Neuronal membrane potential is mildly depolarized in the anoxic turtle cortex

Neuronal membrane potential (E_m) regulates the activity of excitatory voltage-sensitive channels. Anoxic insults lead to a severe loss of E_m and excitotoxic cell death (ECD) in mammalian neurons. Conversely, anoxia-tolerant freshwater turtle neurons depress energy usage during anoxia by altering ionic conductance to reduce neuronal excitability and ECD is avoided. This wholesale alteration of ion channel and pump activity likely has a significant effect on E_m. Using the whole-cell patch clamp technique we recorded changes in E_m from turtle cortical neurons during a normoxic to anoxic transition in the presence of various ion channel/pump modulators. E_m did not change with normoxic perfusion but underwent a reversible, mild depolarization of 8.1 ± 0.2 mV following anoxic perfusion. This mild anoxic depolarization (MAD) was not prevented by the manipulation of any single ionic conductance, but was partially reduced by pre-treatment with antagonists of GABA_A receptors (5.7 ± 0.5 mV), cellular bicarbonate production (5.3 ± 0.2 mV) or K⁺ channels (6.0 ± 0.2 mV), or by perfusion of reactive oxygen species scavengers (5.2 ± 0.3 mV). Furthermore, all of these treatments induced depolarization in normoxic neurons. Together these data suggest that the MAD may be due to the summation of numerous altered ion conductance states during anoxia.     

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     

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.     

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.     

A comparative study on the responses of the gills of two mudskippers to hypoxia and anoxia

A comparison of branchial enzyme profiles indicates that the gills of Periophthalmodon schlosseri would have a greater capacity for energy metabolism through glycolysis than those of Boleophthalmus boddaerti. Indeed, after exposure to hypoxia, or anoxia, there were significant increases in the lactate content in the gills of P. schlosseri. In addition, exposure to hypoxia or anoxia significantly lowered the glycogen level in the gills of this mudskipper. It can be deduced from these results that the glycolytic flux was increased to compensate for the decrease in ATP production through anaerobic glycolysis. Different from P. schlosseri, although there was an increase in lactate production in the gills of B. boddaerti exposed to hypoxia, there was no significant change in the branchial glycogen content, indicating that a reversed Pasteur effect might have occurred under such conditions. In contrast, anoxia induced an accumulation of lactate and a decrease in glycogen in the gills of B. boddaerti. Although lactate production in the gills of these mudskippers during hypoxia was inhibited by iodoacetate, the decreases in branchial glycogen contents could not account for the amounts of lactate formed. The branchial fructose-2,6-bisphosphate contents of these mudskippers exposed to hypoxia or anoxia decreased significantly, leaving phosphofructokinase and glycolytic rate responsive to cellular energy requirements under such conditions. The differences in response in the gills of B. boddaerti and P. schlosseri to hypoxia were possibly related to the distribution of phosphofructokinase between the free and bound states.Abbreviations ADP adenosine diphosphate - ALD aldolase - ALT alanine transaminase - AST aspartate transaminase - ATP adenosine triphosphate - CS citrate synthase - EDTA ethylenediaminetetra-acetic acid - EGTA ethylene glycol tetra-acetic acid - F6P fructose-6-phosphate - F-1,6-P₂ fructose-1,6-bisphosphate - F-2,6-P₂ fructose-2,6-bisphosphate - FBPase fructose-1,6-bisphosphatese - GAPDH glyceraldehyde-3-phosphate dehydrogenase - GDH glutamate dehydrogenase - -GDH -glycerophosphate dehydrogenase - GPase glycogen phosphorylase - HK hexokinase - HOAD 3-hydroxyacyl-CoA dehydrogenase - IDH isocitrate dehydrogenase - IOA iodoacetic acid - LDH lactate dehydrogenase - LO lactate oxidizing activity - MDH malate dehydrogenase - 3-PG 3-phosphoglyceric acid - PEP phosphoenolpyruvate - PEPCK phosphoenolpyruvate carboxykinase - PGI phosphoglucose isomerase - PGK phosphoglycerate kinase - PFK 6-phosphofructo-1-kinase - PIPES piperazine-N, N-bis-(2-ethanesulphonic acid) - PK pyruvate kinase - PMSF phenylmethylsulphonyl fluoride - PR pyrurate reducing activity - SE standard error - SW seawater - TPI triosephosphate isomerase     

