Cryoprotection by urea in a terrestrially hibernating frog
Department of Zoology, Miami University, Oxford, OH 45056, USA
* Author for correspondence (e-mail: costanjp{at}muohio.edu)
Accepted 25 August 2005
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Summary |
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Key words: amphibian, freeze tolerance, osmolyte, hibernation, Rana sylvatica, wood frog, cryoprotection
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Introduction |
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Several species of temperate frogs overwinter beneath forest duff, where
they may be exposed to dehydrating conditions and subzero temperatures, which
they can survive by virtue of their profound tolerances to dehydration
(Hillyard, 1999) and somatic
freezing (Schmid, 1982
).
Notably, the wood frog (Rana sylvatica LeConte), which ranges further
north than any other anuran, recovers from severe dehydration
(Churchill and Storey, 1993
)
and survives the freezing of up to 70% of its body water at temperatures
between -4 and -6°C (Storey and
Storey, 2004
). Freeze tolerance in amphibians is supported by a
host of molecular, biochemical and physiological responses that provide
protection against the stresses associated with the freezing and thawing of
tissues. Foremost among these is an accumulation of newly synthesized
carbohydrate (glucose in R. sylvatica; glycerol and/or glucose in
hylid tree frogs) over the first few hours of freezing. These permeable
osmolytes, or `cryoprotectants', colligatively lower the freezable fraction of
body water and reduce cell dehydration and shrinkage, thereby limiting osmotic
and mechanical injury to membranes and other cellular structures. In addition,
cryoprotectants safeguard cellular functions by stabilizing intracellular
proteins (Carpenter and Crowe,
1988
; Mazur,
1984
). Glucose, purportedly the sole cryoprotectant in R.
sylvatica, contributes to freezing survival at cell, organ and
whole-animal levels of organization
(Costanzo et al., 1995
).
Cryoprotectants employed by freeze-tolerant organisms constitute a diverse
array of organic compounds, although they all share certain attributes,
including low molecular mass, high solubility and permeability, stability,
ready availability, and compatibility with macromolecules
(Storey and Storey, 2004). In
principle, urea accumulating in response to water deficit could also serve a
cryoprotective function in hibernating amphibians, but this contention has not
been tested. Here, we provide evidence that overwintering R.
sylvatica can accumulate substantial quantities of urea and that this
osmolyte protects cells and tissues from freeze/thaw injury.
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Materials and methods |
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Other frogs were permitted to overwinter in an open-air enclosure located in the same deciduous woodlot. This 10-m2 arena was circumscribed by a 1.25-m high wall of plastic mesh. In order to prevent frogs from escaping, the lower edge of the mesh extended 10 cm below ground. The arena contained a small pool and natural cover, such as woodland forbs, a few small shrubs, and detritus from nearby deciduous trees. A data logger (model CR-10; Campbell Scientific, Logan, UT, USA) and sensors permitted us to monitor a suite of environmental variables (solar radiation, wind speed, relative humidity, air temperature, soil temperature, soil moisture, leaf litter moisture) as well as operative environmental temperatures (Te) of `active' and `hibernating' frogs. These Te models were made from painted metal castings of a small frog and were placed on the ground surface or in a shallow depression in the soil beneath leaf litter, respectively.
Tissue sampling and osmolyte assays
Several experiments required us to measure osmolyte concentrations in the
blood or organs and to collect erythrocytes or organ samples for use in
cryoprotection experiments. Frogs were double-pithed and blood was immediately
drawn from an incision in the aortic trunk (or collected from the ventricle of
frozen frogs) into heparinized capillary tubes. The tubes were centrifuged
(2000 g, 5 min) and the plasma was reserved on ice for
analysis. Carcasses were promptly dissected, and various organs, or portions
of organs, were excised and blotted free of surface moisture. In some
experiments, samples were placed in ice-cold phosphate-buffered saline (PBS;
in g l-1; 6.10 NaCl, 0.15 KCl, 0.88 Na2HPO4,
0.15 KH2PO4; 230 mosmol kg-1, pH 7.4 at
23°C) and reserved for later use. In order to assay osmolytes,
deproteinized tissue extracts were prepared by homogenizing pre-weighed
samples in HClO4 and neutralizing the supernatants with KOH.
