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First published online September 5, 2008
Journal of Experimental Biology 211, 2969-2975 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.019695
Urea loading enhances freezing survival and postfreeze recovery in a terrestrially hibernating frog
Department of Zoology, Miami University, Oxford, OH 45056, USA
* Author for correspondence (e-mail: costanjp{at}muohio.edu)
Accepted 21 July 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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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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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Jørgensen, 1997). As a
consequence of environmental water deficit, cold exposure and anuria,
amphibians might also accumulate urea in terrestrial hibernation, although
this possibility has received little study.
Our investigation of the wood frog (Rana sylvatica LeConte), a
northern species that hibernates beneath forest duff, showed that plasma urea
was 
50 µmol ml–1 in autumn and early winter, when
soil moisture was scarce, but only 2 µmol ml–1 in
late winter and spring, after moisture availability increased
(Costanzo and Lee, 2005). In
laboratory experiments, we also determined that hibernating R.
sylvatica can accumulate urea to at least 90µmol ml–1
under relatively dry, warm conditions. Subsequent research showed that urea is
the major solute contributing to increased plasma osmotic pressure during
progressive dehydration (Muir et al.,
2007). In nature, urea accumulation in R. sylvatica
probably is facilitated by their preference for overwintering in relatively
dry, upland habitats (Regosin et al.,
2003). Urea is of obvious importance in the osmotic homeostasis of
terrestrial amphibians, but this solute may play additional roles in
overwintering survival, particularly in freeze-tolerant anurans such as R.
sylvatica.
Freeze tolerance in anurans is supported by a host of molecular,
biochemical and physiological responses that ameliorate the various stresses
induced by the freezing and thawing of water in extracellular compartments of
the body. Foremost among these is the synthesis of low-molecular-mass
carbohydrates (glucose in R. sylvatica; glycerol and/or glucose in
hylids) during the early hours of freezing. These permeable osmolytes, or
`cryoprotectants', colligatively lower the freezable fraction of body water
and reduce cell dehydration and shrinkage, thereby limiting iono-osmotic and
mechanical injuries. They may also act directly to stabilize macromolecules,
membranes and other cellular structures, and possibly have other protective
functions (Carpenter and Crowe,
1988; Storey and Storey,
2004).
Purportedly the sole cryoprotectant in R. sylvatica, glucose
contributes to freezing survival at the cellular, organ and whole-animal
levels of organization (Canty et al.,
1986; Costanzo et al.,
1993). However, recent studies have shown that physiological
levels of urea (40–80 µmol ml–1) enhance tolerance
of R. sylvatica cells and organs to in vitro
freezing–thawing (Costanzo and Lee,
2005; Costanzo et al.,
2008), suggesting that urea also plays a role in amphibian freeze
tolerance. In the present investigation, we tested this hypothesis by
determining whether R. sylvatica experimentally rendered hyperuremic,
via urea injection, demonstrate reduced cryoinjury and an enhanced
tolerance to freezing–thawing.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Costanzo et al., 1993;
Costanzo et al., 1997a).
Urea loading
We prepared frogs for experimentation by draining any urine from the
bladder through a polished glass cannula inserted into the cloaca. Frogs were
then weighed on a digital balance, kept in darkness at 4°C on dry paper
inside covered plastic cups, and reweighed after 2–3 days. Because water
vapor was permitted to escape through perforations in the cover, frogs lost
0.5 g of their standard body mass (mean ± s.e.m.=14.8±0.3
g, N=48) whilst inside the cups. We randomly assigned frogs to either
of two groups to which we administered cold phosphate-buffered saline (PBS, in
grams per liter: 6.10 NaCl, 0.15 KCl, 0.88 Na2HPO4, 0.15
KH2PO4; 230 mOsmol kg–1, pH7.4 at
23°C) or PBS containing 1.5 mol l–1 urea. Using a
27.5-gauge needle, the dorsal lymph pad of each frog was injected with a
volume of the appropriate solution equal to 3.3% of standard body mass. Thus,
the average frog received 0.5 ml of injectate, which restored its initial
hydration level. Frogs remained in darkened cages at 4°C for 3–5 h
prior to being experimentally frozen or euthanized for tissue sampling (see
below).
