Reversible decreases in ATP and PCr concentrations in anoxic turtle brain

A hallmark of anoxia tolerance in western painted turtles is relative constancy of tissue adenylate concentrations during periods of oxygen limitation. During anoxia heart and brain intracellular compartments become more acidic and cellular energy demands are met by anaerobic glycolysis. Because changes in adenylates and pH during anoxic stress could represent important signals triggering metabolic and ion channel down-regulation we measured PCr, ATP and intracellular pH in turtle brain sheets throughout a 3-h anoxic-re-oxygenation transition with ³¹P NMR. Within 30 min of anoxia, PCr levels decrease 40% and remain at this level during anoxia. A different profile is observed for ATP, with a statistically significant decrease of 23% occurring gradually during 110 min of anoxic perfusion. Intracellular pH decreases significantly with the onset of anoxia, from 7.2 to 6.6 within 50 min. Upon re-oxygenation PCr, ATP and intracellular pH recover to pre-anoxic levels within 60 min. This is the first demonstration of a sustained reversible decrease in ATP levels with anoxia in turtle brain. The observed changes in pH and adenylates, and a probable concomitant increase in adenosine, may represent important metabolic signals during anoxia.     

Adenosine as a signal for ion channel arrest in anoxia-tolerant organisms

Certain freshwater turtles and fish are extremely anoxia-tolerant, capable of surviving hours of anoxia at high temperatures and weeks to months at low temperatures. There is great interest in understanding the cellular mechanisms underlying anoxia-tolerance in these groups because they are anoxia-tolerant vertebrates and because of the far-reaching medical benefits that would be gained. It has become clear that a pre-condition of prolonged anoxic survival must involve the matching of ATP production with ATP utilization to maintain stable ATP levels during anoxia. In most vertebrates, anoxia leads to a severe decrease in ATP production without a concomitant reduction in utilization, which inevitably leads to the catastrophic events associated with cell death or necrosis. Anoxia-tolerant organisms do not increase ATP production when faced with anoxia, but rather decrease utilization to a level that can be met by anaerobic glycolysis alone. Protein synthesis and ion movement across the plasma membrane are the two main targets of regulatory processes that reduce ATP utilization and promote anoxic survival. However, the oxygen sensing and biochemical signaling mechanisms that achieve a coordinated reduction in ATP production and utilization remain unclear. One candidate-signaling compound whose extracellular concentration increases in concert with decreasing oxygen availability is adenosine. Adenosine is known to have profound effects on various aspects of tissue metabolism, including protein synthesis, ion pumping and permeability of ion channels. In this review, I will investigate the role of adenosine in the naturally anoxia-tolerant freshwater turtle and goldfish and give an overview of pathways by which adenosine concentrations are regulated.     

Anoxia-induced changes in reactive oxygen species and cyclic nucleotides in the painted turtle

The Western painted turtle survives months without oxygen. A key adaptation is a coordinated reduction of cellular ATP production and utilization that may be signaled by changes in the concentrations of reactive oxygen species (ROS) and cyclic nucleotides (cAMP and cGMP). Little is known about the involvement of cyclic nucleotides in the turtle’s metabolic arrest and ROS have not been previously measured in any facultative anaerobes. The present study was designed to measure changes in these second messengers in the anoxic turtle. ROS were measured in isolated turtle brain sheets during a 40-min normoxic to anoxic transition. Changes in cAMP and cGMP were determined in turtle brain, pectoralis muscle, heart and liver throughout 4 h of forced submergence at 20–22°C. Turtle brain ROS production decreased 25% within 10 min of cyanide or N₂-induced anoxia and returned to control levels upon reoxygenation. Inhibition of electron transfer from ubiquinol to complex III caused a smaller decrease in [ROS]. Conversely, inhibition of complex I increased [ROS] 15% above controls. In brain [cAMP] decreased 63%. In liver [cAMP] doubled after 2 h of anoxia before returning to control levels with prolonged anoxia. Conversely, skeletal muscle and heart [cAMP] remained unchanged; however, skeletal muscle [cGMP] became elevated sixfold after 4 h of submergence. In liver and heart [cGMP] rose 41 and 127%, respectively, after 2 h of anoxia. Brain [cGMP] did not change significantly during 4 h of submergence. We conclude that turtle brain ROS production occurs primarily between mitochondrial complexes I and III and decreases during anoxia. Also, cyclic nucleotide concentrations change in a manner suggestive of a role in metabolic suppression in the brain and a role in increasing liver glycogenolysis.     

