Time-dependent expression of heat shock proteins 70 and 90 in tissues of the anoxic western painted turtle
Department of Zoology, University of Toronto, Toronto, ON, Canada, M5S 3G5
* Author for correspondence (e-mail: buckl{at}zoo.utoronto.ca)
Accepted 26 July 2004
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Summary |
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Key words: stress protein, forced dive, recovery, western blot, Chrysemys picta bellii, western painted turtle.
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Introduction |
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One of the most energetically expensive processes in terms of ATP
in the cell is protein biosynthesis. Consequently, its reduction
represents a valid mechanism of saving energy during limited oxygen supply.
Such a mechanism of anoxia adaptation has been demonstrated in C.
picta and has been termed `translational arrest'
(Jackson, 2000). A 50%
reduction in protein synthesis in heart and a 92% reduction in hepatocytes
have been observed in response to anoxia
(Bailey and Driedzic, 1996
;
Land et al., 1993
).
Interestingly, there was an upregulation in expression of five specific
proteins in turtle hepatocytes under anoxia or heat shock but these proteins
were not rigorously identified (Land and
Hochachka, 1995
). Heat shock proteins (Hsps) have been considered
as possible candidates, since their expression is induced by various stresses
such as hyperthermia, radiation, heavy metals, ischemia, anoxia and
reoxygenation in anoxia-sensitive models
(Airaksinen et al., 1998
;
Manzerra et al., 1997
;
Quraishi and Brown, 1995
).
Chang et al. (2000
) provided
the first evidence of increased Hsp70 expression in painted turtle myocardium
subjected to a 12 h forced dive, but the antibody they used did not
distinguish between the constitutive (Hsp73) and inducible (Hsp72) isoform.
More recently, Scott et al.
(2003
) reported an increase in
the expression of Hsp72 but not Hsp73 in brain, heart and skeletal muscle of
western painted turtles force-dived for 24 h at 17°C.
Hsps are highly conserved proteins classified on the basis of their
molecular mass and function. The 70 kDa family of Hsps is the best
characterized and includes different isoforms. The constitutive (Hsp73) and
inducible (Hsp72) isoforms of Hsp70 are both present in the unstressed cell,
where they prevent premature folding of nascent polypeptides and assist
translocation of other proteins to organelles. Following stress, the
expression of Hsp73 is moderately upregulated whereas Hsp72 is highly induced
(Lopez-Barneo et al., 2001;
Manzerra et al., 1997
). Under
this condition they bind to damaged or misfolded polypeptides, either
facilitating their repair or targeting irreparably damaged proteins for
degradation by the ubiquitin/proteasome-dependent pathway
(Lindquist and Craig, 1988
).
In contrast to the widely described 70 kDa family of stress proteins, the 90
kDa Hsp is less well characterized. Under physiological conditions, Hsp90
specifically regulates the activity of other proteins, including steroid
hormone receptors (Scheibel and Buchner,
1998
), the dioxin or aryl hydrocarbon (Ah) receptor
(Perdew, 1988
), protein
kinases (Pratt et al., 1993
),
calmodulin (Someren et al.,
1999
), actin (Miyata and
Yahara, 1991
) and tubulin
(Garnier et al., 1998
).
Similarly to Hsp70, Hsp90 expression is induced in the stressed cell
(Kawagoe et al., 2001
;
Quraishi and Brown, 1995
),
where it binds to partially unfolded proteins, holding them in a
folding-competent state until other chaperones, such as Hsp70, are recruited
to help restore the original structure of the protein.
Increased Hsp72 and Hsp90 expression in mammalian models correlates with
increased protection from hypoxic/anoxic injury (for Hsp72, see
Kawagoe et al., 2001;
Kirino et al., 1991
;
Kitagawa et al., 1990
; for
Hsp90, see Nayeem et al.,
1997
). More specifically, neurons and cardiomyocytes
overexpressing Hsp72 are more resistant to ischemiareperfusion injury
(Marber et al., 1995
;
Yenari et al., 1999
), whereas
Hsp72 antisense oligonucleotides increase cellular injury in mammalian
cardiomyocytes (Nakano et al.,
1997
). Hsp90 levels increase threefold in cardiomyocytes
preconditioned by mild heat shock, conveying protection from a subsequent
anoxic/ischemic treatment (Nayeem et al.,
1997
). This evidence supports a protective role for Hsps in
conditions of limiting oxygen supply. However, while a cytoprotective
mechanism against such a stress is predictable in anoxia-sensitive species, it
is unclear whether or not anoxia is a stressful event for anoxia-tolerant
vertebrates such as the freshwater turtle.
