Hypoxic responses of Na+/K+ ATPase in trout hepatocytes
1 Institute of Veterinary Physiology, Vetsuisse Faculty, University of
Zurich, Zurich, Switzerland
2 Laboratory of Animal Physiology, Department of Biology, University of
Turku, Turku, Finland
* Author for correspondence (e-mail: annab{at}access.unizh.ch)
Accepted 7 March 2005
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Summary |
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Key words: Na+-K+ ATPase, hypoxia, redox state, hepatocytes
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Introduction |
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Although the activity of Na+/K+ pump is oxygen
sensitive in some species, the mechanisms by which oxygen influences the pump
function remain obscure (e.g. Buck and
Hochachka, 1993; Angermuller et
al., 1995
; Krumschnabel et
al., 2000b
; Lifshitz et al.,
1986
; Lutz and Nilsson,
1997
; Bogdanova et al.,
2003a
,b
;
Clausen, 2003
;
Dada et al., 2003
).
Hypoxia-induced reduction or cessation of Na+/K+ pump
activity in rat liver tissue is fast and can be rapidly reversed upon
reoxygenation (e.g. Angermuller et al.,
1995
). As follows from the data we have obtained on trout
hepatocytes and mouse erythrocytes, hypoxia-induced inhibition of the pump is
independent of the changes in cellular ATP levels (Bogdanova et al.,
2003a
,b
).
In addition, hypoxic response of the Na+/K+ pump in fish
hepatocytes does not require the presence of adenosine, although its release
into the circulation in response to hypoxic insult may somewhat decrease the
activity of the pump in vivo (Krumschnabel
et al., 2000a
). However, reduction in oxygen tension may have a
pronounced effect on cellular redox state. Acute in vivo and in
vitro hypoxic exposure results in shift in redox balance that is
especially pronounced in erythrocytes and myocytes because of high levels of
reactive oxygen species (ROS) production in these cells under normoxic
conditions (Hermes-Lima and Zenteno-Savin,
2002
; Di Meo and Venditti,
2001
; Bogdanova et al.,
2003b
) (A. Bogdanova and O. Ogunshola, unpublished data on in
vivo hypoxic effects on GSH levels in murine red blood cells). The
following increase in cellular reduced glutathione (GSH) levels is the cause
of hypoxia-induced suppression of the Na+/K+ pump
function in these cells (Hermes-Lima and
Zenteno-Savin, 2002
; Bogdanova
et al., 2003b
). Suppression of free radical production by NAD(P)H
oxidases under hypoxic conditions has also been shown in some cell types (e.g.
Porwol et al., 2001
;
Acker and Acker, 2004
).
Alternatively, under conditions of severe hypoxia, gradual reduction of
mitochondrial electron transduction chain components may result in an
uncontrolled formation of ROS with consequent shifts in cellular redox
potential (e.g. Rifkind, 1993; Chandel and
Schumacker, 2000
). In particular, slow build-up in ROS production
can be observed after a 1 h incubation of trout hepatocytes at 1%
O2 (Bogdanova et al.,
2003a
). Recent studies reveal that both upregulation of
mitochondrial ROS and suppression of ROS generation by NADPH oxidases in
response to hypoxia may occur simultaneously both being important for hypoxic
signaling (Aley et al.,
2005
).
Hypoxic response of the Na+/K+ pump in mouse red
blood cells resembles that observed in other cell types, including liver.
However, the presence of high concentrations of heme iron functioning as a
catalyst of the Fenton reaction (in which the most active ROS, hydroxyl
radicals, are produced) and the absence of mitochondria in erythrocytes does
not allow any direct comparisons between the red blood cells and other cell
types. Therefore, in this study we have focused on the mechanisms of the
oxygen sensitivity of the Na+/K+ pump, using primary
cultures of rainbow trout hepatocytes. Earlier, we have shown that active
K+ influx into these cells decreases substantially in response to a
15 min exposure to a medium equilibrated with 1% oxygen
(Bogdanova et al., 2003a). This
observation indicated oxygen sensitivity of the Na+/K+
pump in trout liver cells. Notably, most studies using liver cells have
compared anoxia to `normoxia' corresponding to 21% O2
(Krumschnabel et al., 2000b
).