Metabolic depression in animals: physiological perspectives and biochemical generalizations

Depression of metabolic rate has been recorded for virtually all major animal phyla in response to environmental stress. The extent of depression is usually measured as the ratio of the depressed metabolic rate to the normal resting metabolic rate. Metabolic rate is sometimes only depressed to approx. 80% of the resting value (i.e. a depression of approx. 20% of resting); it is more commonly 5-40 % of resting (i.e. a depression of approx. 60-95% of resting); extreme depression is to 1% or less of resting, or even to an unmeasurably low metabolic rate (i.e. a depression of approx. 99-100% of resting). We have examined the resting and depressed metabolic rate of animals as a function of their body mass, corrected to a common temperature. This allometric approach allows ready comparison of the absolute level of both resting and depressed metabolic rate for various animals, and suggests three general patterns of metabolic depression. Firstly, metabolic depression to approx. 0.05-0.4 of rest is a common and remarkably consistent pattern for various non-cryptobiotic animals (e.g. molluscs, earthworms, crustaceans, fishes, amphibians, reptiles). This extent of metabolic depression is typical for dormant animals with ‘intrinsic’ depression, i.e. reduction of metabolic rate in anticipation of adverse environmental conditions but without substantial changes to their ionic or osmotic status, or state of body water. Some of these types of animal are able to survive anoxia for limited periods, and their anaerobic metabolic depression is also to approx. 0.05-0.4 of resting. Metabolic depression to much less than 0.2 of resting is apparent for some ‘resting’, ‘over-wintering’ or diapaused eggs of these animals, but this can be due to early developmental arrest so that the egg has a low ‘metabolic mass’ of developed tissue (compared to the overall mass of the egg) with no metabolic depression, rather than having metabolic depression of the entire cell mass. A profound decrease in metabolic rate occurs in hibernating (or aestivating) mammals and birds during torpor, e.g. to less than 0.01 of pre-torpor metabolic rate, but there is often no intrinsic metabolic depression in addition to that reduction in metabolic rate due to readjustment of thermoregulatory control and a decrease in body temperature with a concommitant Q₁₀ effect. There may be a modest intrinsic metabolic depression for some species in shallow torpor (to approx. 0.86) and a more substantial metabolic depression for deep torpor (approx. 0.6), but any energy saving accruing from this intrinsic depression is small compared to the substantial savings accrued from the readjustment of thermoregulation and the Q₁₀ effect. Secondly, a more extreme pattern of metabolic depression (to < 0.05 of rest) is evident for cryptobiotic animals. For these animals there is a profound change in their internal environment-for anoxybiotic animals there is an absence of oxygen and for osmobiotic, anhydrobiotic or cryobiotic animals there is an alteration of the ionic/osmotic balance or state of body water. Some normally aerobic animals can tolerate anoxia for considerable periods, and their duration of tolerance is inversely related to their magnitude of metabolic depression; anaerobic metabolic rate can be less than 0.005 of resting. The metabolic rate of anhydrobiotic animals is often so low as to be unmeasurable, if not zero. Thus, anhydrobiosis is the ultimate strategy for eggs or other stages of the life cycle to survive extended periods of environmental stress. Thirdly, a pattern of absence of metabolism when normally hydrated (as opposed to anhydrobiotic or cryobiotic) is apparently unique to diapaused eggs of the brine-shrimp (Artemia spp., an anostracan crustacean) during anoxia. The apparent complete metabolic depression of anoxic yet hydrated cysts (and extreme metabolic depression of normoxic, hypoxic, or osmobiotic, yet hydrated cysts), is an obvious exception to the above patterns. In searching for biochemical mechanisms for metabolic depression, it is clear that there are five general characteristics at the molecular level of cells which have a depressed metabolism; a decrease in pH, the presence of latent mRNA, a change in protein phosphorylation state, the maintenance of one particular energy-utilizing process (ion pumping), and the down-regulation of another (protein synthesis). Oxygen sensing is now the focus of intense investigation and obviously plays an important role in many aspects of cell biology. Recent studies show that oxygen sensing is involved in metabolic depression and research is now being directed towards characterising the proteins and mechanisms that comprise this response. As more data accumulate, oxygen sensing as a mechanism will probably become the sixth general characteristic of depressed cells. The majority of studies on these general characteristics of metabolically depressed cells come from members of the most common group of animals that depress metabolism, those non-cryptobiotic animals that remain hydrated and depress to 0.05-0.4 of rest. These biochemical investigations are becoming more molecular and sophisticated, and directed towards defined processes, but as yet no complete mechanism has been delineated. The consistency of the molecular data within this group of animals suggests similar metabolic strategies and mechanisms associated with metabolic depression. The biochemical ‘adaptations’ of anhydrobiotic organisms would seem to be related more to surviving the dramatic reduction in cell water content and its physico-chemical state, than to molecular mechanisms for lowering metabolic rate. Metabolic depression would seem to be an almost inevitable consequence of their altered hydration state. The unique case of profound metabolic depression of hydrated Artemia spp. cysts under a variety of conditions could reflect unique mechanisms at the molecular level. However, the available data are not consistent with this possibility (with the exception of a uniquely large decrease in ATP concentration of depressed, hydrated Artemia spp. cysts) and the question remains: how do cells of anoxic and hydrated Artemia spp. differ from anoxic goldfish or turtle cells, enabling them so much more completely to depress their metabolism?     

