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.     

The effect of prolonged anoxia at 3°C on tissue high energy phosphates and phosphodiesters in turtles: A 31P-NMR study

Selected tissues (skeletal muscle, heart ventrical, and liver), sampled from turtles (Chrysemys picta bellii) at 3°C either under normoxic conditions or after 12 weeks of anoxic submergence were quantiaatively analysed for intracellular pH and phosphorus metabolites using ³¹P-NMR. Plasma was tested for osmolality and for the concentrations of lactate, calcium, and magnesium to confirm anoxic stress. We hypothesized that, in the anoxic animals, tissue ATP levels would be maintained and that the increased osmolality of the body fluids of anoxic turtles would be accounted for by a corresponding increase in the concentrations of phosphodiesters. The responses observed differed among the three tissues. In muscle, ATP was unchanged by anoxia but phosphocreatine was reduced by 80%; in heart, both ATP and phosphocreatine fell by 35–40%. The reduction in phosphocreatine in heart tissue at 3°C was similar to that observed in isolated, perfused working hearts from turtles maintained at 20°C but no decrease in ATP occurred in the latter tissues. In liver, although analyses of several specimens were confounded by line-broadening, neither ATP nor phosphocreatine was detectable in anoxic samples. Phosphosdiesters were detected in amounts sufficient to account for 30% of normoxic cell osmotic concentration in heart and 11% and 12% in liver and muscle, respectively. The phosphodiester levels did not change in anoxia. Heart ventricular phosphodiester levels in turtles at 3°C were significantly higher than those determined for whole hearts from turtles at 20°C. ¹H, ¹³C and ³¹P NMR analyses of perchloric acid extracts of heart and skeletal muscle from 20°C turtles con firmed that the major phosphodiester observed by NMR in these tissues is serine ethanolamine phosphate. We conclude that the three types of tissues studied differ substantially in their ability to maintain levels of ATP during anoxia, and that liver may continue to function despite NMR-undetectable levels of this metabolite. In addition, we conclude that phosphodiesters do not serve as regulated osmolytes during anoxia, and that the functional significance of their high concentrations in turtle tissues remains uncertain.     

Effects of L-carnitine and its acetyl and propionyl esters on ATP and PCr levels of isolated rat hearts perfused without fatty acids and investigated by means of 31P-NMR spectroscopy

³¹P-NMR in vivo spectroscopy is a non-invasive and non-hazardous technique which investigates chemical composition and metabolism of living objects, for example by determining phosphocreatine (PCr) and ATP concentrations. In the present study we investigated the influence of L-carnitine, acetyl-L-carnitine and propionyl-L-carnitine on the energetic state of the Langendorff rat heart subjected to an ischemic period of 20 min followed by a reperfusion period of 60 min. To avoid an overlapping of the effects of fatty acids and glucose, the hearts were perfused with a Tyrode solution containing no fatty acids. Ischemia causes a rapid decrease in the PCr signal, followed by a decrease in the ATP signal after a prolonged period of ischemia. At the same time, a drastic increase in the P_i signal was observed. A partial recovery of the ATP and PCr signals was observed in the reperfusion period. With L-carnitine a markedly improved recovery of the high energy phosphates (e.g. increased PCr/P_i ratios) was found. With acetyl-L-carnitine this effect was enhanced in the first postischemic phase. It was followed, however, by a more rapid decrease in the PCr/P_i ratio in the late reperfusion period. The effect of propionyl-L-carnitine was not significantly improved in the first minutes of the reperfusion period, but during the whole reperfusion phase a stabilization of the PCr/P_i ratio was observed. Intracellular pH can be calculated from determination of the P_i-chemical shift. This shows that L-carnitine and its derivatives have a protective effect against intracellular pH decrease during ischemia. L-carnitine improves the energetic state of the heart, which leads to increased ischemia tolerance. Hearts under L-carnitine were able to tolerate up to four ischemia-reperfusion periods in succession, whereas the controls were not able to do so. These NMR results confirm the hypothesis that L-carnitine and its esters have a protective effect in the reperfusion period of the ischemic rat heart. This could be of importance for the treatment of ischemic cardiac diseases.     

