Chemical Anoxia Research Articles

Maintained mitochondrial integrity without oxygen in the anoxia-tolerant crucian carp.

Very few vertebrates survive without oxygen (anoxia) for more than a few minutes. Crucian carp (Carassius carassius) are one example, surviving months of anoxia at low temperatures, and we hypothesised that they maintain mitochondrial membrane potential and function. Isolated crucian carp cardiomyocytes indeed maintained mitochondrial membrane potential after blocking complex IV of the electron transport system with cyanide, while those of anoxia-intolerant trout depolarised. When complexes I-III were inhibited, crucian carp mitochondria depolarised, indicating that these complexes need to function during anoxia. Mitochondrial membrane potential depended on reversal of ATP synthase in chemical anoxia, as blocking with cyanide combined with oligomycin to inhibit ATP synthase led to depolarisation. ATP synthase activity was reduced in the heart after 1week of anoxia in crucian carp, together with a downregulation of ATP synthase subunit gene expression. However, the morphology of cardiac mitochondria was not affected by 1week of anoxia, even with a large increase in mitofusin 2 mRNA expression. Cardiac citrate synthase activity was not affected by anoxia, while cytochrome c oxidase activity was increased. We show how mitochondria respond to anoxia. A mechanistic understanding of how mitochondrial function can be maintained in anoxia may provide new perspectives to reduce mitochondrial damage in anoxia-sensitive organisms.

Carrier-mediated serotonin efflux induced by pharmacological anoxia in the rat heart in vivo.

Serotonin (5-HT) accumulates in the heart during myocardial ischaemia and induces deleterious effects on the cardiomyocytes. We aimed to investigate whether carrier-mediated 5-HT efflux contributed to the increase in interstitial 5-HT level during ischaemia. Using microdialysis technique applied to the heart of anaesthetised Wistar rats, myocardial interstitial concentration of 5-HT was measured by electro-chemical detection coupled with high-performance liquid chromatography (HPLC-ECD) while simultaneously various pharmacological agents, which create a similar condition to ischaemia, were locally administered by reverse-microdialysis. Sodium cyanide-induced chemical anoxia increased dialysate 5-HT concentration. A similar increase in dialysate 5-HT concentration was induced by ouabain, an inhibitor of sodium-potassium ATPase and reserpine, an inhibitor of vesicular monoamine transporter. Fluoxetine, a selective serotonin reuptake inhibitor raised the baseline level of 5-HT, and neither sodium cyanide nor the combination of ouabain and reserpine induced further increase in 5-HT in the presence of fluoxetine. The results indicate that reverse transport of 5-HT via SERT, which is caused by an impaired ion gradient, contributes to the rise in interstitial level of 5-HT during ischaemia suggesting carrier-mediated 5-HT efflux occurs in the heart in vivo.

Embryos of an Antarctic zooplankton require anoxia for dormancy, are permeable to lipophilic chemicals, and reside in sediments containing PCBs

Zooplankton in Antarctic maritime lakes face challenges imposed by anthropogenic chemicals. Studies on temperate species suggest that lipophilic chemicals will accumulate in dormant embryos of Antarctic zooplankton and decrease hatching success, thereby threatening centuries of accumulated genetic diversity that would increase population resilience in the face of climate change. We evaluated the potential for lakes to act as sinks for legacy pollutants in the maritime Antarctic by testing sediments for polychlorinated biphenyls (PCBs) previously identified in soil, flora and fauna of lake catchments. Direct tests of embryo permeability to chemicals are confounded by potential adhesion of chemicals to the embryo surface and limited biomass available. Therefore, in order to assess the potential for lipophilic chemicals to penetrate and passively accumulate in dormant embryos of Antarctic lacustrine zooplankton, we evaluated the effect of anoxia on post-diapause development in the calanoid copepod, Boeckella poppei, and then used chemical anoxia induced by rotenone as a reporter for permeability of these embryos to moderately lipophilic chemicals. The data presented demonstrate that embryos of B. poppei from Antarctic lake sediments will passively accumulate moderately lipophilic chemicals while lying dormant in anoxic sediments. Implications for legacy POPs in sediments of Antarctic maritime lakes are discussed.