Freezing–thawing protocol
Following an earlier study (Costanzo et
al., 1991b), our experimental freezing–thawing protocol
simulated chilling episodes to which R. sylvatica are exposed during
hibernation. Briefly, each frog was outfitted with a copper–constantan
thermocouple placed against its abdomen and cooled inside a 50 ml
polypropylene centrifuge tube. Groups of tubes (N=38) were submerged
in a refrigerated ethanol bath programmed to cool from 0 to –4°C
over a 40 h period. During chilling, body temperature was recorded on a
multichannel data logger. Once the frogs had reached –0.5°C,
freezing was initiated by placing small ice crystals against their skin.
Cooling then resumed and, after reaching –4°C, they were kept in
situ for an additional 2 h to ensure they attained thermoequilibrium.
Fully-frozen frogs were transferred to an incubator at 4°C and, after
1–2 h, gently removed from their tubes and individually placed on damp
paper inside plastic cups.
Recovery time course and survival assessment
For both urea-loaded (N=14) and saline-treated (N=14)
frogs, we monitored the time course for restoration of neurobehavioral
functions following freezing–thawing by examining each frog daily, at
08:30 h and 17:00 h, for 1 week. At each observation we recorded whether or
not the subject exhibited each of the following behaviors: corneal reflex,
pulmonary breathing, alert posture (i.e. limbs trunk, and head held in
characteristic manner), retraction of hindlimb within 2 s of manual extension,
and righting reflex (i.e. inversion within 2 s of being placed on dorsum). On
day 7, we assessed survival on the basis of whether or not each frog met the
righting-reflex criterion. Frogs failing to meet this criterion were
double-pithed, dissected, and the blood was sampled and assayed as described
below.
Sublethal cryoinjury
We investigated freeze–thaw injury in separate groups of urea-loaded
(N=5) and saline-treated (N=5) frogs by measuring
circulating levels of three intracellular proteins. Blood samples were
collected (see below) 24 h after thawing was initiated. Hemoglobin (Hb)
concentration in the cell-free plasma, an index of hemolytic damage, was
assayed as cyanmethemoglobin (Sigma, no. 525; St Louis, MO, USA). Plasma
samples were also assayed for the ubiquitous cytoplasmic enzyme, lactate
dehydrogenase (LDH), and creatine kinase (CK), an intracellular enzyme found
primarily in skeletal and cardiac muscle. LDH activity was quantified using a
colorimetric assay kit (Sigma, no. TOX-7), whereas CK activity was assayed at
25°C using a kinetic procedure (Pointe Scientific, C7512; Canton, MI,
USA). In order to establish baseline levels of these proteins, we also
analyzed blood sampled from unfrozen frogs (urea-loaded, N=5;
saline-treated, N=5) 4.5 h after receiving the injections.
Tissue sampling and osmolyte assays
Frogs were double-pithed and quickly dissected. Blood was drawn from an
incision in the aortic trunk into heparinized capillary tubes and centrifuged
(2000 g, 5 min). The resultant plasma was isolated and
reserved on ice for subsequent analysis. We removed and sagittally bisected
the heart, and equally divided a 50–100 mg portion of both the liver and
hindlimb muscle (gastrocnemius). One set of samples was used in osmolyte
assays; remaining samples were weighed to 0.1 mg, thoroughly dried in a
65°C oven, and reweighed in order to determine tissue water content. We
estimated body water content by drying pre-weighed carcasses and determining
the mass of water that had evaporated; values were expressed as a percentage
of fresh mass.
We prepared tissue extracts by homogenizing pre-weighed organ samples in 7% perchloric acid. After removing the proteins by centrifugation (4000 g, 5 min), the clear supernatant was neutralized with potassium hydroxide and the resulting precipitate was removed by a second centrifugation. Glucose and urea in these extracts and in blood plasma were assayed using glucose oxidase (Sigma, no. 510) and urease (Pointe Scientific, no. B7551-120) procedures, respectively. Plasma osmolality was determined on 10 µl samples by vapor-pressure osmometry (Wescor, model 5500, Logan, UT, USA).
