Metabolic arrest and its regulation in anoxic eel hepatocytes.

Some vertebrates depress overall metabolism in an abrupt and reversible fashion when challenged with anoxia, ensuring stabilization of cellular [ATP] and long-term survival, but little is known about the eliciting stimuli (e.g., change in O2, adenylates) and downstream effectors responsible for metabolic arrest. Accordingly, eel (Anguilla anguilla) hepatocytes were treated with inhibitors of putative components of the oxygen/metabolite-sensing pathway(s) and exposed to anoxia (Po2=0 mmHg). Anoxia in untreated cells caused a remarkable 85-fold decrease in ATP production rate, but cellular ATP levels stabilized following an initial steep drop. Reoxygenation of cells after 4 h of anoxia caused a fast metabolization of accumulated lactate and reestablishment of preanoxic ATP levels. Unlike physiological anoxia, pharmacological inhibition of the electron transport chain in the presence of oxygen caused extensive cellular ATP depletion, though no loss in viability. In contrast, cellular lactate (i.e., ATP) production rate was affected similarly by either treatment, suggesting that anaerobic glycolysis is regulated by a stimulus other than oxygen tension per se, whereas the continuous matching of ATP consumption and a rapidly ceasing mitochondrial ATP supply require a physiological relevant change in oxygen tension. Protein kinases, notably kinase C (PKC) and A (PKA), have been proposed as key downstream regulators of stress-induced defense mechanisms, but anoxic cell viability, metabolic rate, and [ATP] were not significantly affected by inhibitors of PKC and PKA. Likewise, inhibition of the upstream PKC-activating enzymes phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI 3-K) had no effect on recorded parameters. Anoxic cell survival in complex organisms may, in vivo, also depend on stress hormones released from distant oxygen-sensing cells. Accordingly, adrenaline elevated anaerobic energy production but, apparently, also elevated ATP consumption because cellular ATP levels during oxygen deprivation were slightly lowered by adrenergic stimulation.

Caudal differential pressure as a predictor of swimming speed of cod (Gadus morhua).

We report the results of an experiment designed to investigate the feasibility of using differential pressure to estimate the swimming speed and metabolic rate of Atlantic cod (Gadus morhua). Seven cod were fitted with a miniature differential pressure sensor mounted on one side of the caudal peduncle immediately anterior to the base of the caudal fin rays. Relationships between differential pressure, tailbeat frequency, tailbeat amplitude, swimming speed and rate of oxygen consumption ((O(2))) were determined as a function of the swimming speed of cod swimming at 5 degrees C in a recirculating 'Brett-style' respirometer. Tailbeat differential pressure, tailbeat amplitude and tailbeat frequency were highly correlated with swimming speed. The average or integrated pressure ranged from 0 to 150 Pa for speeds up to 0.8 m s(-1) (1.1 L s(-1), where L is total body length), while the 'pressure difference' (maximum minus minimum pressure) ranged from 0 to 900 Pa. Small changes in swimming speed of less than 0.05 m s(-1) were readily detected as differences in tailbeat pressure. Burst swimming in the respirometer resulted in huge pressure 'bursts' of up to 5000 Pa 'pressure difference'. The rate of oxygen consumption increased exponentially and was highly correlated with swimming speed (r(2)=0.77). The rate of oxygen consumption was also correlated with tailbeat integrated pressure (r(2)=0.68) and with differential pressure (r(2)=0.43); regression correlations were always greater for individuals than for combined data from all cod. The results detailed in this study indicate that an ultrasonic differential pressure transmitter would enable accurate estimates of the swimming speed, rates of oxygen consumption and activity patterns of free-ranging fish in nature.

Mechanisms of metabolic defense against hypoxia in hibernating frogs.

