We develop a unifying theory of hypoxia tolerance based on information from two cell level models (brain cortical cells and isolated hepatocytes) from the highly anoxia tolerant aquatic turtle and from other more hypoxia sensitive systems. We propose that the response of hypoxia tolerant systems to oxygen lack occurs in two phases (defense and rescue). The first lines of defense against hypoxia include a balanced suppression of ATP-demand and ATP-supply pathways; this regulation stabilizes (adenylates) at new steady-state levels even while ATP turnover rates greatly decline. The ATP demands of ion pumping are down-regulated by generalized "channel" arrest in hepatocytes and by "spike" arrest in neurons. Hypoxic ATP demands of protein synthesis are down-regulated probably by translational arrest. In hypoxia sensitive cells this translational arrest seems irreversible, but hypoxia-tolerant systems activate "rescue" mechanisms if the period of oxygen lack is extended by preferentially regulating the expression of several proteins. In these cells, a cascade ofprocesses underpinning hypoxia rescue and defense begins with an oxygen sensor (a heme protein) and a signaltransduction pathway, which leads to significant gene-based metabolic reprogramming-the rescue process-with maintained down-regulation of energy-demand and energy-supply pathways in metabolism throughout the hypoxic period. This recent work begins to clarify how normoxic maintenance ATP turnover rates can be drastically (10-fold) down-regulated to a new hypometabolic steady state, which is prerequisite for surviving prolonged hypoxia or anoxia. The implications of these developments are extensive in biology and medicine. energy turnover supplies the greatest protection against, and hence, advantage in, hypoxia. The immense advantage of this defense strategy is widely appreciated by many biologists (4, 5, 11-13). In one of his last personal communications to one of the authors (P.W.H.), the great comparative physiologist Kjell Johansen referred to this strategy as "turning down to the pilot light" and he, like many earlier workers, was acutely aware of its relative importance. Although recognized as a kind of hallmark of reversible entry into and return from states of severe 02 deprivation, a number of unexplained problems have remained. In particular, it has not been clear (i) how cells/ tissues "know" when to turn on their hypoxia defense mechanisms, (ii) which pathways of ATP demand and ATP supply are down-regulated or by how much, (iii) how membrane electrochemical gradients are stabilized, and (iv) what geneexpression and protein-expression level adjustments are involved in hypoxic reorganization of cell structure and function. Recent studies of a well-known vertebrate "facultative anaerobe," the aquatic turtle, used brain cortical slices to probe electrophysiological properties of neurons under anoxia (16)(17)(18)(19) and isolated liver hepatocytes to probe cell level biochemical responses to anoxia (20)(21)(22)(23)(24). When integrated with ...

The ability of fishes, amphibians, and reptiles to survive extremes of oxygen availability derives from a core triad of adaptations: profound metabolic suppression, tolerance of ionic and pH disturbances, and mechanisms for avoiding free-radical injury during reoxygenation. For long-term anoxic survival, enhanced storage of glycogen in critical tissues is also necessary. The diversity of body morphologies and habitats and the utilization of dormancy have resulted in a broad array of adaptations to hypoxia in lower vertebrates. For example, the most anoxia-tolerant vertebrates, painted turtles and crucian carp, meet the challenge of variable oxygen in fundamentally different ways: Turtles undergo near-suspended animation, whereas carp remain active and responsive in the absence of oxygen. Although the mechanisms of survival in both of these cases include large stores of glycogen and drastically decreased metabolism, other mechanisms, such as regulation of ion channels in excitable membranes, are apparently divergent. Common themes in the regulatory adjustments to hypoxia involve control of metabolism and ion channel conductance by protein phosphorylation. Tolerance of decreased energy charge and accumulating anaerobic end products as well as enhanced antioxidant defenses and regenerative capacities are also key to hypoxia survival in lower vertebrates.

BackgroundWe describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing.ResultsOur phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented.ConclusionsOur comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle's extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.

The immortalized and proliferative cell line SH-SY5Y is one of the most commonly used cell lines in neuroscience and neuroblastoma research. However, undifferentiated SH-SY5Y cells share few properties with mature neurons. In this study, we present an optimized neuronal differentiation protocol for SH-SY5Y that requires only two work steps and 6 days. After differentiation, the cells present increased levels of ATP and plasma membrane activity but reduced expression of energetic stress response genes. Differentiation results in reduced mitochondrial membrane potential and decreased robustness toward perturbations with 6-hydroxydopamine. We are convinced that the presented differentiation method will leverage genetic and chemical high-throughput screening projects targeting pathways that are involved in the selective vulnerability of neurons with high energetic stress levels.

