Notenboom Sylvia, Miller David S, Smits P, Russel Frans G M, Masereeuw Rosalinde
Department of Pharmacology and Toxicology, University Medical Center Nijmegen, 6500 HB Nijmegen, The Netherlands.
Am J Physiol Renal Physiol. 2004 Jul;287(1):F33-8. doi: 10.1152/ajprenal.00443.2003. Epub 2004 Feb 17.
In killifish renal proximal tubules, endothelin-1 (ET-1), acting through a basolateral ET(B) receptor, nitric oxide synthase (NOS), and PKC, decreases cell-to-lumen organic anion transport mediated by the multidrug resistance protein isoform 2 (Mrp2). In the present study, we examined the roles of guanylyl cyclase and cGMP in ET signaling to Mrp2. Using confocal microscopy and quantitative image analysis to measure Mrp2-mediated transport of the fluorescent drug fluorescein methotrexate (FL-MTX), we found that oxadiazole quinoxalin (ODQ), an inhibitor of NO-sensitive guanylyl cyclase, blocked ET-1 signaling. ODQ was also effective when signaling was initiated by nephrotoxicants (gentamicin, amikacin, diatrizoate, HgCl(2), and CdCl(2)), which appear to stimulate ET release from the tubules themselves. ODQ blocked the effects of the NO donor sodium nitroprusside but not of the phorbol ester that activates PKC. Exposing tubules to 8-bromo-cGMP (8-BrcGMP), a cell-permeable cGMP analog, decreased luminal FL-MTX accumulation. This effect was abolished by bisindoylmaleimide (BIM), a PKC inhibitor, but not by N(G)-methyl-l-arginine, a NOS inhibitor. Together, these data indicate that ET regulation of Mrp2 involves activation of guanylyl cyclase and generation of cGMP. Signaling by cGMP follows NO release and precedes PKC activation.
在鳉鱼的肾近端小管中，内皮素-1（ET-1）通过基底外侧的ET（B）受体、一氧化氮合酶（NOS）和蛋白激酶C（PKC）发挥作用，降低由多药耐药蛋白异构体2（Mrp2）介导的细胞向管腔的有机阴离子转运。在本研究中，我们检测了鸟苷酸环化酶和环磷酸鸟苷（cGMP）在ET向Mrp2信号传导中的作用。利用共聚焦显微镜和定量图像分析来测量Mrp2介导的荧光药物荧光素甲氨蝶呤（FL-MTX）的转运，我们发现，对NO敏感的鸟苷酸环化酶抑制剂恶二唑喹喔啉（ODQ）可阻断ET-1信号传导。当由肾毒性药物（庆大霉素、阿米卡星、泛影酸盐、HgCl₂和CdCl₂）引发信号传导时，ODQ同样有效，这些肾毒性药物似乎能刺激小管自身释放ET。ODQ可阻断NO供体硝普钠的作用，但不能阻断激活PKC的佛波酯的作用。将小管暴露于一种可透过细胞的cGMP类似物8-溴-cGMP（8-BrcGMP）中，可减少管腔中FL-MTX的蓄积。蛋白激酶C抑制剂双吲哚马来酰亚胺（BIM）可消除这种作用，但一氧化氮合酶抑制剂N⁰-甲基-L-精氨酸则不能。这些数据共同表明，ET对Mrp2的调节涉及鸟苷酸环化酶的激活和cGMP的生成。cGMP信号传导在NO释放之后、PKC激活之前发生。