Glucose and urea in tissue extracts and in blood plasma were assayed using
glucose oxidase (no. 510; Sigma, St Louis, MO, USA) and urease (no. 736;
Sigma) procedures, respectively. Plasma osmolality was determined by
vapor-pressure osmometry (model 5500; Wescor, Logan, UT, USA).
Osmolytes in winter frogs
We examined seasonal changes in osmolyte levels and hydroosmotic balance in
R. sylvatica inhabiting the outdoor enclosure by sampling animals at
intervals from November to April. Frogs wore a thin waistband of polyethylene
tubing to which a passive integrative transponder (PIT) tag was attached.
Systematically sweeping a portable reader (model LID 500; Trovan Ltd, North
Humberside, UK) over the ground permitted us to locate frogs hidden beneath
snow and/or leaf litter. Captured frogs were transported under refrigeration
to the laboratory and immediately assayed for plasma osmolality and for plasma
levels of urea and glucose. We estimated the body water content of each frog
by thoroughly drying its carcass in a 65°C oven and determining the mass
of water that had evaporated. Our primary objective in this experiment was to
examine the dynamic physiological state of overwintering frogs in relation to
prevailing microenvironmental conditions.
In the laboratory, we investigated the association between environmental
moisture and urea concentration by manipulating conditions to which frogs were
exposed during simulated hibernation. Frogs were held in opaque plastic boxes
on a thick substratum of absorbent moss. The boxes were closed with a loosely
fitting lid that excluded ambient light and permitted exchange of respiratory
gases whilst also minimizing water loss. Initially, frogs were held at 4°C
on fully hydrated moss. After habituating to these conditions for 6
weeks, some of the frogs were killed and assayed for plasma urea. We then
reduced the amount of moisture available to the remaining frogs by wringing
most of the water from the moss. Frogs were held at 4°C on this substratum
for 10 days or several weeks before additional animals were assayed. The box
was then transferred to a 10°C incubator, and the remaining frogs were
sampled 3 days later.
Osmolytes in frozen/thawed frogs
We compared levels of urea and glucose in laboratory-held frogs sampled
directly from their boxes (control) with those subjected to somatic freezing.
These experiments were performed using frogs rendered moderately hyperuremic
by keeping them for 10 days on slightly damp moss. The experimental freezing
protocol exposed frogs to conditions mimicking a natural chilling episode
(Costanzo et al., 1991a).
Briefly, each frog was outfitted with a copper/constantan thermocouple placed
against its abdomen and cooled inside a 50 ml polypropylene centrifuge tube
submerged in a refrigerated ethanol bath (model 2095; Forma Scientific,
Marietta, OH, USA). During cooling, body temperature was recorded on a
multichannel data logger (model OM-500; Omega, Stamford, CT, USA). Once they
had reached -0.5°C, freezing was initiated by applying small ice crystals
to their skin. Subsequently, the frogs were cooled to an equilibrium
temperature of -2.5°C over the next 24 h, during which time approximately
65% of their body water would have frozen
(Layne and Lee, 1987
).
Portions of the liver, small intestine and muscle from one forelimb (flexor
carpi) and hindlimb (gracilis) were rapidly excised from fully frozen frogs
(N=5), and also from control frogs (N=5), and assayed for
glucose and urea (see below). Replicate sets of organ samples were weighed to
the nearest 0.1 mg and thoroughly dried in a 65°C oven. They were
reweighed and tissue water content was determined from the change in mass.
Cell cryoprotection by urea
Following Costanzo and Lee
(1991), we tested the
cryoprotective efficacy of urea by comparing the viability of R.
sylvatica erythrocytes frozen/thawed in the absence or presence of urea.