龟脑的强抗氧化功能可能与龟类的长寿相关

龟脑防御缺氧和再供氧伤害的过程可能与龟类的长寿相关,研究表明龟体内有特别的保护各种离子通道和受体功能的机制,龟脑在缺氧条件下,可以抑制兴奋性神经递质的毒害作用,其机制也许是通过维持多巴胺和谷氨酸的释放与再吸收之间的平衡来实现的,此外,它通过胞外的γ-氨基丁酸(GABA)浓度的持续升高和其受体密度的相应增加而抵抗活性氧基团的生成,并且免受其伤害．这样的机制可能在缺氧和再供氧的 状态下被选择性的激活,因此龟类可作为研究衰老和抗衰老生理机制的动物模型。     

Examination of Reptilian Erythrocytes as Models of the Progenitor of Mammalian Red Blood Cells

Among the reptile species examined, only loggerhead turtle RBC with their high capacity of anaerobic metabolism and low oxygen uptake possess all the suitable metabolic characteristics as a model for transition from aerobic to anaerobic metabolism of mammalian erythrocytes (RBC). Neither the alligator RBC, which lack a significant level of anaerobic metabolism, nor the savannah monitor lizard RBC with their higher level of temperature-dependent aerobic metabolism, possess all the characteristics suitable as a model for the metabolic evolution of mammalian RBC. In the formation of this metabolic model, no phylogenetic relationships are implied or inferred. The metabolic similarity of loggerhead turtle RBC to mammalian RBC is further indicated by the high activity of the pentose phosphate (PPO₄) pathway, as evidenced by the low thermal sensitivity of their oxygen uptake and by their low ¹⁴C⁶O₂/¹⁴C¹O₂ ratios. By comparison, although the ¹⁴C⁶O₂/¹⁴C¹O₂ ratios of both alligator and monitor lizard RBC are low as compared to loggerhead turtle RBC, only alligator RBC share with loggerhead turtle RBC a low thermal sensitivity of their oxygen uptake. A comparison of hemoglobin concentrations relative to hematocrit for loggerhead turtle, alligator and monitor lizard RBC indicates that RBC hemoglobin concentrations are approximately the same for each of these species. Apart from this similarity, RBC from these three species of reptiles were differentiated in this study with respect to their density and osmotic fragility.     

