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Comparative Biochemistry and Physiology Part A 125 (2000) 299 – 315 www.elsevier.com/locate/cbpa Review Living without oxygen: lessons from the freshwater turtle Donald C. Jackson * Department of Molecular Pharmacology, Physiology and Biotechnology, Brown Uni6ersity, Box G, Pro6idence, RI 02912, USA Received 18 October 1999; accepted 20 December 1999 Abstract Freshwater turtles, and specifically, painted turtles, Chrysemys picta, are the most anoxia-tolerant air-breathing vertebrates. These animals can survive experimental anoxic submergences lasting up to 5 months at 3°C. Two general integrative adaptations underlie this remarkable capacity. First is a profound reduction in energy metabolism to : 10% of the normoxic rate at the same temperature. This is a coordinated reduction of both ATP generating mechanisms and ATP consuming pathways of the cells. Second is a defense of acid – base state in response to the extreme lactic acidosis that results from anaerobic glycolysis. Central to this defense is an exploitation of buffer reserves within the skeleton and, in particular, the turtle’s shell, its most characteristic structure. Carbonates are released from bone and shell to enhance body fluid buffering of lactic acid and lactic acid moves into shell and bone where it is buffered and stored. The combination of slow metabolic rate and a large and responsive mineral reserve are key to this animal’s extraordinary anaerobic capacity. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Acid – base balance; Anaerobiosis; Anoxia; Buffering; Bone; Lactic acid; Metabolic depression; Metabolic rate; Strong ion difference; Turtle shell 1. Introduction Because this review is adapted from an invited address named in honor of the great Danish physiologist, August Krogh, it is fitting to begin with a quotation from his work (Krogh, 1941): Crocodiles, turtles, and many tortoises, living in water, dive regularly and many of them are stated to stay for hours or even days under This invited review is based upon the Krogh Lecture, sponsored by the Comparative Physiology Section of the American Physiological Society, presented at the 1999 Experimental Biology Annual Conference. * Tel.: +1-401-8632373; fax: + 1-401-8633352. E-mail address: donald – [email protected] (D.C. Jackson) water.... It is evident that the metabolism must be either greatly reduced or become mainly anaerobic. No really quantitative work on the diving of reptiles has been made thus far, although some of them should be very suitable for the purpose. In the years since Krogh wrote these words, his predictions have been borne out and we now have considerable quantitative information on the diving of reptiles. In addition, particular species have been identified that are indeed ‘‘very suitable for the purpose.’’ Extensive information is available on the responses and adaptive mechanisms that permit long periods of anoxic submergence in the painted turtle, Chrysemys picta, and the red-eared 1095-6433/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 ( 0 0 ) 0 0 1 6 0 - 4 300 D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315 slider, Trachemys scripta. My own work, which will be emphasized in the paper, has largely been on the former species, in particular the sub-species, C. picta bellii. In its normal range, which extends into north-central United States and into southern Canada, this turtle, the western painted turtle, experiences severe winters when the water in which they hibernate may be ice-covered for many months. They therefore face long periods of apnea under conditions that are very likely severely hypoxic or anoxic (Ultsch, 1989). Because of their natural history, this species has been a logical subject for studies of anoxic tolerance. Early studies, some even before Krogh’s remarks, documented the capacity for turtles to survive periods of anoxia (Johlin and Moreland, 1933; Musacchia, 1959; Belkin, 1963; Robin et al., 1964). Our work (Herbert and Jackson, 1985a) looked at durations of reversible anoxic submergences over a range of temperatures and found a marked temperature effect (Fig. 1). Of particular significance in the present context is the extremely long anoxic period (12 weeks) from which turtles can fully recover when studied at 3°C, a temperature close to the conditions under which they hibernate. In other studies in which recovery was not monitored, turtles remained alive at this temperature while anoxic for as long as 5 months (Ultsch and Jackson, 1982), a duration approaching the maximum a turtle could encounter in the wild. Turtles, like all other vertebrates, are aerobic animals that depend ultimately on oxygen for normal function. But they are also facultative anaerobes, which means that they possess traits that permit them to sustain some minimal level of function with no available oxygen. How can they survive for such long periods in the absence of O2? I will discuss two general classes of responses, metabolic responses and acid–base responses, which can help to provide a basis for understanding this remarkable performance. My emphasis will be at the level of whole animal integrative physiology. A recent review in this journal on the same general topic focused on the biochemical basis of metabolic adaptations (Storey, 1996). 2. Metabolic basis for anoxia tolerance As illustrated in Fig. 1, the length of time an animal can remain anoxic is inversely related to its metabolic rate; i.e. the slower its metabolic rate, the longer the anoxia. A number of factors combine to produce particularly low metabolic rate in a cold anoxic turtle. First, the turtle is an ectotherm and it therefore has a metabolic rate that is low compared to an endotherm of similar size, some five to ten times lower even at the same body temperature. Second, a drop in environmental temperature lowers an ectotherm’s metabolism even further, usually by two to three times for each 10°C fall in body temperature (Q10 = 2–3), and with an even steeper slope at the low end of the temperature range of most reptiles (Bennett and Dawson, 1976), including this turtle (Herbert Fig. 1. Duration of anoxic submergences at various temperatures from which painted turtles (Chrysemys picta bellii ) have been observed to recover fully. Data are adapted from Herbert and Jackson (1985a). D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315 Fig. 2. Schematic diagram illustrating factors that contribute to a drastically lower (to 0.01%) metabolic rate in a cold, anoxic turtle compared to a euthermic mammal of the same approximate body size. and Jackson, 1985b). Third, anoxia and a switch to anaerobic metabolism produces a further lowering of metabolism to about 10% of the aerobic rate at the same temperature. Together these factors result in an exceedingly low rate of metabolism in an anoxic turtle at 3°C that is only 0.5% of its own aerobic metabolism at 20°C and less than 0.01% of a similar-sized mammal at rest in a thermoneutral environment (Fig. 2). 