Statistical inferences
Fisher's exact test was used to compare ratios of urea-loaded and
saline-treated frogs fully recovering from freezing and meeting behavioral
criteria at select time points. We used two-factor analysis of variance
(ANOVA) to test for effects of urea loading and freezing–thawing on
plasma and organ osmolyte levels and water content. Analyses involving
percentage data were performed on values after arcsine-root transformation.
Significance of statistical analyses was accepted at P<0.05. Mean
values are reported as ±s.e.m.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Dynamics of recovery from freezing–thawing differed between urea-loaded and saline-treated frogs (Fig. 1). Most urea-loaded frogs exhibited the limb-retraction reflex and normal posture by 24 h after thawing, all doing so within 48 h. By contrast (P=0.008), only eight of 14 (57.1%) saline-treated frogs demonstrated these behaviors within 48 h after thawing began. Righting reflex, our most rigorous test of neurobehavioral function, was exhibited by all urea-loaded frogs within 56 h after thawing, whereas only four saline-treated frogs (28.6%) could right themselves by this time (P=0.002). The last of the latter group to meet the righting-reflex criterion did so after 80 h of postfreeze recovery.
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Sublethal cryoinjury
Expectedly, we found little of the intracellular marker proteins in plasma
of unfrozen frogs. Free Hb was absent. We detected low levels of LDH activity,
which did not differ (P=0.139) between urea-loaded (0.4±0.08
i.u. ml–1) and saline-treated (0.6±0.1 i.u.
ml–1) individuals. Levels of CK activity in plasma of
unfrozen frogs also were low, but inexplicably differed (P<0.0001)
between urea-loaded and saline-treated frogs (0.02±0.001
versus 1.1±0.1 mi.u. ml–1, respectively).
As evidenced by their rhythmically contracting hearts, all frozen–thawed frogs used for cryoinjury analysis were alive when examined 24 h after thawing commenced. These frogs physically resembled specimens used in the survival experiments at the same stage of recovery, although it is impossible to know whether they would have met our ultimate survival criterion. Experimental freezing–thawing was associated with higher plasma levels of intracellular proteins as compared to those in unfrozen frogs (P<0.01, all cases), indicating that cell damage occurred in both urea-loaded and saline-treated frogs. However, among frozen–thawed frogs, saline-treated animals had markedly higher (1.7- to 3.2-fold) levels of plasma LDH activity (P=0.027), CK activity (P=0.008) and Hb (P=0.005) relative to urea-loaded frogs (Fig. 2).
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Glucose levels in plasma and organs of frozen–thawed frogs were substantially higher (P<0.0001, all cases) than those of unfrozen frogs (Table 1), indicating that experimental freezing–thawing induced the characteristic hyperglycemic response. Among unfrozen saline-treated frogs, glucose levels were uniformly low in plasma (2µmolml–1) and organs (1–4µmolg–1), whereas 5- to 18-fold higher concentrations (P<0.001, all cases) were found in their frozen–thawed counterparts. Glucose levels also differed (P<0.0001, all cases) between unfrozen and frozen–thawed groups of urea-loaded frogs; however, the interaction terms of the two-factor ANOVAs (P<0.006, all cases) indicated that the magnitude of the glycemic response differed between urea-loaded and saline-treated individuals. Among frozen–thawed frogs, glucose levels in plasma and organs of urea-loaded frogs were up to 3.5-fold higher than those of saline-treated animals. Within the liver, the difference, 70 µmol g–1 versus 20 µmol g–1, was especially striking.
Plasma osmolality was strongly influenced by urea loading
(P<0.0001). Relative to the frogs receiving only isotonic saline,
plasma osmotic pressure was 55 mOsmol kg–1 higher in
urea-loaded frogs in both the unfrozen (277±5 versus
220±4 mOsmol kg–1) and frozen–thawed groups
(308±8 versus 255±5 mOsmol kg–1). This
increment reflected the measured increase in plasma urea concentration
(49–60 µmol ml–1) achieved with the injections.
