Wielinga Peter R, van der Heijden Ingrid, Reid Glen, Beijnen Jos H, Wijnholds Jan, Borst Piet
Division of Molecular Biology and Center for Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066 CX, The Netherlands.
J Biol Chem. 2003 May 16;278(20):17664-71. doi: 10.1074/jbc.M212723200. Epub 2003 Mar 13.
Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues. Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3',5'-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles. We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5. The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively. MRP4- and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner. In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport. Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20-600 microm, indicating low affinity transport. In addition to several classic inhibitors of organic anion transport, prostaglandins A(1) and E(1), the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux. The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour. This was also the case when phosphodiesterase-mediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal. These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting.
已知环核苷酸可从培养细胞或分离组织中流出。多药耐药蛋白家族中最近描述的两个成员，即MRP4和MRP5，可能参与了这一过程，因为它们可将3'，5'-环核苷酸、环磷酸腺苷（cAMP）和环磷酸鸟苷（cGMP）转运到内翻膜囊泡中。我们研究了过表达MRP4或MRP5的完整人胚肾293（HEK293）细胞中cGMP和cAMP的流出情况。分别用释放一氧化氮的化合物硝普钠和腺苷酸环化酶刺激剂福斯可林刺激细胞内cGMP和cAMP的产生。过表达MRP4和MRP5的细胞以ATP依赖的方式比亲本细胞流出更多的cGMP和cAMP。与之前的一份报告相反，我们发现环核苷酸转运不需要谷胱甘肽。在计算得出的20 - 600微摩尔范围内，转运与细胞内环核苷酸浓度成比例增加，表明是低亲和力转运。除了几种经典的有机阴离子转运抑制剂、前列腺素A1和E1、类固醇孕酮以及抗癌药物雌莫司汀外，所有这些物质均抑制环核苷酸流出。由MRP4和MRP5介导的流出并未导致细胞内cGMP或cAMP水平成比例下降，但在最初一小时内cGMP最多降低了2倍。当磷酸二酯酶介导的环核苷酸水解被3 - 异丁基 - 1 - 甲基黄嘌呤抑制时，流出最大，情况也是如此。这些数据表明，MRP4和MRP5是低亲和力的环核苷酸转运体，充其量可能起到溢流泵的作用，在cGMP合成被强烈诱导且磷酸二酯酶活性受限的情况下，降低cGMP水平的急剧升高。