2.1. Metabolic depression in anoxic turtles When a turtle is submerged under anoxic conditions, available oxygen is restricted to what it carries within its body. Its major oxygen store resides in its large lungs with a significant additional amount bound to hemoglobin in its blood (Fuster et al., 1997). If freely diving, a turtle will routinely return to the surface to refill its oxygen stores before they fall to critically low levels (Ackerman and White, 1979; Gatten, 1981), but if denied access to air in anoxic water, either by ice or by experimental design, oxygen reserves will become exhausted and cellular metabolism will shift from aerobic oxidative phosphorylation to anaerobic glycolysis. This transition to anaerobiosis is associated with a gradual fall in energy metabolism to about 10 – 15% of the aerobic level as documented in intact turtles both directly, by calorimetry (Jackson, 1968) and indirectly, by lactate accumulation (Herbert and Jackson, 1985b). Metabolic depression of similar magnitude has also been reported in individual organs: in brain, 301 Lutz et al. (1985) estimated from blood lactate levels that metabolism fell to 5–10% of the aerobic rate; Doll et al. (1994), however, using microcalorimetry on turtle brain slices observed only a 40% reduction in rate, but brain slices, even when supplied with oxygen, may already have suppressed metabolism (Lutz and Nilsson, 1997). Isolated liver cells from turtles also undergo a 90% reduction in metabolic rate on the basis of lactate production (Buck et al., 1993). Our focus at the organ level has been the heart. The heart is unusual in that its metabolic requirements are largely determined by whole body demand for blood flow. If this demand falls, then the active metabolic function of the heart, cardiac output, can be correspondingly reduced. But what is the intrinsic response of cardiac tissue to anoxic lack, independent of its reduced obligations to the rest of the body? To understand this we studied both perfused working hearts and isolated ventricular muscle strips and exposed the cardiac tissue to anoxic conditions simulating what it might experience in vivo. Surprisingly, we observed little effect of anoxia on the mechanical performance of perfused hearts (Wasser et al., 1990b; Jackson et al., 1995), a finding similar to what had been reported earlier by Reeves (1963). In contrast, we found a significant fall in contractile force in ventricular strips exposed to anoxia (Wasser et al., 1990a), confirming an earlier report on a similar preparation by Bing et al. (1972). These contradictory results were resolved in a study by Farrell et al. (1994) on an in situ turtle heart preparation that performed at levels comparable to peak values observed in intact animals. In this preparation, anoxia caused a 50% reduction in power output when the heart operated at its maximum level, but no reduction was observed when performance was submaximal. This suggests that the failure to observe an anoxic depressive effect in perfused heart preparations was because they were not operating at peak levels. No ambiguity exists, however, about the depressant effect of combined anoxia and lactic acidosis on cardiac performance, and this combination of stresses is of course the condition that the animal faces during anoxic submergence. Even a perfused heart operating well below its peak power output is weakened significantly by this duel stress (Wasser et al., 1990b; Jackson et al., 1995). To examine this further, we studied force– velocity relations in ventricular strips from turtle 302 D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315 hearts under controlled conditions of normoxia, acidosis, anoxia, and combined anoxia/acidosis (Shi and Jackson, 1997). Strips could be set at optimal length for maximum force. In this preparation anoxia reduced force by about 50% and combined anoxia/acidosis lowered force further to 20 – 25% of normoxic force. Interestingly, maximum velocity of shortening was unchanged by these conditions, although reduced temperature did slow this rate. The maximum power output of the heart (the peak product of its force and velocity) of a cold anoxic heart is therefore reduced by a combination of reduced force due to anoxia and acidosis and reduced velocity due to low temperature. Because overall body metabolic rate and perfusion requirements decrease profoundly during anoxic submergence, however, it is likely that this lowered cardiac power is still adequate to supply the body’s needs and that heart function is not a limiting factor in anoxic survival. The situation in vivo is complex, though, because function is suppressed not only by the intrinsic effects of anoxia and acidosis, but also by reduced venous return and vagal stimulation. In addition, calcium and catecholamines, both cardiac activators, are increased in the circulating blood of anoxic turtles, although their effects on anoxic hearts in vitro are not dramatic (Wasser et al., 1990a; Shi et al., 1999). The reduction in heart activity is dramatically illustrated by heart rates as low as one beat in 10 min in an anoxic turtle at 3°C. 2.2. Metabolic depression of turtles submerged in aerated water Reduced metabolism may also be critical for turtles overwintering in water with high PO2. Some species, such as softshell turtles (Apalone) and musk turtles (Sternotherus), have permeable integuments and can survive indefinitely in aerated water at low temperature (Ultsch et al., 1984). Other turtles, with heavy calcified shells, such as Chrysemys or the map turtle (Graptemys geographica), appear poorly adapted for aquatic respiration, yet Chrysemys benefits significantly from aquatic O2 (Ultsch and Jackson, 1982) and submerged Graptemys can maintain normal blood acid –base state throughout a cold Vermont winter by selecting well-aerated water for a hibernaculum (Crocker et al., 2000). Although the avenue of O2 uptake by Graptemys is not certain (it may involve cloacal and/or buccopharygeal respiration), it is likely that this turtle also depresses its metabolism to reduce its O2 demands. Recently, Boutilier et al. (1997) have found that winter frogs (Rana temporaria) are able to remain aerobic in hypoxic cold water by reducing their metabolism. Remarkably, Graptemys sustains a predominantly aerobic metabolism with a blood PO2 of only about 1 Torr (Crocker et al., 2000). 