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Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, United Kingdom
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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This study examined whether the steady-state hypometabolism seen in overwintering frogs (Rana temporaria) is reflected at the mitochondrial level either by a reduction in their resting (state 4) and active (state 3) respiration rates and/or by increases in O2 affinity. We isolated mitochondria from the skeletal muscle of cold-submerged frogs at different stages during their hibernation in normoxic and hypoxic water. A modest metabolic depression at the whole animal level (normoxic submergence) was not associated with a reduction in mitochondrial state 4 and state 3 respiration rates. However, mitochondria isolated from frogs that were submerged for 1 mo manifested an increase in their O2 affinity compared with controls and with animals submerged for 4 mo. Hypometabolism was more pronounced at the whole animal level during hypoxic submergence and was accompanied by 1) a reduction in mitochondrial state 4 and state 3 rates and 2) an increase in the O2 affinity of mitochondria. These findings demonstrate that metabolic depression can be reflected at all levels of biological organization in hypoxia-tolerant animals.
frog; hypoxia; state 3; state 4; P50
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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MANY SPECIES OF FROGS cope with harsh overwintering conditions each year. The common frog, Rana temporaria, hibernates under water, often in ice-covered ponds (40). Although the selection of an aquatic environment protects against the stresses of freezing and desiccation, overwintering submergence can last for several months and is often associated with severe hypoxia as well as inhibition of normal feeding behavior (2, 5). When R. temporaria are submerged without air access for 3 to 4 mo at 3°C, so as to mimic the overwintering conditions under ice cover, they enter gradually into a state of metabolic depression. The extent of metabolic depression is greater when frogs are exposed to hypoxic environments (75% depression at PO2 = 60 mmHg; Ref. 14) than in normoxia (39% depression at PO2 = 155 mmHg; Ref. 15). The key to their survival during long periods of cold submergence in hypoxic environments is their ability to enter slowly into a hypometabolic state so that their energetic needs can be met aerobically. Indeed, R. temporaria deplete their substrate reserves and perish when they are presented with rapid or prolonged exposure to severe hypoxia (PO2 < 40 mmHg) because they lack the capacity to acutely suppress their metabolic rate to match such drastic reductions in ambient oxygen supply (13).
Metabolic depression is the strategy of choice for many overwintering ectotherms facing energy limitations because it spares substrate reserves and avoids the accumulation of toxic end-products from anaerobic metabolism (31). At the cellular level, metabolic depression may be brought about by decreasing ATP consuming processes and/or by increasing the efficiency of ATP producing pathways (reviewed in Refs. 24, 26, 28, 30, 44). Among the ATP consuming processes, the rates of protein synthesis and of Na+-K+-ATPase are often reduced in metabolically depressed ectotherms (24, 26, 28, 30). For example, when R. temporaria were submerged in normoxic or hypoxic water, the activity of their skeletal muscle Na+-K+-ATPase was reduced by as much as 50% (16). In addition to a reduced ATP demand for protein synthesis and for Na+-K+-ATPase activity, the rates of protein breakdown, ureagenesis, and gluconeogenesis were found to be reduced when normoxic turtle hepatocytes were exposed to anoxia (reviewed in Ref. 30). Most studies concerning the increased efficiency of energy production during metabolic depression have focused on anaerobic metabolism. For example, some invertebrate species display energetically improved fermentative pathways during anoxia (31). In addition, enzyme phosphorylation events can play important roles in regulating glycolytic ATP production (reviewed in Refs. 6, 43, 44). However, despite the fact that many organisms entering into metabolic depression maintain an overall aerobic metabolism, little information exists concerning the intrinsic properties of mitochondria in such hypometabolic states.
During prolonged submergence, frogs hyperperfuse their cutaneous vasculature to facilitate transcutaneous O2 uptake (4). As a consequence of preferentially redistributing the O2-enriched blood at the skin to the hypoxia-sensitive central organs, the largely inactive and hypoxia-tolerant skeletal muscle mass becomes hypoperfused (4, 40). It appears, therefore, that the skeletal muscle of overwintering frogs may become severely hypoxic for prolonged periods during hibernation. Given that experiments on isolated frog muscle show that step decreases in perfusate oxygen lead to step decreases in muscle metabolic rate (3, 49) and given that 35-40% of the frog's body mass is skeletal muscle, we can conclude that blood flow and therefore O2 flow limitations to the oxyconforming muscle mass probably serve to bring about a major proportion of the overall reduction in whole animal metabolic rate (14).