For primary cultures of liver cells these conditions are non-physiological.
Oxygen concentration measured under normoxic conditions in vivo in
mammalian liver tissue ranges between 6 and 3 kPa, and short-term anoxia does
not result in complete tissue deoxygenation (e.g.
Brooks et al., 2004
;
El Desoky et al., 1999
). We
have monitored transport and hydrolytic activities of the
Na+/K+ ATPase as a function of partial O2
pressure (PO2) in the incubation medium in the
range of 21-0.5 kPa. Kinetics of hypoxic response were monitored and the
obtained data related to the changes in cellular ATP levels, ROS formation and
redox state. Responses of the pump to changes in oxygenation were also studied
in cells with manipulated redox state.
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Materials and methods |
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Manipulation of the cellular redox state
To shift cytosolic redox state to more oxidized, cells were treated with a
conjugating agent chloro-dinitrobenzene (CDNB). CDNB penetrates both plasma
and mitochondrial membranes, and when in the cells selectively interacts with
GSH in a reaction catalyzed by glutathione S-transferase, stable adduct
2,4-dinitrophenyl-s-glutathione is formed that is actively transported out of
the cell (Awasthi et al., 1981;
Scott et al., 1990
). Depending
on the concentration of CDNB (0.1 to 3.0 mmol l-1), slight or
almost complete GSH depletion can be achieved within minutes of incubation
with conjugating agent (Han et al.,
2003
; Lauf et al.,
1995
; Bogdanova et al.,
2003b
). Treatment of cells with permeable thiols such as permeable
diethyl ester of GSH (et-GSH) or N-acetyl cysteine (NAC) shifts the
redox balance in the cells towards reduced. These compounds are usually used
at concentrations comparable with intracellular GSH concentration (1-10 mmol
l-1) to achieve significant shift from the physiological reduced
thiol levels. Finally, exposure of cells to virtually impermeable GSH may be
used to study the effects of extracellular reductants on ion transport and
other cellular functions.
Evaluation of the changes in cellular ROS production
To start the experiments, a chamber (fluid volume 200 µl) was
attached to the cover slip, which served as bottom for the chamber, and the
cells were loaded in darkness with 20 µmol l-1
2',7'-dichlorodihydrofluorescein diacetate (H2-DCFDA)
for 20 min at room temperature. The experimental chamber was attached to the
perfusion system (Ismatec Reglo perfusion pump, Glattbrugg, Switzerland; 10 ml
min-1; fluid volume of the experimental chamber was 200 µl,
therefore a 95% change of the perfusion fluid was achieved within 5 s) on an
inverted microscope (Nikon Diaphot 200; 20x fluorescence objective).
Fluorescence intensity measurements were made using Photon Technology
International Imagescan setup (Lawrenceville, NJ, USA) with Deltascan
monochromator unit and IC-100 CCD video camera. For H2-DCFDA the
excitation and emission wavelengths of 502 and 530 nm respectively were used.