2.3. Energy status of anoxic turtles Although the metabolic rate of anoxic turtles decreases dramatically, the decrease is a carefully controlled process that has little effect on the energy status of the cells. It is important to consider metabolic rate in two ways: first, as the rate at which energy is released by intermediary metabolism and stored in high energy bonds of ATP; i.e. ATP production rate; second, as the rate at which an organism utilizes ATP to carry out vital cellular functions such as synthesis, transport, and contraction; i.e. ATP consumption rate. A number of studies have found that cellular ATP concentrations remain constant during anoxia in turtles, demonstrating that these rates are matched. Kelly and Storey (1988), for example, sampled various tissues from anoxic turtles and found that ATP concentration and total adenylates fell somewhat after 1 h anoxia at 20°C in Pseudemys (= Trachemys) scripta, but returned to normal after 5 h anoxia. Energy charge ([ATP]× 0.5[ADP]/([ATP] + [ADP]+[ADP])) was constant throughout in all tissues examined. Constant ATP during anoxia has also been found in studies of specific organs and cells, including brain (Lutz et al., 1985), heart (Wasser et al., 1990a), and liver (Buck et al., 1993). The metabolism of the anoxic turtle, therefore, undergoes a coordinated downregulation of ATP synthesis and ATP hydrolysis (Fig. 3), and the close matching between these processes maintains relatively constant cell levels of ATP. Three important fundamental questions arise from this observation: First, what regulatory mechanisms produce the profound drop in ATP production when this animal switches from aerobic to anaerobic metabolism? Second, how can 90% of the cellular ATP-requiring processes be suspended and the stability and viability of the cells preserved? Third, how is the metabolic reorganization of the animal coordinated? D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315 Depressed anaerobic ATP production in turtles has been termed the ‘reverse Pasteur effect’ because the classical effect of low oxygen on the glycolytic pathway is activation rather than suppression (Hochachka, 1986). The term may be misleading, however, because even with an overall metabolic depression of 90%, glycolytic flux must increase somewhat over the aerobic rate because of low ATP yield of this pathway. The full aerobic oxidation of a mole of glucose gains only 5 – 6% of the generated moles of ATP from glycolysis alone. Nonetheless, the failure of glycolysis to increase in activity to sustain aerobic-level ATP synthesis must require some controlling factors. Major work on this problem has been carried out by Storey and his colleagues, and has been summarized in thorough reviews of the subject (Storey and Storey, 1990; Storey, 1996). Three general anoxia-induced alterations of glycolytic enzymes have been described by these authors. First, alteration of enzyme activity via phosphorylation or dephosphorylation; second, reversible binding of enzymes to cellular macromolecules or organelles; third, allosteric regulation of enzyme function by specific metabolites. In addition, although protein 303 synthesis is usually found to be severely depressed in anoxic tissues, selective proteins are expressed, including members of the heat shock family (Chang et al., 2000) and also possibly glycolytic enzymes (Hochachka et al., 1996). In contrast to these findings, Brooks and Storey (1993) observed sustained or even increased protein synthesis during exposure to nitrogen gas at 6°C and the appearance of specific protein, perhaps of the heat shock family, in animals recovering from anoxia. Matched to the fall in ATP production is a parallel fall in ATP hydrolysis, the process that pays for energy-requiring processes of the cells. Because the anoxic tissues function with only 10% of the aerobic energy input, drastic reductions in the various forms of cell work must occur. Studies on brain in particular, but also on liver cells, have documented reductions in several key processes that drive ATP utilization: transmembrane ion leakage, electrical activity, and protein synthesis. In the language of the field, these have been referred to, respectively, as channel arrest, spike arrest, and translational arrest (Hochachka et al., 1996). Channel arrest is a reduction in the number of functional ion channels and/or in the probabil- Fig. 3. Constant cellular ATP levels of anoxic turtles indicates that a coordinated decrease occurs in both ATP production and ATP utilization when a turtle makes the transition from a normoxic to an anoxic state. 304 D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315 Fig. 4. Cellular processes of isolated turtle hepatocytes that require ATP all decrease when the cells are made anoxic, and the overall cumulative decrease ( :90%) is close to the decrease observed in the whole animal (Jackson, 1968; Herbert and Jackson, 1985b) and in the total metabolic rate of hepatocytes (Buck et al., 1993). Data shown are adapted from Hochachka et al., 1996). ity that ion channels are open. This slows ion leakage and the dissipation of ion gradients and less cell work is required to maintain these gradients via ATP-dependent ion pumps. Evidence for downregulation of Na+ channels in brain (PérezPinón et al., 1992) and liver cells (Buck et al., 1993), for K+ channels in brain (Chih et al., 1989), and for Ca2 + in brain (Bickler and Buck, 1998) have been reported. The reduced channel activity in brain contributes in turn to what Sick et al. (1998) called spike arrest, a reduction in brain electrical activity. Also contributing to spike arrest are increased levels of the inhibitory neurotransmitter, GABA (Nilsson and Lutz, 1991), reduced sensitivity of the NMDA-type glutamate receptor (Buck and Bickler, 1998), and an apparently overall regulating action of adenosine (Lutz and Nilsson, 1997). Doll et al. (1993) attributed all the reduction in anoxic brain ATP expenditure for ion pumping to spike arrest, since they could detect no significant decrease during anoxia in whole cell ionic