Tissue-specific expression of inducible and constitutive Hsp70 isoforms in the western painted turtle
Department of Zoology, University of Toronto, 25 Harbord St, Toronto, ON, Canada, M5S 3G5
* Author for correspondence (e-mail: buckl{at}zoo.utoronto.ca)
Accepted 28 October 2002
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Summary |
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Key words: stress proteins, forced dive, recovery, heat shock, western blot, Chrysemys picta belli, western painted turtle
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Introduction |
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The expression of heat-shock proteins (Hsps) is known to increase in
response to various stresses, including hyperthermia, oxygen limitation
(ischemia), radiation, heavy metals, anoxia and reoxygenation
(Lindquist and Craig, 1988).
Many heat-shock proteins are considered ATP-dependent molecular chaperones and
thus recognize unfolded, misfolded and aggregated proteins
(Welch, 1992
;
Xu and Lindquist, 1993
). As
molecular chaperones, Hsps share an ability to modulate the folding and
unfolding of other proteins and to facilitate the assembly and disassembly of
multisubunit complexes (Lindquist and
Craig, 1988
).
The 70 kDa family of stress proteins is highly conserved, and several
isoforms exist (Creagh et al.,
2000; Lindquist and Craig,
1988
). In mammals, a constitutive isoform of Hsp70 (Hsp73) is
expressed and demonstrates moderate stress inducibility under certain
conditions (Manzerra et al.,
1997
). Under non-stress conditions, Hsp73 proteins are thought to
act as chaperones for other cellular proteins by binding to nascent
polypeptides and preventing premature folding, as well as translocating
proteins into organelles. By contrast, the stress-inducible isoform of Hsp70
(Hsp72) is generally not expressed in unstressed cells; however, upon exposure
to stressful conditions, Hsp72 is highly inducible. During conditions of
stress, both Hsp72 and Hsp73 are thought to bind to damaged and misfolded
polypeptides and to facilitate repair
(Lindquist and Craig,
1988
).
As protein synthesis is a costly process in terms of energetics, requiring
the hydrolysis of four ATP equivalents per peptide bond and one additional ATP
for amino acid transport, the upregulation of specific heat-shock proteins in
energy-compromised anoxic tissues indicates that Hsps may play a role in
promoting anoxia tolerance. Indeed, increased expression of Hsp72 in mammalian
brain and heart does correlate with increased protection from hypoxic/anoxic
injury (Marber et al., 1995;
Yenari et al., 1999
). However,
it is unclear whether Hsp72 expression is responsible for the protective
effect or a phenomenon of it. More recently, the protective role of Hsp70 has
been tested directly in cells made to overexpress Hsp72 and in cells where
expression is blocked with antisense oligonucleotides
(Marber et al., 1995
;
Yenari et al., 1999
). In these
instances, Hsp72 overexpression in heart and neuronal tissue was clearly
protective during ischemia, and blocking Hsp72 expression during hypoxic
stress in mammalian cardiomyocytes with an Hsp72 antisense oligonucleotide
leads to increased cellular injury (Nakano
et al., 1997
). In addition, transgenic mice overexpressing Hsp70
in their myocardium are more resistant to ischemia reperfusion injury
(Marber et al., 1995
).
Therefore, increased Hsp72 expression during anoxia or ischemia is more than
just strongly correlative; increased Hsp72 expression promotes anoxia
tolerance. However, whether increased Hsp72 expression in a non-mammalian
anoxia-tolerant vertebrate, such as the freshwater turtle, is important for
long-term anoxic survival remains unknown.
The main focus of the present study is to investigate the tissue-specific expression of Hsp73 and Hsp72 in response to heat stress and force dive-induced anoxia in the western painted turtle. In an animal adapted for long-term breath-hold diving, it is unclear whether anoxia is stressful to the animal. Thus, the purpose of this study is threefold: first, to expose the western painted turtle to a hyperthermic stress to determine the magnitude of Hsp73 and Hsp72 expression in brain, heart, liver and skeletal muscle; second, to examine Hsp73 and Hsp72 expression in these four tissues after a 24 h forced dive and 1 h recovery; and third, to quantify the relative expression of Hsp73 and Hsp72 in these tissues.
