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.     

Exposure to hypoxia primes the respiratory and metabolic responses of the epaulette shark to progressive hypoxia

The majority of vertebrates are not tolerant to hypoxia but epaulette sharks (Hemiscyllium ocellatum) living on shallow reef platforms appear to tolerate hypoxic periods during tidal fluctuations. The effects of progressive hypoxia on the metabolic and ventilatory responses of these elasmobranchs were examined in a closed respirometer. In order to determine whether repeated exposure to hypoxia primes these sharks to alter their metabolism, one group of sharks was exposed to repeated sub-lethal hypoxia, at 5% of air saturation, prior to respirometry. In response to falling oxygen concentration [O(2)], the epaulette shark increased its ventilatory rate and maintained its O(2) consumption rate (VO(2)) down to 2.2 mg O(2) l(-1) at 25 degrees C. This is the lowest critical [O(2)] ([O(2)](crit)) ever measured for any elasmobranch. After reaching the [O(2)](crit), the shark remained in the respirometer for a further 4-5 h of progressive hypoxia. Only after the [O(2)] fell to 1.0 mg l(-1) was there a decrease in the ventilatory rate followed by a rise in blood lactate levels, indicating that the epaulette shark responds to severe hypoxia by entering a phase of metabolic and ventilatory depression. Interestingly, hypoxia tolerance was dynamic because hypoxic pre-conditioning lowered the VO(2) of the epaulette shark by 29%, which resulted in a significantly reduced [O(2)](crit) (1.7 mg O(2) l(-1)), revealing that hypoxic pre-conditioning elicits an enhanced physiological response to hypoxia.     

Mechanisms of Cyclic AMP Regulation in Cerebral Anoxia and Their Relationship to Glycogenolysis

Abstract: Anoxia elevates levels of cyclic AMP and depresses levels of cyclic GMP in cerebral cortex of mice. Similar effects are also observed in other regions of the brain. Aminophylline inhibits accumulation of cyclic AMP about 50% in hippocampus and cerebellum, but not in cerebral cortex and striaturn; however, this effect requires high doses (²⁵⁰ mgikg). Pretreatment of animals with reserpine, which depletes brain stores of norepinephrine, dopamine, and serotonin, and also produces sedation and mild hypothermia, markedly inhibits accumulation of cyclic AMP in all regions of anoxic brain. Destruction of norepinephrine terminals by treatment of neonatal animals with ⁶-OH- dopamine, which does not sedate or produce hypothermia, has an effect on cyclic AMP levels similar to that of reserpine. None of the above treatments modifies the effect of anoxia on cyclic GMP levels. These data indicate that norepinephrine is a major regulator of cyclic AMP levels in anoxic brain and that adenosine and, perhaps, other unidentified substances have lesser roles in this process. In contrast, biogenic amines and adenosine appear to have no effect on cyclic GMP regulation in anoxic brain. Reserpine slows the activation of phosphorylase and the utilization of ATP, and slightly attenuates the breakdown of glycogen caused by anoxia, but has no effect on the changes in glucose, lactate, or phosphocreatine. In contrast, ⁶-OH-dopamine has no effect on any of these anoxiainduced changes. It is concluded that the effect of reserpine on phosphorylase, glycogen, and ATP is most likely related to the hypothermic and sedative effect of the drug, and that either cyclic AMP is not responsible for initiating glycogenolysis in anoxic brain or only a small rise in cyclic AMP levels is necessary for this process.     

Anoxic survival of the isolated cerebellum of the turtle Pseudemis scripta elegans

Intact turtle brain provides a useful model for the study of anoxia and potential survival strategies, since this tissue maintains transmembrane ion gradients and ATP levels during prolonged anoxia and recovers functional activity afterwards. Since isolated tissues offer experimental advantages, the present study sought to determine effects of anoxia on the isolated turtle cerebellum and to define relationships between anoxia survival and glucose supply. In normoxia, the extracellular potassium ([K+]o) activity and evoked potentials were maintained with 5 mM glucose, but 20 mM glucose was required to maintain adenosine triphosphate (ATP) levels and prevent significant increases in [K+]o during anoxia. Inhibition of glycolysis by iodoacetic acid (IAA) during anoxia provoked large increases in [K+]o at all glucose levels. These results demonstrate the usefulness of the isolated turtle cerebellum for studies of anoxic survival since this tissue can maintain ATP levels and [K+]o during prolonged anoxia with 20 mM glucose in the artificial cerebrospinal fluid medium. They also suggest the presence of a Pasteur effect at least during the transition to a hypometabolic state.     

