Anoxic Response Research Articles

Evaluating the brainstem in children with breath-holding spells.

Breath-holding spells (BHSs) are a non-epileptic paroxysmal phenomenon characterized by frequent apnea episodes, loss of consciousness, and changes in skin tone and postural tone triggered by negative stimuli of childhood. The pathophysiology of the disease remains unclear; autonomic dysregulation caused by delayed myelination is believed to play a role. In this study, we aimed to evaluate the brainstems of children with BHS using diffusion tensor imaging (DTI) and investigate the etiology of this phenomenon. The study group consisted of 16 children with a history of severe breath-holding episodes (accompanied by loss of consciousness and tonic contraction due to prolonged anoxic response) and 18 age-, gender-, and handedness-matched controls. All children underwent systemic, neurologic, and cardiologic evaluation, including complete blood count, blood biochemistry, serum iron and ferritin level, serum vitamin B12 level, electrocardiogram, and electroencephalograms. Magnetic resonance imaging was performed using a 1.5-Tesla Siemens Aera scanner (Siemens, Germany). Evaluation of brainstem (midbrain, pons, and medulla oblongata) volumes revealed no statistically significant differences between the BHS patient and control groups. In a voxel-wise analysis of DTI data, the BHS patient group had significantly lower fractional anisotropy (FA) values than the control group in the bilateral midbrain and medulla, right corticospinal tract, bilateral corpus callosum body and splenium, and left corpus callosum genu. In contrast, there were no significant differences in FA values in the pons, cerebellum, left corticospinal tract, and right corpus callosum genu. Based on our findings, we think that patients with BHS should be treated with an approach similar to other neurodevelopmental diseases and that this study may help elucidate the pathophysiology and establish the groundwork for future studies on its treatment.

Scavenging of reactive oxygen species mimics the anoxic response in goldfish pyramidal neurons.

Goldfish are one of a few species able to avoid cellular damage during month-long periods in severely hypoxic environments. By suppressing action potentials in excitatory glutamatergic neurons, the goldfish brain decreases its overall energy expenditure. Coincident with reductions in O2 availability is a natural decrease in cellular reactive oxygen species (ROS) generation, which has been proposed to function as part of a low-oxygen signal transduction pathway. Using live-tissue fluorescence microscopy, we found that ROS production decreased by 10% with the onset of anoxia in goldfish telencephalic brain slices. Employing whole-cell patch-clamp recording, we found that, similar to severe hypoxia, the ROS scavengers N-acetyl cysteine (NAC) and MitoTEMPO, added during normoxic periods, depolarized membrane potential (severe hypoxia -73.6 to -61.4 mV, NAC -76.6 to -66.2 mV and MitoTEMPO -71.5 mV to -62.5 mV) and increased whole-cell conductance (severe hypoxia 5.7nS to 8.0 nS, NAC 6.0 nS to 7.5 nS and MitoTEMPO 6.0 nS to 7.6 nS). Also, in a subset of active pyramidal neurons, these treatments reduced action potential firing frequency (severe hypoxia 0.18 Hz to 0.03 Hz, NAC 0.27 Hz to 0.06 Hz and MitoTEMPO 0.35 Hz to 0.08 Hz). Neither severe hypoxia nor ROS scavenging impacted action potential threshold. The addition of exogenous hydrogen peroxide could reverse the effects of the antioxidants. Taken together, this supports a role for a reduction in [ROS] as a low-oxygen signal in goldfish brain.

Investigation of legacy industrial mercury in floodplain soils: South River, Virginia, USA