Assessment of energy metabolism in the developing brain following aglycemic hypoxia by1H and31P NMR

The role played by external calcium and calcium channels in the recovery from aglycaemic hypoxia in cortical brain slices from 10-day old rats was investigated by¹H and³¹P NMR. 30 minutes of aglycaemic hypoxia significantly decreased the levels of phosphocreatine (PCr), ATP, lactate and intracellular pH (pH_i). After a 30 minute recovery period there was incomplete recovery of PCr and ATP with lactate increasing by 50% with pH_i normal. When the aglycemic hypoxia was carried out in media which had no added calcium (≈10 μM) the PCr and ATP recovery was significantly greater. Application of diltiazem or verapamil but not nifedipine significantly improved the recovery from the aglycemic hypoxia. These data suggest that calcium influx through L-type voltagegated calcium channels is involved in the ischemic damage in neonatal brain which manifests itself as a decrease in the energy state and an increase in lactate. Dedication This article is dedicated to our friend and colleague Herman Bachelard. We wish to thank him for his comradeship, advice and support over many years. Our hope for him is a long and fruiful retirement and that he will remain active in the neurosciences for many years, even though the establishment has blown for “full time”.     

Effect of anoxia and pharmacological anoxia on whole-cell NMDA receptor currents in cortical neurons from the western painted turtle

The mammalian brain undergoes rapid cell death during anoxia that is characterized by uncontrolled Ca(2+) entry via N-methyl-D-aspartate receptors (NMDARs). In contrast, the western painted turtle is extremely anoxia tolerant and maintains close-to-normal [Ca(2+)](i) during periods of anoxia lasting from days to months. A plausible mechanism of anoxic survival in turtle neurons is the regulation of NMDARs to prevent excitotoxic Ca(2+) injury. However, studies using metabolic inhibitors such as cyanide (NaCN) as a convenient method to induce anoxia may not represent a true anoxic stress. This study was undertaken to determine whether turtle cortical neuron whole-cell NMDAR currents respond similarly to true anoxia with N(2) and to NaCN-induced anoxia. Whole-cell NMDAR currents were measured during a control N(2)-induced anoxic transition and a control NaCN-induced transition. During anoxia with N(2) normalized, NMDAR currents decreased to 35.3%+/-10.8% of control values. Two different NMDAR current responses were observed during NaCN-induced anoxia: one resulted in a 172%+/-51% increase in NMDAR currents, and the other was a decrease to 48%+/-14% of control. When responses were correlated to the two major neuronal subtypes under study, we found that stellate neurons responded to NaCN treatment with a decrease in NMDAR current, while pyramidal neurons exhibited both increases and decreases. Our results show that whole-cell NMDAR currents respond differently to NaCN-induced anoxia than to the more physiologically relevant anoxia with N(2).     

31P-Nuclear magnetic resonance spectroscopy study of the time course of energy metabolism during exercise and recovery

This study evaluated the time courses of intracellular pH and the metabolism of phosphocreatine (PCr) and inorganic phosphate (P) at the onset of four exercise intensities and recoveries. Non-invasive evaluation of continuous changes in phosphorus metabolites has become possible using³¹P-nuclear magnetic resonance spectroscopy (³¹P-MRS). After measurements at rest, six healthy male subjects performed 4 min of femoral flexion exercise at intensities of 0 (loadless), 10, 20 and 30 kg · m · min^–1 in a 2.1 T superconducting magnet with a 67-cm bore. Measurements were continuously made during 5 min of recovery. During a series of rest-exercise-recovery procedures,³¹P-MRS were accumulated using 32 scans · spectrum^–1 requiring 12.8 s each. At the onset of exercise, PCr decreased exponentially with a time constant of 27–32 s regardless of the exercise intensity. The time constant PCr resynthesis during recovery was about 27–40 s. The PCr kinetics were independent of exercise intensity. There were similar Pi kinetics at the onset of all types of exercise, while those of Pi recovery became significantly longer at the higher exercise intensities (P < 0.05). Furthermore, the intracellular pH indicated temporary alkalosis just at the onset of exercise, probably due to absorption of hydrogen ions by PCr hydrolysis, and then decrease at a point about 40%–50% of the preexercise PCr. The pH recovery time was longer than that for the P_i or PCr kinetics. By using a more efficient resolution system it was possible to obtain the phosphorus kinetics during exercise and to follow PCr resynthesis within the first few minutes of recovery. From our results it was concluded that in general the time course of PCr and Pi metabolism were unaffected by the exercise intensity, both at the onset of exercise and during recovery, with the exception of P_i recovery.     