conductance in turtle pyramidal neurons. Finally, reduced protein synthesis, translational arrest, has been observed both in anoxic turtle hepatocytes (Land et al., 1993) and in anoxic turtle heart (Bailey and Driedzic, 1996), although the upregulation of specific proteins, such as stress proteins in heart (Chang et al., 2000) and possibly glycolytic enzymes in liver cells (Hochachka et al., 1996) has been reported. A comprehensive study of the effect of anoxia on various energy-requiring functions in isolated turtle hepatocytes was conducted by Hochachka and his colleagues. The results of their studies, adapted from Hochachka et al. (1996), are summarized in Fig. 4. Interestingly, the overall decrease in hepatocyte ATP hydrolysis, estimated from the sum of the various components, closely matches total hepatocyte metabolic reduction (Buck et al., 1993) and whole animal metabolic reduction (Jackson, 1968). The coordination of metabolic events leading to a matched decrease in ATP production and utilization, long-term survival in an hypoxic state, and eventual recovery is not well understood. One proposal that has been put forward (Hochachka et al., 1996) suggests that anoxia-tolerance in turtles and in other facultative anaerobes is a twophase process: first, a defense phase during which ATP supply and consumption are suppressed in a controlled fashion, as just described; and second, a rescue phase in which selective proteins, including transcription and elongation factors, molecular chaperones, and glycolytic enzymes, are produced to enable the cells to function during anoxia. A heme-based oxygen sensing mechanism, analogous to that described for the regulation of erythropoietin, has been proposed as the trigger for these responses (Hochachka et al., 1996). The unraveling of the molecular and cellular basis for anoxia tolerance in turtles, and in other organisms, is an active and exciting aspect of this field that will doubtless lead to findings of general biological significance in the coming years. For recent more detailed accounts of this work on anoxic turtles, the reader is referred to Hochachka et al. (1996), Storey (1996), Hochachka (1997), Lutz and Nilsson (1997), Bickler and Buck (1998). 2.4. Ad6antages of reduced metabolism The submerged anoxic turtle is to a great extent a closed system, exchanging only a few substances (oxygen if available, CO2, and water) with the surrounding water. At 3°C, loss of CO2 to the water allows blood PCO2 to remain normal or even fall during anoxia thereby avoiding the respiratory acidosis that is characteristic of submergence at higher temperatures. A net uptake of water from the surroundings also occurs as indicated by an increase in body mass during longterm submergence (Ultsch and Jackson, 1982). D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315 Major regulated exchanges, however, that are crucial for normal homeostasis, including feeding and pulmonary gas exchange do not occur, and renal function is either lost or severely curtailed (Warburton and Jackson, 1995; Jackson et al., 1996). As a consequence, the submerged anoxic turtle must support its metabolic needs throughout its submergence time with on-board energy reserves. In addition, metabolic end-products, other than CO2, remain within the body and must be tolerated by the animal. For an anoxic turtle, the principle metabolic storage molecule is glycogen, and the principle end-product is lactic acid. The length of time a turtle can remain anoxic may therefore be critically dependent on glycogen depletion and/or on lactate accumulation. Adaptations that can delay either of these processes reaching critical limits may extend underwater time. Reduced metabolism is clearly a useful adaptation in this regard. By slowing the rate at which glycogen is utilized by 10-fold, the turtle may extend the anoxic period by that magnitude. Furthermore, painted turtles have large glycogen reserves residing principally in liver and skeletal muscle (Daw et al., 1967; Wasser et al., 1991). Based on estimated anaerobic metabolic rate (Herbert and Jackson, 1985b) and body glycogen stores, we calculate that a turtle could sustain anoxic periods of about 5.5 months at 3°C before glycogen became depleted. It is unlikely that a 305 turtle in nature would ever be subjected to continuous anoxia for that long, so that glycogen reserves may not be the limiting factor for anoxic survival, assuming that the turtle begins the winter with an adequate supply. Lactic acid, on the other hand, may pose a more significant threat to the animal. Lactate is produced by the reduction of pyruvate in order to regenerate NAD+ for sustaining flux through the glycolytic pathway. Protons are a product of ATP hydrolysis and, in a situation such as anoxic submergence in which ATP levels are maintained, protons and lactate ions are produced in equal amounts (Hochachka and Mommsen, 1983) making lactic acid, in effect, the overall end-product of anaerobic metabolism. Even though metabolic depression slows the rate at which lactate is produced and accumulates within the body, very high concentrations are reached during long periods of anoxia. Plasma concentrations as high as 200 mM were measured after 5 months of submergence at 3°C (Ultsch and Jackson, 1982), and concentrations between 100 and 150 mM were reached after 3 months anoxia (Jackson and Heisler, 1982; Herbert and Jackson, 1985a), a submergence duration from which these turtles can fully recover (Herbert and Jackson, 1985a). Despite these high lactate concentrations, that are far in excess of what other animals are able to endure, the painted turtles are able to maintain a viable blood pH in the range of 7.0 or higher (Fig. 5). Clearly the Fig. 5. Blood pH and plasma lactate concentration during 125 days of anoxic submergence in the eastern painted turtle (Chrysemys picta picta). Data are re-drawn from Ultsch et al., 1999. 306 D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315 painted turtle possesses specialized mechanisms for dealing with this enormous acid load. The balance of this paper will address these mechanisms. 