Cells were washed twice in PBS and resuspended in PBS (control) or PBS
containing 40 or 80 mmol l-1 urea. Cell suspensions (3-5%
hematocrit) were divided among multiple 80-µl samples held in 0.5-ml
microcentrifuge tubes and incubated for 45 min on ice. Next, the samples were
placed individually in glass tubes immersed in a refrigerated ethanol bath,
chilled to -1°C and inoculated by briefly applying aerosol coolant to the
exterior of the microcentrifuge tube. After the suspensions began to freeze,
they were held at the target temperature, -4 or -6°C, for 30 min.
Cell viability was assessed after passively thawing the samples at 4°C. Cell suspensions were centrifuged (2000 g, 3 min), and the hemoglobin concentration of the supernatant, an index of hemolytic damage, was assayed (no. 525, Sigma) as cyanmethemoglobin. Sample absorbances were referenced to that of a total hemolysis standard produced by freezing a separate aliquot of suspension for 2 h at -80°C. Supernatants were also assayed (TOX-7; Sigma) for the cytoplasmic enzyme lactate dehydrogenase (LDH), whose concentration in the suspension medium indexed membrane damage. Preliminary trials showed that unfrozen erythrocytes tolerated exposure to at least 80 mmol l-1 urea without leaking LDH and at least 200 mmol l-1 urea without lysing.
We also compared the cryoprotective efficacy of urea with that of glucose
and glycerol, cryoprotectants found in freeze-tolerant frogs. This experiment
was carried out as described above, except that the suspensions were held on
ice for 4 h before being frozen. The longer incubation period was needed to
permit the intracellular glucose concentration to attain equilibrium
(Brooks et al., 1999).
Organ cryoprotection by urea
In two separate experiments, we examined urea's efficacy in cryoprotecting
R. sylvatica organs, comparing indices of cryoinjury in samples
frozen/thawed in vitro after incubation in the presence or absence of
urea. Preliminary tests were undertaken to confirm that exogenous urea readily
permeates R. sylvatica tissues. The intact heart, kidneys and 25-50
mg portions of liver and leg muscle (gastrocnemius) were dissected from
freshly killed frogs, rinsed several times in ice-cold PBS and placed in
individual 0.5-ml centrifuge tubes containing 80 mmol l-1 urea in
250 µl of PBS. After incubating on ice for 45 min, the samples were removed
and gently blotted to remove surface moisture. The heart was bisected
sagittally, and the samples of liver and gastrocnemius were divided into equal
portions. We measured urea concentrations in one set of the samples and in an
entire kidney. The other set and the remaining kidney were dried in a 65°C
oven, and tissue water content was determined from the change in mass upon
drying. Tissue urea concentrations were calculated as µmol g-1
dry tissue.
In one experiment, the heart, both kidneys and a portion of the liver were dissected from double-pithed frogs, thoroughly rinsed with ice-cold PBS, and placed in individual 0.5-ml centrifuge tubes containing 250 µl of PBS (control) or PBS containing 80 mmol l-1 urea. Samples were incubated on ice for 45 min before being frozen for 45 min at -4°C and passively thawed at 4°C, as per the erythrocyte experiments.
Viability of the frozen/thawed organ samples was inferred from metabolic
activity, which was assessed using the non-toxic, oxidation-reduction
indicator dye alamarBlue (Alamar Biosciences, Sacramento, CA, USA). This
assay, which yields a colorimetric change in proportion to cellular
respiration, has been used to evaluate cell viability following hypothermic
exposure (Acker and McGann,
2002; Cook et al.,
1995
). Organ samples were transferred 60 min after thawing to a
1.5-ml centrifuge tube containing 90 µl alamarBlue diluted (1:10) with PBS,
or PBS containing 80 mmol l-1 urea, and incubated at 15°C with
gentle orbital agitation for 120 min. We then decanted and centrifuged (700
g, 3 min) the medium, transferred 0.75 ml of the cell-free
supernatant to a cuvette and read the absorbance at 570 and 600 nm using a
spectrophotometer. Organ samples were thoroughly dried in a 65°C oven, and
reduction rates were standardized to dry tissue mass.