The cold submerged frog (Rana temporaria) serves as a useful model for many hibernating ectotherms that take refuge in hypoxic ponds and lakes until more favourable conditions of climate and food availability return. In all such animals, entry into a hypometabolic state effectively extends their survival time by lessening the impact of ATP demands on endogenous substrates. At the cellular level, metabolic depression may be brought about by decreasing energy-consuming processes and/or by increasing the efficiency of energy-producing pathways. Since the mitochondrion is the major contributor to the total energy production during aerobic metabolism and frog survival during winter depends on entry into a hypometabolic state, this review focuses on the respiratory properties of mitochondria that serve to increase the efficiency of energy production in hibernation. Energy conservation during overwintering also occurs through decreases in the ATP demand of the energy-consuming processes. For example, hibernating frogs decrease their ATP demands for Na(+)/K(+)-ATPase activity as part of a coordinated process of energy conservation wherein O(2)-limitation initiates a generalised suppression of ion channel densities and/or channel leak activities. The net result is that cell membrane permeabilities are reduced, thereby lowering the energetic costs of maintaining transmembrane ion gradients.

Mechanisms of cell survival in hypoxia and hypothermia.

Most animals experience some degree of hypoxia and hypothermia during the course of their natural life history either as a consequence of ambient 'exposure' per se or through metabolic, respiratory and/or circulatory insufficiency. A prevailing experimental approach has been to probe tissues from natural models of hypoxia-tolerant and cold-tolerant vertebrates to look for common mechanisms of defence against O(2) lack and hypothermia. The ability to sustain vital cellular functions in severe cases of either condition varies widely amongst the vertebrates. Like humans, the vast majority of mammals are unable to survive prolonged periods of hypothermia or O(2) deprivation owing to irreversible membrane damage and loss of cellular ion homeostasis in vital organs such as the brain and heart. However, numerous hibernating endotherms, neonatal and diving mammals as well as many ectotherms can tolerate prolonged periods that would, in clinical terms, be called asphyxia or deep hypothermia. The key to their survival under such conditions lies in an inherent ability to downregulate their cellular metabolic rate to new hypometabolic steady states in a way that balances the ATP demand and ATP supply pathways.

Unsteady-state gas exchange and storage in diving marine mammals: the harbor porpoise and gray seal.

Breath-by-breath measurements of end-tidal O(2) and CO(2) concentrations in harbor porpoise reveal that the respiratory gas exchange ratio (R(R); CO(2) output/O(2) uptake) of the first lung ventilation in a breathing bout after a prolonged breath-hold is always well below the animal's metabolic respiratory quotient (RQ) of 0.85. Thus the longest apneic pauses are always followed by an initial breath having a very low R(R) (0.6-0.7), which thereafter increases with each subsequent breath to values in excess of 1.2. Although the O(2) stores of the body are fully readjusted after the first three to four breaths following a prolonged apneic pause, a further three to four ventilations are always needed, not to load more O(2) but to eliminate built-up levels of CO(2). The slower readjustment of CO(2) stores relates to their greater magnitude and to the fact that they must be mobilized from comparatively large and chemically complex HCO/CO(2) stores that are built up in the blood and tissues during the breath-hold. These data, and similar measurements on gray seals (12), indicate that it is the readjustment of metabolic RQ and not O(2) stores per se that governs the amount of time an animal must spend ventilating at the surface after a dive.

Strategies of hypoxia and anoxia tolerance in cardiomyocytes from the overwintering common frog, Rana temporaria.

Using ventricular cardiomyocytes of the common frog, Rana temporaria, we investigated the metabolic strategies employed by the heart to tolerate 4 mo of hypoxic submergence (overwintering) as well as acute bouts of anoxia. In contrast to what is observed for the whole animal, there was no change in oxygen consumption in cardiomyocytes isolated from normoxic frogs compared with those isolated from 4-mo hypoxic animals. Furthermore, cells from both normoxic and hypoxic frogs were able to completely recover oxygen consumption following 30 min of acute anoxia. From estimates of ATP turnover, it appears that frog cardiomyocytes are capable of a profound, completely reversible metabolic depression, such that ATP turnover is reduced by >90% of control levels during anoxia but completely recovers with reoxygenation. Moreover, this phenomenon is also observed in frogs that have been subjected to 4 mo of extended hypoxia. We found a significant increase in the stress protein, hsp70, after 1 mo of hypoxic submergence, which may contribute to the heart's remarkable hypoxia and anoxia tolerance and may act to defend metabolism during the overwintering period.

Aerobic capacity of frog skeletal muscle during hibernation.