Plasma osmolality markedly increased (P<0.0001) with
freezing–thawing over that in the corresponding unfrozen frogs, with the
increase being of similar magnitude (33–35 mOsmol kg–1;
P=0.840) in urea-loaded and saline-treated animals. In both groups,
the increment in osmotic pressure was primarily attributed to net differences
in glucose and urea concentrations between unfrozen and frozen–thawed
frogs (Table 1).
Urea loading had no effect (P>0.05, all cases) on the hydration
state of R. sylvatica organs and carcass, and, with the sole
exception of liver, neither did freezing–thawing (P>0.05,
all other cases) (Table 2).
Water concentration in liver of frozen–thawed frogs was 1.5 times
greater (P<0.0001) than that measured in unfrozen frogs, this
difference being unaffected (P=0.892) by urea loading.
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Hematocrit values determined for frozen–thawed frogs (urea-loaded: 36±3%; saline-treated: 45±2%) were substantially higher (P<0.0001) than those determined for unfrozen frogs (urea-loaded: 26±1%; saline-treated: 29±1%). Among frozen–thawed frogs, the mean hematocrit value for saline-treated frogs was higher (P=0.014) than that for urea-loaded frogs. However, among unfrozen animals, this variable did not differ (P=0.121) between the groups.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Because urea accumulation is a universal amphibian response
to osmotic challenge (Shpun et al.,
1992; Jørgensen,
1997), and because low temperature promotes urea retention through
diminished renal function (Nielsen and
Jørgensen, 1990), hyperuremia may be common among
terrestrially hibernating frogs. Indeed, elevated urea has been found in
winter R. sylvatica (Layne and
Rice, 2003; Costanzo and Lee,
2005) and Hyla versicolor (J. R. Layne, personal
communication). MacArthur and Dandy reported that plasma osmolality increased
by 50 mOsmol kg–1 in winter-acclimatized Pseudacris
triseriata (MacArthur and Dandy,
1982). Although they did not assay for specific solutes, we
suspect that urea accounted for much of the osmotic increase.
Long known as an osmoprotectant, urea also serves numerous other functions
in various ectotherms (Griffith,
1991; Withers,
1998). In terrestrially hibernating frogs, urea undoubtedly is
important in osmotic homeostasis, but may also contribute to winter survival
in other ways. For example, urea accumulated during autumn apparently
functions as a metabolic inhibitor, reducing the energetic cost of
overwintering (Muir et al.,
2007; Muir et al.,
2008). We hypothesize that urea also promotes survival of natural
freezing episodes by acting as a cryoprotective agent. This contention draws
support from experiments in which urea pretreatment of R. sylvatica
cells and organs enhanced their tolerance to freezing–thawing in
vitro (Costanzo and Lee,
2005; Costanzo et al.,
2008). The present study extends these findings by demonstrating
that physiological levels of urea enhance freezing survival and postfreeze
recovery in vivo.
Hyperuremia enhances freezing survival and postfreeze recovery
Tissue urea levels in urea-loaded frogs remained high during freezing and
thawing, although concentrations in the frozen–thawed specimens were
lower than those in unfrozen frogs (Table
1), apparently because some of the solute was lost via
renal filtration. We found no evidence that urea loading altered tissue
hydration, plasma osmolality or glycemia, and previous studies
(Shoemaker, 1965;
Taylor et al., 1999) have
shown it affects neither cutaneous water uptake nor behavior. Thus, we are
reasonably confident in ascribing the observed variation in survival and
postfreeze recovery rates of R. sylvatica to differences in uremia
between treatment groups.