The aim of this study was to test whether the metabolic depression observed in overwintering frogs is reflected at the mitochondrial level either through changes in respiration and/or mitochondrial O2 affinity. To do so, we examined the intrinsic properties of respiration in mitochondria isolated from the skeletal muscle of frogs at various stages of their hibernation.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals. All animals used in these experiments were adult male R. temporaria (~25-30 g) collected by a local supplier (Blades Biological) during the winters of 1998 and 1999. At the start of each winter, frogs were acclimated to 3°C water for 4 wk, during which time they had direct access to air. After this acclimation period, 15 frogs were then taken for experiments (control groups) while another 30 were submerged in either normoxic water (PO2 = 155 mmHg; winter 1998) or hypoxic water (PO2 = 60 mmHg; winter 1999) in a temperature-controlled recirculated water system (Living Stream, Frigid Units, Cleveland, OH) maintained at 3°C, as described previously (14). The normoxic and hypoxic submerged frogs were sampled after 1 and 4 mo.
Isolation of mitochondria. Frogs were killed by concussion of the brain followed by its immersion in liquid nitrogen according to Schedule 1 Home Office protocols (UK). Mitochondria were isolated according to a modification of the methods from Hillman et al. (29). The thigh muscles of three frogs were pooled for each mitochondrial preparation. Each thigh muscle was excised rapidly and finely minced with razor blades. The tissue was then placed in a beaker containing the isolation medium and further homogenized with microscissors. The isolation medium contained (in mM) 170 mannitol, 55 sucrose, 5 EGTA, 20 HEPES, and 50 units/ml heparin and 0.5% BSA adjusted to pH 7.3 at room temperature. The homogenate was transferred to a Potter-type glass homogenizer and ground (200 rpm) consecutively with three pestles of increasing size (2 strokes with each pestle). The homogenate was then centrifuged at 755 g for 5 min, and the supernatant was subsequently filtered through medical gauze and centrifuged at 9,800 g for 11 min. The resulting pellet was then washed with isolation medium lacking heparin, resuspended, and centrifuged at 9,800 g for 8 min. The final pellet was resuspended in isolation medium lacking heparin at a concentration of 25-30 mg of mitochondrial protein/ml. Protein concentrations were determined by the Biuret method (23), and sodium deoxycholate was added to disrupt the membranes.
High-resolution respirometry. To measure oxygen consumption rates and the O2 affinity of isolated mitochondria, we used a respirometer (Oroboros Oxygraph, Paar, Graz, Austria) that enables sensitive measurements of oxygen kinetics at low oxygen partial pressure (22, 27). The air-equilibrated assay medium contained (in mM) 55 mannitol, 24 sucrose, 10 HEPES, 10 K2HPO4, 90 KCl, and 0.5% BSA adjusted to pH 7.3 at room temperature. The experiments were carried out at 3 and 20°C. The main purpose of the experiments carried out at 20°C was to facilitate comparisons with previous data. The temperature of the Oxygraph was regulated to ±0.05°C by a Peltier heat pump. The oxygen solubility of the assay medium was considered to be 18.194 and 12.135 µM/kPa at 3 and 20°C respectively. Calibration of the system, including signal correction for electrode response time, blank controls, internal zero calibration as well as data acquisition and analyses, was carried out as described elsewhere (22, 27, 37). The signals from the oxygen electrode were recorded at 1-s intervals on a computer-driven data acquisition system (DatLab software; Oroboros, Innsbruck, Austria).