Frogs submerged at 3 degrees C in hypoxic water (Po2=60 mmHg) depress their metabolic rate to 25% of that seen in control animals with access to air. The hypometabolic state of the skeletal muscle in such cold-submerged frogs is thought to be the most important contributor to the overall metabolic depression. The aim of this study was to determine whether the aerobic capacity of frog skeletal muscle became altered during 1-4 mo of hibernation to match the reduction in adenosine triphosphate (ATP) demand. To this end, the activities of key mitochondrial enzymes were measured in the skeletal muscle and in isolated mitochondria of frogs at different stages during hibernation. We also measured the activity of lactate dehydrogenase (LDH) as an indicator of glycolytic capacity. The activities of cytochrome c oxidase, citrate synthase, and LDH were significantly lower in frog skeletal muscle after 4 mo of hibernation compared with control conditions. The reduction in skeletal muscle aerobic capacity is apparently due to changes in the intrinsic properties of the mitochondria. Overall, these results indicate an important reorganisation of ATP-producing pathways during long-term metabolic depression to match the lowered ATP demand.

Isolation by distance in the Atlantic cod, Gadus morhua, at large and small geographic scales.

Genetic isolation by distance (IBD) has rarely been described in marine species with high potential for dispersal at both the larval and adult life-history stages. Here, we report significant relationships between inferred levels of gene flow and geographic distance in the Atlantic cod, Gadus morhua, at 10 nuclear restriction-fragment-length-polymorphism (RFLP) loci at small regional scales in the western north Atlantic region (< 1,600 km) that mirror those previously detected over its entire geographic range (up to 7,300 km). Highly significant allele frequency differences were observed among eight northwestern Atlantic populations, although the mean FST for all 10 loci was only 0.014. Despite this weak population structuring, the distance separating populations explained between 54% and 62% of the variation in gene flow depending on whether nine or 10 loci were used to estimate Nm. Across the species' entire geographic range, highly significant differences were observed among six regional populations at nine of the 10 loci (mean FST = 0.068) and seven loci exhibited significant negative relationships between gene flow and distance. At this large geographic scale, natural selection acting in the vicinity of one RFLP locus (GM798) had a significant effect on the correlation between gene flow and distance, and eliminating it from the analysis caused the coefficient of determination to increase from 17% to 62%. The role of vicariance was assessed by sequentially removing populations from the analysis and was found to play a minor role in contributing to the relationship between gene flow and distance at either geographic scale. The correlation between gene flow and distance detected in G. morhua at small and large spatial scales suggests that dispersal distances and effective population sizes are much smaller than predicted for the species and that the recent age of populations, rather than extensive gene flow, may be responsible for its weak population structure. Our results suggest that interpreting limited genetic differences among populations as reflecting high levels of ongoing gene flow should be made with caution.

Metabolic depression and enhanced O(2) affinity of mitochondria in hypoxic hypometabolism.

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 O(2) 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 O(2) 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 O(2) affinity of mitochondria. These findings demonstrate that metabolic depression can be reflected at all levels of biological organization in hypoxia-tolerant animals.

Surviving hypoxia without really dying.

In cases of severe O(2) limitation, most excitable cells of mammals cannot continue to meet the energy demands of active ion transporting systems, leading to catastrophic membrane failure and cell death. However, in certain lower vertebrates, hypoxia-induced membrane destabilisation of the kind seen in mammals is either slow to develop or does not occur at all owing to adaptive decreases in membrane permeability (i.e. ion 'channel arrest'), that dramatically reduce the energetic costs of ion-balancing ATPases. Mammalian cells do, however, exhibit a whole host of adaptive responses to less severe shortages of oxygen, which include energy-balanced metabolic suppression, ionic-induced activation of O(2) receptors and the upregulation of certain genes, all of which enhance the systemic delivery of oxygen and promote energy conservation. Accumulating evidence suggests that the mechanisms underlying these protective effects are orchestrated into action by putative members of an O(2)-sensing pathway that most if not all cells share in common. In this review we address three major questions: (i) how do cells detect shortages of oxygen and subsequently set in motion adaptive mechanisms of either energy production or energy conservation; (ii) how do these mechanisms restructure cellular pathways of ATP supply and demand to ensure that ion-motive ATPases are given priority over other cell functions to preserve membrane integrity in energy-limited states; and (iii) what mechanisms of molecular and metabolic defence against acute and long-term shortages of oxygen set hypoxia-tolerant systems apart from their hypoxia-sensitive counterparts?