Experimentally augmenting tissue glucose levels improves freezing survival
of R. sylvatica, providing strong evidence for the cryoprotective
function of this osmolyte (Costanzo et al.,
1991a; Costanzo et al.,
1993). Similarly, urea loading enhanced survival of R.
sylvatica subjected to freezing at a potentially lethal temperature,
–4°C. This finding is biologically relevant because urea levels in
these frogs (50–80 µmol g–1) were within the range
that can be achieved during hibernation (i.e. to at least 90 µmol
ml–1 in blood) (Costanzo
and Lee, 2005). By contrast, mortality occurred among
saline-treated frogs, whose tissues contained more modest levels of urea
(12–16 µmol g–1).
Leakage of intracellular proteins is a useful index of sublethal cryoinjury
in intact organisms (Costanzo et al.,
1991a; Costanzo et al.,
1993; Costanzo et al.,
1997a; Irwin et al.,
2003; Costanzo et al.,
2006). In our experiment, hyperuremia not only enhanced freezing
survival, but also reduced sublethal cryoinjury, as urea-loaded frogs had
lower extracellular levels of the marker proteins LDH, CK and Hb. Minimizing
insult to cells and tissues is an important function of cryoprotectants
because even freeze-tolerant animals die if injury is excessive. This concept
is underscored by the responses of three saline-treated frogs that were alive
after thawing but ultimately failed to meet the survival criterion. We do not
know why these particular individuals (and two other saline-treated frogs,
which were dead upon thawing) succumbed to freezing. Although cryoprotectant
levels were not measured whilst these frogs were frozen, when assayed 7 days
after thawing commenced, their plasma concentrations of urea (11.1±1.4
µmol ml–1) and glucose (2.0±0.5 µmol
ml–1), as well as plasma osmolality (213±5 mOsmol
kg–1), were comparable to those of unfrozen, saline-treated
animals. Thus, possibly they amassed only meager amounts of cryoprotective
solute and thereby incurred greater injury. Indeed, plasma collected from
these moribund frogs contained substantial amounts of LDH (2.9±0.8 i.u.
ml–1), CK (3.3±0.2 mi.u. ml–1) and Hb
(2.9±0.4 mg ml–1) that were 14–58% higher than
those measured in frozen–thawed, saline-treated frogs
(Fig. 2). However, these data
should be interpreted cautiously because the moribund frogs were sampled much
longer (7 days versus 24 h) after thawing commenced; thus, their
higher protein levels could partly reflect protracted leakage, rather than
more extensive damage.
Additional evidence for urea's cryoprotective efficacy is the finding that
recovery from freezing–thawing stress was expedited in urea-loaded
frogs. In R. sylvatica, restoration of complex neurobehavioral
functions following freezing–thawing requires from several hours to many
days, depending on the severity of the freezing exposure with respect to its
rapidity, duration and minimum temperature
(Costanzo et al., 1991a;
Layne, 1992;
Costanzo et al., 1993;
Layne et al., 1998). This
relationship implies that recovery attends resolution of homeostatic
perturbations and repair of injuries. Given that freezing impairs function in
both nerve and muscle (Kling et al.,
1994; Irwin et al.,
2003), complex behaviors, including ones we examined, are highly
sensitive to freezing–thawing stresses. Relative to saline-treated
frogs, hyperuremic frogs recovered rapidly, suggesting that urea somehow
limits freezing-induced perturbations to neurobehavioral function.
Physiological responses to freezing–thawing
Results of the present investigation, like those of earlier studies with
R. sylvatica (Layne and Rice,
2003; Costanzo and Lee,
2005), show that freezing stimulates synthesis of glucose, but not
urea. Mediated by adrenergic stimulation of hepatocytes, somatic freezing
initiates glycogenolysis and production of glucose, which is then distributed
throughout the body (Storey and Storey,
2004). Apparently there is no equivalent system for mobilizing
urea; therefore, frogs must accumulate this osmolyte in anticipation of
freezing.
Because glucose clearance begins soon after thawing
(Layne et al., 1996;
Costanzo et al., 1997a),
glycemic levels in our frogs (sampled 24 h after thawing began) were markedly
lower than those usually found in still-frozen frogs
(Storey and Storey, 2004) [see
also table 4 in our earlier report
(Costanzo and Lee, 2005)].