Evidence of anoxia-induced channel arrest in the brain of the goldfish (Carassius auratus)

The common goldfish (Carassius auratus) is extremely anoxia tolerant and here we provide evidence that "channel arrest" in the brain of these fish contributes to ATP conservation during periods of anoxia. Whole-cell patch-clamp recordings of slices taken from the telencephalon indicated that the N-methyl-d-aspartate (NMDA) receptor, an ionotropic glutamate receptor and Ca(2+)-channel, underwent a 40-50% reduction in activity during 40 min of acute anoxia. This is the first direct evidence of channel arrest in an anoxia-tolerant fish. Because goldfish produce ethanol as a byproduct of anaerobic metabolism we then conducted experiments to determine if the observed reduction in NMDA receptor current amplitude was due to inhibition by ethanol. NMDA receptor currents were not inhibited by ethanol (10 mmol L(-1)), suggesting that channel arrest of the receptor involved other mechanisms. Longer-term (48 h) in vivo exposure of goldfish to anoxic conditions (less than 1% dissolved O(2)) provided indirect evidence that a reduction in Na(+)/K(+)-ATPase activity also contributed to ATP conservation in the brain but not the gills. Anoxia under these conditions was characterized by a decrease in brain Na(+)/K(+)-ATPase activity of 30-40% by 24 h. Despite 90% reductions in the rates of ventilation, no change was observed in gill Na(+)/K(+)-ATPase activity during the 48-h anoxia exposure, suggesting that branchial ion permeability was unaffected. We conclude that rapid "channel arrest" of NMDA receptors likely prevents excitotoxicity in the brain of the goldfish, and that a more slowly developing decrease in Na(+)/K(+)-ATPase activity also contributes to the profound metabolic depression seen in these animals during oxygen starvation.     

Fecal corticosterone assessment in the epaulette shark,Hemiscyllium ocellatum

The present study examined the feasibility of measuring the steroid hormone corticosterone in fecal extracts of epaulette sharks, Hemiscyllium ocellatum. Six immature, captive-raised epaulette sharks (four females and two males) were obtained from two different zoos and were maintained in a closed-system, 530-liter aquarium. After a one-month adaptation, fecal samples were collected daily from each animal for 33 days. Five-day sets of samples were pooled within animals to insure sufficient material for analysis. Fecal hormone extraction was achieved using repeated cycles of dichloromethane and aqueous washes. The levels of corticosterone were measured by reverse-phase high-performance liquid chromatography (HPLC). Corticosterone presence in HPLC eluent peaks from fecal extracts was determined by comparison of the elution pattern of corticosterone standard with the elution patterns of fecal extracts with and without the addition of tritiated corticosterone or exogenous, unlabeled corticosterone. Exclusive presence of corticosterone in HPLC eluent peaks presumed to be corticosterone was determined by nuclear magnetic resonance mass spectrometry. Corticosterone levels, calculated from a 10-point standard curve, ranged from 1.2 to 20.9 ng/g feces across all sharks, with 92.3% of values being < or =13.5 ng/g. Within individuals, the lowest average for corticosterone levels across 33 days was 2.6+/-0.4 ng/g feces, and the highest average was 8.4+/-2.2 ng/g feces. This study demonstrated that corticosterone was extractable from and reliably measurable in fecal extracts of epaulette sharks. This is the first evidence of this hormone in epaulette sharks and the first report of fecal corticosterone in elasmobranchs.     

Ecophysiology of neuronal metabolism in transiently oxygen-depleted environments: Evidence that GABA is accumulated pre-synaptically in the cerebellum

Interactions between coral reef topography, tide cycles, and photoperiod provided selection pressure for adaptive physiological changes in sheltered hypoxic niches to be exploited by specialized tropical reef fish. The epaulette shark Hemiscyllium ocellatum withstands cyclic hypoxia in its natural environment, many hours of experimental hypoxia, and anoxia for up to 5 h. It shows neuronal hypometabolism in response to 5% oxygen saturation. Northern-hemisphere hypoxia- and anoxia-tolerant vertebrates that over-winter under ice alter their inhibitory to excitatory neurotransmitter balance to forestall brain ATP depletion in the absence of oxidative phosphorylation. GABA immunochemistry, HPLC analysis and receptor binding studies in H. ocellatum cerebellum revealed a heterogeneous regional accumulation of neuronal GABA despite no change in its overall concentration, and a significant increase in GABA_A receptor density without altered binding affinity. Increased GABA_A receptor density would protect the cerebellum during reoxygenation when transmitter release resumes. While all hypoxia- and anoxia-tolerant teleosts examined to date respond to low oxygen levels by elevating brain GABA, the phylogenetically older epaulette shark did not, suggesting that it uses an alternative neuroprotective mechanism for energy conservation. This may reflect an inherent phylogenetic difference, or represent a novel ecophysiological adaptation to cyclic variations in the availability of oxygen.     