Interaction of Respiration, Ion Regulation, and Acid-Base Balance in the Everyday Life of Aquatic Crustaceans

Extracellular and intracellular acid-base balance is necessaryfor the maintenance of normal metabolic processes. The primarysource of acid is metabolically produced CO₂, and the CO₂/HCO₃^–system is the most significant buffer. The regulation of acid-basebalance is complex, involving the interaction between respiratorygas exchange and ion transport. In aquatic crustaceans respirationis governed by the need to extract oxygen from water, an O₂-poormedium; thus, acid-base balance is maintained primarily throughion transport mechanisms. These mechanisms include Na⁺/H⁺ andCl^–/HCO₃^– exchange processes that are sensitiveto the extracellular acid-base status of the animal. In marinecrabs, ion regulation and acid-base balance are accomplishedby the posterior gills, while in freshwater species all gillsand the antennal gland perform these functions. Intracellularacid-base balance appears to be maintained primarily by iontransport across the cell membrane. Hemolymph pH varies inverselywith acclimation temperature and salinity. In both cases Pco₂remains nearly constant, and the pH change is a result of changesin hemolymph HCO₃^– concentrations brought about by ionexchange mechanisms. Environmental hypercapnia or hyperoxiainduces a repiratory acidosis characterized by increased Pco₂,low pH, and elevated HCO₃^–; this is partially compensatedfor by ion exchange processes that bring about a further increasein hemolymph HCO₃^–. Exercise causes a mixed respiratoryand metabolic acidosis with compensation via H⁺ ion excretionand hyperventilation.     

Cardiovascular Function in Turtles During Anoxia and Acidosis: In vivo and in vitro Studies

During prolonged experimental submergence, freshwater turtlesbecome anoxic and develop a combined respiratory and non-respiratoryacidosis. Anoxia and acidosis are known to depress cardiac functionin turtles and other vertebrate species. In vitro studies ofventricular and atrial tissue of turtles indicate that increasedextracellular Ca⁺⁺ concentration can reverse these depressantactions. Intact turtlesutilize this compensatory mechanism duringanoxia and other conditions leading to acidosis byincreasingplasma Ca⁺⁺ concentration. In addition, cardiac cells may releaseCa⁺⁺ from cell organelles to compensate for induced respiratoryacidosis. Both mechanisms presumably improve cardiac contractilityby elevating the level of sarcoplasmicCa⁺⁺.     

Proteomic changes in the brain of the western painted turtle (Chrysemys picta bellii) during exposure to anoxia

During anoxia, overall protein synthesis is almost undetectable in the brain of the western painted turtle. The aim of this investigation was to address the question of whether there are alterations to specific proteins by comparing the normoxic and anoxic brain proteomes. Reductions in creatine kinase, hexokinase, glyceraldehyde‐3‐phosphate dehydrogenase, and pyruvate kinase reflected the reduced production of adenosine triphosphate (ATP) during anoxia while the reduction in transitional endoplasmic reticulum ATPase reflected the conservation of ATP or possibly a decrease in intracellular Ca²⁺. In terms of neural protection programed cell death 6 interacting protein (PDCD6IP; a protein associated with apoptosis), dihydropyrimidinase‐like protein, t‐complex protein, and guanine nucleotide protein G_(o) subunit alpha (G_o alpha; proteins associated with neural degradation and impaired cognitive function) also declined. A decline in actin, gelsolin, and PDCD6IP, together with an increase in tubulin, also provided evidence for the induction of a neurological repair response. Although these proteomic alterations show some similarities with the crucian carp (another anoxia‐tolerant species), there are species‐specific responses, which supports the theory of no single strategy for anoxia tolerance. These findings also suggest the anoxic turtle brain could be an etiological model for investigating mammalian hypoxic damage and clinical neurological disorders.     

Modulation of stress proteins and apoptotic regulators in the anoxia tolerant turtle brain

Freshwater turtles survive prolonged anoxia and reoxygenation without overt brain damage by well-described physiological processes, but little work has been done to investigate the molecular changes associated with anoxic survival. We examined stress proteins and apoptotic regulators in the turtle during early (1 h) and long-term anoxia (4, 24 h) and reoxygenation. Western blot analyses showed changes within the first hour of anoxia; multiple stress proteins (Hsp72, Grp94, Hsp60, Hsp27, and HO-1) increased while apoptotic regulators (Bcl-2 and Bax) decreased. Levels of the ER stress protein Grp78 were unchanged. Stress proteins remained elevated in long-term anoxia while the Bcl-2/Bax ratio was unaltered. No changes in cleaved caspase 3 levels were observed during anoxia while apoptosis inducing factor increased significantly. Furthermore, we found no evidence for the anoxic translocation of Bax from the cytosol to mitochondria, nor movement of apoptosis inducing factor between the mitochondria and nucleus. Reoxygenation did not lead to further increases in stress proteins or apoptotic regulators except for HO-1. The apparent protection against cell damage was corroborated with immunohistochemistry, which indicated no overt damage in the turtle brain subjected to anoxia and reoxygenation. The results suggest that molecular adaptations enhance pro-survival mechanisms and suppress apoptotic pathways to confer anoxia tolerance in freshwater turtles.     

