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Interaction of fluorescein with the dicarboxylate carrier in rat kidney cortex mitochondria
R Masereeuw1, W C Saleming, D S Miller
1Department of Pharmacology, Faculty of Medical Sciences, University of Nijmegen, The Netherlands.
Abstract:
The interaction of the organic anion, fluorescein (FL), with mitochondria in renal proximal tubule cells was investigated. Confocal microscopy was used to demonstrate FL accumulation in mitochondria of intact cells. Phenylsuccinate inhibited the mitochondrial accumulation of the FL analog, carboxyfluorescein (CF) indicating that the dicarboxylate carrier may be involved in the intracellular compartmentation of organic anions. To characterize the interaction, radio-tracer uptake and respiration studies with renal mitochondria were carried out using succinate as a substrate. Respiration measurements in freshly isolated kidney cortex mitochondria revealed that FL inhibited ADP-stimulated and uncoupled respiratory rate, indicating that the organic anion inhibited the availability of succinate as a reducing agent. A similar effect on mitochondrial respiration was found for PAH and phenylsuccinate. FL inhibited 14C-succinate uptake concentration-dependently, and Dixon analysis revealed that the nature of interaction between FL and succinate was competitive, Ki values of 0.5 +/- 0.2 and 1.1 +/- 0.8 mM were calculated for respiration experiments and tracer uptake studies, respectively. The data demonstrate that FL competitively interacts with a mitochondrial dicarboxylate transporter.
Insights
Fluorescein (FL) accumulates in kidney cell mitochondria and competitively inhibits succinate transport, suggesting interaction with a mitochondrial dicarboxylate transporter. This impacts organic anion handling in renal cells.
Area of Science:
- Cell Biology
- Mitochondrial Physiology
- Renal Physiology
Background:
- Organic anions are crucial in cellular transport and drug metabolism.
- Mitochondria play a key role in cellular energy production and substrate transport.
- Understanding organic anion interactions with mitochondria is vital for renal cell function.
Purpose of the Study:
- To investigate the interaction of fluorescein (FL) with mitochondria in renal proximal tubule cells.
- To elucidate the mechanism of organic anion accumulation and transport within mitochondria.
- To determine if dicarboxylate carriers are involved in this process.
Main Methods:
- Confocal microscopy to visualize FL localization in intact renal cells.
- Radio-tracer uptake studies using 14C-succinate with isolated renal mitochondria.
- Mitochondrial respiration assays (ADP-stimulated and uncoupled) with succinate as substrate.
- Dixon analysis to characterize the kinetic interaction between FL and succinate.
Main Results:
- Fluorescein (FL) was observed to accumulate within mitochondria of renal proximal tubule cells.
- Phenylsuccinate inhibited carboxyfluorescein (CF) mitochondrial uptake, suggesting dicarboxylate carrier involvement.
- FL competitively inhibited succinate uptake and mitochondrial respiration, with calculated Ki values.
- PAH and phenylsuccinate also affected mitochondrial respiration similarly to FL.
Conclusions:
- Fluorescein (FL) competitively interacts with a mitochondrial dicarboxylate transporter.
- This interaction affects succinate availability as a reducing agent for mitochondrial respiration.
- The findings provide insight into the intracellular compartmentation and transport of organic anions in renal cells.