The oxygen concentration in the Oxygraph chamber was reduced to ~20% of air saturation at each experimental temperature by blowing nitrogen on the surface of the assay medium before the addition of mitochondria at a final concentration of 1 mg mitochondrial protein/ml. First, malate (1.2 mM) was added to spark the Krebs cycle followed by the addition of pyruvate (5 mM). The respiration rate of mitochondria under these conditions is called state 2. The state 3 respiration rate was then obtained by the addition of 0.428 mM ADP. The mitochondria subsequently consumed all of the oxygen and entered anoxia while still in state 3. After 10 min of anoxia, the oxygen concentration in the chamber was raised again to roughly 20% of air saturation by lifting the lid for a few seconds. The state 4 respiration rate was then reached (state 4 is reached when all the ADP is phosphorylated into ATP) and was recorded until the mitochondria entered a second anoxic period. The experimental protocol is represented graphically in Fig. 1. The respiratory control ratio (RCR) was calculated by dividing the state 3 respiration rate by the state 4 respiration rate.
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Calculations and statistical analyses. All data are presented as means ± SE. Statistical analyses were performed with SigmaStat (version 2.0). Comparisons of state 3 rates, state 4 rates, state 3 O2 affinity (1/P50) values, state 4 P50 values, RCR values, and Q10 values between the three groups of frogs for each year were performed with one-way ANOVA and the a posteriori test of Tukey. Comparisons of state 3 rates, state 4 rates, state 3 P50 values, state 4 P50 values, and RCR values between the two groups of control frogs used the Student's t-test. Comparisons of RCR values between experimental temperatures and of P50 values between state 4 and state 3 rates were carried out with Student's paired t-test. The level of significance was P = 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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General characteristics of mitochondria.
For the experiments carried out in 1998 (normoxic hibernation), there
were no significant differences in the RCR values of control,
1-mo-submerged, and 4-mo-submerged groups of frogs at either of the
experimental temperatures (Table 1).
However, there was a general tendency toward higher RCR values at
20°C compared with 3°C, but this only reached statistical
significance for the 1-mo-submerged group of frogs (P = 0.046; Table 1). There was no difference in the Q10 values
for state 3 and state 4 respiration rates between the three groups of
animals (Table 1).
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Respiration rates and O2 affinity of mitochondria from normoxic submerged frogs. At 3°C, the active (state 3) and resting (state 4) oxygen consumption rates were unchanged between the control, 1-mo-submerged and 4-mo-submerged groups of frogs (Fig. 2). The P50 values for state 3 and state 4 tended to decrease in the 1-mo-submerged frogs compared with controls, but the tendency was significant only for the state 3 P50 values (Fig. 2; P = 0.077 for P50 values in state 4). The P50 values for state 3 and state 4 respiration rates from the 4-mo-submerged group were similar to those of the control group (Fig. 2). At 20°C, there was no difference in the state 3 and state 4 rates or their corresponding P50 values between the three groups of animals (Table 3). At both experimental temperatures and for each group of frogs, the P50 values were lower in the resting state compared with the active state, but this difference did not reach statistical significance for the group of control frogs at 20°C (Fig. 2 and Table 3).
The kinetics of resting (state 4) and active (state 3) oxygen consumption rates at 3°C of frog mitochondria from all groups of frogs in the low oxygen range are illustrated in Fig. 3. The decrease in state 3 and state 4 P50 values observed in the 1-mo-submerged frogs is illustrated in Fig. 3 as a shift of the state 3 and state 4 respiration curves to the left compared with the control and 4-mo-submerged ones (see Fig. 3, insets).
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Respiration rates and O2 affinity of mitochondria from
hypoxic submerged frogs.
After 1 and 4 mo of hypoxic submergence at 3°C, the active and
resting oxygen consumption rates were both reduced by ~60% compared
with controls (Fig. 4). This decreased
active respiration rate in mitochondria from the hibernating frogs was
paralleled by a ~60% diminution of the state 3 P50 value
at 1 and 4 mo compared with controls (Fig. 4). On the other hand, there
were no significant differences observed for the P50 values
at state 4 among the three different groups of animals (Fig. 4).