Filtered glucose is not necessarily lost from the body, but can be reabsorbed
by the bladder epithelium and ultimately reconverted to liver glycogen
(Costanzo et al., 1997b). We
found differences in glycemic state between urea-loaded and saline-treated
frogs, tissues of the former containing considerably more glucose. Although we
cannot rule out the possibility, it seems unlikely that urea-loaded frogs had
synthesized more glucose, which could have contributed to their enhanced
survival. Rather, we suspect that hyperuremic and saline-treated frogs
differed in efficacy of glucose clearance subsequent to thawing. Conceivably,
differential clearance rates could result if urea, a well-known perturbant of
many proteins, inhibited glycogenesis through its interaction with one or more
glycolytic enzymes, such as phosphofructokinase
(Hand and Somero, 1982).
Unlike the case with cartilaginous marine fishes, amphibians apparently do not
co-accumulate methylamines to levels that would counteract perturbing effects
of urea (Wray and Wilkie,
1995; Withers and Guppy,
1996). In any case, hyperglycemia persisting longer during
postfreeze recovery could benefit R. sylvatica by fueling repair
processes and limiting injury due to subsequent, rapid freezing
(Costanzo et al., 1991b).
Aside from using protective solutes, freeze-tolerant anurans minimize
freeze–thaw stress by re-compartmentalizing extracellular water in a
manner that reduces the amount of ice forming within the tissues and
microvasculature. During freezing, much of the water inside organs (up to 60%)
is translocated to the coelom and lymph sacs, where it freezes innocuously
(Lee et al., 1992). After
thawing, circulation resumes and organs quickly rehydrate to prefreeze levels
(Costanzo et al., 1997a).
Accordingly, with the exception of the liver (which tends to hyperhydrate,
probably owing to high solute content), the organs of our frozen–thawed
frogs, sampled 24 h after thawing began, resembled those of unfrozen frogs.
Tissue water contents of urea-loaded and saline-treated frogs were similar,
suggesting that hyperuremia has no influence on the organ dehydration
response; however, studies of fully-frozen animals are needed to draw a
definitive conclusion.
Despite the cryolytic loss of some erythrocytes, the hematocrit of
frozen–thawed frogs commonly is higher than that of unfrozen animals
whilst excess water remains within extravascular spaces
(Costanzo et al., 1991b;
Costanzo et al., 1997a;
Irwin et al., 2003).
Accordingly, both urea-loaded and saline-treated frogs were hypovolemic
following thawing. However, hematocrit in the urea-loaded frogs more closely
matched that of unfrozen frogs, suggesting that urea aided restoration of
blood volume. We speculate that this could occur if urea functions as a
vasodilator (Vajragupta et al.,
1996) and/or if urea enhances water flux by mediating increased
expression of water channels (Storm et
al., 2003; Umenishi et al.,
2005).
Possible mechanisms of cryoprotection by urea
Freeze-tolerant organisms commonly use cryoprotectants representing a
diverse array of organic compounds that share certain attributes, including
low molecular mass, high solubility and permeability, stability, ready
availability and compatibility with macromolecules
(Storey and Storey, 2004). By
virtue of the colligative properties of small solutes, these agents reduce
both the osmotic loss of cell water and the amount of ice forming at any given
temperature. Because urea undoubtedly functions in this manner, its
cryoprotective efficacy is, at least to some extent, concentration dependent
(Costanzo et al., 2008).
However, pretreating R. sylvatica erythrocytes with 80 or 40 µmol
urea ml–1 afforded virtually the same margin of protection
from in vitro cryoinjury (Costanzo
and Lee, 2005), suggesting that this solute also has special
protective properties.
Various organic osmolytes function as cryoprotectants by mollifying
freeze–thaw damage to macromolecules and cellular structures
(Carpenter and Crowe, 1988;
Göller and Galinski,
1999). Contrary to its reputation as a destabilizing agent, urea,
especially when present in low concentrations, can actually enhance
hydrophobic interactions, perhaps by increasing the solvent structure, and
thereby stabilize certain proteins (Bhuyan,
2002; Kumar et al.,
2004; Chakraborty et al.,
2005; Gull et al.,
2007). Urea may even counteract deleterious effects of elevated
ionic solutes on biopolymers (Tian and
Cohen, 2001). Contrary to its protein denaturing effect at high
(i.e. molar) concentration, physiological levels of urea may be even less
perturbing than some `compatible' osmolytes (e.g.