Facilitation of distinct inhibitory synaptic inputs by chemical anoxia in neurons in the oculomotor, facial and hypoglossal motor nuclei of the rat

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons in the brainstem and spinal cord. Clinical studies have indicated that there is a distinct region-dependent difference in the vulnerability of motor neurons. For example, the motor neurons in the facial and hypoglossal nuclei are more susceptible to neuronal death than those in the oculomotor nucleus. To understand the mechanism underlying the differential susceptibility to cell death of the neurons in different motor nuclei, we compared the effects of chemical anoxia on the membrane currents and postsynaptic currents in different motor nuclei. The membrane currents were recorded from neurons in the oculomotor, facial and hypoglossal nuclei in brain slices of juvenile Wistar rats by using whole-cell recording in the presence of tetrodotoxin that prevents action potential-dependent synaptic transmission. NaCN consistently induced an inward current and a significant increase in the frequency of spontaneous synaptic inputs in neurons from these three nuclei. However, this increase in the synaptic input frequency was abolished by strychnine, a glycine receptor antagonist, but not by picrotoxin in neurons from the hypoglossal and facial nuclei, whereas that in neurons from the oculomotor nucleus was abolished by picrotoxin, but not by strychnine. Blocking ionotropic glutamate receptors did not significantly affect the NaCN-induced release facilitation in any of the three motor nuclei. These results suggest that anoxia selectively facilitates glycine release in the hypoglossal and facial nuclei and GABA release in the oculomotor nucleus. The region-dependent differences in the neurotransmitters involved in the anoxia-triggered release facilitation might provide a basis for the selective vulnerability of motor neurons in the neurodegeneration associated with ALS.

Decreased sensitivity of palmitoyl protein thioesterase 1-deficient neurons to chemical anoxia.

Infantile CLN1 disease, also known as infantile neuronal ceroid lipofuscinosis, is a fatal childhood neurodegenerative disorder caused by mutations in the CLN1 gene. CLN1 encodes a soluble lysosomal enzyme, palmitoyl protein thioesterase 1 (PPT1), and it is still unclear why neurons are selectively vulnerable to the loss of PPT1 enzyme activity in infantile CLN1 disease. To examine the effects of PPT1 deficiency on several well-defined neuronal signaling and cell death pathways, different toxic insults were applied in cerebellar granule neuron cultures prepared from wild type (WT) and palmitoyl protein thioesterase 1-deficient (Ppt1 -/- ) mice, a model of infantile CLN1 disease. Glutamate uptake inhibition by t-PDC (L-trans-pyrrolidine-2,4-dicarboxylic acid) or Zn2+-induced general mitochondrial dysfunction caused similar toxicity in WT and Ppt1 -/- cultures. Ppt1 -/- neurons, however, were more sensitive to mitochondrial complex I inhibition by MPP+ (1-methyl-4-phenylpyridinium), and had significantly decreased sensitivity to chemical anoxia induced by the mitochondrial complex IV inhibitor, sodium azide. Our results indicate that PPT1 deficiency causes alterations in the mitochondrial respiratory chain.

Effects of opioids on proximal renal tubular cells undergoing ATP depletion

This study investigated the effect of morphine, fentanyl, butorphanol and buprenorphine on viability and caspase-3 activity in renal proximal tubular cells exposed to opioids for 2 h before or 12 h after chemical anoxia. Cell viability decreased regardless the treatment although intracellular ATP content was elevated in morphine and fentanyl pre-treated cells at 12 h. Anoxia increased caspase activity but this effect was significantly reduced in cells treated before or after with morphine, fentanyl and in cell treated with butorphanol for 12 h. No influence of buprenorphine was detected. Morphine, fentanyl and butorphanol might have protective effects during kidney ischemia.

Permeability transition pore-dependent and PARP-mediated depletion of neuronal pyridine nucleotides during anoxia and glucose deprivation.