Role of adenosine in the hypoxia-induced hypothermia of toads

The concept that hypoxia elicits a drop in body temperature (T(b)) in a wide variety of animals is not new, but the mechanisms remain unclear. We tested the hypothesis that adenosine mediates hypoxia-induced hypothermia in toads. Measurements of selected T(b) were performed using a thermal gradient. Animals were injected (into the lymph sac or intracerebroventricularly) with aminophylline (an adenosine receptor antagonist) followed by an 11-h period of hypoxia (7% O(2)) or normoxia exposure. Control animals received saline injections. Hypoxia elicited a drop in T(b) from 24.8 +/- 0.3 to 19. 5 +/- 1.1 degrees C (P < 0.05). Systemically applied aminophylline (25 mg/kg) did not change T(b) during normoxia, indicating that adenosine does not alter normal thermoregulatory function. However, aminophylline (25 mg/kg) significantly blunted hypoxia-induced hypothermia (P < 0.05). To assess the role of central thermoregulatory mechanisms, a smaller dose of aminophylline (0.25 mg/kg), which did not alter hypoxia-induced hypothermia systemically, was injected into the fourth cerebral ventricle. Intracerebroventricular injection of aminophylline (0.25 mg/kg) caused no significant change in T(b) under normoxia, but it abolished hypoxia-induced hypothermia. The present data indicate that adenosine is a central and possibly peripheral mediator of hypoxia-induced hypothermia.     

海马脑片缺氧早期腺苷的作用及其机制研究

本实验采用海马脑片细胞外记录技术,观察了缺氧早期突触功能可逆性抑制中腺苷的作用并初步探讨其作用机制。结果发现：海马脑片缺氧早期突触功能出现可逆性抑制,与外源施加高浓度腺苷反应类同。腺苷Ａ１受体拮抗剂ＣＰＴ以及Ｋ＋通道阻断剂４－ＡＰ可阻断这种抑制作用;而ＴＥＡ以及ＡＴＰ敏感Ｋ＋通道阻断剂ｇｌｉｐｉｚｉｄｅ均未见显著效应。结果提示：缺氧早期突触功能可逆性抑制与内源性腺苷大量释放有关,腺苷通过作用其Ａ１受体,激活４－ＡＰ敏感Ｋ＋通道,从而抑制突触传递,显示其抗缺氧作用。ＡＴＰ敏感性Ｋ＋通道可能不参于这个过程。     

Dramatic increase of nitrite levels in hearts of anoxia-exposed crucian carp supporting a role in cardioprotection

Nitrite (NO(2)(-)) functions as an important nitric oxide (NO) donor under hypoxic conditions. Both nitrite and NO have been found to protect the mammalian heart and other tissues against ischemia (anoxia)-reoxygenation injury by interacting with mitochondrial electron transport complexes and limiting the generation of reactive oxygen species upon reoxygenation. The crucian carp naturally survives extended periods without oxygen in an active state, which has made it a model for studying how evolution has solved the problems of anoxic survival. We investigated the role of nitrite and NO in the anoxia tolerance of this fish by measuring NO metabolites in normoxic, anoxic, and reoxygenated crucian carp. We also cloned and sequenced crucian carp NO synthase variants and quantified their mRNA levels in several tissues in normoxia and anoxia. Despite falling levels of blood plasma nitrite, the crucian carp showed massive increases in nitrite, S-nitrosothiols (SNO), and iron-nitrosyl (FeNO) compounds in anoxic heart tissue. NO(2)(-) levels were maintained in anoxic brain, liver, and gill tissues, whereas SNO and FeNO increased in a tissue-specific manner. Reoxygenation reestablished normoxic values. We conclude that NO(2)(-) is shifted into the tissues where it acts as NO donor during anoxia, inducing cytoprotection under anoxia/reoxygenation. This can be especially important in the crucian carp heart, which maintains output in anoxia. NO(2)(-) is currently tested as a therapeutic drug against reperfusion damage of ischemic hearts, and the present study provides evolutionary precedent for such an approach.     

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.     

The effect of temperature and anoxia of kidney on the subsequent oxidative phosphorylation of mitochondria

1. Kidneys were kept anoxic at 4 degrees , 20 degrees and 38 degrees . Mitochondria were then isolated and their oxidative phosphorylation and respiration were determined. 2. Under all conditions the rate of phosphate esterification was affected to a greater extent, or earlier, than oxygen consumption. 3. Glutamate and succinate were used as substrates. The depression of P/O ratio was greater for glutamate at 4 degrees , and for succinate at 20 degrees . 4. Anoxia abolished the inhibiting effect of fluoride on respiration. 5. Phosphate esterification, after anoxia, was higher in the presence of fluoride than its absence, whereas in control preparations they were the same. 6. The decrease in P/O ratio did not appear to be due to activation of adenosine triphosphatase, as activities of both Mg(2+)-and dinitrophenol-activated adenosine triphosphatases were decreased after anoxia.     