Mercury (Hg) was used during 1929–1950 as a catalyst to produce rayon acetate at the former DuPont plant in Waynesboro, Virginia, and released into the South River. Though the use of Hg ceased in the 1970s, the affected ecosystem is still a matter of concern. Here, high total mercury (THg) and total organomercury, stated as methylmercury (MeHg) are reported in historically contaminated floodplain soils and shallow groundwater from five locations sited 5.6 km downstream from the plant of one river edge site. In soils, THg ranged from 0.1 to 1201.5 mg/kg exceeding the health-based screening Hg levels for residential and industrial soils while MeHg varied between 0.1 and 54 µg/kg. Concentrations decreased sharply with depth indicating the stratification of legacy industrial Hg-rich soils underlain by the pre-industrial soils with minor Hg. Strong linear correlation between THg and MeHg was observed. Highest soil MeHg was associated with total carbon, poorly crystalline and amorphous Fe and/or Mn oxyhydroxides. Sequential extraction analyses indicated that Hg was present mostly in relatively recalcitrant forms, as determined with procedures that targeted β-HgS, HgS, HgSe, HgAu, thiol-bound Hg, Hg0, and some organo-complexed Hg, Hg2Cl2 phases. High Cu (≤ 404.3 mg/kg), Zn (≤ 151.3 mg/kg), Cr (≤ 123.9 mg/kg) were also identified. In shallow groundwater, THg ranged from 28 to 538 ng/L and MeHg varied between 1.2 and 137 ng/L. During the intermittent precipitation, the highest MeHg was linked to the highest Fe2+, Mn, SO42−, total alkalinity, and conductivity, which could be due to either the potential leaching and dissolution of soil minerals and/or the saturation of the vadose zone. A sharp increase in the soil moisture at the top 40–70 cm of the Hg-rich soils after rainfall and overbank flooding was followed by redox gradients from oxidizing (≈ + 600 mV) to reducing (≈ − 300 mV) and a reverse response in a transmissive gravel zone at the base of the bank (≈ − 400 to + 200 mV) with a defined lag with depth. These dynamic seasonal fluctuations at the South River might be crucial for solubilization of Hg, Fe, and Mn redox-sensitive minerals triggering the anoxic response for new MeHg production.

Stellate and pyramidal neurons in goldfish telencephalon respond differently to anoxia and GABA receptor inhibition.

With oxygen deprivation, the mammalian brain undergoes hyper-activity and neuronal death while this does not occur in the anoxia-tolerant goldfish (Carassius auratus). Anoxic survival of the goldfish may rely on neuromodulatory mechanisms to suppress neuronal hyper-excitability. As γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, we decided to investigate its potential role in suppressing the electrical activity of goldfish telencephalic neurons. Utilizing whole-cell patch-clamp recording, we recorded the electrical activities of both excitatory (pyramidal) and inhibitory (stellate) neurons. With anoxia, membrane potential (Vm) depolarized in both cell types from -72.2 mV to -57.7 mV and from -64.5 mV to -46.8 mV in pyramidal and stellate neurons, respectively. While pyramidal cells remained mostly quiescent, action potential frequency (APf) of the stellate neurons increased 68-fold. Furthermore, the GABAA receptor reversal potential (E-GABA) was determined using the gramicidin perforated-patch-clamp method and found to be depolarizing in pyramidal (-53.8 mV) and stellate neurons (-42.1 mV). Although GABA was depolarizing, pyramidal neurons remained quiescent as EGABA was below the action potential threshold (-36 mV pyramidal and -38 mV stellate neurons). Inhibition of GABAA receptors with gabazine reversed the anoxia-mediated response. While GABAB receptor inhibition alone did not affect the anoxic response, co-antagonism of GABAA and GABAB receptors (gabazine and CGP-55848) led to the generation of seizure-like activities in both neuron types. We conclude that with anoxia, Vm depolarizes towards EGABA which increases APf in stellate neurons and decreases APf in pyramidal neurons, and that GABA plays an important role in the anoxia tolerance of goldfish brain.

Effects of pH on the response of peripheral nerve to anoxia

Purpose/Aim: Changes in pH are not infrequently encountered in clinical situations and can be associated with significant effects on ion channels, mitochondria and axon function. The purpose of this paper is to study the modulatory effects of pH on the anoxic response in peripheral nerve. Materials and Methods: A total of 48 rat sciatic nerves were studied in vitro in a perfusion apparatus. Experiments were carried out at 6 pH levels from 6.0 to 7.8. Results: The amplitude of the nerve action potential (NAP) drops more dramatically with repetitive periods of anoxia when the pH is reduced below 6.5. In addition, velocity decreases and duration increases more with each cycle of anoxia at low pH values. Despite these effects of pH on recovery after anoxia, there was no significant effect of pH on the time course of changes during anoxia. During recovery from anoxia, the NAP recovered more slowly when the pH was lowered. Conclusions: The pattern of changes in amplitude, velocity and duration suggest that they may be due to interference of high hydrogen ion concentrations with sodium and potassium channel function.