The maintenance of ion gradients across the plasma membrane by the Na(+)-K(+)-ATPase has been shown to utilize a large fraction of the total cellular energy demand. In view of the importance of ion gradients to cellular function, and the remarkable anoxia tolerance of Chrysemys picta bellii (western painted turtle) and hepatocytes isolated from this species, it was of interest to determine if in response to anoxia 1) ion gradients were maintained and 2) if the activity of the plasma membrane Na(+)-K(+)-ATPase changed to aid in ion gradient maintenance. From normoxic hepatocyte suspensions the ouabain-inhibitable 86Rb+ uptake (a measure of Na(+)-K(+)-ATPase activity) was determined, and the rate of ATP utilization was 19.1 mumol ATP.g cells-1.h-1 or 28% of the total normoxic cellular ATP turnover. In response to anoxic incubation the activity of the pump decreased by 75% to 4.8 mumol ATP.g cells-1.h-1 and this comprised 74% of the total anoxic ATP turnover. Presently, it is not known whether the observed reduction in Na(+)-K(+)-ATPase activity is regulated by 1) allosteric modification, 2) endocytosis from the membrane, or 3) reduced Na+ influx. Plasma membrane potential was measured during anoxia, using the distribution of 36Cl-, and was not significantly different from the normoxic measurement, -30.6 +/- 3.9 and -31.3 +/- 5.8 mV, respectively. Therefore, the plasma membrane ion gradient is maintained during anoxia, and since the activity of the Na(+)-K(+)-ATPase decreases, the influx of ions must also decrease.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypoxia-induced suppression of NMDA receptors (NMDARs) in western painted turtle (Chrysemys picta) cortical neurons may be critical for surviving months of anoxic dormancy. We report that NMDARs are silenced by at least three different mechanisms operating at different times during anoxia. In pyramidal neurons from cerebrocortex, 1-8 min anoxia suppressed NMDAR activity (Ca(2+) influx and open probability) by 50-60%. This rapid decrease in receptor activity was controlled by activation of phosphatase 1 or 2A but was not associated with an increase in [Ca(2+)](i). However, during 2 hr of anoxia, [Ca(2+)](i) in cerebrocortical neurons increased by 35%, and suppression of NMDARs was predicted by the increase of [Ca(2+)](i) and controlled by calmodulin. An additional mechanism of NMDAR silencing, reversible removal of receptors from the cell membrane, was found in cerebrocortex of turtles remaining anoxic at 3 degrees C for 3-21 d. When suppression of NMDARs was prevented with phosphatase inhibitors, tolerance of anoxia was lost. Silencing of NMDARs is thus critical to the remarkable ability of C. picta to tolerate life without oxygen.

Hepatocytes from the western painted turtle (Chrysemys picta bellii) display a profound metabolic suppression under anoxia. Fractional rates of protein synthesis fell by 92% during 12 h anoxia at 25 degrees C and were indistinguishable from the rate obtained with cycloheximide. Normoxic recovery saw protein synthesis increase to 160% of control values and return to normal after 2 h. The GTP-to-GDP ratio, implicated in the control of translation, fell threefold during anoxia. Purine nucleotide phosphate profiles suggest that this change occurs through increasing concentrations of ADP and GDP, with concentrations of ATP and GTP and total purines remaining constant. The normoxic cost for protein synthesis was calculated at 47.6 +/- 6.8 mmol ATP/g protein. Normoxic protein synthesis accounted for 36% of overall ATP turnover rates, close to the extent of O2 consumption inhibitable by cycloheximide (28%). Under anoxia, the proportion of ATP turnover utilized by protein synthesis did not change significantly. ATP turnover rates for urea synthesis reflected a similar pattern, falling 72% under anoxia. These results reflect the cell's ability to suppress protein synthesis under anoxia in a manner that is coordinated with the reduction in total metabolic rate.

Anoxic insults cause hyperexcitability and cell death in mammalian neurons. Conversely, in anoxia-tolerant turtle brain, spontaneous electrical activity is suppressed by anoxia (i.e., spike arrest; SA) and cell death does not occur. The mechanism(s) of SA is unknown but likely involves GABAergic synaptic transmission, because GABA concentration increases dramatically in anoxic turtle brain. We investigated this possibility in turtle cortical neurons exposed to anoxia and/or GABA A/B receptor (GABAR) modulators. Anoxia increased endogenous slow phasic GABAergic activity, and both anoxia and GABA reversibly induced SA by increasing GABA A Rmediated postsynaptic activity and Cl − conductance, which eliminated the Cl − driving force by depolarizing membrane potential (∼8 mV) to GABA receptor reversal potential (∼−81 mV), and dampened excitatory potentials via shunting inhibition. In addition, both anoxia and GABA decreased excitatory postsynaptic activity, likely via GABA B R-mediated inhibition of presynaptic glutamate release. In combination, these mechanisms increased the stimulation required to elicit an action potential >20-fold, and excitatory activity decreased >70% despite membrane potential depolarization. In contrast, anoxic neurons cotreated with GABA A+B R antagonists underwent seizure-like events, deleterious Ca 2+ influx, and cell death, a phenotype consistent with excitotoxic cell death in anoxic mammalian brain. We conclude that increased endogenous GABA release during anoxia mediates SA by activating an inhibitory postsynaptic shunt and inhibiting presynaptic glutamate release. This represents a natural adaptive mechanism in which to explore strategies to protect mammalian brain from low-oxygen insults.western painted turtle | cerebral cortex | channel arrest | pyramidal neurons | natural anesthetic mechanism W hen deprived of oxygen, mammalian neurons are unable to produce sufficient ATP to meet cellular demands (1, 2). As a result, the Na + /K + ATPase (Na + pump) fails and neuronal membrane potential (V m ) becomes unsustainable and anoxic depolarization (AD) follows, causing electrical hyperexcitability, deleterious Ca 2+ influx, and spreading depression in the penumbral region (2, 3). Numerous studies have focused on the role of glutamatergic N-methyl-D-aspartate receptors (NMDARs) in this mechanism, and although NMDAR blockade prevents glutamatergic excitotoxicity (4), it does not prevent AD-mediated injury or postinsult apoptotic cell death (5). Thus, it is not surprising that clinical interventions targeting glutamate receptors alone have been largely ineffective against anoxic or ischemic damage (6), and therefore examination of alternative mechanisms to limit excitability during such insults is necessary.A potential therapeutic alternative to directly antagonizing excitatory pathways is to up-regulate inhibitory mechanisms such as those mediated by γ-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the mature mammalian CNS (7). GABAergic mechanisms are not strongly recr...