Control experiments showed that no reduction occurred in dye solution alone
or in dye solution containing heat-denatured tissue. However, preliminary
tests suggested that urea treatment reduced metabolic activity in certain
organs, so we more thoroughly investigated this phenomenon. We conducted an
experiment similar to that described above, except that the organ samples were
not frozen before being assayed and, in order to improve statistical power, we
compared urea-treated and control samples harvested from the same frog. The
heart was bisected sagittally, and pieces of the excised liver and
gastrocnemius were cubed (1 mm3) and apportioned into two
lots, thus permitting differential treatment of the same organs. Each lot,
plus one intact kidney, was rinsed with several changes of ice-cold PBS and
then placed in indicator dye diluted with PBS (control) or PBS containing 80
mmol l-1 urea. Samples were incubated at 15°C for 60 min
(heart, gastrocnemius) or 150 min (kidney, heart) before being assayed for
metabolic activity, as described above. Results of this experiment were used
to correct the organ cryoprotection data for any reduction in metabolic
activity purely attributed to urea treatment. In a companion experiment, we
tested the specificity of the metabolic inhibition by substituting 80 mmol
l-1 glycerol for urea.
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Statistical inferences
Sample means were compared using Student's t-tests or analysis of
variance (ANOVA) followed by Bonferroni multiple comparisons tests. The
nonparametric Wilcoxan Signed Rank test was substituted where the data did not
meet the assumptions of parametric tests. Analyses involving percentage data
were performed on values after arcsine/square-root transformation.
Significance of statistical analyses was accepted at P<0.05. Mean
values are reported ± S.E.M.
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Results |
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Uremia in laboratory-hibernating frogs was associated with the hydric and thermal conditions to which the frogs were exposed. Frogs maintained at 4°C on fully hydrated moss had relatively low plasma urea levels (3.9±1.3 mmol l-1; N=9). By contrast, frogs held on the (wrung-out) damp moss at 4°C had accumulated urea (14.0±1.4 mmol l-1; N=5) within 10 days and became strongly hyperuremic (58.5±4.8 mmol l-1; N=2) within several weeks. Plasma urea levels rose to 92.3±3.5 mmol l-1 (N=3) when these frogs were exposed to 10°C for 3 days.
Effect of somatic freezing on urea and glucose levels
Somatic freezing induced the characteristic hyperglycemic response in
R. sylvatica, as plasma glucose concentration in the frozen frogs
(77.4±16.6 mmol l-1; N=5) was markedly higher
(P<0.002) than that in unfrozen controls (3.6±0.4;
N=5). Because the organs of frozen frogs contained 11.3% (flexor
carpi) to 51.4% (heart) less water than the organs of unfrozen controls
(ANOVA; P<0.05; see Lee et
al., 1992), it was necessary to express osmolyte concentrations as
µmol g-1 dry tissue in order to allow comparisons between the
groups. Urea concentration did not change with freezing in most organs;
however, glucose concentrations were up to 35-fold higher in frozen frogs as
compared with unfrozen controls (Table
1). In frozen frogs, the liver and gut contained considerably more
glucose than urea. By contrast, in the case of skeletal muscles, which
accumulated relatively little glucose during freezing, concentrations of both
glucose and urea were
50 µmol g-1
(Table 1).
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Rates of cryohemolysis for erythrocytes incubated in PBS, or PBS containing 40 mmol l-1 urea, 40 mmol l-1 glucose or 40 mmol l-1 glycerol, varied both by exposure temperature (P<0.0001) and by incubation medium (P<0.0001). All three osmolytes reduced cell damage in samples frozen at -4 or -6°C (Fig. 3). The level of cryoprotection afforded by urea was equal to that of glycerol, a well-known cryoprotectant. Furthermore, cryoprotection by urea was equal or superior to that of glucose.
