COMMUNICATION
Preferential Transport of Glutathione versus
Glutathione Disulfide in Rat Liver Microsomal Vesicles*
Gábor
Bánhegyi
§¶,
Lorenzo
Lusini¶
,
Ferenc
Puskás
§¶,
Ranieri
Rossi¶
,
Rosella
Fulceri
,
László
Braun§,
Valéria
Mile§,
Paolo
di Simplicio
,
József
Mandl§, and
Angelo
Benedetti
**
From the
Institute of General Pathology, University
of Siena, 53100 Siena, Italy, the § Department of Medical
Chemistry, Semmelweis University of Medicine, 1444 Budapest, Hungary,
and the
Institute for Nervous and Mental Diseases, University of
Siena, 53100 Siena, Italy
 |
ABSTRACT |
A bi-directional, saturable transport of
glutathione (GSH) was found in rat liver microsomal vesicles. GSH
transport could be inhibited by the anion transport blockers flufenamic
acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. A part of
GSH taken up by the vesicles was metabolized to glutathione disulfide (GSSG) in the lumen. Microsomal membrane was virtually nonpermeable toward GSSG; accordingly, GSSG generated in the microsomal lumen could
hardly exit. Therefore, GSH transport, contrary to previous assumptions, is preferred in the endoplasmic reticulum, and GSSG entrapped and accumulated in the lumen creates the oxidized state of
its redox buffer.
 |
INTRODUCTION |
The endoplasmic reticulum
(ER)1 of the cell is the site
of the synthesis, posttranslational modification, and folding of
proteins transported along the secretory pathway. The oxidizing
environment in the lumen of the ER is necessary for the formation of
disulfide bonds and for the proper folding of these proteins (1). The oxidative effects are reflected in and supported by the GSH redox buffer; the ratio of GSH and GSSG is around 2:1 within the lumen of ER
and along the secretory pathway, whereas the cytosolic ratio ranges
from 30:1 to 100:1 (2). However, the primary source(s) of the oxidative
effect has not been demonstrated. Recent observations suggest two
possible mechanisms. First, the preferential uptake of the oxidized
member of a redox couple through the ER membrane and/or the efflux (or
exocytosis) of its reduced form could ensure the oxidative environment.
Alternatively, enzymes resident in the membrane or lumen of the ER
could produce oxidizing compounds (e.g. reactive oxygen
species) toward the lumen. Experimental evidence supports both
mechanisms. Favoring the transport-based hypothesis, the preferential
transport of dehydroascorbate (the oxidized form of ascorbate) has been
described in rat liver microsomal vesicles (3). Similarly, the
selective microsomal transport of GSSG was also reported (2, 4). On the
other hand, several microsomal enzymes (cytochrome P-450s, NADPH
cytochrome P-450 reductase, gulonolactone oxidase, microsomal iron
protein, NADPH-dependent oxidase, sulfydryl oxidase, etc.)
can produce reactive oxygen species (5-10). The recent exploration of
the ER oxidase protein (ERO1) and its role in the protein folding also
support the latter mechanism (11, 12). Because of the conflicting
opinions, the microsomal transport of GSH and GSSG has not been
unequivocally established. The presently available data are based on
the detection of microsome-associated radioactivity by applying a rapid
filtration method and radiolabeled compounds (2, 4); however,
intraluminal GSH or GSSG contents upon transport have not been directly
demonstrated. Therefore, experiments were undertaken to reinvestigate
the transport of GSH and GSSG through the ER membrane.
The main difficulties in the investigation of microsomal transport
processes are deriving from the very small intraluminal space, the
presence of (intraluminal) reactions affecting the transported
compounds and in case of several molecules, the significant binding to
the membrane. To overcome these problems, glutathione transport was
investigated by two different experimental approaches. The light
scattering technique (13, 14) allows the real time detection of the
permeation of the compounds at high concentrations, whereas with the
polyethylene glycol precipitation-rapid sedimentation method (5, 15)
high microsomal protein concentrations can be used, which makes the
direct detection of the intraluminal pools possible. To distinguish the
uptake from the binding to microsomal vesicles the pore-forming
compound alamethicin was used (16-18).
 |
EXPERIMENTAL PROCEDURES |
Preparation of Rat Liver Microsomes--
Rat liver microsomes
were prepared from male Sprague-Dawley rats (180-230 g) as described
in Ref. 19. Microsomal fractions were resuspended in a buffer
containing 100 mM KCl, 20 mM NaCl, 1 mM MgCl2, 20 mM Mops, pH 7.2. The
suspensions were rapidly frozen and maintained under liquid
N2 until required. The latency of mannose 6-phosphatase
(20) and p-nitrophenol UDP-glucuronosyltransferase (17)
activity was greater than 90 and 95%, respectively. Intactness of
microsomal membrane was also ascertained by the sustained light scattering signal upon the addition of the poorly permeant sucrose (14). To measure microsomal water space, microsomes were diluted (10 mg
protein/ml) in the above buffer containing
[3H]H2O (0.2 µCi/ml) or
[3H(C)]inulin (0.17 µCi/ml) and centrifuged,
(100,000 × g for 60 min), and the radioactivity
associated with pellets was measured to enable calculation of
extravesicular and intravesicular water spaces (18, 21).