3. Acid–base adaptations of anoxic turtles 3.1. Body fluid buffering Lactic acid is a relatively strong acid (approximately pK 4.0) that at physiological pH (approximately 7.0) is about 99.9% dissociated to protons and lactate ions (i.e. [lactate]/[lactic acid] : 1000/ 1). During anoxic submergence, turtles neither excrete lactate nor can they convert it to other, less toxic, compounds. To avoid life-threatening acidosis, therefore, the anoxic turtle must neutralize the generated protons using endogenous buffering. The first line of defense against metabolically produced fixed acid is by intracellular and extracellular buffering. In various freshwater turtles, as first reported by Smith (1929), extracellular buffering of lactic acid is particularly effective due to high concentrations of bicarbonate. In the western painted turtle and in related freshwater species, plasma [HCO− 3 ] is in the range of 35 – 45 mM whereas values for other reptiles are rarely above 30 mM and are often below 20 mM (Ultsch and Jackson, 1996). Furthermore, chelonian species including the painted turtles also have specialized pericardial and peritoneal fluids, bathing the heart and abdominal viscera, respectively, that have even higher concentrations of HCO− 3 : approximately 80 mM in the peritoneal fluid and 120 mM in the pericardial fluid (Smith, 1929; Jackson and Silverblatt, 1974). During acidosis, whether imposed by gastric infusion of dilute HCl (Jackson, 1969) or by prolonged anoxic submergence at low temperature (Murdaugh et al., 1962; Jackson and Ultsch, 1982; Jackson and Heisler, 1984), the [HCO− 3 ] in these fluid compartments falls as [lactate] rises. The specialized pericardial and peritoneal fluids of Trachemys scripta elegans are not affected, however, by briefer (4 h) anoxic submergences at 24°C (Jackson and Silverblatt, 1974) so exchange kinetics may be slow. Buffering by intracellular fluids of the painted turtle, as in other organisms, is mainly by non-bicarbonate buffers and [HCO− 3 ] is lower than in the ECF, at about 10 mM (Jackson and Heisler, 1983). A recent measurement of non-bicarbonate buffering capacity in cardiomyocytes of C. picta −1 −1 l cell water bellii was 22.3 mmol HCO− 3 pH (Shi et al., 1997), a value that conforms to published values for various tissues from other organisms (Pörtner, 1990). Lactate, in the anoxic turtle, distributes preferentially to extracellular fluid leading to cell lactate concentrations in muscle and liver that are generally B50% of extracellular concentrations (Jackson and Heisler, 1983). This distribution conforms generally to a transmembrane equilibrium of the undissociated acid form as predicted by the pH difference between the two compartments. Despite the lower levels of intracellular lactate, however, pH falls by similar magnitudes in the cells and in the blood (Jackson and Heisler, 1983). Assuming that the distribution of the associated protons is the same as the lactate, this indicates that the effective buffering in the extracellular compartment is superior. Effective buffering includes the endogenous buffers, such HCO− 3 , as well as any supplemental buffering contributed by exchanges with other body compartments. The magnitude of the acid load experienced by the turtle after several months of anoxia at 3°C far exceeds the endogenous buffering capacity of the extracellular fluid. Even the high levels of HCO− 3 possessed by this animal are not adequate to deal with acid levels that can reach 150–200 mM. Keeping blood pH near 7.0 requires the contribution of additional buffering from elsewhere in the body. The predominant source of this additional buffering, as will now be discussed, is the shell and skeleton of the turtle. 3.2. The turtle’s shell The shell of the turtle is its characteristic structural feature. Its primary function as a protective enclosure for the animal is a familiar one and one that has served it well during the some 200 million years of its existence in essentially its present form (Pough et al., 1996). The shell also serves as an insertion site for muscle and connective tissue. These well-known roles of the shell require a structure that is rigid and strong, but not one that is necessarily metabolically active or that is in dynamic exchange with the extracellular fluid. But like bone, the turtle’s shell is a dynamic structure; it is perfused with blood, it grows and remodels, and it serves as the major reservoir for the body’s minerals (Zangerl, 1969). D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315 The shell of the painted turtle, Chrysemys picta bellii, is large and accounts for about 32% of the animal’s body mass. Included in this shell mass are elements of the skeleton (vertebrae and ribs) that are fused to the shell. Skeletal elements separate from the shell (limb girdles and long bones and skull) represent a further 5.5% of the body mass (unpublished observations). The total mass of bony tissue, therefore, is over 35% of total body mass, some three times the bone mass of a similar sized lizard or mammal (Calder, 1984). The composition of shell is similar to bone and within these mineralized tissues reside over 95% of the body’s calcium, magnesium, phosphate, and CO2, and over 60% of the body sodium (Jackson, 1999). In a recent study (Jackson et al., 2000) the concentration of these elements and the percent of water and ash were determined in the shell and hindlimb bones of Chrysemys (Table 1). The minerals of the turtle’s shell and bone represent an enormous potential buffer reserve for this animal, and these are exploited during anoxia in two ways. First, mineral buffers, principally in the form of carbonates, are released into the extracellular fluid where they supplement existing buffering in that compartment. Second, lactic acid is taken up by shell and bone where the protons are buffered by carbonate and the lactate is stored for the duration of the anoxic period. 3.3. Mechanism 1: release of carbonate buffers Exploitation of bone carbonates as supplemental buffers is a response to sustained acidosis that has been described in humans and other mammals (Irving and Chute, 1932; Burnell, 1971; Bettice, 1984; Bushinsky and Lechleider, 1987), and in a variety of invertebrates (DeFur et al., 1980; Byrne and McMahon, 1991). Its occurrence in the submerged turtle at 3°C was originally indicated by a progressive increase in the plasma concentrations of calcium and magnesium (Jackson and Ultsch, 1982). This study produced results that were initially quite puzzling. Plasma lactate concentrations increased to very high levels, 150 – 200 mM, but analyses of what we regarded as the major plasma ions (Na+, K+, Cl−, and HCO− 3 ) failed to satisfy ion balance and we were left with a large ‘cation gap.’ However, measurements of total Ca2 + and Mg2 + revealed large increases in these elements, to concentratons as high as 50 and 35 mM, respectively, that almost completely ac- 307 counted for the gap. This result was surprising and not anticipated because concentrations of Ca2 + and Mg2 + generally remain very constant and deviations from normal, especially in Ca2 + , can lead to serious disruptions of excitable tissue function. The full implications of these high circulating Ca2 + and Mg2 + levels are still not clear, but two factors appear to blunt any potentially negative impact on cell function. First, Ca2 + and Mg2 + both complex with lactate; in the case of Ca2 + , a cationic complex, CaLact+, is formed (Ghosh and Nair, 1970). Although the association constant for this reaction is relatively low (:20 l mol − 1) and complex formation is insignificant at normoxic levels of the reactants, complex formation is very significant under conditions of prolonged anoxic acidosis, when as much as two-thirds of the total Ca2 + can be bound in this way (Jackson and Heisler, 1982). As a consequence, ionized calcium, the active form, is much lower in concentration than total Ca2 + and the effective gradient for Ca2 + entry into cells is lessened. A second factor that permits the turtle to tolerate high circulating Ca2 + concentrations is that Ca2 + entry into cells is inhibited during anoxia. In cardiac cells, low extracellular pH reduces Ca2 + influx (Orchard and Kentish, 1990); in brain cells, Ca2 + channels, such as the NMDA receptor, are downregulated, thereby protecting the cells from the damaging elevation of intracellular Ca2 + that occurs in anoxic mammalian cells (Bickler and Buck, 1998) even at normal extracellular concentrations. Nonetheless, plasma ionized Ca2 + of anoxic turtles can be many times higher than control values (12.5 mM compared with 1.0 mM), and further studies on possible effects of, and adaptations to, this unusual hypercalcemic condition are certainly needed. Because shell and bone are the turtle’s major sites for Ca2 + and Mg2 + storage, it is logical to postulate that the elevated plasma concentrations of these elements during anoxia derive from these sources. In addition, various lines of direct experimental evidence support this idea. First, analysis of shell composition revealed a significant decrease in [Mg2 + ], although not in [Ca2 + ], during anoxia at 3°C (Warburton and Jackson, 1995). Calcium is some 50 times more abundant than Mg2 + in shell and bone, but estimated Ca2 + release from these structures is only two to three times as great (Jackson and Heisler, 1982; Herbert 308 Water (%) Ash (%) Organic (%) Shell 27.89 0.6 44.79 0.8 27.490.4 Bone 33.69 1.1 41.09 1.0 25.4 9 0.2 a b [P] (mmol g−1 ash−1) [Mg] (mmol g−1 ash−1) [Na] (mmol g−1 ash−1) [K] (mmol g−1 ash−1) [CO2]b (mmol g−1 ash−1) 9420 9116 4800 9 32 228.0 96.6 362.29 8.3 10.79 1.4 1347 9 67 10 3609186 5138 9 38 196.092.4 369.19 11.2 17.89 0.9 [Ca] (mmol g−1 ash−1) All data, except shell CO2 concentration, is from Jackson et al. (2000). Unpublished data. – D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315 Table 1 Shell and bone composition from control painted turtles (Chrysemys picta bellii ) at 3°Ca D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315 309 Fig. 6. Shell sodium concentration in mmol kg − 1 ww − 1 and plasma sodium and chloride concentrations in mmol l − 1 of painted turtles (Chrysemys picta bellii ) submerged in anoxic water for up to 125 days. The top trace shows the absence of any change in shell sodium during 125 days of submergence in turtles in normoxic water. Data are from Jackson et al. (2000). and Jackson, 1985a. Therefore, the relative change in shell Mg2 + is much easier to detect. Second, in vitro incubation of shell powder in simulated extracellular solution (Jackson et al., 1999) resulted in significant release of both Ca2 + and Mg2 + , in proportions similar to in vivo observations, and in direct proportion to solution acidity. Phosphate, the major anion of bone and shell in turtles, was not released in significant amounts from incubating shell powder in vitro, nor did plasma concentration of phosphate increase during prolonged anoxia in vivo (Jackson et al., 2000). This indicates that the major structural mineral of bone, calcium phosphate, is not broken down under these conditions, and that another buffer anion is released instead. Carbonate, as noted above, is the major anion released from bone under acidotic conditions in a variety of organisms. In turtle, several experimental observations confirm a similar role in prolonged acidosis in this animal. First, total CO2 concentration of shell decreases significantly during anoxia in vivo at 3°C (Warburton and Jackson, 1995). Second, shell powder incubated in vitro releases CO2 in proportion to solution acidity and with a stoichiometry that indicates divalent carbonate as the molecular form (Jackson et al., 1999). In mammalian bone, CO2 is thought to exist in three different sites (Rey et al., 1989): as carbonate substituted for hydroxyl ion in hydroxyapatite, as carbonate substituted for phosphate in hydroxyapatite (the major site), and in a rapidly exchanging fraction, possibly as carbonate on the surface of bone crystals or as bicarbonate in the hydration shell of the crystals. It is the rapidly exchanging fraction that is thought to participate in buffering responses. Our observatons on turtle shell in vitro (Jackson et al., 1999) suggest that the exchangeable CO2 in this structure is in the form of carbonate and not bicarbonate. This is also supported by an earlier observation that the shell possesses no heat-labile CO2 (Warburton and Jackson, 1995); in mammalian studies where a heat-labile CO2 fraction is found, this is taken as evidence for bicarbonate release (Poyart et al., 1975). In addition to calcium and magnesium carbonates, turtle shell also releases sodium ions, presumably also accompanied by carbonate (Jackson et al., 2000). As shown in Fig. 6, shell [Na+] fell progressively throughout 4 months of anoxic submergence at 3°C; however, no change in shell [Na+] was observed in turtles submerged for 4 months in aerated water. The participation of 310 D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315 Fig. 7. Schematic model described putative exchanges between blood and shell (and bone) during prolonged anoxia in the turtle Chrysemys picta bellii. In mechanism 1 (to the left), calcium, magnesium, and sodium carbonates move from the shell to the blood in response to lactic acidosis and thereby supplement extracellular buffering capacity. In