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Materials and methods |
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Western painted turtles, Chrysemys picta belli Schneider 1783, weighing between 590 g and 760 g were commercially obtained from Lemberger Co., Inc. (Oshkosh, WI, USA). Animals were housed in an indoor pond (2 mx4 mx1.5 m) with a sloping floor; one end contained water approximately 0.5 m deep, while the other end had a shallow, rocky basking platform with a heating lamp. The aquarium was equipped with a flow-through dechlorinated freshwater system, with water temperature maintained at approximately 17°C. Turtles were given continuous access to food and kept at a room temperature of 20°C on a 12 h: 12 h light:dark cycle.
Experimental setup
Hyperthermic experiment
A Plexiglas container of dimensions 0.5 mx0.5 mx0.25 m (with a
Plexiglas cover) was half filled with water heated to a temperature of
35°C. Four turtles were placed in the heated pool for 1 h, after which
time the container was drained and refilled with room-temperature water
(20°C). The turtles remained in the room-temperature bath for 5 h, at
which point turtles were killed by decapitation. Brain, heart, liver and
skeletal muscle tissue were rapidly dissected and immediately frozen in liquid
nitrogen.
Submergence experiment
Three empty metal cages were placed in the indoor pond for several days to
allow turtles to acclimate to their presence. Water temperature and oxygen
partial pressure (PO2) were 17°C and 5865.2
Pa, respectively. Three turtles were placed in each cage, then gradually
submerged and left undisturbed for 24 h. Following this period, five animals
had their necks clamped underwater (to prevent the possibility of aeration)
and were killed by decapitation. The remaining four turtles were brought to
the surface and allowed access to air for 1 h, after which time they were
sacrificed. Four other turtles, which were not caged and had free access to
air, were sacrificed as controls following the same 24 h period. Animals were
cut open with a bone saw and scalpel, and the brain, heart, liver and skeletal
muscle were dissected in less than 3 min, wrapped in aluminum foil and frozen
in liquid nitrogen. Aortic blood was collected via a glass syringe
for PO2 analysis. Additional aortic blood was
quickly collected and frozen in liquid nitrogen to be used for lactate
analysis.
Experimental protocol
To determine the degree of anoxia in the dived turtles, blood
PO2 and lactate levels were measured.
Blood PO2 measurement
1 ml of aortic blood was collected via a glass syringe for
PO2 analysis using an oxygen meter (OM2000,
Cameron Instruments, Port Aransas, TX, USA) previously calibrated with
nitrogen-bubbled distilled water.
Deproteinization of blood samples
To deproteinize the blood samples, 450 µl of blood was mixed with 50
µl of ice-cold 70% perchloric acid and sonicated for 30 s. The mixture was
then centrifuged at 10 000 g for 10 min at 2°C. A 100
µl volume of neutralizing buffer (3 moll-1 KOH, 0.4
moll-1 Tris, 0.3 moll-1 KCl) was added to the
supernatant and centrifuged at 10 000 g for 10 min at 2°C.
The mixture was allowed to stand for 15 min on ice, at which time the
supernatant was removed and stored at -20°C until used for lactate
assay.
Lactate assay
Samples were analyzed for lactate content using a standard enzyme-coupled
assay (Noll, 1974).
Two-dimensional gel analysis
Isoelectric focusing (IEF) followed the technique of O'Farrell
(1975) with the modifications
described by Rodenhiser et al.
(1985
). Glass tubes (15
cmx4 mm) were rinsed with double-distilled water, dried and one end
covered with parafilm. An acrylamide solution consisting of 5.5 g urea, 1.98
ml H2O, 2 ml 10% (octylphenoxy)polyethoxyethanol (IGEPAL), 300
µl ampholines pH 5.0-8.0, 200 µl ampholines pH 3.5-10, 1.33 ml 40%
acrylamide, 11 µl 10% ammonium persulfate and 13 µl
N,N,N,N'-tetraethylethylenediamine (TEMED) was quickly poured to a
height of 10 cm and carefully overlaid with H2O. After the gels had
polymerized and the parafilm was removed, gels were pre-run at 200 V for 15
min, 300 V for 30 min and 400 V for 1 h. The upper buffer consisted of 50 mmol
l-1 NaOH, and the lower buffer consisted of 138 mmol l-1
phosphoric acid. After the pre-run, the upper buffer was replaced with fresh
50 mmol l-1 NaOH, and samples suspended in 5 µl 10% IPEGAL, 3
µl ampholines pH 5.0-8.0, 2 µl ampholines pH 3.5-10, 5 µl
ß-mercaptoethanol and made up to 100 µl with 9 mol l-1 urea
were placed in the tubes. Electrophoresis consisted of 14 h at 400 V and 4 h
at 800 V. Following IEF separation, gels were removed from the tubes and
either frozen at -20°C or equilibrated for 1 h in 20% glycerol, 5%
ß-mercaptoethanol, 2% sodium dodecyl sulfate (SDS) in a total volume of
100 ml 80 mmol l-1 Tris (pH 6.8). IEF gels were placed over a 16
cmx12 cm 5-15% SDSpolyacrylamide gradient gel and separated in
the second dimension (SDS-PAGE) as described below.