Hypoxic ventilatory response in Tac1−/− neonatal mice following exposure to opioids

Morphine is the dominating analgetic drug used in neonates, but opioid-induced respiratory depression limits its therapeutic use. In this study, we examined acute morphine effects on respiration during intermittent hypoxia in newborn Tac1 gene knockout mice (Tac1-/-) lacking substance P and neurokinin A. In vivo, plethysmography revealed a blunted hypoxic ventilatory response (HVR) in Tac1-/- mice. Morphine (10 mg/kg) depressed the HVR in wild-type animals through an effect on respiratory frequency, whereas it increased tidal volumes in Tac1-/- during hypoxia, resulting in increased minute ventilation. Apneas were reduced during the first hypoxic episode in both morphine-exposed groups, but were restored subsequently in Tac1-/- mice. Morphine did not affect ventilation or apnea prevalence during baseline conditions. In vitro, morphine (50 nM) had no impact on anoxic response of brain stem preparations of either strain. In contrast, it suppressed the inspiratory rhythm during normoxia and potentiated development of posthypoxic neuronal arrest, especially in Tac1-/-. Thus this phenotype has a higher sensitivity to the depressive effects of morphine on inspiratory rhythm generation, but morphine does not modify the reactivity to oxygen deprivation. In conclusion, although Tac1-/- mice are similar to wild-type animals during normoxia, they differed by displaying a reversed pattern with an improved HVR during intermittent hypoxia both in vivo and in vitro. These data suggest that opioids and the substance P-ergic system interact in the HVR, and that reducing the activity in the tachykinin system may alter the respiratory effects of opioid treatment in newborns.

Metabolic regulation analysis of wild-type and arcA mutant Escherichia coli under nitrate conditions using different levels of omics data

It is of practical interest to investigate the effect of nitrates on bacterial metabolic regulation of both fermentation and energy generation, as compared to aerobic and anaerobic growth without nitrates. Although gene level regulation has previously been studied for nitrate assimilation, it is important to understand this metabolic regulation in terms of global regulators. In the present study, therefore, we measured gene expression using DNA microarrays, intracellular metabolite concentrations using CE-TOFMS, and metabolic fluxes using the (13)C-labeling technique for wild-type E. coli and the ΔarcA (a global regulatory gene for anoxic response control, ArcA) mutant to compare the metabolic state under nitrate conditions to that under aerobic and anaerobic conditions without nitrates in continuous culture conditions at a dilution rate of 0.2 h(-1). In wild-type, although the measured metabolite concentrations changed very little among the three culture conditions, the TCA cycle and the pentose phosphate pathway fluxes were significantly different under each condition. These results suggested that the ATP production rate was 29% higher under nitrate conditions than that under anaerobic conditions, whereas the ATP production rate was 10% lower than that under aerobic conditions. The flux changes in the TCA cycle were caused by changes in control at the gene expression level. In ΔarcA mutant, the TCA cycle flux was significantly increased (4.4 times higher than that of the wild type) under nitrate conditions. Similarly, the intracellular ATP/ADP ratio increased approximately two-fold compared to that of the wild-type strain.

QTL Analysis of Anoxic Tolerance at Seedling Stage in Rice

Coleoptile lengths of 7-day-old seedlings under anoxic stress and normal conditions were investigated in two permanently segregated populations and their parents in rice ( Oryza sativa L.). Using anoxic response index, a ratio of coleoptile length under anoxic stress to coleoptile length under normal conditions, as an indicator of seedling anoxic tolerance (SAT), QTLs for SAT were detected. Two loci controlling SAT, designated as qSAT-2-R and qSAT-7-R, were detected in a recombinant inbred line (RIL) population (247 lines) derived from a cross between Xiushui 79 (japonica variety) and C Bao (japonica restorer line). qSAT-2-R, explaining 8.7% of the phenotype variation, was tightly linked with the SSR marker RM525. qSAT-7-R, explaining 9.8% of the phenotype variation, was tightly linked with the marker RM418. The positive alleles of the two loci came from C Bao. Six loci controlling SAT, designated as qSAT-2-B, qSAT-3-B, qSAT-5-B, qSAT-8-B, qSAT-9-B and qSAT-12-B, were detected in a backcross inbred line (BIL) population (98 lines) derived from a backcross of Nipponbare (japonica)/Kasalath (indica)//Nipponbare (japonica). The positive alleles of qSAT-2-B, qSAT-3-B and qSAT-9-B, which explained 16.2%, 11.4% and 9.5% of the phenotype variation, respectively, came from Nipponbare. Besides, the positive alleles of qSAT-5-B, qSAT-8-B and qSAT-12-B, which explained 7.3%, 5.8% and 14.0% of the phenotype variation, respectively, were from Kasalath.

