Transport of a fluorescent cAMP analog in teleost proximal tubules
Transport of a fluorescent cAMP analog in teleost proximal tubules
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DOI：
10.1152/ajpregu.00029.2007
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发表时间：
2007-12-01
影响因子：
2.8
通讯作者：
Fricker, Gert
中科院分区：
文献类型：
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作者：
Reichel, Valeska;Masereeuw, Rosalinde;Fricker, Gert
先前的研究表明，Killifish（Fundulus heteroclitus）肾近端小管表达一种管腔膜转运蛋白，其在功能和免疫学上类似于哺乳动物多药耐药相关蛋白亚型2（Mrp 2，ABCC 2）。在这里，我们使用共聚焦显微镜，调查在鳉鱼小管的运输的荧光cAMP类似物（fluo-cAMP），Mrp 2和Mrp 4（ABCC 4）的推定底物。稳态腔积累的fluo-cAMP浓度，具体的，代谢依赖性，但不减少高K +培养基或哇巴因。转运不受p-氨基马尿酸盐（有机阴离子转运蛋白抑制剂）或p-糖蛋白抑制剂（PSC 833）的影响，但Mrp抑制剂MK 571、白三烯C4（LTC 4）、叠氮胸苷（AZT）、cAMP和阿德福韦以浓度依赖性方式减少细胞至管腔转运;后两种化合物是Mrp 4底物。尽管MK 571和LTC 4减少了Mrp 2底物荧光素-甲氨蝶呤（FL-MTX）的转运，但cAMP、阿德福韦酯和AZT均不影响FL-MTX的转运。当肾小管暴露于内皮素-1、硝普钠（一氧化氮生成剂）或佛波酯（PKC激活剂）时，Fluo-cAMP转运没有减少，所有这些都表明细胞-管腔FL-MTX转运显著减少。氟环腺苷酸运输减少毛喉素，这种减少被PKA抑制剂H-89阻断。最后，在含有人MRP 4的草地贪夜蛾（Sf 9）细胞的膜囊泡中，可以证明fluo-cAMP的ATP依赖性和特异性摄取。因此，基于抑制剂的特异性和调节信号，荧光cAMP在罗非鱼肾小管中的细胞-管腔转运是由不同于Mrp 2的转运蛋白介导的，推测是Mrp 4的硬骨鱼形式。
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.