Exposure of rat cortical neurons to combined oxygen and glucose deprivation results in loss of NAD(P)H autofluorescence that is only partially reversible following restoration of oxygen and glucose, suggesting catabolism of pyridine nucleotides. This study tested the hypothesis that metabolic inhibition caused by cyanide-induced chemical anoxia plus glucose deprivation promotes both release of mitochondrial NAD(H) in response to opening of the permeability transition pore (PTP) and NAD(P)(H) degradation through activation of poly (ADP-ribose) polymerase (PARP). The NAD(P)H autofluorescence of rat neonatal cortical neurons was monitored during and following acute (10-30min) exposure to the respiratory inhibitor, cyanide, in the absence and presence of glucose. Because nitric oxide-derived peroxynitrite is a known activator of PARP, we additionally assessed the effect of a nitric oxide generating agent on the NAD(P)H autofluorescence response to chemical anoxia plus glucose deprivation. Cyanide induced a rapid increase in autofluorescence, followed by a steady decline promoted by the presence of nitric oxide. This decline was primarily due to NAD(H) catabolism, as verified by measurements of total NAD(H) present in cellular extracts. Catabolism was partially blocked by an inhibitor of PARP, by a PTP inhibitor, and by either glucose or pyruvate as a source of reducing power. Overall, data suggest that metabolic, oxidative, and nitrosative stress during in vitro neuronal anoxia and glucose deprivation result in release of mitochondrial pyridine nucleotides in response to PTP opening and rapid, extensive NAD(H) degradation mediated by PARP activation. These events may contribute to the metabolic dysfunction that occurs in vivo during cerebral ischemia and reperfusion and therefore represent prime targets for neuroprotection.

Activity and metabolism-related Ca2+ and mitochondrial dynamics in co-cultured human fetal cortical neurons and astrocytes

Neurons and neighboring astrocytic glia are mostly studied in nervous tissues from rodents whereas less is known on their properties and interactions in the human brain. Here, confocal/multiphoton fluorescence imaging for several hours revealed that co-cultured fetal human cortical neurons and astrocytes show pronounced spontaneous rises of cytosolic Ca2+ which last for up to several minutes without concomitant changes in either movements or membrane potential of mitochondria. Similar Ca2+ rises were evoked mainly in neurons by bath-applied glutamate or γ-aminobutyric acid (GABA) acting via N-methyl-d-aspartate (NMDA)+AMPA/Kainate and GABAA receptors, respectively. Predominantly in astrocytes, Ca2+ baseline was elevated by adenosine diphosphate (ADP) and adenosine triphosphate (ATP) acting via P2Y1 and P2X7 receptors, likely causing the release of glutamate and glutamine. Mainly astrocytes responded to histamine, whereas the activation of muscarinic acetylcholine (ACh) receptors raised Ca2+ in both cell types. Evoked neuronal and astrocytic Ca2+ rises could last for several minutes without affecting mitochondrial movements or membrane potential. In contrast, reversible depolarization of mitochondrial membrane potential accompanied neuronal Ca2+ rises induced by cyanide-evoked chemical anoxia or the uncoupling of mitochondrial respiration with carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP). During such metabolic perturbation, mitochondrial depolarization also occurred in astrocytes, whereas Ca2+ was largely unaffected. In summary, fetal human cortical neurons and astrocytes show distinct patterns of neuro/glio-transmitter- and metabolically-evoked Ca2+ rises and possess active mitochondria. One aspect of our discussion deals with the question of whether the functional mitochondria contribute to cellular Ca2+ homeostasis that seems to be already well-developed in fetal human cortical brain cells.

STK25 Protein Mediates TrkA and CCM2 Protein-dependent Death in Pediatric Tumor Cells of Neural Origin

The TrkA receptor tyrosine kinase induces death in medulloblastoma cells via an interaction with the cerebral cavernous malformation 2 (CCM2) protein. We used affinity proteomics to identify the germinal center kinase class III (GCKIII) kinases STK24 and STK25 as novel CCM2 interactors. Down-modulation of STK25, but not STK24, rescued medulloblastoma cells from NGF-induced TrkA-dependent cell death, suggesting that STK25 is part of the death-signaling pathway initiated by TrkA and CCM2. CCM2 can be phosphorylated by STK25, and the kinase activity of STK25 is required for death signaling. Finally, STK25 expression in tumors is correlated with positive prognosis in neuroblastoma patients. These findings delineate a death-signaling pathway downstream of neurotrophic receptor tyrosine kinases that may provide targets for therapeutic intervention in pediatric tumors of neural origin.

