Dunkern Torsten R, Hatzelmann Armin
Biochemistry 2 Inflammation, ALTANA Pharma AG, Member of the Nycomed Group, Konstanz, Germany.
FEBS J. 2007 Sep;274(18):4812-24. doi: 10.1111/j.1742-4658.2007.06001.x. Epub 2007 Aug 14.
Different inhibitors of the Ca(2+)/calmodulin-stimulated phosphodiesterase 1 family have been described and used for the examination of phosphodiesterase 1 in cellular, organ or animal models. However, the inhibitors described differ in potency and selectivity for the different phosphodiesterase family enzymes, and in part exhibit additional pharmacodynamic actions. In this study, we demonstrate that phosphodiesterase 1C is expressed in the human glioblastoma cell line A172 with regard to mRNA, protein and activity level, and that lower activities of phosphodiesterase 2, phosphodiesterase 3, phosphodiesterase 4 and phosphodiesterase 5 are also present. The identity of the phosphodiesterase 1C activity detected was verified by downregulation of the mRNA and protein through human phosphodiesterase 1C specific small interfering RNA. In addition, the measured K(m) values (cAMP, 1.7 microm; cGMP, 1.3 microm) are characteristic of phosphodiesterase 1C. We demonstrate that treatment with the Ca(2+) ionophore ionomycin increases intracellular Ca(2+) in a concentration-dependent way without affecting cell viability. Under conditions of enhanced intracellular Ca(2+) concentration, a rapid increase in cAMP levels caused by the adenylyl cyclase activator forskolin was abolished, indicating the involvement of Ca(2+)-activated phosphodiesterase 1C. The reduction of forskolin-stimulated cAMP levels was reversed by phosphodiesterase 1 inhibitors in a concentration-dependent way. Using this cellular system, we compared the cellular potency of published phosphodiesterase 1 inhibitors, including 8-methoxymethyl-3-isobutyl-1-methylxanthine, vinpocetine, SCH51866, and two established phosphodiesterase 1 inhibitors developed by Schering-Plough (named compounds 31 and 30). We demonstrate that up to 10 microm 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine had no effect on the reduction of forskolin-stimulated cAMP levels by ionomycin, whereas the more selective and up to 10 000 times more potent phosphodiesterase 1 inhibitors SCH51866, compound 31 and compound 30 inhibited the ionomycin-induced decline of forskolin-induced cAMP at nanomolar concentrations. Thus, our data indicate that SCH51866 and compounds 31 and 30 are effective phosphodiesterase 1 inhibitors in a cellular context, in contrast to the weakly selective and low-potency phosphodiesterase inhibitors 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine. A172 cells therefore represent a suitable system in which to study the cellular effect of phosphodiesterase 1 inhibitors. 8-Methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine seem not to be suitable for the study of phosphodiesterase 1-mediated functions.
已有多种不同的钙（2+）/钙调蛋白刺激的磷酸二酯酶1家族抑制剂被报道，并用于在细胞、器官或动物模型中检测磷酸二酯酶1。然而，所描述的抑制剂对不同磷酸二酯酶家族酶的效力和选择性不同，并且部分还表现出其他药效学作用。在本研究中，我们证明磷酸二酯酶1C在人胶质母细胞瘤细胞系A172中在mRNA、蛋白质和活性水平方面均有表达，并且磷酸二酯酶2、磷酸二酯酶3、磷酸二酯酶4和磷酸二酯酶5也有较低活性。通过人磷酸二酯酶1C特异性小干扰RNA下调mRNA和蛋白质，验证了所检测到的磷酸二酯酶1C活性的一致性。此外，所测得的米氏常数（cAMP为1.7微摩尔；cGMP为1.3微摩尔）是磷酸二酯酶1C的特征。我们证明用钙（2+）离子载体离子霉素处理以浓度依赖的方式增加细胞内钙（2+），而不影响细胞活力。在细胞内钙（2+）浓度升高的条件下，由腺苷酸环化酶激活剂福司可林引起的cAMP水平的快速升高被消除，表明钙（2+）激活的磷酸二酯酶1C参与其中。磷酸二酯酶1抑制剂以浓度依赖的方式逆转了福司可林刺激的cAMP水平的降低。利用这个细胞系统，我们比较了已发表的磷酸二酯酶1抑制剂的细胞效力，包括8-甲氧基甲基-3-异丁基-1-甲基黄嘌呤、长春西汀、SCH51866，以及先灵葆雅公司开发的两种已确立的磷酸二酯酶1抑制剂（命名为化合物31和30）。我们证明高达10微摩尔的8-甲氧基甲基-3-异丁基-1-甲基黄嘌呤和长春西汀对离子霉素降低福司可林刺激的cAMP水平没有影响，而更具选择性且效力高达10000倍的磷酸二酯酶1抑制剂SCH51866、化合物31和化合物30在纳摩尔浓度下抑制离子霉素诱导的福司可林诱导的cAMP下降。因此，我们的数据表明，与选择性弱且效力低的磷酸二酯酶抑制剂8-甲氧基甲基-3-异丁基-1-甲基黄嘌呤和长春西汀相比，SCH51866以及化合物31和30在细胞环境中是有效的磷酸二酯酶1抑制剂。因此，A172细胞代表了一个适合研究磷酸二酯酶1抑制剂细胞效应的系统。8-甲氧基甲基-3-异丁基-1-甲基黄嘌呤和长春西汀似乎不适合用于研究磷酸二酯酶1介导的功能。