Recordings were started 3 min after the onset of perfusion with a standard
incubation saline containing (in mmol l-1) 133 NaCl, 5 KCl, 3
Na2HPO4, 10 Hepes, 1.6 CaCl2, 0.9
MgSO4 and 10 glucose, pH 7.6 at room temperature (20°C). The
medium was not recirculating. Within the first 20 min of perfusion, the
intensity of fluorescent signal increased linearly with time but reached a
steady-state plateau after 30 min of perfusion. This initial linear period
could be used for observations of acute changes in redox state, with changes
in the slope of the linear curves corresponding to a decrease or increase in
oxidation rate. To avoid artifacts caused by increased leakage of the oxidized
dye, viability of cells was controlled using Trypan Blue staining. Increase in
fluorescent intensity monitored over the first 10 min of perfusion was taken
as `control', thereafter perfusion was continued for another 10 min either
with air-equilibrated incubation saline containing 1 mmol l-1 CDNB
or with the standard incubation saline pre-equilibrated with gas mixture
containing 1% O2 and 99% N2. Photon Technology
International Image Master software was used to analyze the data obtained from
the changes in fluorescent intensity for single cells. Data from 35 to 56
cells were pooled and changes in the rate of oxidation of the fluorescent dye
were detected as changes in the slope of the curves during the linear period
(see Fig. 1).
|
Evaluation of hydrolytic and transport activity of Na+/K+ ATPase
Hydrolytic activity of the Na+/K+ pump at optimal
substrate and ligand concentration was quantified as ouabain-sensitive
inorganic phosphate production in cell homogenates. Intact cells were
incubated at room temperature in the standard incubation saline with or
without 5 mmol l-1 GSH, or NAC, or 1 mmol l-1 CDNB for
15 min. Incubation at different PO2 values was
performed in the Cameron (Port Aransas, TX, USA) DEQ-1 tonometers equilibrated
with gas mixtures of fixed oxygen concentrations generated from air and
N2 by the Cameron gas mixing flowmeter. Thereafter, cells were
destroyed by repeated freeze-thaw cycles and cell homogenates or microsomes
were incubated with or without 1 mmol l-1 ouabain in media
containing (in mmol l-1) 130 NaCl, 20 KCl, and 3 MgCl2
for 10 min. After binding of inhibitor was complete, ATP hydrolysis was
measured in the presence of 3 mmol l-1 ATP as the rate of
production of inorganic phosphate. The amount of inorganic phosphate was
determined using the method of Rathbun and Betlach
(1969).
Transport activity of the pump was evaluated in intact cells using
86Rb as a radioactive tracer for K+. To measure
unidirectional K+ (86Rb) influx hepatocytes were
suspended in the standard incubation saline. Cells were preincubated for 15
min with or without 5 mmol l-1 GSH, N-acetyl cysteine
(NAC), MPG or 1 mmol l-1 CDNB. To distinguish between active,
Na+/K+ pump-mediated, and passive K+ influx,
a selective inhibitor of the Na+/K+ pump (ouabain) at
the concentration of 100 µmol l-1 was added to a half of the
samples 15 min prior to the addition of the radioactive tracer. Flux
measurements were started by adding of 5 µl 86Rb (0.1 mCi
ml l-1 stock on distilled water, Perkin Elmer, Boston, MA, USA) per
ml cell suspension. Aliquots of suspension were collected after 3, 5 and 10
min of incubation with the radioactive tracer, and the flux was stopped by
immediate dilution of 0.9 ml aliquot with 10 ml cold washing medium [100 mmol
l-1 Mg(NO3)2 and 10 mmol l-1
imidazole (buffered at pH 7.4)]. Cells were washed twice to eliminate external
tracer and lysed in 5% TCA. Radioactivity of cells (Ac)
and incubation medium (Am) was measured using Microbeta
Wallac liquid scintillation counter (Perkin-Elmer Wallac, Finland) in water
phase (Cherenkov effect). Accumulation of 86Rb in heaptocytes was
linear for the 10 min incubation time. Unidirectional fluxes J were
calculated from the following equation:
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where Ac and Am are radioactivity of cells in 1 ml suspension and 1 ml medium, respectively; m is amount of protein (mg ml-1 of cell suspension), [K+]e is K+ concentration in the incubation medium, and t is the equilibration time with the tracer.
