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.     

Metabolic suppression in anoxic frog muscle

Microcalorimetry is the only direct method for measuring moment-to-moment changes in whole-cell metabolism (as heat output) during anoxia. We have adapted this methodology, in conjunction with standard muscle isolation techniques, to monitor metabolic transitions in isolated frog (Rana temporaria) sartorius muscle during anoxia and recovery (reoxygenation). Anoxia (sustained 1 h, following 2 h progressive hypoxia) suppressed muscle heat output to 20% of the stable normoxic level. This effect was fully reversible upon reoxygenation. Metabolite profiles were consistent with other anoxia-tolerant vertebrates – most notably, adenosine triphosphate (ATP) content during anoxia and reoxygenation remained unchanged from normoxia (pre-anoxic control). In addition, the concentration of K⁺ ions ([K⁺]) in interstitial dialysates remained stable (2–3 mM) throughout anoxia and recovery. Interstitial [lactate⁻] increased slightly, in accord with anaerobiosis supporting suppressed metabolic rates during anoxia. The degree of anoxic suppression of metabolism observed is similar to other vertebrate models of anoxia tolerance. Furthermore, stable ATP concentrations and interstitial [K⁺] in the isolated tissue suggests that intrinsic mechanisms suppress metabolism in a manner that coordinates ATP supply and demand and avoids the severe ion imbalances that are characteristic of hypoxia-sensitive systems. Accepted: 15 January 1998     

Anaerobic energy metabolism of goldfish,Carassius auratus (L.)

Summary The concentrations of pyruvate, lactate, oxalo-acetate, aceto-acetate -hydroxybutyrate, -ketoglutarate, glutamate, NH ₄ ⁺ , NAD⁺ and NADH were measured in goldfish tissues after previous conditioning to normal and anoxic (12h) conditions. For 11 different metabolites efficiency of different extraction methods was tested by means of internal standards. The recoveries were generally over 80%. The substrate/product couples of the reactions catalysed by lactate dehydrogenase, malate dehydrogenase, -hydroxybutyrate dehydrogenase and glutamate dehydrogenase were used as redox parameters. In the lateral red muscle the redox state did not change during 12 h of anoxia. In the dorsal white muscle only the cytoplasmic redox state underwent a change, as indicated by the increase of the lactate/pyruvate ratio from 20 to 110. In liver both cytoplasm and mitochondria were reduced during anoxia. From the measured values the NAD⁺/NADH ratio was found to change only in white muscle, while the calculated free NAD⁺/NADH ratios were reduced in anoxic white muscle cytoplasm, anoxic liver mitochondria, and anoxic liver cytoplasm. Oxalo-acetate concentrations calculated from the equilibrium constants of lactate dehydrogenase and malate dehydrogenase were at least one order of magnitude smaller than the measured values. The data obtained from anoxic goldfish are in contrast to available data on other animals and support earlier reports which indicate that this animal has a special anaerobic metabolism. The results are discussed especially with respect to the role of ethanol as a sink for reducing equivalents.Abbreviations LDH lactate dehydrogenase - MDH malate dehydrogenase - HBDH -hydroxybutyrate dehydrogenase - GIDH glutamate dehydrogenase     

Role of covalent modification in the control of glycolytic enzymes in response to environmental anoxia in goldfish

Summary The effects of environmental anoxia (24 h at 7°C in N₂/CO bubbled water) on the maximal activities, selected kinetic properties, and isoelectric points of phosphofructokinase and pyruvate kinase were measured in eight tissues of the goldfish,Carassius auratus, in order to evaluate the role of possible covalent modification of enzymes in glycolytic rate control and metabolic depression during facultative anaerobiosis. Both enzymes showed modified kinetic properties as a result of anoxia in liver, kidney, brain, spleen, gill, and heart. Effects of anoxia on properties of pyruvate kinase included reducedV _max, increased S_0.5 for phosphoenolpyruvate, increasedK _a for fructose-1,6-bisphosphate, and strongly reduced I₅₀ for alanine; all these effects are consistent with an anoxia-induced phosphorylation of pyruvate kinase to produce a less active enzyme form. Anoxia-induced alterations in phosphofructokinase kinetics included tissue-specific changes in S_0.5 for fructose-6-phosphate, Hill coefficient,K _a values for fructose-2,6-bisphosphate, AMP, and NH ₄ ⁺ , and I₅₀ values for ATP and citrate, the direction of changes being generally consistent with the production of a less active enzyme form in the anoxic tissue. Enzymes from aerobic versus anoxic skeletal muscle (both red and white) did not differ in kinetic properties but anoxic enzyme forms had significantly different pI values than the corresponding aerobic forms. Enzyme phosphorylation-dephosphorylation as the basis of the anoxia-induced changes in the kinetic properties of PFK and PK was further tested in liver: treatment of the aerobic forms of both enzymes with cAMP dependent protein kinase altered enzyme kinetic properties to those typical of the anoxic enzymes while alkaline phosphatase treatment of the anoxic enzyme forms had the opposite effect. The data provide strong evidence that coordinated glycolytic rate control, as part of an overall metabolic rate depression during anoxia, is mediated via anoxia-induced covalent modification of regulatory enzymes.Abbreviations cAMP cyclic 35 adenosine monophosphate - F16P ₂ fructose-1,6-bisphosphate - F26P ₂ fructose-2,6-bisphosphate - F6P fructose-6-phosphate - PEP phosphoenolpyruvate - PFK phosphofructokinase (E.C. 2.7.1.11) - PK pyruvate kinase (E.C. 2.7.1.40) - PMSF phenylmethylsulfonyl fluoride     