Sugar and fructan accumulation during metabolic adjustment between respiration and fermentation under low oxygen conditions in wheat roots.

In terms of gene expression and carbohydrate metabolism, the response of wheat seedlings to hypoxia is dramatically different from the anoxic response. Total carbohydrate content of roots increased 4-fold during 6 days of hypoxia, with a 17-fold increase in fructans. In contrast, anoxically treated roots depleted all soluble carbohydrates and died within 72 h. Gas exchange measurements (CO(2) release vs. O(2) uptake) demonstrate that hypoxia establishes a new balance between fermentation and aerobic respiration in the roots without altering the flux of carbon through glycolysis. Furthermore, the respiratory component of this new balance is 55% higher in roots that have been hypoxically pretreated compared to non-hypoxically pretreated roots. The establishment of this new homeostasis under hypoxia involves the induction of glycolytic (aldolase and enolase) and fermentative enzymes (pyruvate decarboxylase, alcohol dehydrogenase, and lactate dehydrogenase). Enzyme induction is generally complete within 24 h with mRNA induction occurring primarily during Period I (0-6 h of hypoxia), and maximal enzymes activities attained during Period II (6-24 h of hypoxia). Accumulation rates of Suc, hexoses, and fructans also change during Periods I and II. By the start of Period III (24-144 h of hypoxia), the metabolic adjustments are complete and fructans are the major carbohydrate accumulated. In anoxia, the pattern of enzyme induction was dramatically different: aldolase was not induced and declined throughout the treatment. Alcohol dehydrogenase, pyruvate decarboxylase, and lactate dehydrogenase were induced as in hypoxia, but rapidly declined within 72 h of anoxia. Only enolase exhibited a similar expression pattern in both anoxia and hypoxia.

Regulation of a rat VL30 element in human breast cancer cells in hypoxia and anoxia: role of HIF-1.

Novel approaches to cancer gene therapy currently exploit tumour hypoxia to achieve transcriptional targeting using oxygen-regulated enhancer elements called hypoxia response elements. The activity of such elements in hypoxic cells is directly dependent on upregulation of the hypoxia-inducible transcription factor-1 However tumours also contain areas of anoxia, which may be considered a more tumour-selective transcriptional stimulus than hypoxia for targeting gene therapy to tumours. Another element, from the rat virus-like retrotransposon, VL30 (termed the ‘secondary anoxia response element’) has been reported to be more highly inducible in rat fibroblasts under anoxia than hypoxia. To investigate anoxia as a potential transcriptional target in human tumours, we have examined secondary anoxia response element inducibility in two human breast cancer cell lines, MCF-7 and T47D, under anoxia, hypoxia and normoxia. In both cell types, the trimerised secondary anoxia response element showed greater inducibility in anoxia than hypoxia (1% and 0.5% O2). The anoxic response of the secondary anoxia response element was shown to be dependent on hypoxia-inducible transcription factor-1 and the presence of a hypoxia-inducible transcription binding site consensus (5′-ACGTG-3′). Mutational analysis demonstrated that the base immediately 5′ to this modulates the anoxic/hypoxic induction of the secondary anoxia response element, such that TACGTG>GACGTG>>CACGTG. A similar correlation was found for erythropoietin, phosphoglycerate kinase 1, and aldolase hypoxia response elements, which contain these respective 5′ flanking bases.British Journal of Cancer (2002) 87, 1173–1181. doi:10.1038/sj.bjc.6600576 www.bjcancer.com© 2002 Cancer Research UK

Long-term deprivation of substance P in PPT-A mutant mice alters the anoxic response of the isolated respiratory network.