Since this species undergoes `translational arrest' during anoxia
(Jackson, 2000), the specific
upregulation of stress proteins in dived turtles suggests a protective role in
long-term anoxic survival (Chang et al.,
2000
; Scott et al.,
2003
). However, it is not known whether turtle tissues induce Hsp
expression early in a dive period or upregulation of Hsp expression occurs
late in a dive as a part of a rescue mechanism triggered near the limit of
survival.
The primary aim of this study, therefore, was to determine the time frame
in which Hsp72 expression increases in C. picta during a forced dive.
Since Hsp90 expression has also been shown to increase in response to hypoxia
in mammalian brain (Kawagoe et al.,
2001), changes in its time-course of expression during a forced
dive are also determined.
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Materials and methods |
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Male and female turtles Chrysemys picta bellii Schneider 1783, weighing between 250 g and 750 g, were purchased from Lemberger Co., Inc. (Oshkosh, WI, USA). Animals were housed in an indoor pond (2 mx4 mx1.5 m) equipped with basking platform, heating lamp and a flow-through dechlorinated freshwater system. The water temperature was maintained at approximately 17°C and the air temperature at 20°C. Turtles were given continuous access to food and kept on a 12 h:12 h light:dark photoperiod.
Submergence experiment
All animals were left out of the water and allowed to breathe for 1 h prior
to the experiment. Groups of four turtles were placed in nine metal cages. Six
cages containing four animals each were submerged for periods of 2, 6, 12, 18,
24, 30 h. To avoid aeration, all dived animals were removed from their cages
underwater, neck-clamped and decapitated. Another three cages containing four
animals each were first submerged for 12, 24 or 30 h and subsequently removed
from the water and allowed to recover in air for 1 h. Animals were
decapitated; a bone saw and a scalpel were used to separate carapace and
plastron. The pericardium was then incised and a glass syringe used to collect
2 ml of blood from the aortic arc. A sample (200 µl) of blood was
immediately used for determination of blood arterigal
PO2 (PaO2). The
remaining blood was frozen in liquid nitrogen and later used for lactate and
glucose analysis. Brain, heart, liver and pectoralis muscle were quickly
dissected and frozen in liquid nitrogen.
Arterial blood oxygen measurement
Blood was collected via aortic puncture as described above. Blood
PaO2 was determined using an oxygen meter
(OM2000, Cameron Instruments, Yellow Springs, OH, USA) previously calibrated
with distilled water equilibrated with room air (156 mmHg; 1 mmHg=98 kPa) or
100% nitrogen bubbled water (0 mmHg).
Deproteinization and neutralization of blood samples
Deproteinization of blood samples was performed by adding 50 µl of 70%
perchloric acid to 450 µl of blood. The mixture was sonicated on ice for 30
s and subsequently centrifuged at 10 000 g for 10 min at
2°C. The supernatant (300 µl) was collected and a 150 µl volume of
neutralizing buffer (KOH/Tris/KCl) was added to remove the perchlorate. The
mixture was allowed to stand for 15 min on ice. The supernatant was collected,
frozen in liquid nitrogen and stored at 20°C until used for lactate
or glucose assay.
Lactate and glucose assay
Blood samples were analyzed for lactate and glucose content using a
standard enzyme-coupled assay (Noll,
1974) and a modified enzymatic method
(Keppler and Decker, 1974
),
respectively.
Tissue preparation and protein assay
Frozen tissues from control (N=5), 2, 6, 12, 24, 30 h dived
(N=4 each) and 12, 24, 30 h dived and 1 h recovered (N=4
each) turtles were sonicated on ice in 50 mmol l1 Tris, pH
7.4), 0.5 mmol l1 1,4-dithio-DL-threitol (DTT)
and 0.5% Tween 20 at approximately 1 min intervals. The homogenates were
centrifuged at 10 000 g, at 2°C for 10 min. The
supernatant fraction was collected and used for protein analysis.