Intracellular pH in rice and wheat root tips under hypoxic and anoxic conditions

The influence of anoxia and hypoxia on dynamic of intracellurar pH and ATP content in rice and wheat root tips was investigated with ³¹P-NMR spectroscopy. Both cereals responded to hypoxia similarly, by rapid cytoplasmic acidification (from pH 7.6–7.7 to 7.1), which was followed by slow partial recovery (0.3 units). Anoxia led to a dramatic pH_cyt drop in tissues of both species (from pH 7.6–7.7 to less than 7.0) and partial recovery took place in rice only. In wheat, the acidification continued to pH 6.8 after 6 h of exposure. Anoxic wheat root tips were deficient in ADH induction, whereas increased activity of alcoholic fermentation enzymes took place in anoxic rice root tips, as well as in both species after hypoxic treatment. In both plants, NTP content followed the dynamics of pH_cyt. There was a strong correlation between NTP content and cytoplasmic H⁺ activity ([H⁺]_cyt = 10^−pHcyt) for both hypoxic and anoxic conditions. In this addendum we want to focus the reader''s attention on the importance of adequate experimental design when hypoxia is under investigation and on some further perspectives of intracellular pH regulation in plants under anaerobic conditions.Key words: anoxia, hypoxia, rice, wheat, cytoplasmic pH regulation     

The influence of poly(A)-binding proteins on translation of poly(A)+ RNA in a cell-free system from embryo axes of dry pea seeds

Ribonucleoproteins of the ribosomal fraction of germinated pea embryo axes, containing translationally active mRNA, differ from analogous ribonucleoproteins of dry pea seeds, which contain stored mRNA, by the presence of a 60 kDa protein fraction showing affinity to poly(A). The above protein fraction largely affects the activity of poly(A)⁺ RNA translation in cell-free system. An activating effect is clearly seen at a weight ratio of poly(A)-binding proteins:poly(A)⁺ RNA of 3:1, whereas with an increase in the concentration of these proteins the translational activity drops. The effect of poly(A)-binding proteins containing the 60 kDa fraction on poly(A)⁺ RNA dependent cell-free translation can be efficiently reduced by simultaneous addition of synthetic poly(adenylic acid). It was also proved that activation of translation does not influence its products. It is concluded that poly(A)-binding proteins from the ribosomal fraction of embryo axes of pea seeds, especially the 60 kDa fraction, are involved in regulation of the translational activity of poly(A)⁺ RNA.     

Effects of hibernation on mitochondrial regulation and metabolic capacities in myocardium of painted turtle (Chrysemys picta)

Painted turtles hibernating during winter may endure long-term exposure to low temperature and anoxia. These two conditions may affect the aerobic capacity of a tissue and might be of particular importance to the cardiac muscle normally highly reliant on aerobic energy production. The present study addressed how hibernation affects respiratory characteristics of mitochondria in situ and the metabolic pattern of turtle myocardium. Painted turtles were acclimated to control (25 degrees C), cold (5 degrees C) normoxic and cold anoxic conditions. In saponin-skinned myocardial fibres, cold acclimation increased mitochondrial respiratory capacity and decreased apparent ADP-affinity. Concomitant anoxia did not affect this. Creatine increased the apparent ADP-affinity to similar values in the three acclimation groups, suggesting a functional coupling of creatine kinase to mitochondrial respiration. As to the metabolic pattern, cold acclimation decreased glycolytic capacity in terms of pyruvate kinase activity and increased lactate dehydrogenase (LHD) activity. Concomitant anoxia counteracted the cold-induced decrease in pyruvate kinase activity and increased creatine kinase activity. In conclusion, cold acclimation seems to increase aerobic and decrease anaerobic energy production capacity in painted turtle myocardium. Importantly, anoxia does not affect the mitochondrial functional integrity but seems to increase the capacity for anaerobic energy production and energy buffering.     

