EXPRESSION OF TYPE III PHOSPHODIESTERASE IN NIH 3T3 CELLS

环AMP是参与a调控的重要第二信使

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).