Protein concentrations were determined with a bicinchoninic acid kit (BCA protein assay kit; Pierce, Rockford, IL, USA), using bovine serum albumin (BSA) as a standard.
Western blot analysis
Western blot analysis was performed according to Scott et al.
(2003). Samples were boiled
for 5 min in loading buffer (1:1) containing sodium dodecyl sulfate (SDS;
2.5%) and 2-ß-mercaptoethanol (10%). Following a 1 min centrifugation at
10 000 g, equal amounts of protein (50 µg for Hsp72 and
Hsp73 detection; 150 µg for Hsp90 detection) were loaded onto
SDS-polyacrylamide gradient (4%10%) gels and separated by
electrophoresis at 110 V for 1.5 h. Prestained molecular mass markers
(Invitrogen, Burlington, ON, Canada) were used to determine the migration of
the proteins on the gel. In addition, 10 ng of pure Hsp73 (SPP-751; StressGen,
Victoria, BC, Canada) or 10 ng of pure Hsp72 (NSP-555; StressGen) or 30 ng of
pure Hsp90 (SPP-770; Stressgen) were used as internal controls. Proteins were
transferred to nitrocellulose membranes (NitroBind, Westborough, MA, USA) by
electroblotting using a Novex (San Diego, CA, USA) XCell II Mini-Cell and Blot
Module unit set at 25 V or 100 mA for 2.5 h. Membranes were blocked overnight
at 4°C in Tris buffer saline-Tween 20 (pH 7.5) (TBS-T; 20 mmol
l1 Tris, 500 mmol l1 NaCl and 0.05% Tween
20) containing 5% nonfat dry skim milk. Following 2x 5 min washes with
TBS-T, the blots were incubated for 4 h at room temperature with rabbit
polyclonal antibody against Hsp72 (SPA-812; StressGen) diluted 1:5000 in TBS-T
with 2% non-fat dry skim milk, or rat monoclonal antibody against Hsp73
(SPA-815; StressGen) diluted 1:3000 in TBS-T with 2% non-fat dry skim milk, or
rat monoclonal antibody against Hsp90 (SPA-835; StressGen) diluted 1:200 in
TBS-T with 2% non-fat dry skim milk. The primary mammalian antibodies against
Hsp72 and Hsp73 used in this study are specific for the two turtle Hsp70
isoforms as demonstrated via two-dimensional gel electrophoretic
analysis (Scott et al.,
2003
).
Following incubation with the primary antibodies, the blots were washed for 2x 5min with TBS-T and subsequently incubated for 1 h at room temperature with alkaline phosphatase (AP)-conjugated affinity-purified goat anti-rabbit (A3687; Sigma, Oakville, ON, Canada) or goat anti-rat (SAB-201; Stressgen) diluted 1:3000 in TBS-T with 2% non-fat dry skim milk. The blots were washed for 1x 5min with TBS-T and 1x 5min with TBS. Immunoreactivity was visualized by colorimetric reaction using nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP) (Invitrogen) as a chromagenic substrate for AP. Hsp73, Hsp72 and Hsp90 blots (samples and pure proteins) were incubated with NBT/BCIP for 7 min, 5 min and 5 min, respectively. The reaction was arrested by washing the blots with distilled water several times. Membranes were allowed to dry overnight.
To quantify the amount of Hsp73, Hsp72 and Hsp90 expressed in the tissues, standard curves were generated using pure Hsp73, Hsp72 and Hsp90 as standards. The regression equations for the Hsp73, Hsp72 and Hsp90 standard curves are y=4.00x+11.13, r2=0.95; y=3.50x+3.50, r2=0.97; and y=0.42x+0.44, r2=0.95, respectively. Membranes were scanned with a Hewlett Packard DeskScan II scanner and bands were quantified with Lab Works 4.0 Image Analysis Software (Bioimaging System, GDS 8000, UVP Inc., Upland, CA, USA).
Statistical analysis
All data are expressed as mean ±
S.E.M. Statistical analysis for
PO2 measurements was performed using an
unpaired Student's t-test. Differences were considered statistically
significant when P<0.05. Statistical analyses for lactate and
glucose determinations as well as for dive and recovery data were performed
using a one-way analysis of variance (ANOVA) followed by a
StudentNewmanKeuls post-hoc test (P<0.05).
Statistical analyses were performed using SigmaStat version 1.0 (Jandel
Corporation, Chicago, IL, USA).