Isolation of protein and western blot analysis
For protein extraction, tissues were sonicated on ice in 50 mmol
l-1 Tris (pH 7.4), 0.5 mmol l-1 EDTA, 1.25 mmol
l-1 1,4-dithio-DL-threitol (DTT) and 0.5% Tween20 in approximately
1 min intervals until the tissue was completely homogenized. The homogenate
was then centrifuged at 10,000 g for 10 min, and the resulting
supernatant fraction transferred to a fresh tube. Protein concentrations were
determined using a bicinchoninic acid kit (BCA protein assay kit, Pierce,
Rockford, IL, USA) and bovine serum albumin (BSA) standards. Western blot
analysis was performed according to Locke and Tanguay
(1996). Equal amounts of
protein (50 µg, determined with the BCA kit; samples were diluted with
loading buffer) were loaded into each well of an SDSpolyacrylamide
(10%) gel and separated electrophoretically. Prestained molecular mass markers
(Invitrogen, Burlington, ON, Canada) were used to estimate the positions of
various proteins on the gel. Proteins were electro-blotted onto nitrocellulose
membrane (NitroBind, Westborough, MA, USA) using a Novex (San Diego, CA, USA)
mini trans-blot electrophoretic transfer unit set at 25 V or 100 mA for 2.5 h.
Membranes were blocked overnight at 4°C in 5% non-fat dry skim milk in
Tris buffer salineTween 20 (TBS-T; 20 mmol l-1 Tris, pH 7.5,
500 mmol l-1 NaCl and 0.05% Tween 20). The blots were then
incubated for 4 h with rabbit polyclonal antibody against Hsp72, diluted
1:5000 with TBS-T (SPA-812; StressGen, Victoria, BC, Canada) or rat monoclonal
antibody against Hsp73 diluted 1:3000 with TBS-T (SPA-815; StressGen) and
washed for 2x5 min with TBS-T. Following the washes, blots were reacted
for 1 h with affinity-purified goat anti-rabbit (A3687; Sigma, Oakville, ON,
USA) or anti-rat conjugated with alkaline phosphatase diluted 1:3000 with
TBS-T (SAB-201; Stressgen), washed for 1x5 min with TBS-T and
subsequently for 1x5 min with TBS. Immunoreactivity was visualized using
nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP)
western blotting detection reagents (Invitrogen). Hsp73 blots (samples and
standards) were incubated for 10 min with NBT/BCIP, and Hsp72 blots were
incubated for 5 min with NBT/BCIP. All blots were performed in this manner. To
quantify the amount of Hsp73 and Hsp72 expressed in the tissues, standard
curves were generated using commercially obtained pure Hsp72 and Hsp73
standards (SPP-755 and SPP-751, respectively; Stressgen). The regression
equations for the Hsp73 and Hsp72 standard curves are
y=134.97x-60.09, with an r2 value of
0.99, and y=1922.57x+895.38, with an r2
value of 0.99, respectively. In addition, as an internal control, 10 ng of
either Hsp72 or Hsp73 protein standard was loaded onto each experimental gel.
Membranes were dried overnight, scanned using a Hewlett Packard ScanJet
scanner, and quantification of bands was performed using Kodak 1D 2.0 Image
Analysis Software (Kodak Scientific Imaging Systems, New Haven, CT, USA).
Statistical analysis
All data are expressed as means with corresponding S.E.M. The statistical
analysis software program SigmaStat version 1.0 (SPSS, Chicago, IL, USA) was
used to perform the oneway analysis of variance (ANOVA) followed by
StudentNewmanKeuls post hoc test (P=0.05). In
Fig. 4, mean values are plotted
rather than individually paired values, as it is not possible to obtain
control Hsp73 data and dived Hsp72 data from the same animal.
