文（学術雑誌） 出版者・発行元：Biochemical and Biophysical Research Communications
Using a novel method employing a V8 protease digestion coupled with ethyl acetate extraction, we have purified a peptide with C-terminal amide structure from porcine cardiac atrium. The peptide was determined to be Ala-Val-Leu-Gly-Leu-CONH2. According to the sequence, we have raised an antibody and established the radioimmunoassay. Using this radioimmunoassay, we have isolated a novel 14 amino acid peptide where C-terminus was amidated. This peptide was termed amidicin. Amidicin is widely distributed in porcine tissue and is especially abundant in pituitary gland, cardiac ventricle, and spleen. © 2002 Elsevier Science (USA). All rights reserved.
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Expression and immunohistochemical localization of adrenomedullin in the mouse cochlea 査読あり
Tono T., Shimozono M., Kawano H., Asada Y., Kitamura K., Komune S.
ORL 64 ( 3 ) 169 - 172 2002年6月
掲載種別：研究論文（学術雑誌） 出版者・発行元：ORL
Adrenomedullin (AM) is a novel hypotensive and vasodilator peptide. It has been shown that AM is biosynthesized in various organs and cells and is suggested to play multiple roles including electrolyte homeostasis and body fluid control. The present study describes for the first time the presence of mRNA for AM and AM-like immunoreactivity in the cochlea. A reverse transcription-polymerase chain reaction (RT-PCR) performed with gene-specific AM primer on total RNA extracted from mouse cochlea revealed a PCR product of the expected size. Immunohistochemical examination showed positive immunostaining in vascular smooth muscle cells of the modiolar arterioles and in the stria vascularis of the cochlea. These results suggest that AM may play a role in the regulation not only of cochlear hemodynamics but also of cochlear fluid dynamics. Copyright © 2002 S. Karger AG, Basel.
DOI： 10.1159/000058020
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Uemura T., Kato J., Kuwasako K., Kitamura K., Kangawa K., Eto T.
Journal of Hypertension 20 ( 6 ) 1209 - 1214 2002年6月
掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Hypertension
Objective: Both adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP), processed from the same precursor of prepro-adrenomedullin (preproAM), have differential biological properties; AM dilates blood vessels and presumably affects the vascular remodeling, while PAMP inhibits catecholamine secretion. Since aldosterone has been shown to be involved in vascular remodeling, we examined the effects of aldosterone on AM and PAMP secretion and preproAM gene expression in human aortic vascular smooth muscle cells (VSMC). Methods: AM and PAMP secreted from human VSMC incubated with aldosterone were measured by radioimmunoassay, and preproAM gene expression was evaluated by quantitative polymerase chain reaction. Results: Cultured human VSMC secreted both AM and PAMP into the media, while the secretion rate of AM was much higher than that of PAMP. Aldosterone increased preproAM gene expression in the cultured VSMC in a dose-dependent fashion following incubation for 48 h, with a concomitant increase in AM secretion from the cells, but PAMP secretion remained unchanged. Aldosterone-stimulated AM secretion was significantly reduced by spironolactone. Reverse-phase high-performance liquid chromatography analyses showed that immunoreactive AM secreted from the VSMC untreated or treated with aldosterone emerged at the point of human AM(1-52)-NH2. Conclusions: AM production was stimulated by aldosterone in cultured human VSMC without an increase in PAMP secretion, suggesting a possible role of AM in modulating vascular remodeling by aldosterone. © 2002 Lippincott Williams & Wilkins.
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Rat RAMP domains involved in adrenomedullin binding specificity 査読あり
Kuwasako K., Kitamura K., Onitsuka H., Uemura T., Nagoshi Y., Kato J., Eto T.