Organ cryoprotection
Our preliminary experiment confirmed that R. sylvatica organs
readily take up exogenous urea from an incubation medium. Urea concentrations
in the samples treated with 80 mmol l-1 urea ranged from 200 to 300
µmol g-1 dry tissue (Table
2) and were generally 4-6-fold higher than in control frogs (see
Table 1). Therefore, any
difference in freeze/thaw viability of urea-treated organs can be ascribed to
elevated tissue levels of this osmolyte.
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Organs were metabolically active after in vitro freezing at -4°C, although their respiration rates were nominally lower than those of unfrozen organs. The drop was relatively minor (and statistically nonsignificant, P>0.05) for the urea-treated samples as compared with the control samples (Fig. 4). The kidney appeared innately resistant to cryoinjury, as neither urea-treated nor control samples had respiration rates that differed significantly from unfrozen samples.
These findings were corroborated by the experiment in which organ cryoinjury was assessed from LDH leakage. Except for the kidney, which again proved especially tolerant to freezing/thawing, urea-treated organs exhibited less leakage than samples frozen without urea (Fig. 5). With the heart and gastrocnemius, the difference was robust; however, with the liver it lacked statistical significance.
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Discussion |
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In R. sylvatica inhabiting an outdoor enclosure, plasma urea,
which was 25-fold higher in early winter than in spring, generally tracked
seasonal changes in soil moisture, suggesting that this osmolyte is important
in maintaining hydro-osmotic balance (Fig.
1). In early autumn, urea levels were only slightly elevated
despite the soil being very dry; however, during periods of moderate weather,
the frogs became active and may have hydrated from dew collecting on fallen
leaves. Results of this field study are bolstered by reports of hyperuremia in
winter in R. sylvatica (Layne and
Rice, 2003) and the freeze-tolerant tree frog Hyla
versicolor (35-40 mmol l-1; 60 mmol l-1 in some
individuals; J. R. Layne, Jr, unpublished) and by our laboratory experiments
demonstrating that urea can accumulate to at least 90 mmol l-1 in
cold-acclimated frogs. This response would be especially sensitive to warm,
dry conditions, which would both accelerate urea synthesis and stimulate its
retention. In nature, urea accumulation in hibernating R. sylvatica
is probably promoted by their preference for overwintering in relatively dry,
upland habitats (Regosin et al.,
2003
).
Urea: an unlikely cryoprotectant?
Organic solutes commonly have overlapping roles in osmoprotection,
cryptobiosis and freeze tolerance (Somero
and Yancey, 1997; Storey,
1997
; Yancey,
2001
). Stresses imposed by desiccation and freezing are similar.
When tissues freeze, solvent is lost to ice forming in extracellular spaces,
and intracellular structures become exposed to increasing ionic strength and
crowding. Cryoprotective osmolytes defend cell water volume and also fortify
membranes and intracellular macromolecules against ionic and osmotic
perturbations. Stability, low molecular mass, high water solubility and
permeability are characteristics that render certain organic osmolytes,
including urea, well-suited as cryoprotectants. Accordingly, urea functioning
as an osmoprotectant during autumn and early winter could, in principle, also
protect frogs from the damaging effects of subzero temperatures that occur
later in hibernation.
On the other hand, it is commonly understood that urea has deleterious
effects on protein stability and function. Unlike compatible osmolytes, which
are preferentially excluded from the protein surface, thereby favoring its
folded state (Arakawa and Timasheff,
1985; Carpenter and Crowe,
1988
; Timasheff,
1992
), urea can preferentially bind to proteins, dehydrating their
exposed surfaces and promoting unfolding
(Creighton, 1991
;
Wu and Wang, 1999
;
Zou et al., 1998
). However,
because interactions between such `micromolecules' and macromolecules are
strongly governed by their physicochemical environment
(Somero and Yancey, 1997
;
Timasheff and Xie, 2003
),
dynamics observed in artificial urea/enzyme systems may not be germane in
vivo. Furthermore, protein destabilization generally occurs with urea in
high (i.e. molar) concentrations that probably greatly exceed those in R.
sylvatica tissues; in fact, in modest concentrations, urea may be less
perturbing than some compatible osmolytes (e.g.