Exceptional cardiac anoxia tolerance in tilapia (Oreochromis hybrid)

Anoxic survival requires the matching of cardiac ATP supply (i.e. maximum glycolytic potential, MGP) and demand (i.e. cardiac power output, PO). We examined the idea that the previously observed in vivo downregulation of cardiac function during exposure to severe hypoxia in tilapia (Oreochromis hybrid) represents a physiological strategy to reduce routine PO to within the heart's MGP. The MGP of the ectothermic vertebrate heart has previously been suggested to be ∼70 nmol ATP s(-1) g(-1), sustaining a PO of ∼0.7 mW g(-1) at 15°C. We developed an in situ perfused heart preparation for tilapia (Oreochromis hybrid) and characterized the routine and maximum cardiac performance under both normoxic (>20 kPa O(2)) and severely hypoxic perfusion conditions (<0.20 kPa O(2)) at pH 7.75 and 22°C. The additive effects of acidosis (pH 7.25) and chemical anoxia (1 mmol l(-1) NaCN) on cardiac performance in severe hypoxia were also examined. Under normoxic conditions, cardiac performance and myocardial oxygen consumption rate were comparable to those of other teleosts. The tilapia heart maintained a routine normoxic cardiac output (Q) and PO under all hypoxic conditions, a result that contrasts with the hypoxic cardiac downregulation previously observed in vivo under less severe conditions. Thus, we conclude that the in vivo downregulation of routine cardiac performance in hypoxia is not needed in tilapia to balance cardiac energy supply and demand. Indeed, the MGP of the tilapia heart proved to be quite exceptional. Measurements of myocardial lactate efflux during severe hypoxia were used to calculate the MGP of the tilapia heart. The MGP was estimated to be 172 nmol ATP s(-1) g(-1) at 22°C, and allowed the heart to generate a PO(max) of at least ∼3.1 mW g(-1), which is only 30% lower than the PO(max) observed with normoxia. Even with this MGP, the additional challenge of acidosis during severe hypoxia decreased maximum ATP turnover rate and PO(max) by 30% compared with severe hypoxia alone, suggesting that there are probably direct effects of acidosis on cardiac contractility. We conclude that the high maximum glycolytic ATP turnover rate and levels of PO, which exceed those measured in other ectothermic vertebrate hearts, probably convey a previously unreported anoxia tolerance of the tilapia heart, but a tolerance that may be tempered in vivo by the accumulation of acidotic waste during anoxia.

Differential Role for ERK2 in Anoxia-induced Activation of Transcription and Translation of Hsp70 in NIH 3T3 Cells

Hsp70 has the ability to enhance the recovery of stressed cells by its ability to catalyze the reassembly of damaged proteins. Such a chaperoning function is essential for the Hsp70-mediated protection against anoxic stress that causes protein denaturation. We have studied induction of both transcription and translation of Hsp70 during recovery from chemical anoxia and the role of the extracellular signal regulated kinase ERK2 in this induction of Hsp70. 10 mM azide for 30 minutes (chemical anoxia) significantly inhibited the activity of ERK2 (measured as phospho-ERK) but the ERK-2 activity is rapidly increased in a MEK-independen manner, when azide is washed out of the cells. Chemical anoxia and overnight recovery induced Hsp70 expression (analyzed by Western blotting) and this was inhibited by actinomycin D as well as by cycloheximide showing that induction of both translation and transcription was involved. Inhibition of the MAP kinase p38, which was transiently activated during chemical anoxia, had no effect on the increase in Hsp70 expression whereas an inhibitor of reactive oxygen species and inhibition of the phosphatase PP1 and PP2a inhibited the increase in Hsp70 expression. Inhibition of ERK2 by the MEK inhibitor PD98059 resulted in strong inhibition of Hsp70 protein expression and simultaneous stimulation of hsp70 transcription.