Measurements of cellular ATP and GSH levels
Quantification of total non-protein reduced thiol levels, of which GSH is
the most abundant, was performed spectrophotometrically. After 15 min of
incubation in a tonometer equilibrated at 21, 5 or 1% O2 in the
standard incubation medium with or without 1 mmol l-1 CDNB, 5 mmol
l-1 GSH or NAC, cells were lysed in 5% trichloroacetic acid and
protein removed by centrifugation. Reduced thiol levels were evaluated in
supernatants using Ellmann's technique. The optical density of colored
complexes which thiols formed with Ellmann's reagent
(5,5'-dithiobis(2-nitrobenzoic acid) was determined at 412 nm using
Lambda 25 spectrophotometer (Perkin-Elmer). Details of the analytical protocol
are described by Tietze
(1969). Cellular GSH was
depleted by treatment of cells with 1 mmol l-1 CDNB.
Cellular ATP levels were measured using ATP bioluminescent assay kit (Sigma, St Louis, MO, USA). Chemiluminescence measurements were carried out on a Sirius Luminometer (Berthold Detection Systems, Pforzheim, Germany). Measurements were performed in protein-free cell lysates prepared by mixing cell suspensions with equal amount of 5% TCA as described in the standard kit protocol.
Statistics
Data are presented as mean ± S.E.M.
The statistical significance of the obtained data was analyzed with
Mann-Whitney U-test or Wilcoxon matched-pairs, signed-rank test, or
paired and unpaired t-test (depending on the normality of the data)
provided by GraphPad Instat (version 3) program.
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Results |
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To establish if the changes in ROS production caused by oxygen deprivation affected bulk cellular redox state significantly, we have measured the level of reduced non-protein thiols, most of which are represented by GSH, in hepatocytes equilibrated with 21, 5 or 1% oxygen for 15 min. As can be seen from Fig. 2, a slight increase in cellular GSH content was observed in cells incubated at 5% O2 when compared with 21% O2, whereas no difference of GSH levels in cells incubated at 21% and 1% O2 could be detected (Fig. 2). As expected, GSH depletion caused by treatment of cells with 1 mmol l-1 CDNB was oxygen independent. Incubation of cells at 1% O2 up to 30 min was without an effect on cellular GSH levels (data not shown).
|
Cellular ATP levels as a function of oxygen concentration and redox state
Cellular ATP levels were monitored as a function of oxygen concentration
and cellular redox state. No significant differences in ATP levels could be
seen between cells incubated at 21, 5 and 1% for 15 min
(Fig. 3). GSH depletion as well
as treatment of cells with reducing agents such as NAC or GSH had no effect on
ATP levels in trout hepatocytes.
|
Hydrolytic and transport activity of the Na+/K+ ATPase
Hydrolytic activity of the Na+/K+ ATPase was not
altered by hypoxia in the range of concentrations of 21-1% O2
(Fig. 4) and after incubation
for 40 min at 1% O2 (data not shown). Reducing agents did not
affect the hydrolytic function of the Na+/K+ ATPase
whereas GSH depletion was followed by a twofold decrease in hydrolytic
activity independent of the oxygen concentration used
(Fig. 4).
|
In contrast to hydrolytic activity, transport function of the Na+/K+ ATPase was strikingly dependent on the oxygen concentrations. Active K+ influx decreased after 15 min of exposure to 10% O2 in comparison with 21% O2 (Fig. 5A). Passive K+ influx was slightly increased at PO2 of 1 kPa, remaining constant at any other oxygen concentration tested. The lowest values of active transport were observed in cells incubated at 1% oxygen and consequently the kinetics of the pump inhibition was studied at 1% oxygen level. As can be seen in Fig. 5B, the active K+ transport component responded to hypoxic treatment in a time-dependent manner. Ouabain-resistant K+ influx decreased transiently immediately after the onset of hypoxic exposure but recovered within 10 min of hypoxic treatment. Active K+ influx was also transiently suppressed by hypoxic exposure. Hypoxia-induced inhibition of the Na+/K+ ATPase could be observed over 15 min incubation at 1% O2. However, transport activity of the pump was restored if hypoxic exposure lasted for 20 min. Moreover, 30 min treatment was followed by a two- to threefold stimulation of active K+ influx compared with normoxic control (Fig. 5B). Measurements of cellular ATP under identical conditions revealed no correlations with changes in the Na+/K+ ATPase transport activity.