mechanism 2 (to the right) lactic acid enters the shell and is buffered and sequestered there. Na+ in the shell buffering response agrees with earlier observations in mammals where bone Na+ release is an important regulatory response to both acidosis and hyponatremia (Forbes, 1960; Burnell, 1971). The release of Na+ from powdered shell in vitro, in contrast, was inconsistent and unrelated to incubating solution pH (Jackson Fig. 8. Plasma strong ion difference (SID), the sum of the concentrations of strong cations (Na+, K+, Ca2 + , and Mg2 + ) minus the sum of the concentrations of strong anions (Cl− and lactate−) under control normoxic conditions at 3°C (top trace) and during 12 weeks of anoxic submergence at the same temperature (middle trace). The bottom trace depicts the SID that would have resulted from the observed increases in plasma [lactate−] had no changes in other strong ions occurred. The vertical lines therefore represent the magnitude of the compensatory increases in K+, Ca2 + , and Mg2 + concentrations and decreases in Cl− concentration. Data are from Jackson and Heisler, 1982). et al., 1999). The loss of shell Na+ in vivo is significant and, based on the magnitude of the fall in concentration and the mass of shell, should elevate extracellular [Na+] by about 39 mmol l − 1 (Jackson et al., 2000). Paradoxically, though, plasma [Na+] in this same study actually decreased significantly, and although in previous studies plasma [Na+] has generally remained unchanged (e.g. Jackson and Heisler, 1982), an increase in [Na+] has never been observed. The whereabouts of the sodium leaving the shell is therefore uncertain and must await further study. A simplified model for mechanism 1 (Section 3.3) is presented in Fig. 7. The initiating event in this exchange process is the addition of lactic acid to the extracellular fluid (ECF). The first line of defense, and the principal one at low levels of lactic acid, is the endogenous buffers of the ECF, chiefly HCO− 3 . As lactate levels rise, however, the developing acidosis causes a progressive dissolution of shell and bone minerals, specifically calcium, magnesium and sodium carbonates. The carbonates titrate the protons producing CO2 that at 3°C is mostly lost through non-pulmonary pathways to the surrounding water. Calcium released into the ECF reacts with lactate and up to two thirds may bind in this way. ECF levels of both Ca2 + and Mg2 + increase markedly, but Na+ levels either remain unchanged or paradoxically fall. The impact of the released minerals from turtle shell and bone can best be appreciated in terms of their effect on the strong ion difference (SID) of plasma. Strong ion difference, a term popularized by Stewart (1981) in his analysis of acid–base physiology, is the difference in concentration between the strong cations (Na+, K+, Ca2 + , Mg2 + ) and the strong anions (Cl−, lactate−). In turtle plasma, SID is normally about 44 meq l − 1 (Herbert and Jackson, 1985a) and electroneutrality is satisfied by ‘weak’ anions, primarily HCO− 3 and titratable groups on protein. Because the defense of a viable acid–base state requires maintenance of SID in the positive range, the challenge facing a turtle experiencing long-term anoxic submergence is clear. An increase in the concentration of lactate to 150–200 meq l − 1 with no changes in other plasma strong ions would produce a highly negative SID (Fig. 8). All available buffers would be fully titrated and severe, probably fatal, acidosis would ensue. But release of shell and bone buffers helps counteract this by providing D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315 Fig. 9. Lactate concentration of shell and plasma of painted turtles (Chrysemys picta bellii ) sampled at intervals during 9 days of anoxic submergence and 9 days of recovery at 10°C (Jackson, 1997). carbonate to titrate the protons and by providing strong cations (Ca2 + , Mg2 + , and Na+) to sustain a positive SID. Additional plasma ion changes, apparently unrelated to shell function, that contribute to the defense of SID include increased ECF [K+] and decreased ECF [Cl−]. 3.4. Mechanism 2: uptake of lactic acid by shell and bone The second way that the shell and skeleton of the anoxic turtle contribute to acid – base balance is by sequestering lactic acid. Unlike the buffer release mechanism, the lactate storage mechanism has not previously been described in other organisms. The first evidence for its occurrence was obtained when shell powder, collected from submerged anoxic turtles previously injected with 14 C-labeled lactate, was found to exhibit significant 14C activity. Subsequent systematic investigation, in which lactate was measured by both 14C activity and by conventional enzymatic assay, revealed a substantial and uniform distribution of lactate throughout the entire shell of the turtles during anoxia at 20°C (Jackson et al., 1996). More recently, the phenomenon has been documented at 10 and 3°C as well (Jackson, 1997). In the study at 10°C, turtles were submerged in anoxic water for 9 days and then allowed to 311 recover with access to air for an additional 9 days. Blood and shell samples, taken periodically throughout this period, revealed a parallel rise in lactate in both plasma and shell during submergence, and then a parallel fall in lactate during recovery (Fig. 9). In a more recent study at 3°C (Jackson et al., 2000), bone was shown to accumulate lactate as well, to an even greater extent than shell, during 4 months of anoxic submergence. The extent to which this mechanism contributes to management of total body lactate is considerable. After 4 months anoxia at 3°C, shell and bone concentrations reached 135.6 and 163.6 mmol g − 1 ww − 1, respectively, when plasma lactate concentrations averaged 155.2 mmol l − 1 (Jackson et al., 2000). Based on estimated lactate distribution in the body fluids, the size of body fluid compartments, and the mass of the shell and skeleton, we estimated that 47% of the total body lactate resided in the shell and bone at this time. A similar fraction of body lactate was calculated to be stored in shell and bone after 9 days anoxia at 10°C. The entry of lactate into shell and bone does not, however, prove that these structures are contributing to lactic acid buffering. Lactate cannot enter the shell alone; it must either be accompanied by a cation or must exchange with another anion in order to maintain electroneutrality. The identity of this other ion is crucial for understanding the