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Results |
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Lactate and glucose measurement
To confirm the anoxic status of the animals during the dive, anaerobic
metabolism was assessed by measuring the level of the glycolytic end-product
lactate in the blood of control, dived and recovery turtles
(Fig. 1). Lactate
concentrations significantly increased from a control level of
2.7±0.4mmol l1 to 56.6±3.6mmol
l1 after a 30 h dive. To establish whether the level of
metabolic substrate was a limiting factor during the dive, blood glucose
concentrations were determined under control conditions, after the dive and
following recovery (Fig. 1).
Glucose concentrations significantly increased from a control level of
2.3±0.5 mmol l1 to 20.4±0.8 mmol
l1 after a 24 h dive and 11.5±2.8 mmol
l1 after a 30 h dive. Recovery for 1 h had no significant
impact on lactate and glucose levels (data not shown).
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Control experiment
To test the ability of Hsp expression to increase above baseline levels,
painted turtles were exposed to a 40°C heat shock for 1 h followed by 1 h
recovery at room temperature as previously described
(Scott et al., 2003). The
expression of Hsp73, 72 and 90 in turtle brain, heart, liver and skeletal
muscle followed the expected trend reported by Scott et al.
(2003
). Hsp72 expression
increased in all tissues, Hsp73 expression increased only in heart and Hsp90
expression increased in brain and liver but not in heart and muscle (data not
shown).
Hsp73, 72 and 90 expression following dive and recovery
To determine if the level of heat shock protein expression changes in
turtles from the onset of anoxia through a 30 h dive and after normoxic
recovery, the expression of Hsp73, 72 and 90 was determined by western blot
analysis in brain, heart, liver and skeletal muscle of normoxic, anoxic and
recovery turtles.
Brain
Hsp73 expression in brain increased significantly from a basal level of
39±1.1 pg µg1 tissue to 88±6.6 pg
µg1 tissue after a 30 h dive
(Fig. 2) and remained elevated
following recovery (Fig.
3).
|
|
Similarly, brain Hsp72 expression increased from a control value of 12±1.9 pg µg1 tissue to 23±1.2 pg µg1 tissue after a 30 h dive (Fig. 4) and up to 36±2.1 pg µg1 tissue following recovery (Fig. 5).
|
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Basal Hsp90 expression in brain was 4.6±0.3 pg µg1 tissue but significantly increased to 7.7±0.7 pg µg1 tissue after the 18 h dive and to 15±0.6 pg µg1 tissue after the 24 h dive (Fig. 6) and following recovery (Fig. 7).
|
|
Heart
Constitutive expression of Hsp73 in heart remained at control levels
(5.3±0.7 pg µg1 tissue) throughout the 30 h dive
period (Fig. 2) and following
recovery (Fig. 3).
Heart Hsp72 expression increased significantly from a control level of 19.9±2.9 pg µg1 tissue to 32±1.8 pg µg1 tissue following the 24 h dive and further increased to 70±1.9 pg µg1 tissue following the 30 h dive (Fig. 4). The expression of Hsp72 in heart significantly increased throughout recovery (Fig. 5).
Similar to Hsp73, expression of Hsp90 in heart remained at control levels (2.2±0.1 pg µg1 tissue) throughout the 30 h dive period (Fig. 6) but increased up to 8.2±0.4 pg µg1 tissue following recovery (Fig. 7).
Liver
Basal expression of Hsp73 in liver increased from a control level of
9.0±1.4 pg µg1 tissue to 27±0.6 pg
µg1 tissue following a 30 h dive
(Fig. 2) and recovery
(Fig. 3).
Hsp72 expression in liver followed a rather peculiar trend. It decreased from 26.3±2.7 pg µg1 tissue under normoxic conditions to 10.8±0.6 pg µg1 tissue following the 24 h dive but it significantly increased to 44±5.5 pg µg1 tissue following a 30 h dive (Fig. 4) and up to 60±8.5 pg µg1 tissue following 1 h recovery (Fig. 5).
Similar to the trend observed for brain Hsp90, basal expression of Hsp90 in liver (2.7±0.4 pg µg1 tissue) increased to 4.3±0.2 pg µg1 tissue after the 18 h dive and further increased to 5.6±0.7 pg µg1 tissue after the 24 h and 30 h dive (Fig. 6). The expression of Hsp90 in the liver of recovery turtles was significantly higher than control levels (Fig. 7).