Pigment alterations in the brown mussel Perna perna

Potential sex and/or gametogenic stage differences in the metabolism of chlorophyll-a and carotenoids in the brown mussel Perna perna of southern Brazil were studied using high performance liquid chromatography (HPLC). Carotenoids derived directly from diet (phytoplankton) were fucoxanthin plus diatoxanthin (diatoms), alloxanthin (cryptophytes) and zeaxanthin (mainly cyanobacteria). Females accumulated carotenoid-diols and epoxides (~ 3–4 mg/g-dry wt.) while males had much lower concentrations (~ 0.7 mg/g-dry wt.). An antioxidant/free radical scavenging role is proposed for carotenoids in females. Mean ratios of chlorophyll plus derivatives (Chlns-a) to carotenoids for male and female P. perna were 50:1 and 4:1, respectively. The higher ratio in males relates to both higher carotenoid contents in females plus higher total Chlns-a in males (~ 22 mg/g-dry wt.), relative to the females (~ 4 mg/g-dry wt.). Chlorophyll-a metabolism in both sexes followed two distinct pathways. First, cyclization of pyropheophorbide-a gave 13², 17³-cyclopheophorbide-a-enol (CPPaE) which was further oxidized to hydroxy-chlorophyllone. Second, chlorophyll-a derivatives retaining the 13²-carbomethoxy moiety were oxidized to purpurin-18 which was hydrolyzed to chlorin-p₆. In both cases, metabolism of dietary chlorophyll-a was oxidative and derivatives could either serve as antioxidants or merely be the results of non-specific digestive processes.     

Second messenger and cAMP-dependent protein kinase responses to dehydration and anoxia stresses in frogs

The effects of whole body dehydration (up to 40% of total body water lost) or anoxia exposure (up to 2 days under N₂ gas) at 5 °C on tissue levels of adenosine 3′–5′ cyclic monophosphate (cAMP) and the percentage of cAMP-dependent protein kinase present as the free catalytic subunit (PKAc), as well as the levels of the protein kinase C (PKC) second messenger, inositol 1,4,5-trisphosphate (IP₃), were assessed in two anurans, the freeze-tolerant wood frog, Rana sylvatica, and the freeze-intolerant leopard frog, Rana pipiens. Dehydration of wood frogs resulted in a rapid elevation of liver cAMP and PKAc; cAMP was 3.4-fold greater than control values in animals that had lost 5% of total body water, whereas PKAc was elevated threefold in 20% dehydrated frogs. These results indicate protein kinase A mediation of the liver glycogenolysis and hyperglycemia that is induced by dehydration in this species. Skeletal muscle PKAc content also rose with dehydration but neither cAMP nor PKAc was affected by dehydration in leopard frog tissues. Anoxia exposure had different effects on signal transduction systems. PKAc was elevated after 1 h anoxia in R. sylvatica brain and was sustained over time but the enzyme was unaffected in other organs; by contrast, R. pipiens showed variable responses by PKAc to anoxia in three organs. Both species showed rapid (within 30 min) and large (3 to 7.8-fold) increases in IP₃ in liver of anoxic frogs that decreased slowly with continued anoxia. IP₃ also increased quickly in heart of anoxia-exposed wood frogs. This suggests that PKC may mediate various metabolic adjustments that promote hypoxia/anoxia resistance such as coordinating metabolic rate depression. A progressive rise in liver IP₃ during dehydration in wood frogs (reaching fourfold higher than controls in 40% dehydrated animals) may also mediate similar hypoxia resistance adaptations under this stress since anurans experience progressive hypoxia due to increased blood viscosity when water loss reaches high values. The patterns of second messenger and PKAc changes in wood frog liver during dehydration closely parallel the changes seen in these same parameters during natural freezing suggesting that the freeze tolerance of selected terrestrially hibernating anurans may have evolved out of various anuran mechanisms of dehydration resistance. Accepted: 2 January 1997     

Analysis of <Emphasis Type="Italic">EphA4</Emphasis> in the lesser spotted catshark identifies a primitive gnathostome expression pattern and reveals co-option during evolution of shark-specific morphology

The Eph family is the largest known group of structurally related receptor tyrosine kinases (RTKs). Each Eph receptor has a specific Ephrin ligand, and these function to define spatial boundaries during development. Analyses of EphA4 in mouse, chick, frog and zebrafish embryos have implicated this gene in a number of developmental processes, including maintenance of segmental boundaries, axon guidance, limb development, neural crest migration and patterning of the ear. In order to determine which components of EphA4 function may be primitive for gnathostomes, we cloned EphA4 from the lesser spotted catshark (Scyliorhinus canicula) and examined its expression pattern during shark embryonic development. Consistent with the patterns reported for bony fish and tetrapods, we observed segmental expression of EphA4 in the developing hindbrain and later in the pharyngeal arches of shark embryos. EphA4 was also detected during sensory organogenesis, in the developing ear, eye, nasal pits and lateral line. A dynamic pattern of EphA4 expression occurs during shark fin development, suggesting an early role in outgrowth and patterning of the fin buds and a later role in tissue differentiation. We also observed several novel domains of EphA4 expression that have not been reported in other vertebrates, including external gill buds, dermal denticles, median fins and claspers. While some of these domains may reflect co-option of EphA4 expression to novel sites for development of shark-specific characters, others are more likely to be ancestral patterns of expression that were lost in other vertebrate lineages.Edited by R. P. Elinson     

Expression of heat shock proteins in turtle and mammal hearts: relationship to anoxia tolerance