Effects of anoxia, aglycemia, and acidosis on cytosolic Mg2+, ATP, and pH in rat sensory neurons

Sensory neurons can detect ischemia and transmit pain from various organs. Whereas the primary stimulus in ischemia is assumed to be acidosis, little is known about how the inevitable metabolic challenge influences neuron function. In this study we have investigated the effects of anoxia, aglycemia, and acidosis upon intracellular Mg(2+) concentration [Mg(2+)](i) and intracellular pH (pH(i)) in isolated sensory neurons. Anoxia, anoxic aglycemia, and acidosis all caused a rise in [Mg(2+)](i) and a fall in pH(i). The rise in [Mg(2+)](i) in response to acidosis appears to be due to H(+) competing for intracellular Mg(2+) binding sites. The effects of anoxia and aglycemia were mimicked by metabolic inhibition and, in a dorsal root ganglia (DRG)-derived cell line, the rise in [Mg(2+)](i) during metabolic blockade was closely correlated with fall in intracellular ATP concentration ([ATP](i)). Increase in [Mg(2+)](i) during anoxia and aglycemia were therefore assumed to be due to MgATP hydrolysis. Even brief periods of anoxia (<3 min) resulted in rapid internal acidosis and a rise in [Mg(2+)](i) equivalent to a decline in MgATP levels of 15-20%. With more prolonged anoxia (20 min) MgATP depletion is estimated to be around 40%. With anoxic aglycemia, the [Mg(2+)](i) rise occurs in two phases: the first beginning almost immediately and the second after an 8- to 10-min delay. Within 20 min of anoxic aglycemia [Mg(2+)](i) was comparable to that observed following complete metabolic inhibition (dinitrophenol + 2-deoxyglucose, DNP + 2-DOG) indicating a near total loss of MgATP. The consequences of these events therefore need to be considered in the context of sensory neuron function in ischemia.     

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.     

Smooth muscle and NMR review: An overview of smooth muscle metabolism

Nuclear magnetic resonance (NMR) is a non-invasive technique which allows us to examine the biochemical, physiological and metabolic events occurring inside living tissue; such as vascular and other smooth muscles.It has been found that the smooth muscle metabolism is compartmented such that mitochondrial function fuels contraction and that much glycolytic ATP production is used for membrane pumps. Using NMR we have been able to observe the ATP and phosphocreatine (PCr) concentrations and estimate the ADP concentration, as well as flux through the creatine kinase (CK) system. It has also been found that the smooth muscle metabolism is able to maintain ATP concentration in the absence of mitochondrial function (cyanide inhibition). Therefore, the vessels are able to adapt to metabolic demands as necessary.NMR is versatile in the information it can provide because it has also yielded important contributions with regard to the intracellular pH and ionic status. For example, the intracellular free Mg²⁺ ([Mg²⁺]_i) can be measured with NMR simultaneously with ATP concentrations and NMR has shown us that the [Mg²⁺]_i is highly protected in the muscle (within confined range), but also responds to the environment around it.In this review we conclude that NMR measurements of smooth muscle research is a useful technique for assessing chronic and acute changes that occur in the tissue and during diseases.     

Ischemic changes in myocardial ionic contents of the isolated perfused rat hearts as studied by NMR

Using³¹P-,²³Na- and³⁹K-NMR, we assessed ischemic changes in high energy phosphates and ion contents of isolated perfused rat hearts continuously and systematically. To discriminate intra- and extracellular Na⁺, a shift reagent (Dy(TTHA)^3–) was used in²³Na-NMR study. In³⁹K-NMR study, the extracellular K⁺ signal was suppressed by inversion recovery pulse sequence in order to obtain intracellular K⁺ signal without using shift reagnets. During the early period of ischemia, increases in intracellular Na⁺ and inorganic phosphate (Pi) were observed in addition to the well-documented decreases in creatine phosphate and ATP and a fall of intracellular pH, suggesting an augmented operation of Na⁺–H⁺ exchange triggered by a fall of the intracellular pH resulted from breakdown of ATP. At around 15 min of ischemia, a second larger increase in intracellular Na⁺ and a decrease in intracellular K⁺ were observed in association with a second increase in Pi. This was accompnanied by an abrupt rise of the ventricular end-diastolic pressure. As there was a depletion of ATP at this time, the increase in intracellular Na⁺ and associated decrease in intracellular K⁺ may be explained by inhibition of the Na⁺–K⁺ ATPase due to the depletion of ATP. A longer observation with³¹P-NMR revealed a second phosphate peak (at lower magnetic field to ordinary Pi peak) which increased its intensity as ischemic time lengthened. The pH of this 2nd peak changed in parallel with the changes in pH of the bathing solution, indicating the appearance of a compartment whose hydrogen concentration is in equilibrium with that of the external compartment. Thus, the peak could be used as an index of irreversible membrane damage of the myocardium.     