The protective effects of hypoxia-induced hypometabolism in the Nautilus.

Specimens of Nautilus pompilius were trapped at depths of 225-300 m off the sunken barrier reef southeast of Port Moresby, Papua New Guinea. Animals transported to the Motupore Island laboratory were acclimated to normal habitat temperatures of 18 degrees C and then cannulated for arterial and venous blood sampling. When animals were forced to undergo a period of progressive hypoxia eventually to encounter ambient partial pressure of oxygen (PO2) levels of approximately 10 mmHg (and corresponding arterial PO2's of approximately 5 mmHg), they responded by lowering their aerobic metabolic rates to 5-10% of those seen in resting normoxic animals. Coincident with this profound metabolic suppression was an overall decrease in activity, with brief periods of jet propulsion punctuating long periods of rest. Below ambient PO2 levels of 30-40 mmHg, ventilatory movements became highly periodic and at the lowest PO2 levels encountered, ventilation occasionally ceased altogether. Cardiac output estimated by the Fick equation decreased during progressive hypoxia by as much as 75 80%, and in the deepest hypometabolic states heart rates slowed to one to two cycles of very low amplitude per minute. By the end of 500 min exposure to ambient PO2 levels of 10 mmHg or less, the anaerobic end products octopine and succinate had increased significantly in adductor muscle and heart, respectively. Increased concentrations of octopine in adductor muscle apparently contributed to a small intracellular acidosis and to the development of a combined respiratory and metabolic acidosis in the extracellular compartment. On the other hand, increases in succinate in heart muscle occurred in the absence of any change in cardiac pHi. Taken together, we estimate that these anaerobic end products would make up less than 2% of the energy deficit arising from the decrease in aerobic metabolism. Thus, metabolic suppression is combined with a massive downregulation of systemic O2 delivery to match metabolic supply to demand.

Mitochondria as ATP consumers: cellular treason in anoxia.

In anoxia, mitochondria change from being ATP producers to potentially powerful ATP consumers. This change occurs, because the mitochondrial F(1)F(0)-ATPase begins to hydrolyze ATP to avoid the collapse of the proton motive force. Species that can survive prolonged periods of O(2) lack must limit such ATP use; otherwise, this process would dominate glycolytic metabolism and threaten ATP delivery to essential ATP-consuming processes of the cell (e.g., ion-motive ATPases). There are two ways to limit ATP hydrolysis by the F(1)F(0)-ATPase, namely (i) reduction of the proton conductance of the mitochondrial inner membrane and (ii) inhibition of the enzyme. We assessed these two possibilities by using intact mitochondria isolated from the skeletal muscle of anoxia-tolerant frogs. Our results show that proton conductance is unaltered between normoxia and anoxia. However, ATP use by the F(1)F(0)-ATPase is limited in anoxia by a profound inhibition of the enzyme. Even so, ATP use by the F(1)F(0)-ATPase might account for approximately 9% of the ATP turnover in anoxic frog skeletal muscle.

Effects of ambient PO2 and temperature on oxygen uptake in Nautilus pompilius.

This study employs closed-circuit respirometry to evaluate the effect of declining ambient oxygen partial pressure (PO2) and temperature on mass specific rates of oxygen uptake (VO2) in Nautilus pompilius. At all temperatures investigated (11, 16, and 21 degrees C), VO2 is relatively constant at high PO2 (oxyregulation) but declines sharply at low PO2 (oxyconformation). The critical PO2 below which oxyconformation begins (Pc) is temperature dependent, higher at 21 degrees C (49 mmHg) than at 11 degrees C or 16 degrees C (21.7 mmHg and 30.8 mmHg respectively). In resting, post-absorptive animals, steady-state resting VO2 increases significantly with temperature resulting in a Q10 value of approximately 2.5. The metabolic strategy of N. pompilius appears well suited to its lifestyle, providing sufficient metabolic scope for its extensive daily vertical migrations, but allowing for metabolic suppression when PO2 falls too low. The combination of low temperatures and low PO2 may suppress metabolic rate 16-fold (assuming negligible contributions from anaerobic metabolism and internal O2 stores), enhancing hypoxia tolerance.

The effect of metabolic depression on proton leak rate in mitochondria from hibernating frogs.

