Abstract

The uptake and accumulation of the organic anion fluorescein-sodium (Flu-Na) was investigated in freshly isolated proximal tubular cells (PTC) of the rat kidney. Furthermore, the influence of other organic anions on Flu-Na uptake was studied in order to characterize Flu-Na transport in PTC. Flu-Na showed concentration-dependent, saturable and probenecid-sensitive transport. Comparing the transport parameters with para-aminohippurate (PAH), Flu-Na exhibited a higher affinity, but lower capacity to the organic anion transport system. The apparent Km for Flu-Na transport was 59 +/- 15 microM with a Vmax of 186 +/- 26 pmol/mg of protein/min, and for PAH 207 +/- 11 microM and 740 +/- 46 pmol/mg of protein/min, respectively. Dose-dependent inhibition of Flu-Na uptake with PAH resulted in an apparent inhibition constant Ki of 249 microM. This is in good agreement with the apparent Km of PAH, indicating that Flu-Na uptake is regulated by the PAH transport system. It is suggested that cellular uptake of both organic anions is mediated by a carrier at the basolateral membrane. However, after incubating cells with different concentrations of phenol red the percentage of maximum inhibition was 84%, which was significantly different from the 32% with PAH, suggesting that another transport system may be involved in Flu-Na uptake. Experiments with confocal laser scanning microscopy showed cellular uptake of Flu-Na and accumulation in subcellular structures. After superfusion of PTC with rhodamine 123 these structures were identified as mitochondria.