FEBS Letters 519 ( 1-3 ) 113 - 116 2002年5月
掲載種別：研究論文（学術雑誌） 出版者・発行元：FEBS Letters
When coexpressed with receptor activity-modifying protein (RAMP)2 or -3, calcitonin receptor-like receptor (CRLR) functions as an adrenomedullin (AM) receptor (CRLR/RAMP2 or -3). Coexpression of rat (r)CRLR with rRAMP deletion mutants in HEK293T cells revealed that deletion of residues 93-99 from rRAMP2 or residues 58-64 from rRAMP3 significantly inhibits high-affinity [125I]AM binding and AM-evoked cAMP production, despite full cell surface expression of the receptor heterodimer. Apparently, these two seven-residue segments are key determinants of high-affinity agonist binding to rAM receptors and of receptor functionality. Consequently, their deletion yields peptides that are able to serve as negative regulators of AM receptor function. © 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
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Hirano S., Imamura T., Onitsuka H., Matsuo T., Kitamura K., Koiwaya Y., Eto T.
Circulation Journal 66 ( 4 ) 397 - 402 2002年5月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Circulation Journal
To determine whether acute pressure overload (POL) can stimulate adrenomedullin (AM) production, the response of ventricular AM gene expression and plasma AM concentration to aortic banding was investigated in the rat. Furthermore, any link between AM expression and the renin-angiotensin system (RAS) enhanced by acute POL was examined using: a Ca channel blocker (manidipine), an angiotensin II type I receptor antagonist (candesartan), and an angiotensin-converting enzyme inhibitor (quinapril). Rats with acute POL produced by suprarenal aortic banding were studied 1, 5 and 14 days after surgery. Plasma AM concentrations in banded rats at day 1 increased 1.49-fold (p<0.01), then gradually declined to near the control level at day 14. Plasma AM concentrations correlated with plasma renin activity (PRA) (p<0.001). Adrenomedullin mRNA expression in the left ventricle (LV) increased 1.35-fold (p<0.05) at day 1. This increase was not significant at either 5 or 14 days after surgery. Adrenomedullin mRNA expression in the right ventricle on days 1 and 5 increased by 1.46-fold (p<0.05) and 1.52-fold (p<0.05), respectively. Candesartan, quinapril and manidipine reduced systolic blood pressure equally and activated PRA at day 1. However, augmented LV AM gene expression was suppressed completely by candesartan and quinapril, but remained unaffected by manidipine. In conclusion, POL induces a rapid increase in cardiac AM gene expression and in plasma AM concentrations. Cardiac AM transcription could therefore be partly regulated by RAS in suprarenal aortic banding rats.
DOI： 10.1253/circj.66.397
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Recovery process of arthritis induced by 6-sulfanilamidoindazole (6SAI) in rats 査読あり
Y Ohmachi 1, H Fujimura, E Otsuka, T Miyazaki, W Toriumi, K Kitamura, K Doi
Histol Histopathol 17 ( 2 ) 437 - 444 2002年4月
掲載種別：研究論文（学術雑誌）
DOI： 10.14670/HH-17.437
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Adrenomedullin and PAMP: Discovery, structures, and cardiovascular functions 査読あり
Kitamura K., Kangawa K., Eto T.
Microscopy Research and Technique 57 ( 1 ) 3 - 13 2002年4月
掲載種別：研究論文（学術雑誌） 出版者・発行元：Microscopy Research and Technique
We discovered adrenomedullin (AM) from human pheochromocytoma tissue by monitoring the elevating activity of intracellular cyclic AMP (cAMP) in rat platelets in 1993. Since the discovery of AM, it has attracted intense interest from cardiovascular researchers because AM elicits multiple biological activities, including a potent and powerful hypotensive activity caused by dilatation of resistance vessels. AM is biosynthesized and secreted from tissues, including cardiovascular organs. In addition to AM, "proadrenomedullin N-terminal 20 peptide (PAMP)," another biologically active peptide, was found to be processed from the AM precursor. Plasma AM levels are increased in various cardiovascular and renal diseases. AM, therefore, seems to function as a novel system that controls circulation and body fluid, and may be involved in pathophysiological changes in cardiovascular diseases. Therefore, in this review we will focus on the structure of AM and its gene, distribution, receptor, and the physiological and pathological roles of AM in cardiovascular disease. © 2002 Wiley-Liss, Inc.