Futile cycling of protons across the mitochondrial inner membrane accounts for 20 % or more of the total standard metabolic rate of a rat. Approximately 15 % of this total is due to proton leakage inside the skeletal muscle alone. This study examined whether the rate of proton leak is down-regulated as a part of a coordinated response to energy conservation during metabolic depression in cold-submerged frogs. We compared the proton leak rate of skeletal muscle mitochondria isolated from frogs at different stages of hibernation (control, 1 month and 4 months of submergence in normoxia and hypoxia). The kinetics of mitochondrial proton leak rate was unaltered throughout normoxic and hypoxic submergence. The state 4 respiration rates did not differ between control animals and frogs hibernating in normoxia. In contrast, the state 4 respiration rates obtained from frogs submerged in hypoxic water for 4 months were half those of control animals. This 50 % reduction in respiration rate in hypoxic hibernation was due to a reduction in electron transport chain activity and consequent decrease in mitochondrial membrane potential. We conclude that proton leak rate is reduced during metabolic depression as a secondary result of a decrease in electron transport chain activity, but that the proton conductance is unchanged. In addition, we show that the rate of proton leakage and the activity of the electron transport chain are lower in frogs than in rats, strengthening the observation that mitochondria from ectotherms have a lower proton conductance than mitochondria from endotherms.

Gas exchange and heart rate in the harbour porpoise, Phocoena phocoena.

The respiratory physiology, heart rates and metabolic rates of two captive juvenile male harbour porpoises (both 28 kg) were measured using a rapid-response respiratory gas analysis system in the laboratory. Breath-hold durations in the laboratory (12 +/- 0.3 s, mean +/- SEM) were shorter than field observations, although a few breath-holds of over 40 s were recorded. The mean percentage time spent submerged was 89 +/- 0.4%. Relative to similarly-sized terrestrial mammals, the respiratory frequency was low (4.9 +/- 0.19 breaths.min-1) but with high tidal volumes (1.1 +/- 0.011), enabling a comparatively high minute rate of gas exchange. Oxygen consumption under these experimental conditions (247 +/- 13.8 ml O2.min-1) was 1.9-fold higher than predicted by standard scaling relations. These data together with an estimate of the total oxygen stores predicted an aerobic dive limit of 5.4 min. The peak end-tidal O2 values were related to the length of the previous breath-hold, demonstrating the increased oxygen uptake from the lung for the longer dives. Blood oxygen capacity was 23.5 +/- 1.0 ml.100 ml-1, and the oxygen affinity was high, enabling rapid oxygen loading during ventilation.

Factors affecting membrane permeability and ionic homeostasis in the cold-submerged frog.

Frogs (Rana temporaria) were submerged at 3 degrees C in either normoxic (P(O2)=155 mmHg, P(O2)=20 kPa) or hypoxic (P(O2)=60 mmHg; P(O2)=8 kPa) water for up to 16 weeks, and denied air access, to mimic the conditions of an ice-covered pond during the winter. The activity of the skeletal muscle Na(+)/K(+) pump over the first 2 months of hibernation, measured by ouabain-inhibitable (22)Na(+) efflux, was reduced by 30 % during normoxia and by up to 50 % during hypoxia. The reduction in Na(+)/K(+) pump activity was accompanied by reductions in passive (22)Na(+) influx and (86)Rb(+) efflux (effectively K(+) efflux) across the sarcolemma. This may be due to a decreased Na(+) permeability of the sarcolemma and a 75 % reduction in K(+) leak mediated by ATP-sensitive K(+) channels ('K(ATP)' channels). The lowered rates of (22)Na(+) and (86)Rb(+) flux are coincident with lowered transmembrane ion gradients for [Na(+)] and [K(+)], which may also lower Na(+)/K(+) pump activity. The dilution of extracellular [Na(+)] and intracellular [K(+)] may be partially explained by increased water retention by the whole animal, although measurements of skeletal muscle fluid compartments using (3)H-labelled inulin suggested that the reduced ion gradients represented a new steady state for skeletal muscle. Conversely, intracellular ion homeostasis within ventricular muscle was maintained at pre-submergence levels, despite a significant increase in tissue water content, with the exception of the hypoxic frogs following 4 months of submergence. Both ventricular muscles and skeletal muscles maintained resting membrane potential at pre-submergence levels throughout the entire period of hibernation. The ability of the skeletal muscle to maintain its resting membrane potential, coincident with decreased Na(+)/K(+) pump activity and lowered membrane permeability, provided evidence of functional channel arrest as an energy-sparing strategy during hibernation in the cold-submerged frog.