Yancey and Burg, 1990).
Our results suggest that urea also provides cryoprotection at the cellular
(and perhaps higher) level of biological organization, although its precise
mode(s) of action remains to be determined. Pretreatment with urea protects
certain cells from hyperionic stress leading to apoptotic death
(Santos et al., 1998;
Zhang et al., 2000). Urea,
which is used in the clinical treatment of hyponatremia, also prevents brain
damage and neurobehavioral aberrations caused by fluctuations in ion levels,
such as may occur with freezing–thawing
(Soupart et al., 2007). That
the R. sylvatica heart is particularly amenable to cryoprotection by
urea (Costanzo and Lee, 2005)
is consistent with this solute's high capacity to scavenge certain reactive
oxygen species, thereby protecting cardiocytes from post-ischemic reperfusion
injury (Wang et al., 1999).
Although urea may be less effective against hydroxyl radicals
(Halliwell, 1978), its high
permeability, ubiquity and ease of mobilization are advantages over other
natural antioxidants.
A compound cryoprotectant system
Like many freeze-tolerant animals, R. sylvatica apparently uses a
mixture of osmolytes in its cryoprotectant system. This may be advantageous
because each component offers unique benefits, but also has certain
limitations. For example, urea accrues in autumn, chiefly in response to
environmental water deficit, such that all tissues become laden before
freezing begins. On the other hand, frogs may fail to amass much urea if
environmental moisture is abundant. Glucose accumulation, triggered directly
by tissue freezing, is far more predictable and potentially more robust, but
tissue levels rise meagerly if freezing proceeds rapidly or if hepatic
glycogen reserves are low (Costanzo and
Lee, 2005). Furthermore, levels of glucose, which must be
synthesized in the liver, exported to distant organs, and transported into
cells whilst freezing proceeds, vary markedly between core and peripheral
organs (Storey and Storey,
2004). The net effect is that cells in some tissues could contain
as much (or more) urea as glucose (Table
1) (Costanzo and Lee,
2005).
Another consideration is that the combination of glucose and urea may
provide benefits not bestowed individually. Furthermore, certain osmolytes may
excel at performing functions to which others are not particularly well
suited. For example, in one experiment, urea was better than glucose at
reducing freeze–thaw injury to R. sylvatica erythrocytes,
perhaps because it was superior at raising the fraction of unfreezeable cell
water and/or protecting macromolecules and cellular structures
(Costanzo and Lee, 2005).
Future studies aimed at identifying specific mechanisms of protection
conferred by these osmolytes, both individually and in combination, would be
instructive.
Our present results, together with findings of recent studies
(Costanzo and Lee, 2005;
Costanzo et al., 2008),
marshal compelling evidence for a role of urea as a natural cryoprotective
osmolyte in R. sylvatica. Collectively, this work establishes a
previously undocumented role for this `waste product' of nitrogen metabolism,
and also identifies a new class of natural cryoprotectant. In addition, the
notion that an osmolyte accumulated preparatory to freezing contributes to
freezing survival challenges the longstanding view that freezing-induced
mobilization of cryoprotective solute is the hallmark physiological adaptation
of amphibian freeze tolerance.
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Acknowledgments |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Bhuyan, A. K. (2002). Protein stabilization by urea and guanidine hydrochloride. Biochemistry Mosc. 41,13386 -13394.[CrossRef]
Canty, A., Driedzic, W. R. and Storey, K. B. (1986). Freeze tolerance of isolated ventricle strips of the wood frog, Rana sylvatica. Cryo Letters 7, 81-86.
Carpenter, J. F. and Crowe, J. H. (1988). The mechanism of cryoprotection of proteins by solutes. Cryobiology 25,244 -255.[CrossRef][Medline]
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