The aim of this study was to elucidate the role of the neuromodulator substance P and its related tachykinin neurokinin A (NKA) in the homeostasis of respiratory activity. Respiratory activities, in form of fictive eupneic and sigh activities, were recorded extracellularly from the preBötzinger complex (PBC) in normoxic and anoxic conditions using medullary slice preparations. The effect of a blockade of endogenous substance P was assessed by an acute pharmacological blockade of the receptors with spantide in wild-type animals and by the use of preprotachykinin-A (PPT-A) mutants. These mutants lack from birth the PPT-A gene, which codes for the precursor of substance P and NKA. Spantide treatment reduced frequency (-37%, n = 9) and regularity (twofold) of eupneic-like respiratory activity under normoxic conditions, whereas in PPT-A mutants, eupneic-like activity was under normoxic conditions not significantly different from the wild-type mice (WT). The response to short anoxic episodes (5 min) was characterized in the WT by an increase in respiratory frequencies at the onset of anoxia (ratio anoxic/control frequency = 1.9 +/- 0.2, n = 18). This anoxic ratio was unaltered in the presence of spantide (ratio = 2.3 +/- 0.4, n = 8) but increased in the mutant (ratio = 4.1, n = 15). We conclude that endogenously released substance P is important for the maintenance of regular respiratory activity. Short-term blockade of substance P receptors decreases the frequency and regularity of rhythmic activity. Long-term deficiency in substance P leads to compensatory mechanisms that result in an apparently normal respiratory activity under normoxic conditions but a significantly altered response of the respiratory network during anoxia.

Genomic analyses of anaerobically induced genes in Saccharomyces cerevisiae: functional roles of Rox1 and other factors in mediating the anoxic response.

DNA arrays were used to investigate the functional role of Rox1 in mediating acclimatization to anaerobic conditions in Saccharomyces cerevisiae. Multiple growth conditions for wild-type and rox1 null strains were used to identify open reading frames with a statistically robust response to this repressor. These results were compared to those obtained for a wild-type strain in response to oxygen availability. Transcripts of nearly one-sixth of the genome were differentially expressed (P < 0.05) with respect to oxygen availability, the majority (>65%) being down-regulated under anoxia. Of the anaerobically induced genes, about one-third (106) contain putative Rox1-binding sites in their promoters and were significantly (P < 0.05) up-regulated in the rox1 null strains under aerobiosis. Additional promoter searches revealed that nearly one-third of the anaerobically induced genes contain an AR1 site(s) for the Upc2 transcription factor, suggesting that Upc2 and Rox1 regulate the majority of anaerobically induced genes in S. cerevisiae. Functional analyses indicate that a large fraction of the anaerobically induced genes are involved in cell stress (approximately 1/3), cell wall maintenance (approximately 1/8), carbohydrate metabolism (approximately 1/10), and lipid metabolism (approximately 1/12), with both Rox1 and Upc2 predominating in the regulation of this latter group and Upc2 predominating in cell wall maintenance. Mapping the changes in expression of functional regulons onto metabolic pathways has provided novel insight into the role of Rox1 and other trans-acting factors in mediating the physiological response of S. cerevisiae to anaerobic conditions.

The effect of prolonged anoxia on enzyme activities in oysters (Crassostrea virginica) at different seasons

Abstract The effect of prolonged anoxia (96 h under a N2 atmosphere) during either winter (November) or summer (July) was investigated by measuring the maximal activities of 20 metabolic enzymes in gill, mantle, hepatopancreas, and phasic and catch adductor muscles of the oyster, Crassostrea virginica. The enzymes analyzed are involved in carbohydrate and amino acid metabolism, the pentose phosphate shunt, anaplerotic reactions of the TCA cycle, and phosphagen/adenylate metabolism. The data demonstrate that oyster metabolism is influenced by both long-term seasonal change and by shorter-term environmental insult (anoxia). Seasonal changes were concentrated among enzymes involved in glycogen metabolism whereas the prominent response to anoxia was suppression of PK activity. Anoxia exposure induced tissue-specific changes in enzyme activities suggesting a substantial metabolic reorganization involving both coarse controls on enzyme amount and reversible covalent modification. In addition, the effects of anoxia on enzymes of intermediary metabolism were seasonally dependent and more widespread in the winter. These results demonstrate the interaction of two environmental variables (season, anoxia) and suggest the importance of season as a modifying factor in the anoxic response.