|
Relation between redox-and oxygen-sensitivity of the Na+/K+ ATPase
Depletion of GSH in cells incubated at 21% O2 resulted in
suppression of Na+/K+ pump transport activity
(Fig. 6A). Active K+
influx did not differ for CDNB-treated cells exposed to 21 or to 1% oxygen for
15 min. Addition of hydroxyl radical scavenger MPG at concentration of 5 mmol
l-1 15 min prior to the onset of hypoxia abolished hypoxia-induced
inhibition of the pump function (Fig.
6A).
|
Passive K+ influx decreased significantly (from 0.052±0.019 to 0.014±0.002 mmol mg-1 protein h-1) during the first 3-5 min of exposure to 1% O2 and, thereafter, showed slightly higher values than those at 21% O2. CDNB treatment resulted in upregulation of ouabain-resistant K+ influx at PO2 of 21 kPa but not at 1 kPa, which was in agreement with the data in Fig. 1 showing reduction in free radical production under hypoxic conditions as compared to air-equilibrated `normoxic' control. MPG had no effect on passive K+ influx either at 21 or at 1% O2 (Fig. 6B).
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Discussion |
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It is known that mammalian hepatocytes respond to hypoxic challenge (1 kPa
and lower) almost instantaneously by reduction in cellular ATP levels,
increase in cellular Na+ concentrations and cell death
(Aw and Jones, 1985;
Carini et al., 1997
). Compared
with this extreme oxygen sensitivity, trout hepatocytes seem to be rather
hypoxia tolerant, although the species is known to be oxygen sensitive among
fish. However, oxygen consumption rates for mammalian and trout hepatocytes
are strikingly different, making 20-40 nmol oxygen per 106 cells
min-1) for rat cells compared with 0.6-0.7 nmol oxygen per
106 cells min-1) for trout liver cells
(Rissanen et al., 2003
;
Aw et al., 1987
). Environmental
temperature range is also different, being 15-20°C for the trout and
37°C for the rat liver cells. Therefore, lack of ATP depletion in trout
hepatocytes exposed for 30 min or less to low oxygen concentration is not an
unexpected finding.
Decrease in transport activity of the Na+/K+ pump in
response to 15 min hypoxic exposure can be seen already at 10% O2
with a most pronounced effect at 1% O2. Unfortunately, no data are
available on the in vivo oxygen partial pressure in rainbow trout liver
tissue. In mammalian liver tissue, oxygen levels detected are in the range of
4-6 kPa, whereas at concentrations of 1.5-1.7 kPa, oxygen availability no
longer matches consumption (Soller et al.,
2001; Brooks et al.,
2004
). `Physiologically normoxic' and `critical' oxygen tensions
correspond with 5-3% and 1% O2 levels in gas phase, respectively.
Our data shows that Na+/K+ pump transport function in
this range of oxygen concentrations is significantly lower than at 21%
O2. Reversible inhibition of the active K+ uptake was
shown for trout hepatocytes in response to hypoxia (1% O2) and
reoxygenation (Krumschnabel et al.,
2000b
). As a possible mechanism of hypoxia-induced inhibition of
active K+ influx, Krumschnabel et al. suggest downregulation of ATP
production, postulating existence of transport-metabolic coupling
(Krumschnabel et al., 2000b
).
Our data do not support this hypothesis as hypoxia-induced inhibition of the
pump is almost instantaneous and occurs at oxygen concentrations of 10-5%, at
which no changes in ATP content occur.