However, the state 4 P50 values obtained at 3°C were
extremely low (~0.0034 kPa) and probably near the limit of detection
of the apparatus, thus making it difficult to rule out any possible
decrease in mitochondrial P50 during submergence. At
20°C, the state 3 and state 4 respiration rates were reduced by ~70
and ~30%, respectively, in both the 1- and 4-mo-submerged groups
compared with controls (Table 4). Moreover, the state 3 P50
values of the two submerged groups were ~75% lower than the control
values (Table 4). In contrast, the state 4 P50 value after
4 mo of hibernation was higher than those of both the control and
1-mo-submerged groups of frogs (Table 4). As was the case for normoxic
hibernation, the P50 values were lower in the resting state
compared with the active state at both experimental temperatures and
for each group of frogs, but this difference did not reach statistical
significance for the 1- and 4-mo-submerged groups of frogs at 3°C and
for the 4-mo-submerged group of frogs at 20°C (Figs. 4 and Table 4).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The results presented in this paper show for the first time that an increase in the in vitro O2 affinity of mitochondria can occur after chronic in vivo exposure to cellular hypoxia. Moreover, the results demonstrate that metabolic depression at the whole animal level can be reflected at the mitochondrial level. Taken together, these findings illustrate the plasticity of mitochondria under physiological constraints.
The moderate metabolic depression at the whole animal level in frogs submerged in normoxic water (15) was not reflected at the mitochondrial level (Fig. 3). In fact, the active and resting respiration rates of mitochondria stayed more or less constant throughout normoxic submergence. However, the mitochondria from the 1-mo-submerged group of frogs showed an increase in their state 3 O2 affinity. This increase in O2 affinity would act to promote, rather than suppress, aerobic metabolism in a microoxic intracellular environment (Fig. 3A, inset). However, when metabolic depression becomes fully developed after 4 mo of submergence, the mitochondrial state 3 P50 value reverts to the level observed in control animals (Fig. 3). The corresponding state 4 results (respiration rate and P50) reveal similar trends without reaching statistical significance (Fig. 3B, inset).
The increase in mitochondrial O2 affinity after 1 mo of submergence in normoxic water might not be physiologically relevant. In fact, we have no indication to believe that the intracellular PO2 inside frog skeletal muscle during normoxic submergence are around mitochondrial state 3 or state 4 P50 values. Frogs submerged in normoxic water do not display an increase in plasma or skeletal muscle lactate levels, thereby indicating that blood supply (i.e., oxygen supply) to the skeletal muscle, despite being drastically reduced, is sufficient to maintain aerobic metabolism (15). Moreover, the ATP, ADP, AMP, phosphocreatine, and creatine levels inside the skeletal muscle stay constant throughout normoxic submergence, indicative of a balance between ATP supply and demand (15).
The O2-dependent properties of mitochondria isolated from hypoxic animals show a more profound reorganization of function. For example, mitochondria isolated from frogs submerged in hypoxic water for 1 and 4 mo display reduced resting and active respiration rates and thus metabolic rate at any given intracellular PO2 (Fig. 5). The mechanism responsible for the decrease in mitochondrial state 4 respiration rates during submergence in hypoxic water is a reduction in the activity of the electron transport chain (45). This reduction in electron transport chain activity is at least partly, and might be even entirely, responsible for the decrease in state 3 respiration rates. However, it is also possible that a reduction in the activity of the phosphorylation system plays a role in the decrease of mitochondrial state 3 respiration rates. The intracellular PO2 inside frog skeletal muscle during hypoxic submergence are probably around mitochondrial state 3 and state 4 P50 values and even lower. In fact, frogs recruit anaerobic metabolism during the first 2 mo of submergence in hypoxic water as indicated by the marked increase in plasma lactate concentration (14). A large proportion of the increase in plasma lactate concentration is thought to come from the hypoperfused muscle mass (13). The plasma lactate concentration decreases steeply after 1 mo of submergence in hypoxic water and returns to presubmergence values after 2 mo when the metabolic rate of the frog is dramatically reduced compared with controls (14). In fact, the metabolic depression after 2 mo of submergence is so profound that the energetic needs can now be met through an entirely aerobic metabolism (14).