Effects of K+ channel blockers on the anoxic response of CNS myelinated axons.

Compound action potentials (CAPs) from adult rat optic nerves were recorded in vitro. The area under the CAP was compared before and after 1 h anoxia/reoxygenation. Resting compound membrane potential was measured using the cold grease-gap technique. The acute reduction of CAP magnitude by anoxia was unaffected by TEA (20 mM), 4-AP (300 microM), or the KATP blockers glibenclamide (300 microM) and tolbutamide (2 mM). Neither these K+ channel blockers, nor glipizide (100 microM) or the KATP activator diazoxide (500 microM) altered post-anoxic CAP area recovery. In contrast, although Cs+ (5 mM) accelerated anoxic CAP failure and membrane depolarization, this cation significantly increased CAP area recovery post-anoxia from 22+/-10% (s.d.) to 60+/-22% (p < 0.0001). The unique effects of Cs+ suggest that inward rectifier channels may play an important role in the induction of anoxic injury in optic nerve axons.

Effect of chemical anoxia on protein kinase C and Na+, K+-ATPase in hepatocytes of goldfish (Carassius auratus) and rainbow trout (Oncorhynchus mykiss)

Protein kinase C (PKC) and Na+/K+-ATPase in hepatocytes from the anoxia-tolerant goldfish (Carassius auratus) and the anoxia-intolerant rainbow trout (Oncorhynchus mykiss) were studied to determine their role in the anoxic response of these cells. PKC and Na+/K+-ATPase activities were measured for up to 90 min in the absence (normoxia) and presence (chemical anoxia) of 2 mmol l-1 sodium cyanide. PKC activity of normoxic cells from both species remained constant for the entire experimental period. Addition of cyanide had no effect on PKC activity of trout cells, which was maintained at 25 % of maximal PKC activity. In goldfish hepatocytes, PKC activity remained constant at 56 % of maximal PKC activity for 30 min but fell to 27 % after 90 min of anoxic exposure. ATPase activity was measured in hepatocytes exposed to 100 nmol l-1 phorbol-12,13-dibutyrate (PdBu), a treatment which enhanced PKC activity to its maximum level. In trout cells, there was no significant change in Na+/K+-ATPase activity whereas in goldfish hepatocytes a significant increase to about 150 % of the respective controls was observed. On the basis of the experimental evidence that in hepatocytes of goldfish (1) PKC and Na+/K+-ATPase activities decreased in parallel during chemical anoxia and (2) a stimulation of PKC activity by PdBu increased Na+/K+-ATPase activity, we postulate that PKC activity in goldfish, but not in trout, may be implicated in the Na+/K+-ATPase inhibition observed under anoxia.

Radiosensitization of mouse skin by oxygen and depletion of glutathione

Purpose : To determine the oxygen enhancement ratio (OER) and shape of the oxygen sensitization curve of mouse foot skin, the extent to which glutathione (GSH) depletion radiosensitized skin, and the dependence of such sensitization on the ambient oxygen tension. Methods and Materials : The feet of WHT mice were irradiated with single doses of 240 kVp x-rays while mice were exoposed to carbogen or gases with oxygen/nitrogen mixtures containing 8–100% O 2. The anoxic response was obtained by occluding the blood supply to the leg of anesthetized mice with a tourniquet, surrounding the foot with nitrogen, and allowing the mice to breathe 10% O 2. Further experiments were performed to assess the efficacy of this method to obtain an anoxic response. Radiosensitivy of skin was assessed using the acute skin-reaction assay. Glutathione levels were modified using two schedules of dl-buthionine sulphoximine (BSO) and diethylmaleate (DEM), which were considered to produce extensive and intermediate levels of GSH depletion in the skin of the foot during irradiation. Results : Carbogen caused the greatest radiosensitization of skin, with a reproducible enhancement of 2.2 relative to the anoxic response. The OER of 2.2 is lower than other reports for mouse skin. This may indicate that the extremes of oxygenation were not produced, although there was no direct evidence for this When skin radiosensitivity was plotted against the logarithm of the oxygen tension in the ambient gas, a sigmoid curve with a K value of 17–21% O 2 in the ambient gas was obtained. Depletion of GSH caused minimal radiosensitization when skin was irradiated under anoxic or well-oxygenated conditions. Radiosensitization by GSH depletion was maximal at intermediate oxygen tensions of 10–21% O 2 in the ambient gas. Increasing the extent of GSH depletion led to increasing radiosensitization, with sensitization enhancement ratios of 1.2 and 1.1, respectively, for extensive and intermediate levels of GSH depletion. In mice exposed to 100% O 2, a significant component of skin radiosensitivity was due to diffsuin of oxygen directly through the skin. Pentobarbitone anesthesia radiosensitized skin in mice exposed to 100% O 2 by a factor of 1.2, but did not further sensitize skin in mice exposed to carbogen. Conclusions : Glutathione levels and the local oxygen tension at the time of irradiation were important determinants of mouse foot skin radiosensitivity. The extent to which GSH levels altered the radiosensitivity of skin was critically dependent on the local oxygen tension. These results have significant implications for potential clinical application of GSH depletion.

