Systematic search on a smooth muscle stimulant peptide in mammalian kidney.

哺乳动物肾脏中平滑肌刺激肽的系统搜索。

• 批准号: 04454566
• 负责人: KANGAWA Kenji
• 金额: $ 4.1万
• 依托单位: NATIONAL CARDIOVASCULAR CENTER RESERCH INSTITUTE (1993)宮崎医科大学 (1992)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04454566/
• 关键词: Smooth muscle stimulant peptide; Mammalian kidney; Human monocytic cell line (THP-1); Human CNP-29; Human pheochromocytoma; Rat platelet cAMP; Adrenomedullin; Hypotensive peptide; ラット血小板; cAMP; cDNAクローニング; pro AM-N20; 降圧活性ペプチド; ブタ腎臓; ヒト単球由来白

In a survey for unknown bioactive peptides in mammalian cell lines, we detected the most potent chick rectum relaxant activity in the culture medium of phorbol ester-treated human monocytic cell line, THP-1. Thus, we started purification from the culture medium of THP-1 and isolated four peptides exhibiting strong relaxant effects on chick rectum. By microsequence analysis, amino acid sequences of these peptides were deduced to be a series of human C-type natriuretic peptides (CNPs) with several amino acid elongations from the N-terminus of CNP-22. Furthermore, CNP mRNA was detected in the THP-1 cells, and expression of CNP gene and CNP concentration in the culture medium were shown to be greatly increased by the stimulation of phorbol ester.The demonstration of CNP production in THP-1 monocytic cell line suggests that CNP also functions as a local chemical mediator in the immune and vascular system, although CNP has been recognized to function as a neuropeptide exclusively in the cent … More ral nervous system.We have also been searching for peptides which may be relevant to circulation control, using an assay system which monitors the elevating activty of platelet cAMP.In the present study, we discovered a novel hypotensive peptide in human pheochromocytoma extract. Since this peptide is abundant in normal adrenal medulla as well as in pheochoromocytoma tissue arising from adrenal medulla, it was designated "adrenomedullin". The peptide, consisting of 52 amino acids, has one intramolecular disulfide bond and shows slight homology with calcitonin gene related peptide. It was found to elicit a potent and long lasting hypotensive effect in anesthetized rat.Since adrenomedullin was abundant in pheochromocytoma tissue, the cDNA library of pheochromocytoma was constructed, and the cDNA clone concoding an adrenomedullin precursor was isolated and sequencd. The precursor for human adrenomedullin (human preproadrenomedullin) is 185 amino acids in length, including an adrenomedullin sequence, followed by a glycine (the amide donor). Proadrenomedullin (proAM) contains a unique twenty amino acid sequence followed by Gly-Lys-Arg in the N- terminal region. It is possible that a novel 20 residues peptide "proadrenomedullin N- terminal 20 peptide" (proAM-N20) with an amidated carboxy teminus is processed from the precursor. By RNA blot anaylysis, human adrenomedullin mRNA was found to be highly expressed in several tissues including adrenal medulla, ventricle, lung and kidney as well as pheochromocytoma.Adrenomedullin was widely distributed in several tissues and circulates in blood in a considerable concentration. These data suggest that adrenomedullin is a new hormone participating in circulation control. Occurrence of adrenomedullin indicates the possible existence of a novel system for circulation control. Less