Transport Measurements by Light Scattering
Technique--
Osmotically induced changes in microsomal vesicle size
and shape (13) were monitored at 400 nm at a right angle to the
incoming light beam, using a fluorimeter (Perkin-Elmer model 650-10S)
equipped with a recorder, a temperature-controlled cuvette holder
(22 °C), and a magnetic stirrer, as described elsewhere (14).
Briefly, microsomal vesicles (50 µg protein/ml) were equilibrated in
a hypotonic medium (5 mM K-Pipes, pH 7.0), and the
osmotically induced changes in light scattering were measured after the
addition of a small volume (<10% of the total incubation volume) of
the concentrated and neutralized solutions of the compounds to be tested.
Transport Measurements by Rapid Sedimentation Method--
The
rapid separation of polyethylene glycol-aggregated microsomes was
performed as described previously (5, 15). Briefly, microsomes (10 mg
protein/ml) were incubated in the presence of various concentrations of
GSH or GSSG in a buffer containing 100 mM KCl, 20 mM NaCl, 1 mM MgCl2, 20 mM Mops, pH 7.2 at 22 °C. To distinguish the
intravesicular and the bound GSH/GSSG, the pore-forming antibiotic
alamethicin (16-18) was added at the end of the incubations. At the
indicated times 0.5-ml samples were taken and precipitated with 0.1 ml
of 25% polyethylene glycol. Microsomal vesicles were rapidly
sedimented by centrifugation (20 s at 13000 rpm). Pellets were washed
two times with the buffer containing 5% polyethylene glycol. The final
pellet was deproteinized with perchloric acid.
Metabolite Measurements--
GSH and GSSG contents were measured
by HPLC according to Ref. 22. A Waters Alliance HPLC apparatus equipped
with autosampler was used; results were analyzed by the Milleneum 2000 software.
Materials--
Glutathione, glutathione disulfide, flufenamic
acid, alamethicin, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid
were obtained from Sigma. [3H]H2O (1 mCi/g)
and [3H(C)]inulin (500 mCi/g) were from NEN Life Science
Products. All other chemicals were of analytical grade. Bondapak
aminopropyl column (average particle size 10 µm, 300 × 3.9 mm
inner diameter) was bought from Waters Millipore (Milford, MA).
 |
RESULTS |
First, the permeability of rat liver microsomal membrane toward
GSH and GSSG was tested with the light scattering method. Surprisingly,
the addition of GSSG resulted in a sustained signal, indicating that
microsomal membrane was impermeable toward GSSG, whereas GSH entered
the vesicles (Fig. 1). Flufenamic acid, a known anion transport inhibitor (23), hindered GSH permeation in a
concentration-dependent way; 2.5 mM flufenamic
acid almost completely prevented the influx (Fig. 1).

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Fig. 1.
Influx of GSH, its inhibition by flufenamic
acid, and lack of influx of GSSG in rat liver microsomal vesicles
monitored by light scattering. Light scattering increase is
assumed to reflect shrinkage of microsomal vesicles. The addition of
poorly permeable solutes results in a sustained shrinkage, and the
recovery of the initial signal (swelling phase) after the addition of
solutes is assumed to reflect their entry into vesicles (13). Rat liver
microsomes (70 µg/ml of protein) were equilibrated in a low
osmolarity buffer (5 mM K-Pipes, pH 7) until a stable light
scattering base line was obtained. Concentrated solutions of GSH or
GSSG (0.5 M, in the K-Pipes buffer, pH 7;
arrows) were added to 2.0 ml of the microsomal suspensions
giving 20 mM final concentration for both compounds.
