The low rate of metabolism is functionally important to the turtle because it drastically delays the depletion of stored substrate and slows the build‐up of acid endproducts; nevertheless, the depressed metabolism must still supply all the cellular energy requirements of the animal during its long submergence. Clearly, these requirements are profoundly depressed. The animals are responsive to stimuli and periodically move about but are generally extremely sluggish. Systolic arterial blood pressure is only 10 cmH 2 O and heart rates average one beat in 2–3 min, and in extreme cases only every 5–10 min ( Herbert & Jackson, 1985 b ). Cardiac output has not been measured under these circumstances, but flow must essentially stop during the long diastolic pauses. Blood stasis in euthermic mammals can result in clot formation, but clotting rates at low temperature are greatly slowed ( Valeri et al . 1995 ). A practical advantage this affords the experimenter is that chronic catheters remain patent with minimal attention. At the low prevailing blood pressures, renal function is presumably minimal ( Warburton & Jackson, 1995 ; Jackson et al . 1996 ), although Ca 2+ and lactate concentrations are both elevated in urine collected from the urinary bladder after 3–4 months of anoxia at 3 °C ( Jackson & Ultsch, 1982 ).

A low metabolic rate is central to the turtle's ability to tolerate long‐term anoxia. As an ectothermic reptile, its energy metabolism is only 10–20 % that of a mammal of similar size even at the same body temperature ( Bennett & Ruben, 1979 ). At lower temperatures, metabolism falls still further in the thermally conforming ectotherm, typically at a rate of 2‐ to 3‐fold per 10 °C decrease in temperature ( Q 10 = 2–3). Moreover, the painted turtle, like other reptiles ( Bennett & Dawson, 1976 ), exhibits an exaggerated Q 10 effect at temperatures below 10 °C ( Herbert & Jackson, 1985 b ), so that at 3 °C aerobic metabolism is depressed to about 0.1 % of the euthermic mammalian level. Finally, the anoxic state is characterized by a further sharp fall in metabolism by about 90 % ( Jackson, 1968 ; Buck et al . 1993 ), so that the metabolic rate of the anoxic turtle at its usual hibernating temperature is over 10 000 times lower than that of a similarly sized mammal resting at its normal body temperature. Values of metabolic rate towards the end of a long submergence, estimated on the basis of lactate accumulation in the body fluids ( Jackson et al . 2000 ), are estimated to be about 0.01 cal (0.04 J) kg −1 min −1 (0.7 mW kg −1 ). To illustrate how slowly the metabolic fires are burning in this situation, consider that if all of the turtle's metabolic heat was stored, it would take almost 7 days for its body temperature to rise by 0.1 °C. Figure 1 depicts the pattern of metabolic depression that occurs during anoxia at 3 and 24 °C.

Cellular responses.

Experimental evidence from anoxic turtles indicates a coordinated downregulation in the rates of both ATP utilization and ATP production. The mechanisms involved are still being resolved, but it is clear that cellular ATP levels remain stable during long periods of anoxia (Kelly & Storey, 1988).

ATP utilization. Major consumers of cellular energy are the ion pumps that maintain transmembrane ionic gradients and protein synthesis. Evidence exists for sharp reductions in both these cellular functions during anoxia. The energy requirement for maintaining transmembrane ionic gradients is reduced by slowing the passive flux of ions through membrane channels, via so‐called ‘channel arrest’ (Hochachka, 1986), although the mechanisms whereby this occurs are not well understood. Channel arrest potentially has general importance in circumstances involving reduced metabolism. In a warm reptile, for example, transmembrane ion gradients are similar to those in a mammal of the same size, yet the metabolic rates of these animals are greatly different (Hulbert & Else, 1981). Assuming that the cost of maintaining these gradients requires a significant fraction of each animal's total metabolism, then the cost to the reptile must be less. Furthermore, when the reptile's temperature falls, so does its metabolic rate, but ion concentrations remain essentially unchanged (Herbert & Jackson, 1985a). The interpretation is that membrane ion leakage through ion channels is less in the reptile, and that it falls further when the animal's temperature falls. Anoxia induces a further reduction in ion channel activity.