在对哺乳动物细胞系中未知生物活性肽的调查中，我们在佛波酯处理的人单核细胞系 THP-1 的培养基中检测到最有效的小鸡直肠松弛活性。因此，我们开始从THP-1的培养基中进行纯化，并分离出四种对鸡直肠表现出强烈松弛作用的肽。通过微序列分析，这些肽的氨基酸序列被推断为一系列人C型利尿钠肽(CNP)，并且从CNP-22的N末端有多个氨基酸延伸。此外，在THP-1细胞中检测到CNP mRNA，并且通过佛波酯的刺激，CNP基因的表达和培养基中的CNP浓度显着增加。THP-1单核细胞系中CNP产生的证明表明，尽管CNP已被认为作为神经肽发挥作用，但CNP还在免疫和血管系统中充当局部化学介质。 专门存在于中枢神经系统中。我们还一直在使用监测血小板 cAMP 升高活性的测定系统来寻找可能与循环控制相关的肽。在本研究中，我们在人嗜铬细胞瘤提取物中发现了一种新型降血压肽。由于该肽在正常肾上腺髓质以及由肾上腺髓质产生的嗜铬细胞瘤组织中大量存在，因此将其命名为“肾上腺髓质素”。该肽由52个氨基酸组成，具有1个分子内二硫键，与降钙素基因相关肽有轻微同源性。由于嗜铬细胞瘤组织中富含肾上腺髓质素，因此构建了嗜铬细胞瘤的cDNA文库，分离并测序了编码肾上腺髓质素前体的cDNA克隆。人肾上腺髓质素的前体（人前肾上腺髓质素原）长度为 185 个氨基酸，包括肾上腺髓质素序列，后面是甘氨酸（酰胺供体）。肾上腺髓质素原 (proAM) 包含独特的 20 个氨基酸序列，其后的 N 端区域为 Gly-Lys-Arg。具有酰胺化羧基末端的新型 20 残基肽“肾上腺髓质素原 N 端 20 肽”(proAM-N20) 可能是由前体加工而成。通过RNA印迹分析，发现人肾上腺髓质素mRNA在肾上腺髓质、心室、肺、肾以及嗜铬细胞瘤等多种组织中高表达。肾上腺髓质素广泛分布于多种组织中，并以相当浓度在血液循环中。这些数据表明肾上腺髓质素是一种参与循环控制的新激素。肾上腺髓质素的出现表明可能存在一种新的循环控制系统。较少的

项目成果

K.Kitamura, K.Kangawa, M.Kojima, Y.Ichiki, H.Matsuo and T.Eto: "Complete amino acid sequence of porcine adrenomedullin and cloning of cDNA encoding its precursor." FEBS Lett.338. 306-310 (1994)

K.Kitamura、K.Kangawa、M.Kojima、Y.Ichiki、H.Matsuo 和 T.Eto：“猪肾上腺髓质素的完整氨基酸序列和编码其前体的 cDNA 克隆。”

矢崎 義雄編 (分担)児島 将康,寒川 賢治 他: "循環調節ペプチドと関連疾患 (分担)ナトリウム利尿ペプチドファミリー(ANP・BNP・CNP)の構造と発現" 羊土社, 167 (1992)

由Yoshio Yazaki（撰稿人）Masayasu Kojima、Kenji Samukawa 等编辑：“循环调节肽和相关疾病（撰稿人）利钠肽家族（ANP、BNP、CNP）的结构和表达” Yodosha，167（1992）

K.Kitamura, K.Kangawa, M.Kawamoto, Y.Ichiki, H.Matsuo and T.Eto: "Isolation and characterization of peptides which act on rat platelets, from a pheochromocytoma." Biochem.Biophys.Res.Commun.185. 134-141 (1992)

K.Kitamura、K.Kangawa、M.Kawamoto、Y.Ichiki、H.Matsuo 和 T.Eto：“从嗜铬细胞瘤中分离和表征作用于大鼠血小板的肽。”

H.Tateyama, J.Hino, N.Minamino, K.kangawa, T.Minamino, K.Sakai, T.Ogihara and H.Matsuo: "Concentrations and molecular forms of human brain natriuretic peptide in plasma." Biochem.Biophys.Res.Commun.185. 760-767 (1992)

H.Tateyama、J.Hino、N.Minamino、K.kankawa、T.Minamino、K.Sakai、T.Ogihara 和 H.Matsuo：“血浆中人脑钠尿肽的浓度和分子形式”。

E.Suzuki, Y.Hirata, H.Hayakawa, M.Omata, M.Kojima, K.Kangawa, N.Minamino, and H.Matsuo: "Evidence for C-type nautriuretic peptide production in the rat kidney." Biochem.Biophys.Res.Commun.192. 532-538 (1993)

E.Suzuki、Y.Hirata、H.Hayakawa、M.Omata、M.Kojima、K.Kankawa、N.Minamino 和 H.Matsuo：“大鼠肾脏中产生 C 型利钠肽的证据”。