Yancey and Burg, 1990
).
To our knowledge, urea has not been reported as a natural cryoprotectant in
any organism, yet amphibians readily tolerate urea in concentrations that
could provide significant protection against freeze/thaw injury. We tested
this hypothesis using the erythrocyte suspension as a model system because
earlier studies had characterized the responses of these cells to freeze/thaw
and osmotic stresses (Costanzo and Lee,
1991; Costanzo et al.,
1993
) and also had demonstrated the modest protection that urea
afforded human erythrocytes (Doebbler and
Rinfret, 1962
). In our experiments, physiological concentrations
of urea improved the viability of R. sylvatica erythrocytes
frozen/thawed at temperatures of ecological relevance to the species (Figs
2,
3). The apparent absence of a
concentration-dependent effect suggests that urea's action is not purely
colligative but also involves protection of macromolecules and cellular
structures (Carpenter and Crowe,
1988
; Mazur,
1984
). Recent findings (Bhuyan,
2002
; Kumar et al.,
2004
) of protein renaturation in the presence of urea at
relatively low concentrations support this notion.
Comparing the results presented in Figs 2 and 3 suggests that erythrocyte tolerance to freeze/thawing varied between experiments. The heightened sensitivity of cells apparent in Fig. 3 could have resulted because this work was done in late winter, after R. sylvatica usually arouses from hibernation. Seasonal variation in tolerance to osmotic and freeze/thaw stress can potentially stem from changes in the structure and chemical composition of the plasma membrane. An ongoing study suggests that cholesterol levels in the erythrocyte membrane, which have profound effects on fluidity and thermal adaptation, vary seasonally in the hatchling painted turtle, Chrysemys picta, another cold-hardy species (M. R. Polin, J. P. Costanzo and R. E. Lee, unpublished).
Assessing post-thaw viability of R. sylvatica organs from rates of
metabolic activity or LDH leakage demonstrated that urea markedly improved
freezing tolerance of intact tissues. Cardiac muscle was particularly amenable
to cryoprotection by urea. In preliminary studies, we found that isolated
R. sylvatica hearts frozen for 30 min at -4°C spontaneously
resumed autonomic contractions upon thawing when the incubation medium (PBS)
contained 100 mmol l-1 urea (N=2) but not when urea was
absent (N=2). We observed no discernable benefit of urea treatment
with kidney, but for unknown reasons this organ proved particularly resistant
to cryoinjury under the conditions of our experiment. Urea treatment of liver
tissue curbed freeze/thaw injury, as thawed samples exhibited no appreciable
reduction in metabolic activity. Urea treatment also reduced hepatocyte
membrane damage (indexed by LDH leakage) in six of the eight experimental
replicates; overall, however, the effect lacked statistical significance. We
suspected that in some cases the protection afforded liver by urea was masked
by the presence of high concentrations of glucose, which also reduces LDH
leakage from frozen/thawed hepatocytes
(Storey and Mommsen, 1994).
This could have occurred only if our in vitro liver samples had
autogenerated copious glucose during the experiment. We confirmed this
supposition by assaying portions of R. sylvatica liver immediately
upon dissection and after incubation in 80 mmol l-1 urea and
subsequent freezing at -4°C. In one trial, liver glucose concentration
increased with freezing, from 13 to 56 µmol g-1, and in another
it rose from 19 to 47 µmol g-1. By contrast, glucose levels in
gastrocnemius,
5 µmol g-1, were unchanged. The results for
liver were probably confounded by the presence of a second cryoprotectant;
however, on the whole, our data marshal sound evidence that urea cryopreserves
both isolated cells and intact tissues.