Pectoralis muscle
The expression of Hsp73 followed the same trend in skeletal muscle as in
heart. It remained at normoxic level (2.5±0.1 pg
µg1 tissue) throughout the 30 h dive period and following
recovery (Figs 2,
3).
Hsp72 expression followed the same trend in skeletal muscle as in heart. Muscle Hsp72 expression remained at a control level of 9.3±0.7 pg µg1 tissue throughout the 30 h dive period but increased up to 49±4.7 pg µg1 tissue following the 30 h dive (Fig. 4) and up to 70±3.8 pg µg1 tissue following 1 h recovery (Fig. 5).
The basal level of Hsp90 expression in skeletal muscle (1.1±0.2 pg µg1 tissue) significantly increased to 2.6±0.4 pg µg1 tissue following the 24 h and 30 h dives (Fig. 6) and increased to 6.1±1.6 pg µg1 tissue following recovery (Fig. 7).
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Discussion |
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The ability of anoxia-tolerant species to survive long periods without
oxygen is partly dependent on `translational arrest'
(Jackson, 2000). Since protein
biosynthesis is an expensive process in terms of ATP usage, its depression is
essential to anoxic survival. In anoxic painted turtles, protein synthesis has
been reported to be only 10% of the normoxic level in liver and 50% of the
normoxic level in heart (Bailey and
Driedzic, 1996
; Land et al.,
1993
). In light of these findings, it is unclear whether or not
Hsps would be produced during anoxia. Interestingly, two recent studies
reported an increase in the expression of Hsps in tissues from the western
painted turtle exposed to long-term anoxia
(Chang et al., 2000
;
Scott et al., 2003
). However,
it is still unknown whether Hsp expression increases early, at the onset of
anoxia, and remains at high levels throughout the anoxic period, or if it is
only upregulated late during anoxia as part of a rescue mechanism triggered at
the limit of survival. To understand whether or not increased Hsp expression
is critical to anoxic survival, Hsp73, 72 and 90 expression was monitored in
four tissues of the western painted turtle from the onset of anoxia throughout
a 30 h forced dive and following reoxygenation.
The results of this study show that, with one exception, the expression of the Hsps examined in turtle brain, heart, liver and skeletal muscle did not change significantly from normoxic levels at the onset of anoxia but rapidly increased late during the dive. The exception was the liver, where basal Hsp72 expression decreased significantly throughout the dive before raising above normoxic values at the end of the anoxic period. As expected, expression of all Hsps was generally sustained or even increased upon reoxygenation. In addition, these results indicate a tissue-specific expression pattern during both anoxia and recovery.
Anaerobic status and substrate availability
As indicated by arterial blood PO2
(PaO2) levels, turtles force-dived for 30 h
were anoxic throughout the dive and had returned to normoxic levels following
1 h recovery. These findings agree with previously reported values, where
blood PaO2 levels dropped from normoxic values
of 88 mmHg to 1.4 mmHg after a 6 h dive at 20°C
(Crocker et al., 1999;
Herbert and Jackson,
1985a
).
Blood lactate levels were significantly higher than normoxic levels in
turtles exposed to a 2 h dive and continued to increase throughout the longer
dives, reaching 50 mmol l1 after 30 h of submergence. This
is a good indication that force-dived turtles were relying on anaerobic
metabolism (glycolysis). The data are supported by previous studies where
lactate levels in painted turtles increased from control levels of about 2
mmol l1 to as high as 200 mmol
(Chang et al., 2000;
Crocker et al., 1999
;
Herbert and Jackson, 1985a
;
Ultsch and Jackson, 1982
).
Together with an increase in the level of blood lactate during the dive,
the present study also shows increases in blood glucose, the main metabolic
substrate of anaerobic glycolysis. Blood glucose levels were significantly
higher than normoxic levels in turtles exposed to a 2 h dive and continued to
increase up to 20 mmol l1 after a 24 h dive. These results
are supported by studies where plasma glucose concentrations increased above
normoxic levels to about 10 mmol l1 and 16 mmol
l1 after 2 and 6 h anoxic dives at 22°C, respectively
(Clark and Miller, 1973;
Daw et al., 1967
;
Keiver and Hochachka, 1991
;
Keiver et al., 1992
). Blood
glucose levels reached a peak at 24 h in dived turtles but significantly
decreased at 30 h.Although this is only one time point and a descending trend
cannot be extrapolated, the same pattern has been observed previously
(Daw et al., 1967
).