Constant set points for pH and P(CO2) in cold-submerged skin-breathing frogs.

The low temperatures encountered by overwintering frogs result in a large downregulation of metabolism and behaviour. However, little is known about acid-base regulation in the extreme cold, especially when frogs become exclusive skin-breathers during their winter submergence. Blood and muscle tissue acid-base parameters (pH, P(CO2), bicarbonate and lactic acid concentrations) were determined in submerged frogs exposed to a range of low temperatures (0.2-7 degrees C). At overwintering temperatures between T = 0.2 and 4 degrees C plasma pH and P(CO2) were maintained constant, whereas intracellular pH regulation resulted in larger pH-temperature slopes occurring in the presumably more active heart muscle (deltapH/deltaT = -0.0313) than in the gastrocnemius muscle (deltapH/deltaT = -0.00799). Although blood pH was not significantly affected by submergence between 0.2 and 4 degrees C (pH = 8.220-8.253), it declined in the 7 degrees C frogs (pH = 8.086), a decrease not linked to the recruitment of anaerobiosis. Plasma P(CO2) and pH in the cold appear to be regulated at constant levels, implying that cutaneous CO2 conductance in submerged frogs is adjusted within the range of overwintering temperatures. This is likely geared toward facilitating the uptake of oxygen under conditions of greater metabolic demand, however there remains the possibility that acid-base balance itself is maintained at a constant set point at the frog's natural overwintering temperatures.

The use of extracellular lactate as an oxidative substrate in the oxygen-limited frog.

The aim of this paper was to determine the contribution of anaerobic respiration to metabolism in hibernating frogs exposed to progressive hypoxia. Previous studies on acute exposure to hypoxia had shown that even at ambient PO2 levels of 60 mmHg, cold-submerged frogs were obliged to recruit anaerobic pathways to provide enough energy to maintain the ATP and phosphocreatine concentrations of tissues perfectly homeostatic. In the current experiments, we exposed frogs to hypoxic conditions gradually to reveal that 30 mmHg probably represents a 'threshold PO2' at which survival is still possible, at least for 1 week. However, by the time this threshold was reached, liver glycogen reserves were exhausted and the frog must rely thereafter on its quantitatively large store of skeletal muscle glycogen. The lactate produced as a by-product of glycolytic ATP production did not accumulate in the muscle but was preferentially exported to the plasma where it was held against a sizeable extracellular to intracellular gradient. The results suggest that the exported lactate was 'shuttled' between a poorly-perfused skeletal muscle and a more highly-perfused and oxygenated core of the animal where it could act as both a substrate for direct oxidation or for gluconeogenesis.

Metabolic suppression in anoxic frog muscle.

Microcalorimetry is the only direct method for measuring moment-to-moment changes in whole-cell metabolism (as heat output) during anoxia. We have adapted this methodology, in conjunction with standard muscle isolation techniques, to monitor metabolic transitions in isolated frog (Rana temporaria) sartorius muscle during anoxia and recovery (reoxygenation). Anoxia (sustained 1 h, following 2 h progressive hypoxia) suppressed muscle heat output to 20% of the stable normoxic level. This effect was fully reversible upon reoxygenation. Metabolite profiles were consistent with other anoxia-tolerant vertebrates--most notably, adenosine triphosphate (ATP) content during anoxia and reoxygenation remained unchanged from normoxia (pre-anoxic control). In addition, the concentration of K+ ions ([K+]) in interstitial dialysates remained stable (2-3 mM) throughout anoxia and recovery. Interstitial [lactate-] increased slightly, in accord with anaerobiosis supporting suppressed metabolic rates during anoxia. The degree of anoxic suppression of metabolism observed is similar to other vertebrate models of anoxia tolerance. Furthermore, stable ATP concentrations and interstitial [K+] in the isolated tissue suggests that intrinsic mechanisms suppress metabolism in a manner that coordinates ATP supply and demand and avoids the severe ion imbalances that are characteristics of hypoxia-sensitive systems.