Reply:
Authors: Jose C. FernÁndez-Checa, Carmen Garcia-Ruiz
Journal: Hepatology (2004)

In the preceding letter, Estrela and coworkers challenge the conclusion from our recent study1 indicating that the 2-oxoglutarate carrier functions as a glutathione (GSH) transporter. Our work showed the functional expression of the 2-oxoglutarate carrier in mitochondria from Xenopus oocytes, its sensitivity to altered mitochondrial membrane fluidity, and competition with GSH for transport.1 Their argument is based on the discrepancy between the kinetic parameters we reported with those previously published2 and their inability to reproduce our findings. This comparison, however, is misleading, as the work referred to by Estrela et al. describes the kinetics of the 2-oxoglutarate carrier from bovine heart mitochondria reconstituted in liposomes2 that differed from those of isolated rat-liver mitochondria.3 Since our findings in Xenopus oocytes indicated the ability of the 2-oxoglutarate carrier to transport GSH into mitochondria, we examined the uptake of 2-oxoglutarate in mitochondria from rat liver in exchange with GSH instead of malate; rat-liver mitochondria display a concentration of GSH about 9–12 mmol/L4. We would like to clarify that all transport measurements of 2-oxoglutarate as well as GSH in mitochondria from rat liver or Xenopus oocytes were done at 25°C, as detailed previously.5 In attempting to reproduce our findings, it was unfortunate that Estrela et al. examined the transport of 2-oxoglutarate at 0°C, what most likely reflects the binding of 2-oxoglutarate to mitochondria rather than its transport. In our study1 we characterized the kinetics of 2-oxoglutarate in relationship with those of GSH in isolated rat-liver mitochondria that were previously reported by Martensson et al.6 and Colell et al.7 While these published kinetic parameters of GSH were in mutual agreement and showed two components, the kinetics of 2-oxoglutarate in rat-liver mitochondria exhibited a single Michaelis-Menten component1 with KM and Vmax similar to the high-capacity, low-affinity transport site for GSH.6, 7 Thus, while the functional expression in oocytes unequivocally demonstrates the role of 2-oxoglutarate carrier as a GSH transporting polypeptide, its sensitivity to appropriate membrane fluidity and kinetics provide compelling evidence that the 2-oxoglutarate carrier may account for the low-affinity transport of GSH. While we agree with Estrela et al. about the critical importance of mitochondrial GSH in controlling the rate of reactive oxygen generation within mitochondria and the fate of cells in response to stress, as shown by our own work8-10 as well as the work of others,11, 12 the identification of the mitochondrial GSH carrier responsible for the high-affinity transport of GSH clearly will require more work. In this regard, the suggestion by Estrela and coworkers that the mitochondrial transport of GSH may occur through the voltage-dependent anion channel (see their letter), referring to the work of Cummings et al.13 is unfounded, since these authors demonstrated that voltage-dependent anion channel does not play any role in the mitochondrial transport of GSH.