EXPRESSION OF TYPE III PHOSPHODIESTERASE IN NIH 3T3 CELLS

环磷酸腺苷是一种重要的第二信使，参与调节 生理过程的数量，包括心肌收缩能力， 血小板聚集和脂解。在脂肪组织中，胰岛素是一种 生理上的强效和重要的脂解抑制剂。虽然 它的抗脂作用的确切机制尚不清楚，它已经 研究表明，大鼠脂肪细胞与胰岛素孵育会导致 III型CGI PDE的磷酸化和激活。在心脏 组织中，CGI PDE的特异性抑制是其主要作用机制 一些正性肌力药。 为了加深我们对分子机制的理解 最近克隆了两个参与CGI PDE调控的cDNA 来自大鼠脂肪细胞(RcGIP1)和人心脏(HcGIP2)的cDNA 图书馆。我们已经在NIH-3006成纤维细胞和 表征了这两种重组酶的特性。 两种重组蛋白的几种稳定的转染体 分离并扩增了高cAMP水解酶活性。两者都有 酶表现出CGI PDE的预期特征；分子 质量大，对cAMP(Km)亲和力高，对选择性抑制剂敏感 OPC 3689和cGMP。此外，重组HcGIP2 PDE的反应性更强 强与抗血小板CGI PDE抗体和RcGIP1 PDE是 在更大程度上被识别的抗肽抗体提出 针对受调控的多肽和 RcGIP1 PDE序列的催化结构域。 我们的结果有力地表明，重组RcGIP1和HcGIP2 PDE 代表两种相似的CGI PDE异构体，并与 先前的观察表明，这两个DNA是不同但相关的产物 基因(Taira等人，1993，J.Biol.化学268：18573-9)。

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).