Notably, the hypoxic inhibition of the pump at
PO2 of 1 kPa is transient with activity
recovering within 25-30 min of incubation under hypoxic conditions. Data we
have obtained previously on oxygen sensitivity of Na+/H+
exchanger reveal that this transporter is activated by hypoxic treatment
(Tuominen et al., 2003). If
this were the case, hypoxic conditions should result in gradual Na+
accumulation as passive Na+ uptake is increased and active efflux
downregulated. Increases in intracellular Na+ cause activation of
Na+/K+ pump in mouse red blood cells that overrides
hypoxic deactivation (Bogdanova et al.,
2003b
). Therefore, re-activation of the
Na+/K+ ATPase most probably results from intracellular
Na+ accumulation.
Interestingly, the hypoxic inhibition of the transport activity of
Na+/K+ pump can be abolished by pretreatment of cells
with the scavenger of hydroxyl radicals, MPG. The selectivity of MPG to
scavenging hydroxyl radicals but not H2O2 and superoxide
anion has been proven elsewhere (Sekili et
al., 1993; Sun et al.,
1993
). This observation makes it tempting to suggest that this
radical species is the second messenger involved in transferring information
from so far unknown primary oxygen-binding protein(s) to ion transporters.
Notably, treatment of hepatocytes with MPG suppresses proton extrusion
mediated by Na+/H+ exchanger
(Tuominen et al., 2003
). The
latter finding suggests common regulatory pathways for oxygen-induced
regulation of both ion transporters. Interestingly, similar coupling of two
oxygen-sensitive ion transport systems, the K+/Cl-
cotransporter and Na+/H+ exchanger, mediated by hydroxyl
radicals has been demonstrated in rainbow trout erythrocytes
(Bogdanova and Nikinmaa, 2001
;
Nikinmaa et al., 2003
).
Earlier, it was shown that the transport function of the
Na+/K+ pump in ischemic kidneys could be restored by
application of MPG (Kato and Kako,
1987
).
Paradoxically, measurements using the fluorescent dye H2-DCFDA
did not show upregulation but, instead, a modest decrease in total ROS
formation immediately after the onset of hypoxic exposure. A small but
significant increase in cellular GSH levels could also be observed after a 15
min incubation at a PO2 of 5 kPa, confirming
that the bulk level of oxidants decreased upon acute hypoxic treatment. Such a
dual response has recently been shown in venous endothelial cells where
hypoxia triggers upregulation in free radical production by mitochondria, but
reduces ROS levels originating from NADPH oxidase (Kang et al., 2005). Our
data suggest that, together with decreased bulk ROS levels under hypoxic
conditions, a fraction of responsible
for ion transport modulation appears to be upregulated. Hydroxyl radicals are
produced in Fenton reaction from hydrogen peroxide exclusively in the presence
of a catalyst (Fe2+ or Cu+) and one could assume that a
rate-limiting step in the production of `signaling' hydroxyl radicals is
probably the availability of the catalytic ferrous or cuprous ion, which
becomes accessible for H2O2 upon delocalization only
under hypoxic conditions.
The fluorescent dye used to monitor ROS production is predominantly
oxidized by H2O2 in the presence of
Fe2+-containing enzymes rather than by direct interaction with
hydroxyl radicals or superoxide anion
(LeBel et al., 1992). However,
when formed, hydroxyl radicals cause H2O2 production and
one cannot rule out the impact of
on
the dye oxidation when abnormally high amounts of hydroxyl radicals are
produced (as in CDNB-treated cells). As the reduced form of the dye
H2-DCF is negatively charged, it cannot cross the mitochondrial
membranes and therefore responds only to bulk changes in
H2O2 levels in the cytosol. Acute hypoxic exposure
decreases bulk H2O2 production in the cytosol,
suggesting presence of sufficient amounts of enzymes able to bind and reduce
oxygen. Longer exposure to low oxygen (e.g. 1 kPa) have been shown to cause a
slight increase in cytosolic ROS that may be attributed to leakage of
mitochondrial H2O2 as uncoupling in electron
transduction chain occurs (Bogdanova et
al., 2003a
; Rifkind,
1993
).