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Results |
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Hsp73 and Hsp72 expression following heat shock
In mammals, the induction of stress proteins occurs upon exposure to a
variety of stressors, including heat shock. Therefore, as a positive control
for Hsp73 and Hsp72 expression, the effect of a 35°C heat shock on the
expression of Hsp73 and Hsp72 was examined in turtle brain, heart, liver and
skeletal muscle.
After western blot analysis, quantification by densitometry showed that Hsp73 expression remained unchanged in all tissues examined, except in liver where a significant reduction in Hsp73 was observed (Fig. 2A). By contrast, Hsp72 expression was significantly increased (P<0.05, N=4) in all tissues examined (Fig. 2B).
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Metabolic status
To determine whether turtles were relying on anaerobic metabolism during
the dive, blood lactate levels were measured in control animals, in animals
dived for 24 h and in 1 h recovery animals. Blood lactate concentrations were
1.74±0.34 mmol l-1 (N=4) in controls, increased
significantly to 53.14±7.60 mmol l-1 (N=5) after 24
h of submergence (P<0.05) and remained significantly raised after
a recovery period of 1 h (53.74±3.32 mmol l-1; N=4,
P<0.05).
To confirm the anoxic status and to ascertain whether animals were obtaining oxygen via extrapulmonary respiratory routes, arterial blood oxygen (PaO2) levels were measured under control conditions, after the 24 h dive and after the 1 h normoxic recovery. PaO2 levels dropped significantly from 2492.7±4.0 Pa (N=4) in controls to 29.3±21.3 Pa (N=5) after the 24 h dive (P<0.05) but recovered to 7991.3±485.2 Pa (N=4, P<0.05) after the 1 h recovery.
Hsp73 and Hsp72 expression and quantitative analysis after dive and
recovery
Using mammalian antibodies specific for Hsp73 and Hsp72, Hsp70 isoform
expression was determined by western blot analysis
(Fig. 3A) and quantified by
densitometry (Fig. 3B) in
selected tissues of normoxic control animals (N=4) and animals
following a 24 h dive (N=5) and 1 h recovery (N=4).
Brain
After the 24 h dive and 1 h recovery, basal Hsp73 expression in brain
(32.50±3.80 pg µg tissue-1) remained unchanged from
control values (Fig. 3Bi). By
contrast, Hsp72 expression increased significantly from 7.20±0.70 pg
µg tissue-1 in controls to 14.50±1.00 pg µg
tissue-1 after the 24 h dive and further increased to
30.50±3.50 pg µg tissue-1 after 1 h normoxic recovery
(P<0.05; Fig.
3Bi).
Heart
Similar to the results for brain, heart Hsp73 expression (control
13.30±0.20 pg µg tissue-1;
Fig. 3Bii) did not change
following the 24 h dive and 1 h recovery. Hsp72 expression was detectable in
heart (7.20±0.70 pg µg tissue-1) and increased
significantly following the 24 h dive to 32.00±4.40 pg µg
tissue-1 (P<0.05). Following 1 h recovery, Hsp72
expression increased further to 37.80±4.00 pg µg tissue-1
(P<0.05; Fig.
3Bii).
Liver
The constitutive expression of Hsp73 in liver (73.20±2.10 pg µg
tissue-1) was the highest of all the tissues examined
(Fig. 3Biii). After the 24 h
dive, Hsp73 expression decreased to 45.70±6.10 pg µg
tissue-1 and further decreased following 1 h recovery to
17.20±3.90 pg µg tissue-1 (P<0.05).
By contrast, basal Hsp72 expression (Fig. 3Biii) was only 18.90±4.30 pg µg tissue-1 and remained unchanged after a 24 h dive (9.60±1.20 pg µg tissue-1). However, after 1 h recovery, Hsp72 expression was significantly increased to 38.80±7.40 pg µg tissue-1 (P<0.05).