31P nuclear magnetic resonance study on changes in phosphocreatine and the intracellular pH in rat skeletal muscle during exercise at various inspired oxygen contents

We measured ATP, phosphocreatine (PCr), inorganic phosphate (P_i), and the intracellular pH in rat hindlimb muscles during submaximal isometric exercise with various O₂ deliveries using³¹P nuclear magnetic resonance spectroscopy (³¹P NMR) to evaluate changes in energy metabolism in relation to O₂ availability. Delivery of O₂ to muscles was altered by controlling the fractional concentration of inspired oxygen (F _IO₂) at 0.50, 0.28, 0.21, 0.11 and 0.08 with monitoring partial pressure of oxygen and carbon dioxide, and bicarbonate at the femoral artery. The steady-state ratio of PCr : (PCr + P_i) during exercise decreased as a function ofF _IO₂ even at 0.21. Significant acidification of the intracellular pH during exercise occurred at 0.08F _IO₂. Change in the PCr : (PCr + P_i) ratio demonstrated that the oxidative capacity, i.e. the maximal rate of the oxidative phosphorylation reaction, in muscle was not limited by O₂ delivery at 0.50F _IO₂, but was significantly limited at 0.21F _IO₂ or below. Change in the intracellular pH at 0.08F _IO₂ could be interpreted as an increase in lactate, suggesting activation of glycolysis. Correlation between the PCr : (PCr + P_i) ratio and the intracellular pH revealed the existence of a critical PCr : (PCr + P_i) ratio and pH for glycolysis activation at around 0.4 and 6.7, respectively.     

Comparison of in vivo energy metabolism in the brain of rainbow trout, Salmo gairdneri and bullhead catfish, Ictalurus nebulosus during anoxia

1. Rainbow trout and bullhead catfish (Ictalurus nebulosus) were exposed to anoxic water inside a plastic tube until death (12 min for trout; 62 min for catfish). Immediately upon death, the brain was removed and analyzed for metabolites, high energy phosphate compounds, and metabolic fuel while the blood was analyzed for metabolites. 2. Control bullhead brains had higher concentrations of glycogen, ATP, creatine phosphate (CrP), and glucose than control trout. 3. After anoxia bullheads showed a significant decrease in ATP, CrP, and glycogen while lactate more than doubled in concentration. 4. After anoxia trout showed a doubling in brain lactate and a decline in glycogen, but no decline in ATP or CrP. There were no changes in brain glucose, ketone bodies, or alternative anaerobic end-products in either species although an elevation in blood isobutyrate was noted. 5. Brain death in the catfish may be due to depletion of fuel for anaerobiosis and a subsequent loss of ATP. In the trout there may be other causes such as a greater permeability of its neuronal membranes and alterations in intracellular free calcium stores.     

Manipulating cytoplasmic pH under anoxia: A critical test of the role of pH in the switch from aerobic to anaerobic metabolism