Podocyte Injury and Albuminuria in Mice with Podocyte-Specific Overexpression of the Ste20-Like Kinase, SLK

SLK expression and activity are increased during kidney development and recovery from renal ischemia-reperfusion injury. In cultured cells, SLK promotes F-actin destabilization as well as apoptosis, partially via the p38 kinase pathway. To better understand the effects of SLK in vivo, a transgenic mouse model was developed where SLK was expressed in a podocyte-specific manner using the mouse nephrin promoter. Offspring of four founder mice carried the SLK transgene. Among male transgenic mice, 66% developed albuminuria at approximately 3 months of age, and the albuminuric mice originated from three of four founders. Overall, the male transgenic mice demonstrated about fivefold greater urinary albumin/creatinine compared with male non-transgenic mice. Transgenic and non-transgenic female mice did not develop albuminuria, suggesting that females were less susceptible to glomerular filtration barrier damage than their male counterparts. In transgenic mice, electron microscopy revealed striking podocyte injury, including poorly formed or effaced foot processes, and edematous and vacuolated cell bodies. By immunoblotting, nephrin expression was decreased in glomeruli of the albuminuric transgenic mice. Activation-specific phosphorylation of p38 was increased in transgenic mice compared with non-transgenic animals. Glomeruli of SLK transgenic mice showed around 30% fewer podocytes, and a reduction in F-actin compared with control glomeruli. Thus, podocyte SLK overexpression in vivo results in injury and podocyte loss, consistent with the effects of SLK in cultured cells.

Bromocriptine, an ergot alkaloid, inhibits excitatory amino acid release mediated by glutamate transporter reversal

Bromocriptine, a dopamine D 2 receptor agonist, has widely been used for patients with Parkinson's disease. The aim of the present study was to investigate the effect of bromocriptine on glutamate transporter. Since the astroglial glutamate transporter GLT-1 (EAAT2) is the predominant isoform in the forebrain, we generated EAAT2-expressing human embryonic kidney cells and immortalized mouse astrocytes. In the present studies, we observed a GLT-1-immunoreactive band and significant Na +-dependent d-[ 3H] aspartate uptake. Furthermore, the glutamate transporter inhibitors, dl- threo-β-benzyloxyaspartic acid (TBOA) and dihydrokainate (DHK), displayed a dose-dependent reduction of d-[ 3H] aspartate uptake in both types of cells. In contrast, cells exposed to either chemical anoxia or high KCl elicited a marked release of d-[ 3H] aspartate, and the release was inhibited by TBOA and DHK, implying the contribution of glutamate transporter reversal. Interestingly, we found that bromocriptine dose-dependently inhibits d-[ 3H] aspartate release elicited by chemical anoxia or high KCl, while no changes occurred in the uptake. The inhibitory action of bromocriptine was not affected by sulpiride, a dopamine D 2 receptor antagonist. On the other hand, bromocriptine had no effect on swelling-induced d-[ 3H] aspartate release, which is mediated by volume-regulated anion channels. In vivo studies revealed that bromocriptine suppresses the excessive elevation of glutamate levels in gerbils subjected to transient forebrain ischemia in a manner similar to DHK. Taken together, these results provide evidence that bromocriptine inhibits excitatory amino acid release via reversed operation of GLT-1 without altering forward transport.

Differences in in vitro cerebellar neuronal responses to hypoxia in eider ducks, chicken and rats