acid–base significance of lactate entry. If, for example, a strong cation such as Na+ enters with lactate, or a strong anion such as Cl− leaves as lactate enters, then the lactate sequestration will have no impact on extracellular acid–base status. This is because the process will not effect the ECF strong ion difference (see above). If, however, lactate enters with an H+ or if two lactates enter in exchange for a carbonate, then this would have great significance for the ECF acid–base status. Both indirect and direct evidence support the latter possibility. The indirect evidence is that no strong ion among the major ECF ions appears to be a likely candidate. It is known that calcium, magnesium, and sodium all leave the shell during anoxia, and that ECF calcium and magnesium concentrations rise significantly. Also, ECF [Cl−] falls sharply during anoxia making it unlikely that Cl− serves as a counterion for lactate. The direct evidence comes from in vitro incubation of either shell powder in 312 D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315 solutions with high lactate, and of shell powder containing high lactate in solutions without lactate (Jackson et al., 1999). These experiments reveal that lactate entry into shell alkalinizes the bathing solution whereas lactate release from shell acidifies the solution. This supports the proposition that protons, in effect, accompany lactate movement, and that this mechanism of lactate sequestration exploits the buffering capacity of the shell and bone. A simple model for this lactate uptake mechanism is depicted in Fig. 7. As in mechanism 1, the first step is the addition of lactic acid to the ECF. Assuming that lactate entry into the shell is a passive process, elevation of ECF [lactate] will initiate diffusion of lactate into the shell and this will continue as further lactate is produced anaerobically and enters the ECF. The equilibrium state between the two compartments is not certain, but it seems to be approached when the shell concentration in mmol kg − 1 ww − 1 approximates the plasma lactate concentration in mmol l − 1 (Jackson, 1997, 1999). As depicted by the model, each lactate is accompanied, in effect, by a proton, and the proton is buffered within the shell substance by carbonate. The CO2 produced thereby diffuses back into the ECF and is lost by diffusion to the surrounding water. The physical state of lactate within the shell is uncertain, but it probably exists primarily in combined form, most likely complexed with Ca2 + , as has already been described to occur in the ECF. The high concentration of lactate and the small volume of shell water (only : 30% of shell mass) makes it unlikely that lactate could exist in simple solution; the estimated concentration would have to be too high. The mechanism as described occurs in the skeleton of the turtle as well as in the shell (Jackson et al., 2000). A remarkable feature of this mechanism is that the lactic acid sequestered in this fashion may have no impact whatever on extracellular acid – base state. Both the lactate and proton are processed within the shell itself. The only product coming out (other than water) is CO2, and at low temperature this is lost to the environment and ECF PCO2 is the same as or even lower than the normoxic value. Because, as already noted, shell and bone lactate storage can account for over 45% of the total body lactate, this mechanism greatly minimizes the effect of anaerobic acid production on body fluid acid – base status. It is likely that other organisms employ this same mechanism under circumstances when circulating lactate is elevated. In non-chelonian vertebrates, however, its quantitative importance is almost certainly less for the following reasons. First, no vertebrates other than turtles have a comparable mass of mineralized tissue. As noted earlier, skeletal mass in mammals and lizards of similar size to the painted turtle have only one third the bone mass. Second, no other vertebrate experiences or can tolerate extracellular lactate concentrations as high as observed in the turtle, nor are elevated lactate levels sustained for such extended periods of time. The kinetics of bone exchange are generally slow so that acute elevations of lactate, such as those that occur during and following strenuous exercise, may not provide adequate time for significant lactate uptake by bone. More promising candidates for utilizing this mechanism to a significant degree are invertebrates, such as crustaceans and mollusks, which possess large shells or exoskeletons with abundant calcium carbonate. Many of these organisms also experience hypoxia-induced elevations of lactate, and mobilization of calcium carbonate to the hemolymph for supplemental buffering is well-established. In addition, in a recent study (Jackson, D.C., Wang, T. and Taylor, E.W., unpublished observations), lactate uptake by the exoskeleton of the freshwater crayfish, Austropotamobius pallipes, was observed during emersion hypoxia. 3.5. O6erall contribution of shell and bone to lactic acid buffering The two buffering mechanisms occurring in turtle shell and bone-carbonate release and lactic acid uptake-may together account for as much as 70% of the total lactic acid buffering in the anoxic turtle. This calculation refers to the acid–base state that exists after 3 months of anoxic submergence at 3°C. About two thirds of the shell and bone contribution is contributed by uptake and buffering of lactic acid within the shell and bone. The balance, buffer release, is estimated from the calculated increases in the extracellular concentrations of calcium and magnesium (Jackson, 1997). The calculation of the buffer release mechanism, however, did not take into account the possible contribution of sodium carbonate release, so it may actually be an underestimate. An accurate quantitative assessment of the whole body picture D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315 is further complicated by uncertainty regarding possible changes in body fluid compartment volumes during anoxia. Clearly, though, the mineralized tissues are crucial to the successful survival of turtles during prolonged anoxic submergence. Not only is the turtle shell a protective shield against the hazards of life and a structural framework for muscle attachments, but it also is a enormous reserve of mineral that the turtle can tap to counteract acidosis during extended periods of anoxia. 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