Analysis of action mechanism of atrial matri-uretic peptide in perfused heart and tissue-cultured myocytes -in vivo analysis using a muti emission-reflexion analyzer and NMR-

心房泌尿肽在灌注心脏和组织培养的肌细胞中的作用机制分析-使用多发射反射分析仪和NMR进行体内分析-

• 批准号: 01480156
• 负责人: HATASE Osamu
• 金额: $ 0.9万
• 依托单位: Kagawa Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-01480156/
• 关键词: Atrial Natriuretic Peptide; Mitochondria; Perfused Rat Heart; CPAE Cells; Protein Phosphorylation; Calcium Ion; 心房性利尿ペプチド; 瞬間マルチ測光システム; 初代培養心筋細胞; 酸化防元反応; 酸化還元反応; 虚血心; 灌流心

The effect of rANP(rat atrial natriuretic peptide) on mitochondrial respiratory state in perfused rat heart.Purified mitochondria was used for the assay of oxidative phosphorylation using oxygraph. rANP at the concentration of 10ng/ml did not affect the respiratory function of heart mitochondria in vitro. Perfused rat heart with Langendorf apparatus was then used for the assay of mitochondrial respiratory function. A multi-light-emission-reflection monitoring system was used for this assay, and oxidation-reduction state of cytochromes c, al-a3, b, myoglobin was monitored through the experiment. rANP at 10ng/ml significantly prolonged the transient time from oxidative state to reduced state which was induced by ischemic condition.This indicates that rANP made heart cells more resistant to ischemic damage through the protection of mitochondrial respiratory function. Electro-microscopic study also supported these results.The effect of hANP(human atrial natriuretic peptide) on phosphorylation and intracellular concentration of calcium ion of CPAE cells derived from bovine pulmonary artery endothelium.Cultured CAPE cells were used for the phosphorylation assay under aerobic and anerobic condition using [32P orthophosphate in the culture medium. hANP at a concentration of 10ng/ml was added to the medium and the phosphorylation was analyzed and compared. hANP did not affect the phosphorylation under the aerobic condition. In contrast, hANP recovered to some extent the reduced phosphorylation which occurred under anerobic condition.The concentration of intracellular calcium ion([Ca i) was monitored by a calcium-specific fluorescent dye, Fura 2, and a calcium imaging analysis was performed. Under the anerobic condition, [Ca i raised by 30-50% as compared to that under aerobic condition. hANP(10ng/ml) reduced the incraese of [Ca i to 20-30%. hANP itself did not stimulate the increase of [Ca i.

rANP（大鼠心房钠尿肽）对灌注大鼠心脏线粒体呼吸状态的影响。纯化的线粒体用于氧描记仪测定氧化磷酸化。 10ng/ml浓度的rANP不影响体外心脏线粒体的呼吸功能。然后用兰根多夫装置灌注大鼠心脏来测定线粒体呼吸功能。本实验采用多重发光反射监测系统，通过实验监测细胞色素c、al-a3、b、肌红蛋白的氧化还原状态。 10ng/ml的rANP显着延长了缺血条件下从氧化态到还原态的瞬时时间。这表明rANP通过保护线粒体呼吸功能使心脏细胞更能抵抗缺血损伤。电镜研究也支持了这些结果。hANP（人心房钠尿肽）对牛肺动脉内皮来源的CPAE细胞磷酸化和细胞内钙离子浓度的影响。培养的CAPE细胞在有氧和无氧条件下使用[32P正磷酸盐在培养基中进行磷酸化测定。将浓度为10ng/ml的hANP添加到培养基中并分析和比较磷酸化。 hANP在有氧条件下不影响磷酸化。相比之下，hANP在一定程度上恢复了缺氧条件下发生的磷酸化减少。通过钙特异性荧光染料Fura 2监测细胞内钙离子（[Ca i）的浓度，并进行钙成像分析。无氧条件下[Ca i 比有氧条件下升高30-50%。 hANP(10ng/ml)使[Ca i 的增加降低至20-30%。 hANP 本身并不刺激[Ca i 的增加。

项目成果

Taizo Tasaka, Masaaki Tokuda, Teruhisa Taoka, Toshifumi Itano, Hideki Matsui, Seiji Etoh, Hajime Nishio, Osamu Miyamoto, Shozo Irino, Osamu Hatase: "Mechanism of transient increase in intracellular concentration of free calcium ions in HL-60 cell differen

Taizo Tasaka、Masaaki Tokuda、Teruhisa Taika、Toshifumi Itano、Hideki Matsui、Seiji Etoh、Hajime Nishio、Osamu Miyamoto、Shozo Irino、Osamu Hatase：“HL-60 细胞中游离钙离子浓度短暂增加的机制不同

Teruhisa Taoka, Masaaki Tokuda, Osamu Hatase, Shozo Irino, Anthiny W. Norman: "Induction of differentiation of HL60 cells by a protein kinase C inhibitor, K252a." Biochem. Biophys. Res. Commun.170. 1151-1156 (1990)

Teruhisa Taika、Masaaki Tokuda、Osamu Hatase、Shozo Irino、Anthiny W. Norman：“蛋白激酶 C 抑制剂 K252a 诱导 HL60 细胞分化。”

T.Taoka: "Induction of differentiation of HL60 cells by a protein kinase C inhibitor,K252a." Biochemical Biophysical Research Communication. 170. 1151-1156 (1990)

T.Taoka：“蛋白激酶 C 抑制剂 K252a 诱导 HL60 细胞分化。”

Seiji,Etoh et al.: "Isolation and sequence of novel rat testis cDNA coding for calcineurin βーsubunit,Ca^<2+>ーbinding regulatory subunit of a calmodulin stimulated protein phosphatase." Biophys.Biochem.Res.Commun.

Seiji, Etoh 等人：“编码钙调磷酸酶β-亚基、钙调蛋白刺激的蛋白磷酸酶的 Ca^2+-结合调节亚基的新型大鼠睾丸 cDNA 的分离和序列。”

H.Nishio: "The evidence for post-meiotic expression of a testis-specific isoform of a regulatory subunit of calcineurin using a monoclonal antibody." Biochemical Biophysical Research Communication. 187. 828-831 (1992)

H.Nishio：“使用单克隆抗体证明钙调神经磷酸酶调节亚基的睾丸特异性亚型在减数分裂后表达的证据。”