Ducks are well-known to be more tolerant to asphyxia than non-diving birds, but it is not known if their defences include enhanced neuronal hypoxia tolerance. To test this, we compared extracellular recordings of spontaneous activity in the Purkinje cell layer of 400 mum thick isolated cerebellar slices from eider ducks, chickens and rats, before, during and after 60 min hypoxia (95%N(2)-5%CO(2)) or chemical anoxia (hypoxia + 2 mM NaCN). Most slices rapidly lost activity in hypoxia, with or without recovery after rinse and return to normoxia (95%O(2)-5%CO(2)), but some maintained spontaneous activity throughout the insult. Proportions of 'surviving' (i.e. recovering or active) duck slices were significantly higher than for chickens in anoxia, and relative activity levels were higher for ducks than for chickens during hypoxia, anoxia and recovery. Survival of rat slices was significantly poorer than for birds under all conditions. Results suggest that (1) duck cerebellar neurons are intrinsically more hypoxia-tolerant than chicken neurons; (2) avian neurons are more hypoxia-tolerant than rat neurons, and (3) the enhanced hypoxic tolerance of duck neurons largely depended on efficient anaerobiosis since it mainly manifested itself in chemical anoxia. Mechanisms underlying the observed differences in neuronal hypoxic responses remain to be elucidated.

The Ste20-like kinase SLK promotes p53 transactivation and apoptosis

Expression and activity of the germinal center kinase [corrected] SLK are increased during kidney development and recovery from renal ischemia-reperfusion injury. SLK promotes apoptosis, in part, via pathway(s) involving apoptosis signal-regulating kinase-1 and p38 mitogen-activated protein kinase. This study addresses the role of p53 as a potential effector of SLK. p53 transactivation was measured after transient transfection of a luciferase reporter plasmid that contains a p53 cis-acting enhancer element. Overexpression of SLK in COS-1 cells and cotransfection of SLK and p53-wild type (wt) cDNAs in glomerular epithelial cells (GECs) stimulated p53 transactivational activity, as measured by a p53 response element-driven luciferase reporter. In GECs, chemical anoxia followed by glucose reexposure (in vitro ischemia-reperfusion) increased p53 reporter activity, and this increase was amplified by overexpression of SLK. Expression of SLK induced p53 phosphorylation on serine (S)-33 and S315. In GECs, cotransfection of SLK with p53-wt, p53-S33A, p53-S315A, or p53-S33A+S315A mutants showed that only the double mutation abolished the SLK-induced increase in p53 reporter activity. SLK-induced stimulation of p53 reporter activity was attenuated by inhibition of JNK. Overexpression of SLK amplified apoptosis induced by subjecting cells to in vitro ischemia-reperfusion injury, while ectopic expression of a dominant negative SLK mutant attenuated the ischemia-reperfusion-induced apoptosis. The p53 transactivation inhibitor pifithrin-alpha significantly attenuated the amount of apoptosis after ischemia-reperfusion and SLK overexpression. Thus SLK induces p53 phosphorylation and transactivation, which enhances apoptosis after in vitro ischemia-reperfusion injury.

3-Nitropropionic acid depresses spinal reflexes involving GABAergic and glycinergic transmission in neonatal rat spinal cord in vitro

Aims 3-Nitropropionic acid (3-NPA) is a naturally occurring fungal toxin that leads to ATP-depletion by inhibiting mitochondrial succinate dehydrogenase and produces chemical anoxia. The present study was conducted to identify the involvement of inhibitory system in 3-NPA-induced depression of spinal reflexes. Methods The monosynaptic (MSR) and polysynaptic reflex (PSR) potentials were recorded at ventral root by stimulating the corresponding dorsal root in hemisected (sagitally) spinal cord from 4–8 day old rats. Effect of 3-NPA in the absence and presence of antagonists was evaluated on the reflexes. Key findings Superfusion of 3-NPA (3.4 mM) depressed the reflexes in a time-dependent manner abolishing them by 35 min. The T-50 values were around 18 and 16 min for MSR and PSR, respectively. An NMDA receptor antagonist, DL-2-amino-5-phosphonovaleric acid (10 μM) failed to block the 3-NPA (3.4 mM)-induced depression of reflexes. Superfusion of bicuculline (GABA A receptor antagonist; 1 μM), or strychnine (glycine A receptor antagonist; 1 μM) antagonized the 3-NPA-induced depression of reflexes significantly. The T-50 values were 26 and 30 min in bicuculline and strychnine pretreated groups, respectively and were significantly greater than 3-NPA only group. Significance The results indicate that 3-NPA-induced depression of spinal reflexes is partially mediated by GABAergic and glycinergic inhibitory transmission.