Increased plasma glucose levels are probably the result of hepatic
glycogenolysis; however, the importance of this is unclear. It could be
essential to sustain glycolysis in the brain where the level of glycogen is
low (1% w/w) compared to that of liver (15% w/w)
(Clark and Miller, 1973;
Sick et al., 1993
).
Hsp70 and Hsp90 expression following dive and recovery
The expression of constitutive Hsp73, inducible Hsp72 and Hsp90 in C.
picta brain, heart, liver and skeletal muscle did not increase
significantly from normoxic levels during the first 12 h of a forced dive. One
exception to this trend was liver Hsp72, where expression followed a curious
pattern. During the dive it showed an early drop from normoxic values,
reaching a significant threefold decrease by 24 h. Interestingly, forced-dive
expression of Hsp73 and 72 in brain and liver increased two- to three-fold
above normoxic levels at 30 h. The same forced-dive-mediated increase was
detected for brain and liver Hsp90 but it occurred earlier, at 18 h. While the
expression of heart and muscle Hsp73 as well as heart Hsp90 remained at
normoxic levels throughout the entire dive, muscle Hsp90 as well as heart and
muscle Hsp72 increased two- to fourfold at the 24 h and 30 h dive times.
Lack of early induction of Hsp73, 72 and 90 in anoxic painted turtle
tissues suggests that increased expression of these Hsps is not critical in
this species' adaptation to tolerate anoxia. It is unlikely that lack of early
Hsp induction results from an increased basal level of Hsp expression in
anoxia-tolerant species compared to anoxia-sensitive species, since it has
been demonstrated that normoxic levels of heart Hsp72/73 were not
significantly different in the highly anoxia-tolerant painted turtle, the
relatively less anoxia-tolerant softshell turtle and the anoxia-sensitive rat
and rabbit (Chang et al.,
2000). Given the lower metabolic rate, lower ion channel densities
and lower enzyme levels of an ectotherm such as C. picta compared to
those of an endothermic mammal, the lack of a difference in Hsp72/73
expression actually suggests that Hsp levels are somewhat elevated in C.
picta. This may represent an important survival strategy for
anoxia-tolerant species, although our results indicate that further increases
in Hsp expression are also important.
The only previous study on the effect of short-term anoxia on the
expression of Hsps in anoxia-tolerant species is that of Clegg et al.
(2000), who monitored changes
in the expression level of combined Hsp72/73 and Hsp90 in encysted embryos of
the brine shrimp Artemia franciscana exposed to 38 days and 2.6 years
of anoxia. Since this crustacean is able to survive almost 7 years of anoxia,
a 2.6 year period could be considered a relatively short-term anoxia. They
reported no changes from normoxic levels in the expression of these Hsps
during either anoxic period. Their findings support the results from this
study.
A surprising result of the present study was the decrease in the expression
of liver Hsp72 observed during short-term anoxia. Although not confirmed by
this study, such a pattern has been observed in our laboratory for painted
turtle liver Hsp73 expression following heat shock and a 24 h forced dive
(Scott et al., 2003). The
reduced levels of basal Hsp73 expression could result from reduced levels of
protein synthesis, especially in the liver. Protein synthesis decreases by 92%
in C. picta hepatocytes during anoxia (Land et al., 1995). This has
also been demonstrated in the anoxia-tolerant crucian carp Carassius
carassius where, following a 48 hanoxic exposure, protein synthesis in
liver decreased by 95% of normoxic levels
(Smith et al., 1996
).