Role of neuroglobin in regulating reactive oxygen species in the brain of the anoxia-tolerant turtle Trachemys scripta

Neuroglobin (Ngb) is an oxygen binding heme protein found in nervous tissue with a yet unclear physiological and protective role in the hypoxia-sensitive mammalian brain. Here we utilized in vivo and in vitro studies to examine the role of Ngb in anoxic and post-anoxic neuronal survival in the freshwater turtle. We employed semiquantitative RT-PCR and western blotting to analyze Ngb mRNA and protein levels in turtle brain and neuronally enriched cultures. Ngb expression is strongly up-regulated by hypoxia and post-anoxia reoxygenation but increases only modestly in anoxia. The potential neuroprotective role of Ngb in this species was analyzed by knocking down Ngb using specific small interfering RNA. Ngb knockdown in neuronally enriched cell cultures resulted in significant increases in H₂O₂ release compared to controls but no change in cell death. Cell survival may be linked to activation of other protective responses such as the extracellular regulated kinase transduction pathway, as phosphorylated extracellular regulated kinase levels in anoxia were significantly higher in Ngb knockdown cultures compared to controls. The greater expression of Ngb when reactive oxygen species are likely to be high, and the increased susceptibility of neurons to H₂O₂ release and external oxidative stress in knockdown cultures, suggests a role for Ngb in reducing reactive oxygen species production or in detoxification, though it does not appear to be of primary importance in the anoxia tolerant turtle in the presence of compensatory survival mechanisms.     

Inactivation and Calcium-Dependent Reactivation of Oxygen Evolution in Photosystem II Preparations Treated at pH 3.0 or with High Concentrations of NaCl

A brief treatment at pH 3.0 of Photosystem II (PS II) membranescontaining two bound Ca²⁺ from rice resulted in strong suppressionof oxygen evolution concomitant with extraction of one Ca²⁺and the lost activity was restored on addition of 50 mM Ca²⁺.However, inactivation of oxygen evolution by low pH-treatmentof oxygen-evolving PS II complexes containing only one Ca²⁺from a rice chlorophyll b-deficient mutant was not associatedwith extraction of the bound Ca²⁺, although oxygen evolutionwas markedly enhanced by the addition of Ca²⁺ to the treatedcomplexes. Thus, the acid-inactivation of oxygen evolution cannotbe related to extraction of Ca²⁺. On the other hand, low pH-treatmentwas found to share the following common features with NaCl-treatmentwhich also causes a Ca²⁺-reversible inactivation of oxygen evolution.(1) Exposure of PS II membranes to pH 3.0 resulted in solubilizationof the 23 and 17 kDa extrinsic proteins, although the releasedproteins rebound to the membranes when pH was raised to 6.5.(2) There was an apparent heterogeneity in the binding affinityof Ca²⁺ effective in restoration of the oxygen-evolving activity.(3) Low pH-treated preparations required a higher concentrationof Ca²⁺ for the maximum reactivation of oxygen evolution thandid NaCl-washed preparations. This was also the case with Sr²⁺,which stimulated oxygen evolution of both low pH-treated andNaCl-washed PS II membranes to smaller extents. When the extrinsic23 and 17 kDa proteins had been removed, however, Ca²⁺ concentrationdependence of oxygen evolution in low pH-treated membranes becamesimilar to that in NaCl-washed PS II preparations and the changeswere largely reversed by rebinding of the two proteins. Theseresults strongly suggest that low pH-treatment and NaCl-washinvolve similar mechanisms of Ca²⁺-dependent reactivation. ¹ Present address: Solar Energy Research Group, The Instituteof Physical and Chemical Research (RIKEN), Wako, Saitama, 351-01Japan (Received August 27, 1990; Accepted February 12, 1991)