Ethanol production by maize (Zea mays L.) root tips, measured by an enzymic assay of the suspending medium, was correlated with changes in the cytoplasmic pH, determined by in-vivo ³¹P nuclear magnetic resonance (NMR) spectroscopy, following the onset of anoxia. Strong evidence for the role of the cytoplasmic pH in triggering the switch to ethanol production under anoxia was obtained by: (i) varying the pH of the suspending medium between pH 4 and pH 10; and (ii) using the permeant weak base methylamine to combat the acidification of the cytoplasm induced by the anoxic conditions. Experimentally, it proved to be much easier to manipulate the cytoplasmic pH under anoxia after the pH had stabilised, rather than during the initial rapid acidification that occurred following the onset of anoxia, and in the presence of methylamine, it was possible to impose a normal aerobic cytoplasmic pH value on tissue that was metabolising anaerobically. By this means it was possible to demonstrate the reversibility of the pH effect on ethanol production under anoxia and thus to provide good evidence in support of the biochemical pH-stat model of the anoxic response. The NMR measurement of the cytoplasmic pH in the presence of methylamine was achieved by using a manganese pretreatment technique to eliminate interference between the cytoplasmic and vacuolar P_i signals, and it seems likely that the experimental approach used here will have further applications in studies of the metabolic response to anoxia.Abbreviations Caps 3-(cyclohexylamino)-1-propane sulphonic acid - Mes 2-(N-morpholino)-ethane sulphonic acid - NMR nuclear magnetic resonance - Pi inorganic phosphate We acknowledge the financial support of the Agricultural and Food Research Council and G.G.F. acknowledges the receipt of a Research Fellowship from the Royal Commission for the Exhibition of 1851.     

Calcium and protein phosphatase 1/2A attenuate N-methyl-D-aspartate receptor activity in the anoxic turtle cortex

Excitotoxic cell death (ECD) is characteristic of mammalian brain following min of anoxia, but is not observed in the western painted turtle following days to months without oxygen. A key event in ECD is a massive increase in intracellular Ca(2+) by over-stimulation of N-methyl-d-aspartate receptors (NMDARs). The turtle's anoxia tolerance may involve the prevention of ECD by attenuating NMDAR-induced Ca(2+) influx. The goal of this study was to determine if protein phosphatases (PPs) and intracellular calcium mediate reductions in turtle cortical neuron whole-cell NMDAR currents during anoxia, thereby preventing ECD. Whole-cell NMDAR currents did not change during 80 min of normoxia, but decreased 56% during 40 min of anoxia. Okadaic acid and calyculin A, inhibitors of serine/threonine PP1 and PP2A, potentiated NMDAR currents during normoxia and prevented anoxia-mediated attenuation of NMDAR currents. Decreases in NMDAR activity during anoxia were also abolished by inclusion of the Ca(2+) chelator -- BAPTA and the calmodulin inhibitor -- calmidazolium. However, cypermethrin, an inhibitor of the Ca(2+)/calmodulin-dependent PP2B (calcineurin), abolished the anoxic decrease in NMDAR activity at 20, but not 40 min suggesting that this phosphatase might play an early role in attenuating NMDAR activity during anoxia. Our results show that PPs, Ca(2+) and calmodulin play an important role in decreasing NMDAR activity during anoxia in the turtle cortex. We offer a novel mechanism describing this attenuation in which PP1 and 2A dephosphorylate the NMDAR (NR1 subunit) followed by calmodulin binding, a subsequent dissociation of alpha-actinin-2 from the NR1 subunit, and a decrease in NMDAR activity.     

The role of adenosine in the anoxic survival of the epaulette shark, Hemiscyllium ocellatum

The epaulette shark (Hemiscyllium ocellatum) is among the few vertebrates that can tolerate extreme hypoxia for prolonged periods and, as shown here, anoxia. We examined how anoxia affected this shark's level of responsiveness, concentration of brain ATP and adenosine -- an endogenous neuronal depressant. In addition, we investigated how these variables were affected by aminophylline, an adenosine receptor antagonist. Epaulette sharks placed in an anoxic environment (<0.02 mg O2 l(-1)) lost their righting reflex after 46.3 +/- 2.8 min, but immediately regained vigilance upon return to normoxia. Then 24 h later, the same sharks were injected with either saline or aminophylline (30 mg kg(-1)) in saline and re-exposed to anoxia. In this second anoxic episode, controls sharks showed a 56% decrease in the time taken to lose their righting reflex but maintained their brain ATP levels; conversely, aminophylline-treated epaulette sharks displayed a 46% increase in the time to loss of righting reflex and had significantly lower brain ATP levels. Since anoxia also caused a 3.5-fold increase in brain adenosine levels, these results suggest that adenosine receptor activation had a pre-emptive role in maintaining brain ATP levels during anoxia. Perhaps because adenosine receptor activation initiates metabolic depression, indicated by the early loss of responsiveness (righting reflex), such a mechanism would serve to reduce ATP consumption and maintain brain ATP levels.