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.     

Effects of Anoxia on the Stimulated Release of Amino Acid Neurotransmitters in the Cerebellum In Vitro

Abstract: The effect of anoxia and ischemia on the release of amino acid transmitters from cerebellar slices induced by veratridine or high [K⁺] was studied. Synaptic specificity was tested by examining the tetradotoxin (TTX)-sensitive and the Ca²⁺-dependent components of stimulated release. Evoked release of endogenous amino acids was investigated in addition to more detailed studies on the stimulated efflux of preloaded [¹⁴C]GABA and d -[³H]aspartate (a metabolically more stable anologue of acidic amino acids).[¹⁴C]GABA release evoked by either method of stimulation was unaffected by periods of up to 35 min of anoxia and declined moderately by 45 min. In contrast, induced release of d -[³H]Asp increased markedly during anoxia to a peak at about 25 min, followed by a decline when anoxia was prolonged to 45 min. Evidence was obtained that the increased evoked efflux of d -[³H]Asp from anoxic slices was not due to impaired reuptake of the released amino acid and that it was completely reversible by reoxygenation of the slices. Results of experiments examining the evoked release of endogenous amino acids in anoxia were consistent with those obtained with the exogenous amino acids. Only 4 of the 10 endogenous amino acids studied exhibited TTX-sensitive veratridine-induced release under aerobic conditions (glutamate, aspartate, GABA, and glycine). Anoxia for 25 min did not affect the stimulated efflux of these amino acids with the exception of glutamate, which showed a significant increase. Compared with anoxia, effects of ischemia on synaptic function appeared to be more severe. Veratridine-evoked release of [¹⁴C]GABA was already depressed by 10 min and that of d -[³H]Asp showed a modest elevation only at 5 min. Stimulated release of d -Asp and labelled GABA declined progressively after 5 min. These findings were compared with changes in tissue ATP concentrations and histology. The latter studies indicated that in anoxia the earliest alterations are detectable in glia and that nerve terminals were the structures by far the most resistant to anoxic damage. The results thus indicated that evoked release of amino acid transmitters in the cerebellum is compromised only by prolonged anoxia in vitro. In addition, it would appear that the stimulated release of glutamate is selectively accentuated during anoxia. This effect may have a bearing on some hypoxic behavioral changes and, perhaps, also on the well-known selective vulnerability of certain neurons during hypoxia.     

Anoxia-Reoxygenation Regulates Mitochondrial Dynamics through the Hypoxia Response Pathway,SKN-1/Nrf,and Stomatin-Like Protein STL-1/SLP-2

Many aerobic organisms encounter oxygen-deprived environments and thus must have adaptive mechanisms to survive such stress. It is important to understand how mitochondria respond to oxygen deprivation given the critical role they play in using oxygen to generate cellular energy. Here we examine mitochondrial stress response in C. elegans, which adapt to extreme oxygen deprivation (anoxia, less than 0.1% oxygen) by entering into a reversible suspended animation state of locomotory arrest. We show that neuronal mitochondria undergo DRP-1-dependent fission in response to anoxia and undergo refusion upon reoxygenation. The hypoxia response pathway, including EGL-9 and HIF-1, is not required for anoxia-induced fission, but does regulate mitochondrial reconstitution during reoxygenation. Mutants for egl-9 exhibit a rapid refusion of mitochondria and a rapid behavioral recovery from suspended animation during reoxygenation; both phenotypes require HIF-1. Mitochondria are significantly larger in egl-9 mutants after reoxygenation, a phenotype similar to stress-induced mitochondria hyperfusion (SIMH). Anoxia results in mitochondrial oxidative stress, and the oxidative response factor SKN-1/Nrf is required for both rapid mitochondrial refusion and rapid behavioral recovery during reoxygenation. In response to anoxia, SKN-1 promotes the expression of the mitochondrial resident protein Stomatin-like 1 (STL-1), which helps facilitate mitochondrial dynamics following anoxia. Our results suggest the existence of a conserved anoxic stress response involving changes in mitochondrial fission and fusion.