Representative traces out of five to eight similar experiments are
shown. F.A., flufenamic acid (2.5 mM).
|
|
Thereafter the transport was also detected by measuring the
intravesicular GSH and GSSG contents upon their addition by using the
polyethylene glycol precipitation-rapid sedimentation method. GSH
addition (5 mM, a cytosol-like concentration) resulted in the time-dependent increase of intraluminal GSH content
(Fig. 2). An initial rapid phase of
uptake was followed by a second slower phase, which in accordance with
the light scattering observations did not reach the level of the
complete equilibrium estimated on the basis of the measured total
intravesicular water space of the microsomes (3.46 ± 0.91 µl/mg
protein; mean ± S.D., n = 4). GSH associated with
the microsomal vesicles really occupied an intravesicular space,
because the addition of the pore-forming alamethicin rapidly released
it. GSH taken up by the vesicles was partially oxidized to GSSG. This
oxidation process occurred mainly in the first 2 min of the uptake
(Fig. 2) and resulted in an approximately 0.3 mM estimated
intravesicular concentration of GSSG. The extravesicular GSSG
concentration (presumably due to the contamination of added GSH) never
exceeded 0.05 mM and did not increase during the
incubation. Therefore, at least a 6-fold concentration gradient between
the intra- and extravesicular GSSG pools was established, indicating
that the oxidation of GSH was predominantly intravesicular.
Intravesicularly formed GSSG could also be released from the lumen by
alamethicin addition (Fig. 2). Upon the addition of GSSG (5 mM), as expected on the basis of light scattering
experiments, a negligible amount of the added compound entered the
vesicles (Fig. 2, open squares).

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Fig. 2.
Influx of GSH and accumulation of GSSG in rat
liver microsomal vesicles. Microsomes were incubated in the
presence of 5 mM GSH (filled symbols) or 5 mM GSSG (open symbols). At 15 min, alamethicin
(0.1 mg/mg microsomal protein) was added to permeabilize the vesicles
(dotted lines). Intraluminal GSH (circles) and
GSSG (squares) contents were measured as described under
"Experimental Procedures." The triangles indicate the
total uptake expressed as GSH (GSH + 2GSSG) in the presence of 5 mM GSH. Means ± S.E. of four to eight experiments are
shown. Error bars are not visible when they are smaller than
the symbol size.
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The redox potentials of the intravesicular GSH/GSSG redox system were
calculated by the Nernst equation from the measured intraluminal GSH
and GSSG contents using
0.24 V standard potential for glutathione
(2). Concentrations were calculated from the measured contents on the
basis of intravesicular water space of rat liver microsomal vesicles
(see above). At the second, slower phase of the GSH uptake (after 6 min) the intraluminal redox potential of the GSH/GSSG redox system was
stabilized around
0.183 mV, which is close to the value reported in
the secretory pathway of an intact cell (2).
The initial rate of GSH uptake was protein
concentration-dependent; the addition of ATP in accordance
with previous observations (4) did not stimulate GSH transport (data
not shown). The influx of GSH was saturable (Fig.
3). No increase in the initial rate (measured after 2 min of incubation) of accumulation of GSH or GSSG was
detected above 5 mM extravesicular GSH concentrations (Fig.
3). The apparent Michaelis constant for the total uptake of GSH (GSH + 2GSSG) was 1.65 mM, and the apparent maximal rate was 2.66 nmol/min/mg protein. The correlation coefficient for the linear
Lineweaver-Burk plot was r = 0.9904. However, these kinetic data cannot be the exact parameters for the transport alone,
because it is also affected by the intravesicular metabolism.

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Fig. 3.
Initial rates of the uptake of GSH and
accumulation of GSSG in rat liver microsomal vesicles as a function of
extravesicular GSH concentration. The intravesicular content of
GSH (circles) and GSSG (squares) were measured
after 2 min of incubation in the presence of the indicated
concentration of extravesicular GSH. The triangles indicate
the total uptake expressed as GSH (GSH + 2GSSG). Means ± S.E. of
three experiments are shown.
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The inhibitory effect of flufenamic acid could be demonstrated in the
polyethylene glycol precipitation-rapid sedimentation experimental
system. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid, another anion
transport inhibitor, also decreased the initial rate of GSH uptake
(Table I).
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Table I
Inhibition of GSH influx and GSSG accumulation by flufenamic and
4,4'-diisothiocyanostilbene-2,2'-disulfonic acids in rat liver
microsomal vesicles
Microsomes were incubated in the presence of 5 mM GSH for 2 min. Intraluminal GSH and GSSG contents were measured as described
under "Experimental Procedures." Flufenamic acid and
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid were added
simultaneously with GSH.
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The microsomal GSH transport was bi-directional. After 15 min of
incubation in the presence of GSH (5 mM), microsomes were precipitated with polyethylene glycol and were taken up in a GSH-free buffer. GSH release was detected as the decrease of its intravesicular content; the measurements indicated that GSH could leave the lumen of
microsomes with a rate comparable with that of influx and flufenamic acid was inhibitory also on the efflux (Fig.