The reduction in the resting and active respiration rates of frog skeletal muscle mitochondria during hypoxic submergence will lead to a decrease in the rate of ATP production. Even so, the rate of ATP consuming processes seems to be reduced accordingly, as indicated by the maintenance of ATP, phosphocreatine, and creatine levels similar to controls throughout hypoxic submergence (14). However, there is an increase in the adenylate energy charge inside the skeletal muscle during hypoxic submergence owing to significant decreases in ADP and AMP levels (14). This might indicate lowered ATP demand by the skeletal muscle. In fact, the activity of the skeletal muscle Na+-K+-ATPase is reduced by 50% during hibernation in hypoxic water (16). The profound metabolic depression observed in frogs after 2 mo of submergence also has the advantage of reducing the rate of depletion of glycogen inside the skeletal muscle (14). Overall, the steady-state metabolic depression at the whole animal level during hypoxic submergence (14) is reflected at the cellular level by a maintenance of energy balance; a situation similar to that during normoxic hibernation.
Modifications in mitochondrial properties also occur in mammals during chronic exposure to hypoxia. Hypoxia in mammalian cells is often correlated with a reduction in cytochrome levels and mitochondrial enzyme activities. In addition, respiration rates decrease in brain mitochondria of rats and mice exposed to intermittent hypobaric hypoxia (10, 17, 35, 38). However, the resting and active respiration rates of liver and heart mitochondria isolated from rats acclimatized to hypobaric hypoxia did not differ from those of control rats (11), not even when measured at more physiological low oxygen concentrations (12). Previous studies concerning the affinity of cells and of mitochondria for oxygen have produced contrasting results. On the one hand, studies carried out on liver mitochondria isolated from hypoxic and control rats revealed no change in P50 values (12, 32). On the other hand, the cellular P50 values for hepatocytes isolated from hypoxic rats were lower than those from control rats (32). Such changes in cellular P50 values have been ascribed to a redistribution of mitochondria within the cell (11; reviewed in Ref. 32). Similarly, the P50 values of rat mitochondria isolated from hypoxia-tolerant newborns are not different than those of their hypoxia-sensitive adults, whereas hepatocytes isolated from newborn rats manifest lower P50 values than those of their adult counterparts (1). Again the differences in cellular P50 values were ascribed to differences in the density and distribution of mitochondria within the cellular network (32). The differences in mitochondrial and cellular responses to hypoxia in the ectotherm and mammal may reflect fundamental differences in their tolerance to hypoxia and/or their relative capacity to exploit metabolic suppression at the whole animal level.
Entering into a new viable hypometabolic state implies that energy supply remains balanced with energy demand. Many studies have looked at cellular adaptations, focusing mainly on the energy demand processes that can be turned down during metabolic depression (reviewed in Refs. 24, 26, 28, 30). However, few studies have looked at the intrinsic properties of mitochondria during hypometabolic states. From the information available, we know that mitochondrial state 3, but not state 4, respiration rates are reduced in hibernating mammals (7-9, 19, 34, 36, 39). We also know that respiration rates of hepatocytes isolated from hibernating ground squirrels are the same as the rates obtained from hepatocytes isolated from summer "cold-acclimated" animals (41). Indeed, there is no evidence that state 4 rates become altered during hibernation in mammals. With regard to ectotherms, citrate synthase (CS) activities are unchanged during metabolic depression in most tissues of the terrestrial snail, with the exception of the hepatopancreas (47). Similarly, cytochrome c oxidase activity is considerably reduced in the hepatopancreas of estivating snails compared with control animals (46). Also, mitochondrial protein synthesis is markedly decreased during anoxia-induced quiescence in brine shrimp embryos (33). Other studies have shown intrinsic reductions in metabolic rate at the tissue level (18) and at the cellular level (25). The present study demonstrates metabolic depression at the mitochondrial level (Fig. 5), supporting the view that hypometabolism can be reflected at all levels of biological organization in hypoxia-tolerant animals.