Skeletal muscle
Hsp73 expression in skeletal muscle tissue
(Fig. 3Biv) was relatively low
in controls (6.40±1.30 pg µg tissue-1) and was not
significantly different from values obtained after the 24 h dive
(4.50±0.70 pg µg tissue-1) or 1 h recovery
(4.30±0.40 pg µg tissue-1). Basal Hsp72 expression
(Fig. 3Biv) was also relatively
low (5.40±0.90 pg µg tissue-1). However, after the 24 h
dive, Hsp72 expression increased significantly to 20.20±1.10 pg µg
tissue-1 (P<0.05). An additional increase to
34.40±3.50 pg µg tissue-1 was observed after 1 h recovery
(P<0.05).
Taken together, these results show that the expression of Hsp72, but not Hsp73, is increased in all tissues examined, except liver, after a 24 h dive and increased in all tissues following a 1 h recovery.
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Discussion |
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Hsp70 expression following heat shock
Exposing tissues to elevated temperatures has been shown to induce
heat-shock proteins in almost all organisms examined to date
(Morimoto et al., 1997). In
the present study, it was shown that turtles are no exception, as an ambient
temperature increase from 17°C to 35°C induced Hsp72 expression in all
four tissues of the western painted turtle examined
(Fig. 2). Brain, heart and
muscle exhibited an approximately sixfold increase and liver a twofold
increase in Hsp72 levels after heat shock, while Hsp73 expression was
unchanged in all tissues except liver, which showed a sixfold decrease
(Fig. 2). Similar increases in
Hsp70 expression have been shown in non-mammalian species, including the
rainbow trout (Oncorhynchus mykiss;
Airaksinen et al., 1998
), the
African clawed toad (Xenopus laevis;
Phang et al., 1999
) and the
toad-headed agamid lizard (Phrynocephalus interscapularis;
Ulmasov et al., 1999
).
Airaksinen et al. (1998
)
demonstrated that a temperature increase from 18°C to 26°C induced the
synthesis of 67 kDa and 69 kDa proteins (members of the Hsp70 family of stress
proteins) in cultured O. mykiss hepatocytes, gill epithelial cells
and gonadal fibroblasts. In cultured X. laevis kidney epithelial
cells, Phang et al. (1999
)
showed that a temperature increase from 22°C to 35°C induced the
synthesis of Hsp70 family members. Additionally, Ulmasov et al.
(1999
) demonstrated that
induction of Hsp70 family members occurs at 39°C and proceeds up to
47-50°C in P. interscapularis. In mammalian species, heat shock
also results in induction of Hsp70 proteins
(Bechtold et al., 2000
).
Metabolic status
In the present study, turtles that had dived for 24 h were shown to be
relying on anaerobic metabolism (glycolysis), as indicated by increased
lactate levels. Blood lactate levels of this magnitude (53 mmol
l-1) are associated with severe anoxia in the turtle
(Jackson et al., 1996). These
data are supported by previous studies showing lactate levels in the painted
turtle increasing from low control levels (<2 mmol l-1) to 25
mmol l-1 after a 12 h dive in anoxic water at 22°C
(Chang et al., 2000
) and to 14
mmol l-1 after a 6 h dive in normoxic water at 20°C
(Crocker et al., 1999
).
In addition to lactate, blood PO2 levels
indicate that turtles were anoxic during the 24 h dive but had returned to
normoxic levels after 1 h of recovery. Similar results were obtained by
Crocker et al. (1999), where
blood PO2 was observed to drop from control
levels of 11,730.4 to 186.6 Pa after a 6 h dive at 20°C. In addition,
Herbert and Jackson (1985
)
demonstrated that by 1.5 h of submergence, blood
PO2 was minimal, O2 stores were
essentially exhausted and all turtles were relying primarily on anaerobic
metabolism.
In the present study, blood oxygen levels of control animals were
relatively low (2492.7±533.2 Pa). A possible reason for the low
PO2 level observed in control animals may be
that turtles were voluntarily diving prior to capture and sacrifice. As
turtles respire intermittently, and resting blood
PO2 levels can vary greatly, another
explanation could be that the animals were sacrificed when blood
PO2 was naturally low. This observation is
supported by Ultsch et al.
(1999), where turtles that had
access to air had variable blood PO2 levels,
ranging from approximately 666.5 to 11,997.0 Pa.
