Metabolite anion carriers mediate the uptake of the anionic drug fluorescein in renal cortical mitochondria

The fluorescent organic anion fluorescein (FL) accumulates in proximal tubu lar cells of the kidney during renal secretion. In freshly isolated and per meabilized proximal tubular cells, the uptake was reduced but still sensiti ve to probenecid, suggesting a concentrative mechanism that is associated w ith intracellular compartments. Previous studies have shown that one of the se compartments may be mitochondrial. In this study, we further investigate d the transport characteristics of FL in isolated rat kidney cortex mitocho ndria. Mitochondrial uptake of 100 mu M FL was rapid, with an initial rate of 60 pmol/mg protein.min, and reached equilibrium after 5 min. To characte rize the transport system(s) involved, FL uptake was studied in the absence and presence of substrates or inhibitors specific for the various mitochon drial anion carriers. Phenylsuccinate (10 mM), an inhibitor of the alpha-ke toglutarate carrier, reduced uptake significantly with a maximum inhibition of 33% and an inhibitory constant (-log IC50) of 4.0 +/- 0.4 (P < .05). Th e apparent K-m for the phenylsuccinate-corrected FL uptake was 1.3 +/- 0.3 mM with a V-max of 260 +/- 26 pmol/mg protein.15 s. Substrates for the tric arboxylate and glutamate-aspartate carriers significantly reduced the uptak e of 100 muM 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 monocarboxyla te and dicarboxylate carriers were without effect. The anionic drugs, valpr oate, indomethacin, and salicylate, significantly reduced FL uptake, wherea s cephaloglycin and cephaloridine had no effect. Finally, a combination of phenylsuccinate, glutamate, and citrate reduced the uptake by 66%, indicati ng that at least three metabolite carriers contribute concomitantly to intr amitochondrial FL transport.