Role of NO in endothelin-regulated drug transport in the renal proximal tubule.
We previously demonstrated in intact killifish renal proximal tubules that endothelin (ET), acting through an ET(B) receptor and protein kinase C (PKC), reduced transport mediated by multidrug resistance-associated protein 2 (Mrp2), i.e., luminal accumulation of fluorescein methotrexate (FL-MTX) (Masereeuw R, Terlouw SA, Van Aubel RAMH, Russel FGM, and Miller DS. Mol Pharmacol 57: 59-67, 2000). In the present study, we used confocal microscopy and quantitative image analysis to measure Mrp2-mediated transport of FL-MTX in killifish tubules as an indicator of the status of this ET-fired, intracellular signaling pathway. Exposing tubules to sodium nitroprusside (SNP), a nitric oxide (NO) donor, signaled a reduction in luminal accumulation of FL-MTX, which suggested pathway activation. N(G)-monomethyl-L-arginine (L-NMMA), an NO synthase inhibitor, blocked the action of ET-1 on transport. Because SNP effects on transport were blocked by bisindoylmaleide, a PKC-selective inhibitor, but not by RES-701-1, an ET(B)-receptor antagonist, generation of NO occurred after ET(B) receptor signaling but before PKC activation. NO generation was implicated in the actions of several nephrotoxicants, i.e., diatrizoate, gentamicin, amikacin, HgCl(2), and CdCl(2), each of which decreased Mrp2-mediated transport by activating ET signaling. For each nephrotoxicant, decreased FL-MTX transport was prevented when tubules were exposed to L-NMMA. ET-1 and each nephrotoxicant stimulated NO production by the tubules, as determined by a fluorescence-based assay. Together, the data show that NO generation follows ET binding to the basolateral ET(B) receptor and that, in activating the ET-signaling pathway, nephrotoxicants produce NO, a molecule that could contribute to subsequent toxic effects.
Notenboom
Sylvia
S
Department of Pharmacology and Toxicology, University Medical Center Nijmegen, 6500 HB Nijmegen, The Netherlands.
Miller
David S
DS
Smits
Paul
P
Russel
Frans G M
FG
Masereeuw
Rosalinde
R
D000583:Amikacin / Q000493:pharmacokinetics / Q000633:toxicity; D000818:Animals; D000900:Anti-Bacterial Agents / Q000493:pharmacokinetics* / Q000633:toxicity; D019256:Cadmium Chloride / Q000493:pharmacokinetics / Q000633:toxicity; D002118:Calcium / Q000378:metabolism; D019742:Chemokines, CC; D003287:Contrast Media / Q000493:pharmacokinetics / Q000633:toxicity; D016207:Cytokines / Q000378:metabolism*; D003973:Diatrizoate / Q000493:pharmacokinetics / Q000633:toxicity; D004202:Disinfectants / Q000493:pharmacokinetics / Q000633:toxicity; D019332:Endothelin-1 / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D023781:Fundulidae; D005839:Gentamicins / Q000493:pharmacokinetics* / Q000633:toxicity; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D019402:Macrophage Inflammatory Proteins; D008627:Mercuric Chloride / Q000493:pharmacokinetics / Q000633:toxicity; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D019323:omega-N-Methylarginine / Q000494:pharmacology
D000900:Anti-Bacterial Agents; C095218:Ccl9 protein, mouse; D019742:Chemokines, CC; D003287:Contrast Media; D016207:Cytokines; D004202:Disinfectants; D019332:Endothelin-1; D004791:Enzyme Inhibitors; D005839:Gentamicins; D019402:Macrophage Inflammatory Proteins; D020030:Nitric Oxide Donors; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; D003973:Diatrizoate; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D008627:Mercuric Chloride; D000583:Amikacin; D011493:Protein Kinase C; D019256:Cadmium Chloride; D002118:Calcium