An endogenous atp-sensitive glutathione s-conjugate efflux
mechanism in xenopus laevis oocytes.
Ballatori, Nazzareno, Wei Wang, Liqiong Li, Anh T. Truong.
Department of Environmental Medicine, University of Rochester
School of Medicine, Rochester, NY 14642
APStracts 2:0352R, 1995.
Constitutive efflux mechanisms for reduced glutathione (GSH) and the
glutathione S-conjugates S-ethylglutathione (ethyl-SG) and S-(2,4
-dinitrophenyl)-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 (1.2 mM
intracellular concentration) and microinjected 0.5 mM [3H]DNP-SG was
a linear function of time, with 20% released in 3 h at 25oC.
[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, inhibited after ATP depletion, but was 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 Km 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, by 0.25 mM DIDS, 0.5 mM
sulfobromophthalein, and 0.5 mM dibromosulfophthalein, 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.
Received 12 October 1995; accepted in final form 11 December
1995.
APS Manuscript Number R645-5.
Article publication pending Am. J. Physiol. (Regulatory Integrative
Comp. Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 23 December 95