Hsp73 and Hsp72 expression after dive and recovery
The pattern of Hsp73 and Hsp72 expression in response to a 24 h forced dive
and 1 h recovery showed tissue- and stress-specificity, an observation that is
common for stress protein and mRNA expression in both mammalian and
non-mammalian species (Airaksinen et al.,
1998; DiDomenico et al.,
1982
; Manzerra et al.,
1997
; Rodenhiser et al.,
1985
). In the present study, Hsp73 expression was relatively high
in brain and liver under control conditions as well as after the 24 h dive and
recovery (Fig. 3Bi,iii) as
compared with heart and skeletal muscle
(Fig. 3Bii,iv). In contrast to
Hsp73 expression, Hsp72 expression was very low in all tissues under control
conditions (Fig. 3B). Hsp72
expression in liver was also very low after a 24 h dive
(Fig. 3Biii), and, in brain,
Hsp72 expression was induced twofold (Fig.
3Bi). By comparison, heart and skeletal muscle Hsp72 expression
was induced approximately fourfold after the 24 h dive
(Fig. 3Bii,iv). Furthermore,
all tissues showed a significant increase in Hsp72 expression after the 1 h
recovery period. These results correlate well with studies examining stress
protein expression in various species where tissue- and stress-specific
responses have been demonstrated
(Airaksinen et al., 1998
;
DiDomenico et al., 1982
;
Manzerra et al., 1997
;
Rodenhiser et al., 1985
).
Correlation between Hsp73 and Hsp72 expression
Interestingly, a strong correlation was observed between the expression of
the two Hsp70 isoforms (Fig.
4). There is a strong correlation (r2=0.82)
between basal Hsp73 and dive-induced Hsp72 expression. High levels of Hsp73
expression, such as in brain and liver, correlated with low levels of
dive-induced Hsp72 expression. Alternatively, low basal levels of Hsp73
expression, such as in heart and skeletal muscle, correlated with high levels
of dive-induced Hsp72 expression. Liver displayed high Hsp73 expression under
control conditions, with no induction of Hsp72 after a 24 h dive
(Fig. 3iii). Brain showed
intermediate Hsp73 expression under control conditions with a twofold
induction of Hsp72 during the 24 h dive
(Fig. 3i). By contrast, heart
and skeletal muscle expressed low basal Hsp73 expression and, following a 24 h
dive, exhibited an approximately fourfold increase in Hsp72 expression
(Fig. 3ii,iv). Previous studies
have shown similar patterns of expression correlating a high constitutive
Hsp73 expression to low inducible Hsp72 expression and vice versa.
Manzerra et al. (1997)
demonstrated that rabbit cerebrum showed high expression of constitutive Hsp73
and correspondingly low expression of inducible Hsp72 upon hyperthermic
conditions. Manzerra and colleagues
(1997
) also showed that other
tissues, such as liver, heart and muscle, had an opposite expression pattern,
with low Hsp73 expression and a large induction of Hsp72 expression when
stressed. Carpenter and Hofmann
(2000
) demonstrated a similar
result, showing a higher constitutive Hsp70 expression in brain tissue as
compared with white muscle tissue in several different notothenid fish
species. Several other studies also showed tissue-specific differences in
Hsp72 induction following hyperthermia; however, these studies did not examine
the constitutive levels of Hsp73 and basal levels of Hsp72 in relation to the
induction response (Flanagan et al.,
1995
; Hotchkiss et al.,
1993
; Lu and Das,
1993
). In addition, it was shown that when Hsp73 was directly
injected into Xenopus oocytes prior to an increase in temperature
that would normally result in Hsp72 induction, an attenuation of the stress
response was observed (Mifflin and Cohen,
1994
). Generally, when levels of Hsp73 or Hsp72 are elevated, the
stress response appears to be diminished
(DiDomenico et al., 1982
;
Mosser et al., 1993
). Taken
together, these studies demonstrate that the amount of pre-existing Hsp73
and/or Hsp72 can influence the inducible expression of Hsp72 upon exposure to
stressful conditions.