In some experiments the viability of tissues following experimental
freezing/thawing was assessed using an index of metabolic activity. Our
preliminary experiments showed that physiological levels of urea markedly
reduced aerobic respiration in some tissues. Although incidental to the focus
of our present report, this result merits consideration. In principle,
elevated urea could induce metabolic depression in overwintering frogs if, as
has been postulated for various estivating organisms, transition between
arousal and dormancy is modulated by shifts between the active/inactive states
of key regulatory enzymes in response to changes in urea concentration, pH and
temperature (Somero, 1986;
Withers and Guppy, 1996
;
Yancey et al., 1982
). The
relatively low levels of urea found in R. sylvatica tissues might be
generally nonperturbing and yet inhibitory to certain key enzymes (e.g.
phosphofructokinase; Hand and Somero,
1982
). Because urea's effects are strongly potentiated by low pH
(e.g. Hand and Somero, 1982
)
and dormant frogs are acidotic (Pinder et
al., 1992
), hyperuremia, acidosis and cold may be potent,
synergistic effectors of hypometabolism in overwintering R.
sylvatica. The few available data suggest that hyperuremic amphibians do
not accumulate methylamines to levels that might counteract the urea
inhibition (Pinder et al.,
1992
; Wray and Wilkie,
1995
; Withers and Guppy,
1996
), however additional study is needed before definitive
conclusions can be drawn.
The urea-hypometabolism hypothesis garners support from in vitro
studies of enzyme kinetics (Cowan and
Storey, 2002; Grundy and
Storey, 1994
; Stewart et al.,
2000
) but heretofore was not demonstrated at higher levels of
organization. Our present findings should be regarded as tentative until
confirmed through additional research. However, microrespirometry experiments
have shown that treatment with 80 mmol l-1 urea reduces oxygen
consumption in R. sylvatica liver and skeletal muscle by
33%,
whereas increasing the osmolality without adding urea has no effect (T. J.
Muir, J. P. Costanzo and R. E. Lee, unpublished). Curiously, in the present
study, urea treatment suppressed in vitro metabolism in R.
sylvatica liver and muscle but not in heart or kidney. Urea sensitivity
of enzyme systems varies among tissues
(Cowan and Storey, 2002
) and,
indeed, tissues differ in their capacity for metabolic depression
(Boutilier and St-Pierre, 2002
;
Flanigan et al., 1991
). A
marked hypometabolism in only the liver and skeletal muscle could profoundly
reduce energy consumption inasmuch as these organs constitute >50% of the
total tissue mass. Additional study is needed to assess the depth of metabolic
depression in intact, hyperuremic frogs.
Urea and glucose: a dual cryoprotectant system?
The freezing-induced mobilization of carbohydrate cryoprotectant (glucose
in R. sylvatica; glycerol and/or glucose in some tree frogs) has long
been touted as the hallmark physiological adaptation in amphibian freeze
tolerance. Our present findings indicate that urea accumulated before freezing
can contribute to freezing survival, raising the possibility that
freeze-tolerant frogs rely on more than one class of cryoprotective agent.
Moreover, in the case of R. sylvatica, one could reasonably argue
that urea plays a key role in reducing freeze/thaw injury.
Given that equimolar solutions of urea and glucose have the same
equilibrium freezing/melting point, as a colligative cryoprotectant the value
of either depends solely on its concentration within the tissues. Glucose
accumulates in tissues during somatic freezing whereas urea does not
(Table 1; see also
Layne and Rice, 2003).