Treatment of cells with CDNB results in a rapid depletion of both cytosolic
and mitochondrial GSH pools with a consequent massive increase in cytosolic
ROS in hepatocytes (Han et al.,
2003; Deneke and Fanburg,
1989
). This response differs significantly from that we observed
in CDNB-treated neurons. The difference can be explained by lower amount of
ROS generating systems in neurons where no upregulation in cytosolic ROS
production could be observed using H2-DCFDA in response to CDNB
treatment (I. Petrushanko and A. Bogdanova, unpublished). Depletion of both
cytosolic and mitochondrial GSH pool with CDNB in neurons did not cause
inhibition of hydrolytic activity of the Na+/K+ ATPase
whereas in CDNB-treated hepatocytes the latter was observed independent on
oxygen concentration. Moreover, in neurons CDNB treatment resulted in rapid
ATP depletion and burst in mitochondrial free radical production (I.
Petrushanko and A. Bogdanova, unpublished). ATP levels in hepatocytes were not
affected by CDNB treatment. Since we did not measure mitochondrial ROS
production rate in GSH-depleted trout hepatocytes, we can only speculate on
two possible reasons for this difference. Either for some reasons antioxidant
defense is more powerful in mitochondria of hepatocytes than in neurons, or
other metabolic pathways compensate for suppressed mitochondrial function.
Slight acidification of the cytosol observed in CDNB-treated hepatocytes (the
pH decreases to 7.25±0.03 from 7.40±0.04 in non-treated control)
suggests that lactate production may be upregulated. The latter suggests a
general susceptibility of the Na+/K+ ATPase in trout
hepatocytes to oxidative stress, which does not occur in response to acute
hypoxic insult, despite local increase in hydroxyl radical production.
Oxidative stress induced by GSH depletion or treatment with oxidants was
shown to cause rapid suppression of the transport and hydrolytic
Na+/K+ ATPase function in different tissues, including
neurons, cardiac myocytes and erythrocytes (e.g. (Haddock et al.,
1995a,b
;
Bilgin et al., 1999
;
Cheng et al., 1984
;
Boldyrev and Bulygina, 1997
;
Bogdanova et al., 2003b
) (I.
Petrushanko and A. Bogdanova, unpublished). The only study in which no effect
of CDNB treatment on active K+ transport in human red cells was
reported is the work of Muzyamba and Gibson
(Muzyamba and Gibson, 2003
).
The reasons of particular resistance of the Na+/K+
ATPase to oxidation remains unclear, especially as CDNB treatment of human red
blood cells resulted total disappearance of intracellular GSH, which has never
been the case in other studies where 1 mmol l-1 CDNB was used to
deplete GSH in mammalian erythrocytes
(Lauf et al., 1995
;
Bogdanova et al., 2003b
).
In conclusion, the data we have obtained for hypoxia-induced changes in K+ transport across hepatocyte plasma membrane reveal that active transport is oxygen sensitive. Acute hypoxic exposure resulted in a rapid transient decrease in the transport function of the Na+/K+ ATPase without affecting its hydrolytic activity. The reduction of active K+ uptake could be abolished by treatment of cells with hydroxyl radical scavenger, revealing the importance of this radical species in acute hypoxic signaling and coordination of two ion transport systems: the Na+/K+ pump and Na+/H+ exchanger. Oxygen-induced responses in the transport function of the Na+/K+ ATPase did not directly correlate with changes of cellular ATP or pH. By contrast, oxidative stress induced by treatment of trout hepatocytes with the GSH-conjugating agent CDNB was followed by a marked increase in cytosolic ROS production and resulted in inhibition of both transport and hydrolytic activity of the Na+/K+ ATPase without any significant changes in cellular ATP levels.
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Acknowledgments |
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