DOI： 10.1002/jemt.10052
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Adrenomedullin, an endogenous peptide, counteracts cardiovascular damage 査読あり
Shimosawa T., Shibagaki Y., Ishibashi K., Kitamura K., Kangawa K., Kato S., Ando K., Fujita T.
Circulation 105 ( 1 ) 106 - 111 2002年1月
掲載種別：研究論文（学術雑誌） 出版者・発行元：Circulation
Background - Adrenomedullin (AM), a potent vasodilator peptide, is produced by posttranslational splicing of proadrenomedullin together with proadrenomedullin N-terminal 20 peptide (PAMP), another hypotensive peptide. Although both AM and PAMP have the potential not only to decrease blood pressure but also to protect organs from damage, there is no direct evidence for their individual physiological roles in vivo. Methods and Results - Using knockout mice with the disruption of AM peptide alone, we investigated the organ-protective effect of AM. Although the AM-/- mutation in mice was embryonic lethal without any apparent phenotypic changes, AM+/- mice were viable and fertile; plasma and organ AM concentrations were almost half of those in AM+/+ mice. With the administration of angiotensin II (Ang II) on a high-salt diet for 12 days, marked perivascular fibrosis and intimal hyperplasia were found in coronary arteries of Ang II/salt-treated AM+/- mice, without the AM upregulation that was observed in Ang II/salt-treated AM+/+ mice. In AM+/- mice, Ang II/salt loading increased both urinary excretion of 8-hydroxydeoxyguanosine and isoprostane, markers of oxidative stress. Consistently, immunostaining of both p67phox and gp91phox, subunits of NAD(P)H oxidase and 3-nitrotyrosine, the metabolites of reactive oxygen species (ROS), and the generation of ROS measured by electron spin resonance spectroscopy apparently increased in the Ang II/salt-treated heart. These data suggested that the overproduction of oxidative stress might be involved in the cardiovascular changes induced by Ang II/salt loading. Conclusions - The evidence presented supports the hypothesis that endogenous AM possesses a protective action against cardiovascular damage, possibly through the inhibition of oxidative stress production.
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Yuchi H., Suganuma T., Sawaguchi A., Ide S., Kawano J.I., Aoki T., Kitamura K., Eto T.
Histochemistry and Cell Biology 118 ( 3 ) 259 - 265 2002年
掲載種別：研究論文（学術雑誌） 出版者・発行元：Histochemistry and Cell Biology
We reappraised the precise immunohistochemical localization of adrenomedullin (AM) by means of the combined use of the catalyzed signal amplification (CSA) system and plunge freezing (PF)/freeze substitution (FS) for light microscopy or high-pressure freezing (HPF)/FS for electron microscopy, focusing on the rat adrenal gland and heart. In the case of adrenal glands, the PF processing showed that almost all medullary cells were intensively immunoreactive, while the cortical cells showed weak immunoreaction. In the heart, almost all cardiac muscle cells of the atria were also vividly stained with the PF/FS and the CSA enhancement. On the contrary, traces of immunoreactions were seen in most of the ventricular cells. These results are consistent with the previous reports of AM radioimmunoassays and the expression of AM mRNA. However, the chemical fixation processing revealed heterogeneous immunostaining in the atrial and ventricular myocardium as well as the adrenal medulla. Intensity of the immunostaining in the chemically fixed tissues was not likely to correspond with that of AM radioimmunoassays. The HPF/FS processing clearly demonstrated the immunogold labeling on secretory granules of adrenal medullary cells as well as cardiac muscle cells of the right auricles. Immunogold labeling intensity of the cryofixed specimens was 3- to 25-fold higher than that of the chemically fixed ones.