4). GSSG produced by the intraluminal
oxidation of GSH could hardly exit from the vesicles (Fig. 4); the
half-time of the efflux was eventually longer than 1 h (data not
shown).

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Fig. 4.
Efflux of GSH from rat liver microsomal
vesicles and its inhibition by flufenamic acid. Microsomes were
preincubated in the presence of 5 mM GSH for 15 min, and
then they were taken up in a GSH-free buffer in the presence or absence
of flufenamic acid (2.5 mM). Intraluminal GSH
(circles) and GSSG (squares) contents were
measured. Open circles indicate efflux experiments performed
in the presence of flufenamic acid. Means ± S.E. of four to eight
experiments are shown.
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 |
DISCUSSION |
In this study, we demonstrate that GSH is the preferentially
transported form of glutathione in the hepatic endoplasmic reticulum. The features of the transport meet the requirements of a facilitative transport process: it is bi-directional, time-, concentration-, and
protein-dependent, saturable, and inhibitable. On the other hand, the very slow permeation of GSSG can be regarded as simple diffusion; the influx and efflux of GSSG are slower than those of
sucrose, which is widely used as a nonpermeant test compound for the
investigation of the integrity of microsomal membrane. Our results are
in contradiction with the conclusion of previous reports indicating the
preferential transport of GSSG (2, 4). This discrepancy can be due to
the differences in the preparation of microsomes. In one of the studies
(2), microsomes were prepared and stored in reducing buffer
(i.e. in the presence of 1 mM dithiothreitol). The permeability of the microsomal membranes to other low molecular mass compounds was not presented. A possible interpretation might be
that an increased nonspecific permeability together with mixed disulfide formation between the reduced microsomal proteins and added
GSSG led to the underestimation of GSH and overestimation of GSSG
transport. The other study (4) cannot be reconciled with our results
due to shortage of data. However, in these reports, the intravesicular
GSH and GSSG pools were not directly detected. The major novelties of
the present report are (i) the estimation of these intravesicular
pools, (ii) the use of alamethicin to distinguish the uptake and
binding, and (iii) demonstration of the bi-directional feature of the
transport. Our results, gained by two different methods, are consonant.
Although the relatively long time required for the
precipitation-sedimentation method might allow a partial efflux of the
transported compound, the real time detection of the transport with the
light scattering technique rules out the possible fast efflux of the
investigated compound (especially GSSG) during the washing procedure.
GSH taken up by microsomal vesicles was partially oxidized in the
lumen. Because the oxidation was restricted to the early phase of the
uptake, it can be attributed to a thiol-disulfide exchange with protein
disulfides present in the lumen rather than a continuous
oxidation.
As for the transport system(s) involved in the permeability of ER to
GSH, it appears to meet the feature of a facilitate transport, i.e. there is no energy requirement; it is saturable,
bi-directional, and inhibitable. The inhibitors used here, however, do
not allow a more precise characterization because they are known to be
active on the transport of various anionic compounds (14, 23). Because the transporters of ER have not been completely characterized both
functionally and structurally, suggestions cannot be made on the basis
of analogies.
In summary, the primary source of the oxidizing environment in the
lumen of the endoplasmic reticulum is not the preferential GSSG
transport. The intraluminal GSH/GSSG ratio can be generated by the
retention of GSSG derived from GSH imported into the ER. GSSG can be
formed in the lumen either by GSH-dependent reduction of
imported dehydroascorbate (3) catalyzed by protein disulfide isomerase
(24) or by other oxidative processes mediated by local enzymes.
 |
FOOTNOTES |
*
This work was supported Ministry of Welfare Grants ETT 448 and 449, Ministry of Education Grant FKFP 0652/97, and Orzagos Tudomanyos Kutatasi Alap Grants T017404 and T019907, by Italian Telethon Grant E.638 (to A. B.), by Hungarian-Italian
Intergovernmental Science and Technology Co-operation Programme
Grant I-44/95, and a NATO linkage grant (to A. B. and G. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
These authors contributed equally to this work.
**
To whom correspondence should be addressed. Tel.: 39-577-227021;
Fax: 39-577-227009; E-mail: benedetti{at}unisi.it.
 |
ABBREVIATIONS |
The abbreviations used are:
ER, endoplasmic
reticulum;
GSH, glutathione;
GSSG, glutathione disulfide;
Mops, 4-morpholinepropanesulfonic acid;
Pipes, 1,4-piperazinediethanesulfonic
acid;
HPLC, high pressure liquid chromatography.
 |
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