Studies of channel arrest in the anoxic turtle have focused largely on the brain because of the critical importance of maintaining this organ's function and because of the brain's normally large commitment of energy to ionic regulation. In addition, the susceptibility of the mammalian brain to hypoxic damage is linked to the collapse of ion gradients, and destructive Ca2+ influx (Hochachka, 1986; Sattler & Tymianski, 2000). Evidence exists for reduced channel function for Na+, K+ and Ca2+ in the turtle brain, although the evidence for the first two ions is indirect. Reduced Na+ channel density was found in isolated cerebellum exposed to anoxic perfusion for 4 h at room temperature using the channel ligand brevetoxin (Pérez‐Pinzón et al. 1992). The magnitude of the reduction (42 %) is much less than the estimated fall in brain metabolism, so it is possible that passive Na+ flux is further lowered by decreasing the open probability of remaining channels. Downregulation of K+ channels was inferred from a slowing of K+ efflux from anoxic neurones, compared to normoxic neurones, in anaesthetized animals treated with ouabain to poison Na+‐K+‐ATPase (Chih et al. 1989). The mammalian brain responds to anoxia and the attendant fall in ATP by opening ATP‐sensitive K+ channels (K ATP channels). The resultant hyperpolarization reduces electrical activity and serves as a short‐term defence mechanism, but persistent anoxia leads in minutes to massive failure due to a rapid increase in extracellular K+, membrane depolarization, rapid influx of Ca2+ through voltage‐dependent channels and Ca2+‐induced cellular damage. In contrast, the turtle brain apparently lacks K ATP channels and instead reduces activity in other K+ channels. Coupled with the similar effect on Na+ leakage, the energy required for Na+‐K+‐ATPase can thereby be significantly reduced.

Direct evidence for inactivation of Ca2+ channels in anoxic slices of turtle cortex has been obtained by Bickler and colleagues (Bickler & Buck, 1998). Initial studies demonstrated that the intracellular Ca2+ concentration ([Ca2+] i ) of cortical brain slices of turtle fell slightly during anoxia in contrast to a rapid and large increase in [Ca2+] i in rat brain slices (Bickler, 1992). Inhibition of glycolysis with iodoacetate caused rapidly increased [Ca2+] i in the acutely anoxic turtle brain, but this effect was greatly attenuated after prolonged anoxic exposure, suggesting a time‐dependent arrest of Ca2+ channels. Because glutamate receptors are implicated in mediating lethal neuronal Ca2+ fluxes in anoxia‐sensitive animals (Bickler & Hansen, 1994), these receptors were postulated as potential sites of downregulation in turtle neurones. Patch‐clamp studies of NMDA receptors from turtle cortex revealed a 65 % decrease in the open probability of Ca2+ channels and further established that the downregulation was mediated by adenosine (Buck & Bickler, 1998), a molecule previously implicated in turtle brain anoxia tolerance (Nilsson & Lutz, 1992). Furthermore, normal [Ca2+] i persisted in turtle brain slices removed from animals after 6 weeks of experimental anoxia at 2–3 °C, even in the presence of elevated solution [Ca2+] simulating the in vivo state (Bickler, 1998). In recent studies, time‐dependent mechanisms for NMDA‐receptor downregulation have been identified, including an acute reduction of channel open probability mediated by phosphatase 1 or 2A, a delayed suppression of receptors associated with elevated [Ca2+] i and controlled by calmodulin, and a longer term removal of NMDA receptors from the cell membrane (Bickler et al. 2000). Of particular significance is that prevention of NMDA‐receptor downregulation eliminated the anoxia tolerance of the brain tissue.

Utilization of ATP for protein synthesis is also suppressed during anoxia in turtle hepatocytes (Land et al. 1993) and in the heart (Bailey & Driedzic, 1996), although increased synthesis of selected proteins has been reported (Brooks & Storey, 1993; Hochachka et al. 1996; Chang et al. 2000).

ATP production. During anoxia, ATP production occurs via glycolysis, and modulation of the flux rate is considered to be via control of key enzymes in this pathway. The reduced rate goes counter to the usual hypoxia‐induced activation and has been termed the ‘reverse Pasteur effect’ (Hochachka, 1986). Storey and co‐workers (Storey & Storey, 1990; Storey, 1996) suggest three effects of anoxia on glycolytic enzymes: alteration of activity via phosphorylation and dephosphorylation, reversible binding of enzymes to macromolecules or organelles and allosteric regulation via specific metabolites.

The low ATP yield of this pathway requires a large commitment of substrate in the form of glucose or glycogen to supply the energy needs of prolonged anoxia. However, the extremely low rate of glycolysis in the anoxic turtle and the large initial stores of glycogen in liver and muscle (Daw et al. 1967) probably prevent this from being a limiting factor for the animal.