Abstract
The fluorescent organic anion fluorescein (FL) accumulates in proximal tubular cells of the kidney during renal secretion. In freshly isolated and permeabilized proximal tubular cells, the uptake was reduced but still sensitive to probenecid, suggesting a concentrative mechanism that is associated with intracellular compartments. Previous studies have shown that one of these compartments may be mitochondrial. In this study, we further investigated the transport characteristics of FL in isolated rat kidney cortex mitochondria. Mitochondrial uptake of 100 μM FL was rapid, with an initial rate of 60 pmol/mg protein·min, and reached equilibrium after 5 min. To characterize the transport system(s) involved, FL uptake was studied in the absence and presence of substrates or inhibitors specific for the various mitochondrial anion carriers. Phenylsuccinate (10 mM), an inhibitor of the α-ketoglutarate carrier, reduced uptake significantly with a maximum inhibition of 33% and an inhibitory constant (−log IC50) of 4.0 ± 0.4 (P < .05). The apparentKm for the phenylsuccinate-corrected FL uptake was 1.3 ± 0.3 mM with a Vmax of 260 ± 26 pmol/mg protein·15 s. Substrates for the tricarboxylate and glutamate-aspartate carriers significantly reduced the uptake of 100 μM FL with −log IC50 values of 4.6 ± 0.4 (citrate), 5.5 ± 0.3 (glutamate), and 4.1 ± 0.4 (aspartate). Substrates for the monocarboxylate and dicarboxylate carriers were without effect. The anionic drugs, valproate, indomethacin, and salicylate, significantly reduced FL uptake, whereas cephaloglycin and cephaloridine had no effect. Finally, a combination of phenylsuccinate, glutamate, and citrate reduced the uptake by 66%, indicating that at least three metabolite carriers contribute concomitantly to intramitochondrial FL transport.
Footnotes
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Send reprint requests to: Dr. Frans G. M. Russel, Department of Pharmacology and Toxicology 233, University of Nijmegen, P.O. Box 9101, 6500 HB, Nijmegen, the Netherlands. E-mail:F.Russel{at}farm.kun.nl
- Abbreviations:
- PTC
- proximal tubular cells
- FL
- fluorescein
- OAT1
- organic anion transport system
- RCR
- respiratory control ratio
- Received June 11, 1999.
- Accepted November 22, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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