Adenosine A2 receptors are involved in the activation of ATP-sensitive K+ currents during metabolic inhibition in guinea pig ventricular myocytes

It has been hypothesized that an interaction among adenosine A(1) receptors, protein kinase C (PKC) activation, and ATP-sensitive potassium channels (K(ATP)) mediates ischemic preconditioning in experiments on different animal species. The purpose of this study was to determine if activation of K(ATP) is functionally coupled to A(1) receptors and (or) PKC activation during metabolic inhibition (MI) in guinea pig ventricular myocytes. Perforated-patch using nystatin and conventional whole-cell recording methods were used to observe the effects of adenosine and adenosine-receptor antagonists on the activation of K(ATP) currents during MI induced by application of 2,4-dinitrophenol (DNP) and 2-deoxyglucose (2DG) without glucose, in the presence or absence of a PKC activator, phorbol 12-myristate 13-acetate (PMA). Adenosine accelerated the time course activation of K(ATP) currents during MI under the intact intracellular condition or dialyzed condition with l mmol/L ATP in the pipette solution. The accelerated effect of adenosine activation of K(ATP) under MI was not reversed by a nonselective Al adenosine receptor antagonist, 8-(p-sulfophenyl)theophylline (SPT), or a specific Al adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). However, the adenosine A(2) receptor antagonist alloxazine reversed the time course activation of the K(ATP) current under MI. An adenylate cyclase activator, forskolin, did not further abbreviate the time course activation of K(ATP) with or without adenosine. Application of a PKC blocker, chelerythrine, reversed the time course activation of K(ATP) by adenosine under MI. In addition, pretreatment with a PKC activator, PMA, had similar effects to adenosine, while adenosine did not further shorten the time required for activation of K(ATP) currents during MI with PMA pretreatment. There is no direct evidence of activation of K(ATP) currents by adenosine A(1) receptor during metabolic inhibition under our experimental condition. However, adenosine A(2) receptor activation is involved in the K(ATP) channel activation in the guinea pig ventricular myocytes, of which effect is not mediated through the increase in intracellular cAMP. Adenosine seems to interact with PKC activation to open K(ATP) during MI, but a possible link between the adenosine A(2) receptor and PKC activation in this process needs further elucidation.     

Primary cortical neuronal cultures reduce cellular energy utilization during anoxic energy deprivation

It has been widely hypothesized that neurons reduce cellular energy use in response to periods of energy deprivation. To test this hypothesis, we measured rates of energy use under normoxia and anoxia in immature (6 days in vitro) and mature (13 days in vitro) neuronal cultures. During anoxic incubation immature and mature cultures reduced cellular energy use by 80% and 45%, respectively. Reduced cellular energy use dramatically affected ATP depletion in neuronal cultures under anoxia. Intracellular ATP stores were expected to deplete within 3 min of anoxia. However, ATP was maintained at decreased but stabilized concentrations for at least 3 h. The capacity of neuronal cultures to reduce cellular energy use during anoxia correlated with their sensitivity towards simulated ischemia. Immature cultures, with the largest capacity to reduce cellular energy use, survived simulated ischemia 2.5 times longer than mature cultures. The addition of glutamate receptor antagonists to mature cultures further decreased cellular energy use during anoxia and significantly extended their survival time under simulated ischemia. This study verifies that primary cortical neuronal cultures reduce cellular energy use during energy deprivation. Additionally, we show that maturation of glutamate receptor activity increases non-depressible energy demand in neuronal cultures.     

Effects of hypoxia, anoxia, and endogenous ethanol on thermoregulation in goldfish, Carassius auratus

Effects of hypoxia, anoxia, and endogenous ethanol (EtOH) on selected temperature (T(sel)) and activity in goldfish were evaluated. Blood and brain EtOH concentrations ([EtOH]) and brain oxygen partial pressure (PO(2)) were quantified at crucial ambient oxygen pressures. Below a threshold value near 31 Torr, T(sel) decreased as a function of environmental PO(2). T(sel) of 15 degrees C-acclimated fish was approximately 10 degrees C at the onset of anoxia and changed little over 2 h. Activity showed a similar response pattern. Brain [EtOH] was significantly elevated above control levels after 1 h anoxia. In normoxic water, T(sel) remained different in previously anoxic and normoxic control fish for approximately 20 min. Blood [EtOH] of previously anoxic fish remained significantly elevated ([EtOH] >4.0 micromol/g blood), and activity was significantly depressed at 20 min. Brain PO(2) reached normal levels in <3 min. We conclude that [EtOH] (brain or blood) and brain PO(2) are not proximal causes of either behavioral anapyrexia (hypothermia) or inactivity in goldfish exposed to oxygen-depleted environments.