While the expression of Hsps (with the exception of liver Hsp72) remained
at normoxic levels for most of the dive, a rapid increase was observed late in
the dive protocol. These findings are supported by a previous study where the
combined expression of Hsp72/73 was measured in anoxia-tolerant painted
turtles and the less anoxia-tolerant softshell turtle heart exposed to a 12 h
anoxic dive and recovery at 22°C (Chang
et al., 2000). While the level of normoxic Hsp72/73 did not differ
significantly between the two species, it increased in painted turtle heart
from basal levels of 2.8 mg g1 to 3.9 mg
g1 following the dive but decreased in softshell turtle
heart from basal level of 2.4 mg g1 to 1.3 mg
g1 following the dive. We recently extended this study
(Scott et al., 2003
) to brain,
liver and skeletal muscle tissues, distinguishing between the constitutive
(Hsp73) and the inducible (Hsp72) isoform of Hsp70. Western painted turtles
exposed to a 24 h forced dive and 1 h recovery at 17°C exhibited a pattern
of Hsp72 and Hsp73 expression that was tissue-specific under both normoxic and
anoxic conditions. Following the dive, expression of Hsp73 remained at
normoxic levels in all tissues but the liver, where it decreased significantly
from 70 pg µgg1 tissue to 46 pg µg1
tissue. In contrast, Hsp72 expression increased in all tissues but the liver,
where a downward trend was observed. With the exception of liver Hsp73, these
results support the findings from the present study. The level of expression
of liver Hsp73 previously measured was generally higher than that observed in
this study (Scott et al.,
2003
). Additionally, the previously observed dive-induced decrease
in liver Hsp73 expression was not detected in this study.
While the expression of Hsp72 and Hsp73 has been investigated in the
anoxia-tolerant freshwater turtle (Chang et
al., 2000; Scott et al.,
2003
), this is the first time that the expression of Hsp90 has
been monitored in this species. The pattern of Hsp90 expression in turtle
tissues is similar to that of Hsp73 and Hsp72; it remains at control levels
early during anoxia and increases above normoxic levels during long-term
anoxia. However, the upregulation of Hsp90 expression occurs earlier in the
dive than Hsp73 or Hsp72. As mentioned in the introduction, Hsp90 works in
concert with other Hsps by binding to partially unfolded proteins in
preparation for the binding of other Hsps (Hsp72) to complete the refolding
process. Thus, the early increase in Hsp90 expression that we observed may
occur for this purpose.
One well-documented change in turtles exposed to anoxia is a decrease in
extracellular and intracellular pH (Clark
and Miller, 1973; Herbert and Jackson,
1985a
,b
;
Jackson and Heisler, 1983
;
Ultsch and Jackson, 1982
;
Wasser et al., 1991
). Blood pH
of normoxic turtles is 7.8; following 2 h submergence in aerated water at
20°C it decreases by 0.4 units, by 0.6 units at 6 h and by 0.7 units at 12
h (Herbert and Jackson,
1985a
,b
).
Intracellular pH also decreases: after 6 h submergence at 20°C, brain
intracellular pH decreases from 7.5 to 6.9, heart pH from 7.2 to 7.0, liver pH
from 7.5 to 6.9 and skeletal muscle pH from 7.2 to 6.8. Ultsch and Jackson
(1982
) monitored changes in
arterial pH in turtles submerged at 3°C. The last pH value recorded before
death ranged from 6.7 to 7.0 and they suggest that this could represent the
lower limit to survival. Although changes in pH have not been monitored in
turtles force-dived at 17°C, it is possible to extend the findings from
previous work to this study and assume that turtles force-dived at 17°C
experienced acidosis by 12 h of anoxia. Furthermore, lactate, an indicator of
cellular acidosis, increased significantly at this temperature and time
frame.
High concentrations of hydrogen ions interact with proteins and disrupt
non-covalent bonds that hold proteins in a proper folded state. Interestingly,
changes in intracellular pH can trigger Hsp induction
(Narasimhan et al., 1996;
Nishimura et al., 1989
).
Indeed, exposing cultured rat astrocytes to acidic medium (pH 5.5) for 3 h
induced the expression of a protein belonging to the Hsp70 (68 kDa) family of
stress proteins (Nishimura et al.,
1989
). Also, brief exposure of cultured cortical rat astrocytes to
acid (pH 5.2 for 40 min) markedly induced Hsp70 mRNA and protein expression
(Narasimhan et al., 1996
). In
addition, they showed that heat pretreatment enhanced astrocyte survival
against acidosis. More recently, the rate of survival of crucian carp exposed
directly to acidic water (pH 4.5) for 2 h was compared with the rate of
survival of carp pretreated with a 2 h period of heat shock at 33°C before
a longer (24 h) acidic shock (Martin et
al., 1998
). Preconditioning increased acidosis resistance in
pretreated fish. In addition, the expression of Hsp70 in nervous tissues
(brain and spinal cord) from pretreated carp increased by 40% of control
levels.