Internal Ca2+ stores involved in anoxic responses of rat hippocampal neurons.

1. During whole-cell recordings from CA1 neurons of rat brain slices with electrodes containing only KMeSO4 and Hepes, brief anoxia (2-3 min) consistently evoked a hyperpolarization (delta V approximately 14 mV) and reduction in input resistance (delta R approximately -20%). 2. As in previous intracellular recordings, Dantrolene sodium (10 microM) suppressed the anoxic delta V and delta R, confirming the release of internal Ca2+ is a major component of the anoxic response. 3. To identify the relevant intracellular Ca2+ store, other blockers of Ca2+ release were applied either externally (in the bath) or internally, by addition to the contents of the recording electrode. 4. The anoxic hyperpolarization was abolished or much reduced by heparin (10-20 micrograms ml-1, internal), thapsigargin (10 microM, external), Ruthenium Red (50 microM, internal) and external procaine (0.5-2 mM), but not by internal procaine (0.5-1 mM) or ryanodine (10 microM, external). 5. The anoxic fall in resistance was also abolished or reduced by heparin, thapsigargin and external procaine, but not by ryanodine, internal procaine or Ruthenium Red. 6. In addition, external procaine (0.5-2 mM) eliminated the early (transient) depolarization and reduced the post-anoxic hyperpolarization by 60 +/- 22%. 7. None of these agents consistently changed the resting potential, but the input resistance was significantly increased by Dantrolene and external procaine. 8. In view of the marked effects of heparin and thapsigargin, but not ryanodine and internal procaine, we conclude that the anoxic response seen in such whole-cell recordings is initiated predominantly by Ca2+ release from an internal store that is InsP3 sensitive rather than Ca2+ sensitive. 9. Comparable but less pronounced effects of external procaine were seen during intracellular recordings with 3 M KCl-containing electrodes. The dose-dependent suppression of various features of the anoxic response by external procaine (EC50 approximately 0.2 mM) is presumed to be mediated by a superficial membrane trigger or modulating site.

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 Pi signals, and it seems likely that the experimental approach used here will have further applications in studies of the metabolic response to anoxia.

Central control of breathing in mammals: neuronal circuitry, membrane properties, and neurotransmitters

Central control of breathing in mammals: neuronal circuitry, membrane properties, and neurotransmitters

Anoxia, wound healing, VL30 elements, and the molecular basis of malignant conversion

Although VL30 retrotransposable elements have been associated with certain cancers for nearly twenty years, because of their expression in rodent malignancies and recombination into murine sarcoma viruses, their causative role, if any, in cancer has been uncertain and enigmatic. Recent findings suggest loss of normal transcriptional control of specific VL30 element expression may make a critical contribution to tumor progression at a step associated with malignant conversion, by bringing into play a cellular program normally involved in wound healing. This program, the fibroblast anoxic response system, includes an adaptation to glycolytic metabolism, secretion of metalloproteinases, and activation of an endonuclease. While appropriate for facilitating debris removal during wound healing, loss of control of this program in a cell which has already progressed to the benign neoplastic state has the potential to simultaneously produce the invasiveness and genomic instability characteristic of malignancy. Examination of tumors and tumor derived cell lines has confirmed that key aspects of this system are in fact activated in cancer.