This study reports state 3 and state 4 P50 values from an ectotherm using high-resolution respirometry. The average state 3 and state 4 P50 values for the two control groups of frogs are ~0.077 and 0.017 kPa, respectively, at 20°C. These values are similar to the state 3 and state 4 P50 values of rat liver mitochondria (0.057 and 0.020 kPa) and rat heart mitochondria (0.035 and 0.016 kPa) at 30°C (20). The mitochondrial P50 values for all groups of frogs increase during transition from a resting state (state 4) to an active one (state 3). Other studies have shown similar increases in P50 values in active states in isolated mitochondria (12, 20, 48). The present study shows that state 3 and state 4 P50 values can change with metabolic rate. By varying the mitochondrial concentration in their experimental chamber, Steinlechner-Maran et al. (42) noted an incidental positive correlation between respiration rate and P50 in endothelial cells. However, the P50 values of isolated mitochondria were shown to be constant at various mitochondrial concentrations in the same experimental chamber as we used (20). This, in conjunction with the fact that we carried out all our experiments at the same concentration of mitochondria (see MATERIALS AND METHODS), supports the conclusion that the parallel decrease in state 3 respiration rates and state 3 P50 values during hypoxic submergence (Fig. 5) was due to different active metabolic states of the mitochondria.
There is an inverse relationship between the turnover rate of cytochrome c oxidase and the O2 affinity of mitochondria that can explain the differences in P50 values between state 3 and state 4 respiration rates and between different types of mitochondria (21). The turnover rate of cytochrome c oxidase is reduced under state 4 conditions, which leads to a decrease in P50 value compared with state 3 conditions. Rat heart mitochondria have a higher excess capacity of cytochrome c oxidase compared with rat liver mitochondria, which leads to a lower turnover rate of cytochrome c oxidase at high flux through the electron transport chain (state 3) by distributing electron input flux through a higher number of enzymes. This result correlates with the higher O2 affinity of heart mitochondria compared with liver mitochondria under state 3 conditions (21).
Perspectives
During submergence in normoxic water, the state 3 P50 values were lower in 1-mo-submerged frogs compared with control and 4-mo-submerged groups of animals, whereas respiration rates stayed more or less constant throughout submergence (Fig. 3A). This result suggests an increase in the catalytic efficiency [maximal velocity (Vmax)/P50] of mitochondria from the 1-mo-submerged group of frogs compared with control and 4-mo-submerged groups of frogs. On the other hand, the diminution in the active respiration rates during submergence in hypoxic water was proportional to the decrease in state 3 P50 values, indicating that mitochondria from frogs hibernating in hypoxic water maintain the same catalytic efficiency as the control frogs (Fig. 5A). The ratio Vmax to P50, which is based on the Kcat-to-Km ratio used for isolated enzymes, has been used in the past as an indicator of the catalytic efficiency of mitochondria when using a given type of mitochondria (for a review, see Ref. 21). However, because we are comparing mitochondria from animals in different metabolic states, we must interpret this result cautiously. In fact, the values we measured are a complex result of different factors (e.g., substrate and electron transport capacities as well as cytochrome c oxidase concentration), which can vary between the three groups of frogs. It will be interesting to see if these modifications in intrinsic properties of mitochondria during hibernation in frogs are accompanied by alterations in the number or distribution of mitochondria within the muscle fibers. Mitochondrial and cellular changes could act in concert in the skeletal muscle of frogs to bring about important functional modifications in face of severe hypoxia. We know very little about the properties of mitochondria during metabolic depression. Further studies are required to gain a better understanding of the role that mitochondria play in the development of hypometabolic states.| |
ACKNOWLEDGEMENTS |
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The authors thank Dr. T. G. West and Dr. M. D. Brand for critical reading of the manuscript.
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FOOTNOTES |
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This work was funded by the Natural Environment Research Council. J. St-Pierre was recipient of Natural Sciences and Engineering Research Council of Canada (NSERC), Fonds pour la formation de chercheurs et l'aide à la recherche and Trinity College (UK) scholarships. G. J. Tattersall was recipient of a 1967 Centennial NSERC scholarship.
Present address of G. J. Tattersall: Department of Biological Sciences, Kent State University, Kent, OH 44242
Address for reprint requests and other correspondence: J. St-Pierre, Dept. of Zoology, Univ. of Cambridge, Downing St., Cambridge CB2 3EJ, United Kingdom (E-mail: js264{at}cam.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 January 2000; accepted in final form 8 May 2000.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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O2
(%max)=[PO2/(P50 + PO2)]*100 (
Significant difference from the
respiration rate of the control group; P < 0.05 (1-way
ANOVA and a posteriori test of Tukey).