Consistent with our previous findings from C. picta
(Scott et al., 2003) as well
as from other non-mammalian (Airaksinen et
al., 1998
) and mammalian species
(Krueger et al., 1999
), this
study shows a tissue-specific pattern in the expression of the Hsps examined.
Remarkable is the relatively high level of expression of Hsp73 in the brain of
normoxic turtles with respect to heart, liver and muscle basal Hsp73 levels.
As previously suggested (Manzerra et al.,
1997
), high levels of Hsp73 could reflect the involvement of this
stress protein in axonal transport of neuronal proteins and synaptic vesicle
recycling. Hsp73 is also thought to interact with PDZ-like domains, in the
organization of postsynaptic structures and clustering of neurotransmitter
receptors and ion channels (Feng and
Gierasch, 1998
). In addition, observation of stress-induced
nuclear relocalization of Hsp73 has suggested a possible role as a nuclear
shuttle for various proteins (Hayashi et
al., 1991
).
Perspectives
Hochachka et al. (1996)
proposed that the response of hypoxia-tolerant systems to oxygen lack occurs
in two phases: `defense' and `rescue'. The first line of `defense' against
hypoxia includes a coordinated suppression of ATP-consuming and ATP-producing
pathways to reach a new steady state level of ATP, even when energy turnover
is suppressed by a factor of almost tenfold
(Buck and Hochachka, 1993
).
While ATP demands of ion pumping are downregulated by generalized `channel
arrest' in cells (Hochachka,
1986
) and by `spike arrest' in neurons
(Sick et al., 1993
), ATP
demands of protein synthesis are downregulated by `translational arrest'
(Jackson, 2000
). In
hypoxia-sensitive cells `translational arrest' seems to be irreversible. In
contrast, if the period of oxygen lack is extended, hypoxia-tolerant systems
activate `rescue' mechanisms by preferentially regulating the expression of
several proteins. The theory of a molecular `rescue' phase was proposed based
on the observation that, under conditions of prolonged O2 lack, the
expression of five proteins in painted turtle hepatocytes was preferentially
upregulated whereas the expression of four proteins was preferentially
downregulated (Land and Hochachka,
1995
).
In this study, lack of early induction of Hsp expression followed by a late
upregulation of stress proteins during anoxia strongly supports the theory of
`defense' and `rescue' phases in the adaptation to anoxic survival. Two time
points are essential in determining the chronological development of this
adaptation. First, the transition from normoxia to anoxia must be detected by
oxygen sensing mechanism(s) and the message delivered by signal transduction
pathways that will eventually trigger the `defense' phase. Second, the
transition from short-term anoxia to long-term anoxia must be detected to
induce the `rescue' phase. Although various oxygen sensing mechanisms have
been proposed (reviewed in Lopez-Barneo et
al., 2001), the theory of a transition from short- to long-term
anoxia is new and the sensors upstream from the `rescue' phase are still
unknown.
The definition of short- and long-term anoxia is relative to the length of
time a given species can survive without oxygen and is generally
temperature-dependent. The freshwater turtle Chrysemys picta bellii
has been shown to fully recover from anoxia after 12 h at 20°C, 3 days at
15°C, 10 days at 10°C and 90 days at 3°C (Herbert and Jackson,
1985a,b
).
However, experiments that monitored survival time in this species reported
that painted turtles can live for approximately 24 h at 26° and 155 days
at 1.5°C (Musacchia,
1959
). In the present study, painted turtles were force-dived for
30 h at 17°C. Although no report has described the survival time of
turtles at this temperature, 30 h of anoxia is probably close to their limit
of survival at 17°C. We propose that changing pH is the challenge that
signals the transition from the short-term to the long-term anoxia.
In summary, we show no early induction of Hsp73, 72 and 90 expression in four tissues from force-dived western painted turtles, suggesting that increased Hsp expression is not critical in the early adaptation to anoxic survival and that short-term anoxia is probably not a stress for species adapted to survive long periods without oxygen. However, a rapid increase in the expression of all stress proteins examined occurs late during the forced dive, suggesting that increased expression of stress proteins could be part of a rescue mechanism triggered at the limit of survival.
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References |
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