First published online May 5, 2005
Journal of Experimental Biology 208, 1793-1801 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01572
Hypoxic responses of Na+/K+ ATPase in trout hepatocytes
A. Bogdanova1,*,
B. Grenacher1,
M. Nikinmaa2 and
M. Gassmann1
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
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Fig. 1. Changes in H2O2 levels in trout hepatocytes in
response to hypoxia, MPG and CDNB. (A) Original traces of the changes in
intensity of DCF fluorescence from individual cells over time. (B) Average
changes in the rate of increase in fluorescence during the first 300 s and an
interval of 800 to 1100 s of recording. Onset of hypoxic exposure or addition
of 5 mmol l-1 MPG or 1 mmol l-1 CDNB corresponded to the
400 s time point. When analyzed, data for the corresponding intervals of time
were fitted using linear regression and slope of each curve used to
characterize increase in fluorescence for single cells with time. Presented
values are means ± S.E.M. for 28-35
individual cells. * indicates P<0.05.
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Fig. 2. Cellular reduced glutathione levels as a function of oxygen concentration
and treatment with the conjugating agent CDNB. GSH levels in cells incubated
for 15 min at 21, 5 or 1% O2 in the presence or in the absence of 1
mm CDNB. Data are means ± S.E.M. of
four independent experiments. *P<0.05 and
**P<0.01.
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Fig. 3. ATP levels in cells incubated for 15 min at 21, 5 or 1% O2 alone
or in the presence of 1 mmol l-1 CDNB, 5 mmol l-1
N-acetyl cysteine or 5 mmol l-1 GSH. Data are means
± S.E.M. of five independent
experiments.
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Fig. 4. Hydrolytic activity of Na+/K+ ATPase after exposure
to different oxygen concentrations and manipulation of cellular redox state.
Cells were exposed for 15 min to 21, 5 or 1% O2 in the presence or
in the absence of 1 mmol l-1 CDNB, 5 mmol l-1
N-acetyl cysteine or GSH. After incubation, the cells were
immediately destroyed by repetitive freezing-thawing and ouabain-sensitive
production of Pi measured in cell homogenate in the presence of 3
mmol l-1 ATP. Data are means ±
S.E.M. of five independent experiments.
*P<0.05.
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Fig. 5. K+ influx into trout hepatocytes as a function of oxygen
concentration. (A) Active and passive components of K influx as a function of
oxygen concentration. Active K+ influx was calculated as difference
in K+(86Rb) uptake in the presence or in the absence of
100 µmol l-1 ouabain. Cells were incubated at different oxygen
concentrations for 15 min. Data are means of five independent experiments
± S.E.M. According to the results of
Dunnett multiple comparison test (one-way ANOVA),
*P<0.05 and **P<0.01 when
compared with 21% O2 `control'. (B) Kinetics of the changes in
active K+ influx and cellular ATP levels in trout hepatocytes
exposed to 1% O2. Data are means ±
S.E.M. of four or five independent
experiments. *P<0.05.
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Fig. 6. Redox-sensitivity of active (A) and passive (B) K+ influx in
cells incubated at 21 or 1% O2. Cells were exposed to hypoxic or
normoxic conditions in the presence or in the absence of 1 mmol l-1
CDNB or 5 mmol l-1 MPG for 15 min. Data are means ±
S.E.M. of four independent experiments.
*P<0.05; **P<0.01.
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© The Company of Biologists Ltd 2005
