An endogenous ATP-sensitive glutathione S-conjugate efflux mechanism in Xenopus laevis oocytes.
Constitutive efflux mechanisms for reduced glutathione (GSH) and the glutathione S-conjugates S-ethylglutathione (ethyl-SG) and S-(2,4-dinitrophenol)-glutathione (DNP-SG) were examined in Xenopus laevis oocytes. Oocytes were loaded by either microinjection with 50 nl of the 3H-labeled compounds or were exposed to unlabeled 1-chloro-2,4-dinitrobenzene and efflux of DNP-SG synthesized within the oocytes measured spectrophotometrically. Efflux of unlabeled DNP-SG (approximately 1.2 mM intracellular concentration) and microinjected 0.5 mM [3H]DNP-SG was a linear function of time, with approximately 20% released in 3 h at 25 degrees C. [3H] ethyl-SG, 0.5 mM, was released at a comparable rate, whereas only 4% of a tracer dose of [3H]GSH (2.5 mM intracellular GSH) was released in 3 h. Efflux of all three compounds was temperature sensitive and inhibited after ATP depletion but unaffected when Na+ in the culture medium was replaced with K+ or when the pH was changed from 7.5 to either 6.8 or 8.0. Efflux was saturable, with apparent Michaelis constant values of 0.91 +/- 0.19, 0.44 +/- 0.25, and 5.3 +/- 2.2 mM for DNP-SG, ethyl-SG, and GSH, respectively. Bilirubin ditaurate, 0.5 mM, cis-inhibited efflux of 0.5 mM [3H]DNP-SG, 0.5 mM [3H]ethyl-SG, and 2.5 mM [3H]GSH. DNP-SG and ethyl-SG efflux was also cis-inhibited by other glutathione S-conjugates, 0.25 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 0.5 mM sulfobromophthalein, and 0.5 mM dibromosulfophthalin, but not by 0.25 mM taurocholate. [3H]GSH release (2.5 mM) was unaffected by these compounds or by 10 mM extracellular GSH or methionine. These findings indicate that Xenopus oocytes have an endogenous ATP-sensitive mechanism for extruding glutathione S-conjugates, with properties comparable to ATP-dependent glutathione S-conjugate/organic anion transport systems described in a variety of cell types. However, in contrast to mammalian cells, GSH and ethyl-SG release from Xenopus oocytes was also inactivated after cellular ATP depletion but was not sensitive to membrane depolarization in high-K+ medium or trans-stimulated by extracellular GSH, indicating that efflux of these organic anions from Xenopus laevis oocytes is also mediated by an ATP-sensitive mechanism.
Ballatori
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Department of Environmental Medicine, University of Rochester School of Medicine, New York 14642, USA.
Wang
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W
Li
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L
Truong
A T
AT
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001692:Biological Transport; D004137:Dinitrochlorobenzene / Q000378:metabolism; D005240:Feasibility Studies; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism*; D014982:Xenopus laevis
D004137:Dinitrochlorobenzene; C030190:S-(2,4-dinitrophenyl)glutathione; C042431:S-ethyl glutathione; D000255:Adenosine Triphosphate; D005978:Glutathione