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アドレノメデュリン中枢投与による昇圧反応における交感神経系の関与および心血管反射調節系としての延髄最後野の役割 査読あり
斉田光彦、北村和雄、江藤胤尚、河南洋
日本内分泌学会雑誌 78 ( 2 ) 454 - 454 2002年
掲載種別：研究論文（学術雑誌）
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培養骨芽細胞, 軟骨細胞におけるアドレノメデュリンの成長因子としての作用 査読あり
濱田浩朗、北村和雄、田島直也、帖佐悦男
日本内分泌学会雑誌 78 ( 2 ) 452 - 452 2002年
掲載種別：研究論文（学術雑誌）
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CRLR/RAMP1複合体はCGRP-(8-37)に感受性の高いアドレノメデュリン受容体として機能する 査読あり
名越康子、桑迫健二、加藤丈司、北村和雄、江藤胤尚
日本内分泌学会雑誌 78 ( 2 ) 452 - 452 2002年
掲載種別：研究論文（学術雑誌）
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アドレノメデュリンと心血管病 査読あり
北村和雄、北俊弘、加藤丈司、江藤胤尚
日本内分泌学会雑誌 78 ( 2 ) 444 - 444 2002年
掲載種別：研究論文（学術雑誌）
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慢性糸球体腎炎患者における血漿・尿中Adrenomedullin(AM),Proadrenomedullin N-temina120peptide(PAMP)の動態 査読あり
藤元昭一、木下浩、戸倉健、北村和雄、江藤胤尚
Therapeutic Research 23 ( 5 ) 799 - 804 2002年
掲載種別：研究論文（学術雑誌）
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心筋梗塞後リモデリングに対するアドレノメデュリン長期投与の有効性 査読あり
中村亮斉、加藤丈司、北村和雄、鬼塚久充、今村卓郎、寒川賢治、江藤胤尚
日本内分泌学会雑誌 78 ( 2 ) 451 - 451 2002年
掲載種別：研究論文（学術雑誌）
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高食塩食によるラットアドレノメデュリンとその受容体遺伝子発現の上昇 査読あり
曹遠寧、北村和雄、加藤丈司、桑迫健二、江藤胤尚
日本内分泌学会雑誌 78 ( 2 ) 452 - 452 2002年
掲載種別：研究論文（学術雑誌）
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Kuwasako K., Kitamura K., Ito K., Uemura T., Yanagita Y., Kato J., Sakata T., Eto T.
Journal of Biological Chemistry 276 ( 52 ) 49459 - 49465 2001年12月
掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Biological Chemistry
When co-expressed with a receptor activity-modifying protein (RAMP) accessory protein, calcitonin receptor-like receptor (CRLR) can function as a calcitonin gene-related peptide receptor (CRLR-RAMP1) or an adrenomedullin (AM) receptor (CRLR-RAMP2/3). Here we report on the structural domain(s) involved in selective AM binding that were examined using various RAMP chimeras and deletion mutants. Co-expression of chimeric RAMPs and CRLR in HEK293 cells revealed that residues 77-101, situated in the extracellular N-terminal domain of human RAMP2 (hRAMP2), were crucial for selective AM-evoked cAMP production. More detailed analysis showed that deletion of hRAMP2 residues 86-92 significantly attenuated high-affinity 125I-AM binding and AM-evoked cAMP production despite full cell surface expression of the receptor heterodimer and that deletion of hRAMP3 residues 59-65 had a similar effect. There is little sequence identity between hRAMP3 residues 59-65 and hRAMP2 residues 86-92; moreover, substituting alanine for Trp86 (Ala 87), Met88, Ile89, Ser90, Arg 91, or Pro92 of hRAMP2 had no effect on AM-evoked cAMP production. It thus seems unlikely that any one amino acid residue is responsible for determining selective AM binding or that AM binds directly to these peptide segments. Instead these findings suggest that the respective seven-amino acid sequences confer selectivity either by directly contributing to the structure of ligand binding pocket or by allosteric modulation of the conformation of CRLR.