Reichel Valeska, Masereeuw Rosalinde, van den Heuvel Jeroen J M W, Miller David S, Fricker Gert
Institute of Pharmacy and Molecular Biotechnology, INF 366, 69120 Heidelberg, Germany.
Am J Physiol Regul Integr Comp Physiol. 2007 Dec;293(6):R2382-9. doi: 10.1152/ajpregu.00029.2007. Epub 2007 Sep 12.
Previous studies have shown that killifish (Fundulus heteroclitus) renal proximal tubules express a luminal membrane transporter that is functionally and immunologically analogous to the mammalian multidrug resistance-associated protein isoform 2 (Mrp2, ABCC2). Here we used confocal microscopy to investigate in killifish tubules the transport of a fluorescent cAMP analog (fluo-cAMP), a putative substrate for Mrp2 and Mrp4 (ABCC4). Steady-state luminal accumulation of fluo-cAMP was concentrative, specific, and metabolism-dependent, but not reduced by high K+ medium or ouabain. Transport was not affected by p-aminohippurate (organic anion transporter inhibitor) or p-glycoprotein inhibitor (PSC833), but cell-to-lumen transport was reduced in a concentration-dependent manner by Mrp inhibitor MK571, leukotriene C4 (LTC4), azidothymidine (AZT), cAMP, and adefovir; the latter two compounds are Mrp4 substrates. Although MK571 and LTC4 reduced transport of the Mrp2 substrate fluorescein-methotrexate (FL-MTX), neither cAMP, adefovir, nor AZT affected FL-MTX transport. Fluo-cAMP transport was not reduced when tubules were exposed to endothelin-1, Na nitroprusside (an nitric oxide generator) or phorbol ester (PKC activator), all of which signal substantial reductions in cell-to-lumen FL-MTX transport. Fluo-cAMP transport was reduced by forskolin, and this reduction was blocked by the PKA inhibitor H-89. Finally, in membrane vesicles from Spodoptera frugiperda (Sf9) cells containing human MRP4, ATP-dependent and specific uptake of fluo-cAMP could be demonstrated. Thus, based on inhibitor specificity and regulatory signaling, cell-to-lumen transport of fluo-cAMP in killifish renal tubules is mediated by a transporter distinct from Mrp2, presumably a teleost form of Mrp4.
先前的研究表明，鳉鱼（Fundulus heteroclitus）的肾近端小管表达一种管腔膜转运蛋白，其在功能和免疫方面与哺乳动物多药耐药相关蛋白亚型2（Mrp2，ABCC2）类似。在此，我们使用共聚焦显微镜研究了鳉鱼小管中荧光环磷酸腺苷类似物（fluo-cAMP）的转运情况，fluo-cAMP是Mrp2和Mrp4（ABCC4）的一种假定底物。fluo-cAMP在管腔中的稳态积累是浓缩性、特异性且依赖代谢的，但不受高钾培养基或哇巴因的影响。转运不受对氨基马尿酸（有机阴离子转运抑制剂）或P-糖蛋白抑制剂（PSC833）的影响，但Mrp抑制剂MK571、白三烯C4（LTC4）、叠氮胸苷（AZT）、环磷酸腺苷（cAMP）和阿德福韦以浓度依赖的方式降低了从细胞到管腔的转运；后两种化合物是Mrp4的底物。尽管MK571和LTC4降低了Mrp2底物荧光素甲氨蝶呤（FL-MTX）的转运，但cAMP、阿德福韦和AZT均不影响FL-MTX的转运。当小管暴露于内皮素-1、硝普钠（一种一氧化氮供体）或佛波酯（蛋白激酶C激活剂）时，fluo-cAMP的转运并未降低，而所有这些物质都会使从细胞到管腔的FL-MTX转运显著减少。福斯可林降低了fluo-cAMP的转运，且这种降低被蛋白激酶A抑制剂H-89阻断。最后，在含有人类MRP4的草地贪夜蛾（Sf9）细胞的膜囊泡中，可证明存在ATP依赖的fluo-cAMP特异性摄取。因此，基于抑制剂特异性和调节信号，鳉鱼肾小管中fluo-cAMP从细胞到管腔的转运由一种不同于Mrp2的转运蛋白介导，推测是硬骨鱼形式的Mrp4。
