EXPRESSION OF TYPE III PHOSPHODIESTERASE IN NIH 3T3 CELLS

NIH 3T3 细胞中 III 型磷酸二酯酶的表达

• 批准号: 3757611
• 负责人: M J LEROY
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3757611
• 关键词: 3'5' cyclic nucleotide phosphodiesterase; 3T3 cells; complementary DNA; cyclic AMP; enzyme activity; gene expression; hormone regulation /control mechanism; laboratory rat; phosphodiesterase inhibitors; phosphorylation; plasmids; protein isoforms; recombinant proteins; second messengers; transfection

Cyclic AMP is an important second messenger involved in regulation of a number of physiological processes, including myocardial contractility, platelet aggregation and lipolysis. In adipose tissue, insulin is a physiologically potent and important inhibitor of lipolysis. Although the precise mechanism for its antilipolytic action is unknown, it has been shown that incubation of rat adipocytes with insulin results in phosphorylation and activation of the type III cGI PDE. In cardiac tissue, specific inhibition of cGI PDE is a primary mechanism of action of some positive inotropic agents. In order to further our understanding of the molecular mechanisms involved in regulation of cGI PDEs, two cDNAs have been recently cloned from rat adipocyte (RcGIP1 cDNA) and human cardiac (HcGIP2 cDNA) cDNA libraries. We have expressed these two cDNAs in NIH-3006 fibroblasts and characterized both recombinant enzymes. Several stable transfectants for both recombinant proteins that exhibited elevated cAMP hydrolytic activity were isolated and amplified. Both enzymes displayed the expected characteristics for cGI PDEs; molecular mass, high affinity for cAMP (Km), sensitivity to the selective inhibitor OPC 3689 and to cGMP. Furthermore, recombinant HcGIP2 PDE reacted more strongly with the anti-platelet cGI PDE antibody and RcGIP1 PDE was recognized to a greater extent by the anti-peptide antibodies raised against peptides of the regulatory and the "additional region" of the catalytic domain of the RcGIP1 PDE sequence. Our results strongly suggest that recombinant RcGIP1 and HcGIP2 PDEs represent two similar cGI PDE isoforms and are consistent with the previous observation that both cDNAs are product of different but related genes (Taira et al., 1993, J. Biol. Chem.268: 18573-9).

环磷酸腺苷是一种重要的第二信使， 许多生理过程，包括心肌收缩力， 血小板聚集和脂解。 在脂肪组织中，胰岛素是 生理上有效的和重要的脂解抑制剂。 虽然 其抗脂肪分解作用精确机制尚不清楚， 已经表明，用胰岛素孵育大鼠脂肪细胞， III型cGI PDE的磷酸化和活化。 心脏 组织特异性抑制cGI PDE是主要作用机制 一些正性肌力药物 为了加深我们对分子机制的理解 最近克隆了两种cDNA，它们参与cGI PDE的调节 从大鼠脂肪细胞（RcGIP 1 cDNA）和人心脏（HcGIP 2 cDNA）cDNA 图书馆. 我们已经在NIH-3006成纤维细胞中表达了这两种cDNA， 对两种重组酶进行了表征。 两种重组蛋白的几种稳定转染子表现出 升高的cAMP水解活性被分离和扩增。 两 酶显示cGI PDE的预期特征;分子 质量，对cAMP的高亲和力（Km），对选择性抑制剂的敏感性 OPC 3689和cGMP。 此外，重组HcGIP 2 PDE反应更多， 与抗血小板cGI PDE抗体和RcGIP 1 PDE强烈相关， 在更大程度上被所产生的抗肽抗体识别 针对调节肽和调节肽的“额外区域”， RcGIP 1 PDE序列的催化结构域。 我们的研究结果强烈表明，重组RcGIP 1和HcGIP 2 PDE 代表两种相似的cGI PDE亚型，并与 先前的观察表明，这两种cDNA是不同但相关的产物， 基因（Taira等人，268：18573-9）。