Heat shock proteins (HSPs) may play a cardioprotective role during hypoxia or ischemia. We hypothesized that cardiac tissue from hypoxia-tolerant animals might have high levels of specific HSPs. We measured myocardial HSP60 and HSP72/73 in painted and softshell turtles during normoxia and anoxia (12 h) and after recovery (12 or 24 h). We also measured myocardial HSPs in normoxic rats and rabbits. During normoxia, hearts from the most highly anoxia-tolerant species, the painted turtle, expressed the highest levels of HSP60 (22.6+/-2.0 mg/g total protein) followed by softshells (11.5+/-0.8 mg/g), rabbits (6.8+/-0.9 mg/g), and rats (4.5+/-0.5 mg/g). HSP72/73 levels, however, were not significantly different. HSP60 levels in hearts from both painted and softshell turtles did not deviate significantly from control values after either 12 h of anoxia or 12 or 24 h of recovery. The pattern of changes observed in HSP72/73 was quite different in the two turtle species. In painted turtles anoxia induced a significant increase in myocardial HSP72/73 (from 2.8+/-0.1 mg/g normoxic to 3.9+/-0.2 mg/g anoxic, P<0.05). By 12 h of recovery, HSP72/73 had returned to control levels (2.7+/-0.1 mg/g) and remained there through 24 h (2.6+/-0.2 mg/g). In softshell turtles, HSP72/73 decreased significantly after 12 h of anoxia (from 2.4+/-0.4 mg/g normoxic to 1.3+/-0.2 mg/g anoxic, P<0.05). HSP72/73 levels were still slightly below control after 12 h of recovery (2.1+/-0.1 mg/g) and then rose to significantly above control after 24 h of recovery (4.1+/-0.7 mg/g, P<0.05). We also conclude that anoxia-tolerant and anoxia-sensitive turtles exhibit different patterns of myocardial HSP changes during anoxia and recovery. Whether these changes correlate with their relative degrees of anoxia tolerance remains to be determined.     

Endogenous reductions in N-methyl-d-aspartate receptor activity inhibit nitric oxide production in the anoxic freshwater turtle cortex

Increased nitric oxide (NO) production from hypoxic mammalian neurons increases cerebral blood flow (CBF) but also glutamatergic excitotoxicity and DNA fragmentation. Anoxia-tolerant freshwater turtles have evolved NO-independent mechanisms to increase CBF; however, the mechanism(s) of NO regulation are not understood. In turtle cortex, anoxia or NMDAR blockade depressed NO production by 27+/-3% and 41+/-5%, respectively. NMDAR antagonists also reduced the subsequent anoxic decrease in NO by 74+/-6%, suggesting the majority of the anoxic decrease is due to endogenous suppression of NMDAR activity. Prevention of NO-mediated damage during the transition to and from anoxia may be incidental to natural reductions of NMDAR activity in the anoxic turtle cortex.     

Innate immune response and disease resistance in Carassius auratus by triherbal solvent extracts

This study reports the effect of aqueous, ethanol and methanol triherbal solvent extract from Azadirachta indica, Ocimum sanctum and Curcuma longa on innate immune mechanisms such as phagocytosis activity, respiratory burst activity, alternative complement activity and lysozyme activity and disease resistance in goldfish (Carassius auratus) against Aeromonas hydrophila. Fish were intraperitoneally injected with different doses of 0, 5, 50 and 100 mg kg⁻¹ body weight of each triherbal solvent extracts. The functional immunity in terms of percentage mortality and Relative Percent Survival (RPS) and innate immune response was assessed on week 1, 2 and 4 by challenging with live A. hydrophila (1 × 10⁷ cells ml⁻¹). All the chosen innate immune parameters were enhanced in the ethanol and methanol triherbal solvent extract treatment after week 2. However, the aqueous triherbal extract was enhanced only after week 4. The ethanol and methanol triherbal solvent extracts administration preceding the challenge with live A. hydrophila decreased the percentage mortality in the experimental groups with the consequence increase in RPS values. The study indicates that all the doses of ethanol or methanol triberbal solvent extracts could be positively influence the immune response and protect the heath status of goldfish against A. hydrophila infection.     

Trace fossils of the arthropod Camptophyllia from the Westphalian (Carboniferous) rocks of Lancashire, UK and their palaeoenvironmental context

Two arthropod trace fossils are described and analysed from the Carboniferous Lower Westphalian (C. communis and basal A. modiolaris chronozones) coal-bearing strata of Lancashire. The biserial trackway Diplichnites triassicus consists of five overlapping en echelon sets of 7–9 tracks preserved as epichnia and hypichnia in lacustrine siltstones. The trackway suggests subaqueous in-phase walking by a multi-segmented producer with a body length of 35–40 mm, width 17–22 mm, and 7–9 appendages. Curved, clustered, or laterally repeated, hypichnial lobes with transverse striations on the base of ripple cross-laminated sandstone are identified as Rusophycus versans. This trace fossil is interpreted as shallow resting or furrowing burrows of a homopodous arthropod, 30–60 mm long, 15–30 mm wide, and probably the same kind of arthropod as produced D. triassicus.A review of contemporary arthropod body fossils from Lagerstätten in Lancashire favours the onisciform, or Arthropleura like arthropod Camptophyllia as a potential producer of both of these trace fossils in a lacustrine palaeoenvironment.This study integrates the analysis of sediments, trace fossils and body fossils for reconstructing the arthropod biota and ecology in Westphalian lacustrine and crevasse splay fluvial palaeoenvironments.