The tissue-specific expression of Hsp73 is probably attributable to
differences in tissue protein synthetic rates. In studies where protein
synthesis was measured in rat and fish liver, brain, heart and skeletal
muscle, the highest rates were found in liver (rat,
Garlick et al., 1975; fish,
Smith et al., 1996
). The liver
protein synthesis rate was 2-3-fold higher than those measured in heart,
muscle and brain, which all had similar rates. Protein synthesis in turtle
hepatocytes consumes 36% of the total cellular ATP turnover
(Land et al., 1993
), which is
a much greater proportion than the 2-3% measured in heart, brain and muscle
(measurements from fish, Smith et al.,
1996
). As Hsp73 is an important chaperone protein, it follows that
tissues with high protein synthetic rates, such as liver, would have higher
basal levels of Hsp73. An exception to this reasoning is found in brain, where
the rate of protein synthesis is similar to the rate in heart and muscle but
basal Hsp73 expression is much higher. As Manzerra et al.
(1997
) point out, high Hsp73
levels detected in mammalian brain are probably a result of its involvement in
axonal transport of neuronal proteins and as a clathrin-uncoating ATPase in
vesicle-mediated cycling events. These additional functions probably account
for the high level of Hsp73 expression we detect in turtle brain.
One curious result that we cannot explain is the decrease in liver Hsp73
expression following heat shock and the 24 h forced dive. To our knowledge,
this is the first time that this has been reported. We have no reason to
believe that our control Hsp73 data are artifactually high. This value is a
mean of measurements collected from four separate animals, and the S.E.M. is
very small. Furthermore, Hsp73 expression decreased throughout the control, 24
h dive and recovery protocol, suggesting a trend. Anoxia-tolerant animals,
such as the western painted turtle, reduce metabolism dramatically when faced
with anoxia. One cell function that decreases is protein synthesis, which in
liver decreases by 92% (Land et al.,
1993). We propose that reduced levels of protein synthesis result
in reduced levels of basal Hsp73 expression. This is consistent with changes
in protein synthesis observed in crucian carp (Carassius carassius)
tissues following a 48 h anoxic exposure
(Smith et al., 1996
); protein
synthesis was found to decrease 95% in liver, 53% in heart, 54% in muscle, and
no change in brain. As mentioned above, protein synthesis is such a small
proportion of total energy metabolism in heart, muscle and brain that one
would not expect large changes in basal Hsp73 levels resulting from decreased
synthetic rates. Even though protein synthetic rates are relatively low in
brain, the lack of an anoxia-mediated decrease supports our finding that basal
Hsp73 levels do not change in anoxic turtle brain.
As discussed above, hyperthermia is well established as a cellular stress
that results in protein denaturation; however, oxidative stress such as anoxic
exposure and reoxygenation after anoxia can also lead to the denaturation of
proteins. During hypoxia and subsequent reoxygenation, changes in the cellular
redox status occur, altering the ratio of oxidized and reduced forms of
glutathione. This, in turn, can alter the redox state of cysteine residues on
various cellular proteins, resulting in partial loss of tertiary structure or
denaturation (Piacentini and Karliner,
1999). Although the exact nature of the trigger for Hsp70
induction is not established, it is thought that under unstressed conditions
Hsp70 members are complexed with heat shock factor (HSF) monomers. An
accumulation of denatured protein displaces Hsp70, which allows HSF monomers
to form a trimer, resulting in the activation of HSF and the subsequent
transcription of Hsp70 (Morimoto et al.,
1997
). Although Hsps have not been extensively studied in the
western painted turtle, it is presumed that the same mechanism of Hsp70
induction may occur.
We have shown that Hsp73 and Hsp72 expression in turtle tissues does not follow a simple model of low basal Hsp73 and/or Hsp72 expression and induction of Hsp72 upon anoxic stress but rather three complex tissue-specific strategies. Firstly, we conclude that liver maintains a very high basal level of Hsp73 expression and minimal, if any, induction of Hsp72 upon anoxic stress. High Hsp73 levels may be a result of the high normoxic protein synthetic rates in liver and the concomitant need for increased chaperone levels. Secondly, heart and skeletal muscle display an alternative strategy, maintaining low levels of Hsp73 expression and undergoing a strong induction of Hsp72 under anoxic stress. Lastly, brain maintains intermediate basal levels of Hsp73 and undergoes a moderate induction of Hsp72 upon anoxic exposure.
A further examination of the precise molecular mechanisms underlying the anoxia tolerance of the western painted turtle and the possible role that stress proteins play in this tolerance is no doubt required. As protein synthesis is a costly process in terms of energetics, the upregulation of heat-shock proteins in anoxic tissues suggests that they may play a role in promoting anoxia tolerance.
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Acknowledgments |
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References |
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