Nevertheless, in some circumstances, frozen frogs could have as much (or more)
urea as glucose inside their cells. Frogs often generate only modest amounts
of glucose during freezing (Table
4). This statement contradicts the conventional wisdom that
freezing R. sylvatica amass 0.3-0.5 mol l-1 glucose;
however, it is important to realize that these often-cited values are extremes
and are uncharacteristic of the glycemic response. In fact, not only has the
popular and scientific literature exaggerated the glycemic response, but,
because tissues dehydrate profoundly while freezing
(Lee et al., 1992
), the common
convention of reporting metabolite concentrations as per-unit-mass of fresh
tissue has grossly overestimated the capacity for de novo glucose
synthesis. Glucose synthetic capacity apparently varies with age, sex, body
size, cooling rate and myriad other factors. In particular, frogs can achieve
markedly higher glycemic levels in autumn, when glycogen is abundant, than
they can in spring (Costanzo and Lee,
1993
; Layne, 1995
;
Storey and Storey, 1987
), and
glucose production capacity may be unusually well-developed in northern
populations (Layne, 1995
). The
literature attests that the blood glucose concentrations achieved during
freezing by temperate R. sylvatica are typically <100 mmol
l-1 in autumn and <25 mmol l-1 by the end of winter
(Table 4). Under conducive
environmental conditions, blood urea levels could be as high or higher.
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Another important consideration is that cryoprotectant must actually enter
cells in order to exert its full effect. Whereas virtually all cells are laden
with urea before freezing commences, glucose must be synthesized in the liver,
circulated throughout the body and transported into cells while the tissues
are freezing. Because freezing is initiated where the body contacts ice in the
environment, peripheral organs, such as the skin and skeletal muscles, quickly
become isolated from the blood supply and thus accumulate little glucose
(Rubinsky et al., 1994;
Storey and Storey, 1988
).
Glucose uptake by muscle fibers is further hampered by their low permeability
(King et al., 1993
;
Storey and Storey, 2004
), and
nervous tissues also accumulate relatively little glucose with freezing
(Costanzo et al., 1992
;
Kling et al., 1994
). As
demonstrated in our experiment (Table
1), even in moderately hyperuremic frogs, cells in some tissues
could contain as much urea as glucose because equilibrium levels of the former
are attained in advance of freezing.
Our assertion about urea's relative importance as a cryoprotectant draws
additional support from findings that urea was as good or better than glucose
at reducing freeze/thaw injury to R. sylvatica erythrocytes
(Fig. 3). Arguably, this result
could simply reflect differential permeabilities and intracellular
concentrations of the two solutes. However, this was probably not the case
because glucose quickly penetrates R. sylvatica erythrocytes and is
not metabolized at the low temperature used in our experiments
(Brooks et al., 1999). Rather,
this finding suggests that urea is more effective than glucose in increasing
the fraction of unfreezeable cell water and/or in preserving the integrity of
macromolecules and cellular structures. Caution should be used in interpreting
the results of in vitro experiments, which cannot accurately
replicate in vivo conditions. In the live frog, for example, both
osmolytes could be present and working in concert. We found no evidence for
synergism in cryoprotective efficacy when we tested erythrocytes preincubated
with 40 mmol l-1 urea and 40 mmol l-1 glucose (data not
shown), although undoubtedly the colligative effects in reducing ice content
would be additive.
Evolutionary perspectives
Long known as a balancing osmolyte, urea also serves myriad other
physiological functions in diverse animal taxa
(Withers, 1998). Our present
findings not only suggest that urea plays a key, previously undocumented role
in amphibian cold hardiness but they also identify a novel class of natural
cryoprotectant. In freeze-tolerant frogs, both dehydration and somatic
freezing initiate molecular events that increase glycemia, suggesting that
amphibian cryoprotectant systems derive from rudimentary mechanisms of water
conservation (Storey and Storey,
2004
). Among amphibians, osmotic stress universally stimulates
urea retention, which defends against cellular dehydration. Our contention
that urea is a key cryoprotective agent in R. sylvatica (and probably
other taxa) supports the tenet of overlapping adaptations in cold hardiness
and dehydration tolerance in ectothermic animals
(Churchill and Storey, 1993
;
Ring and Danks, 1994
). In
addition, finding a cryoprotective role for urea in R. sylvatica
resolves the apparent enigma of why osmolytes in freezing adaptation should be
exclusively carbohydrates, whereas organisms facing other osmotic stresses use
a mixture of osmolytes, often of different classes
(Yancey, 2001
).
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Acknowledgments |
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References |
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