Metabolic Challenge to Glia Activates an Adenosine-Mediated Safety Mechanism that Promotes Neuronal Survival by Delaying the Onset of Spreading Depression Waves

In a model of glial-specific chemical anoxia, we have examined how astrocytes influence both synaptic transmission and the viability of hippocampal pyramidal neurons. This relationship was assessed using electrophysiological, pharmacological, and biochemical techniques in rat slices and cell cultures, and oxidative metabolism was selectively impaired in glial cells by exposure to the mitochondrial gliotoxin, fluoroacetate. We found that synaptic transmission was blocked shortly after inducing glial metabolic stress and peri-infarct-like spreading depression (SD) waves developed within 1 to 2 h of treatment. Neuronal electrogenesis was not affected until SD waves developed, thereafter decaying irreversibly. The blockage of synaptic transmission was totally reversed by A(1) adenosine receptor antagonists, unlike the development of SD waves, which appeared earlier under these conditions. Such blockage led to a marked reduction in the electrical viability of pyramidal neurons 1 h after gliotoxin treatment. Cell culture experiments confirmed that astrocytes indeed release adenosine. We interpret this early glial response as a novel safety mechanism that allocates metabolic resources to vital processes when the glia itself sense an energy shortage, thereby delaying or preventing entry into massive lethal ischemic-like depolarization. The implication of these results on the functional recovery of the penumbra regions after ischemic insults is discussed.

SOK1 Translocates from the Golgi to the Nucleus upon Chemical Anoxia and Induces Apoptotic Cell Death

SOK1 is a Ste20 protein kinase of the germinal center kinase (GCK) family that has been shown to be activated by oxidant stress and chemical anoxia, a cell culture model of ischemia. More recently, it has been shown to be localized to the Golgi apparatus, where it functions in a signaling pathway required for cell migration and polarization. Herein, we demonstrate that SOK1 regulates cell death after chemical anoxia, as its down-regulation by RNA interference enhances cell survival. Furthermore, expression of SOK1 elicits apoptotic cell death by activating the intrinsic pathway. We also find that a cleaved form of SOK1 translocates from the Golgi to the nucleus after chemical anoxia and that this translocation is dependent on both caspase activity and on amino acids 275-292, located immediately C-terminal to the SOK1 kinase domain. Furthermore, SOK1 entry into the nucleus is important for the cell death response since SOK1 mutants unable to enter the nucleus do not induce cell death. In summary, SOK1 is necessary to induce cell death and can induce death when overexpressed. Furthermore, SOK1 appears to play distinctly different roles in stressed versus non-stressed cells, regulating cell death in the former.

Comparison of two anoxia models in rainbow trout cells by a 2‐DE and MS/MS‐based proteome approach

In the literature, a variety of ways have been used to obtain anoxia, and most often results are compared between studies without taking into consideration how anoxia has been obtained. Here, we provide a comprehensive study of two types of anoxia, using a proteomics approach to compare changes in protein expression. The two investigated situations were 30 min of chemical anoxia (10 mM NaN(3)) followed by reoxygenation overnight (CR) and 2 h of N(2)-induced anoxia (achieved by flushing with N(2)) followed by reoxygenation overnight (NR), after which samples were resolved by 2-DE. Forty-five protein spots changed their abundance in response to CR and 35 protein spots changed their abundance in response to NR, but only six proteins changed their abundance in response to both stimuli. By the means of MS/MS, 40 protein spots were identified including proteins involved in processes like cell protection and protein synthesis. It was also revealed that the level of a number of keratins was down-regulated. This study therefore provides a valuable comparison of two different anoxia models and shows that great care should be taken when comparing the effects of anoxia in studies that have used different types and durations of anoxia.

Role of calcium-independent phospholipase A2in complement-mediated glomerular epithelial cell injury

In experimental membranous nephropathy, complement C5b-9-induced glomerular epithelial cell (GEC) injury leads to morphological changes in GEC and proteinuria, in association with phospholipase A(2) (PLA(2)) activation. The present study addresses the role of calcium-independent PLA(2) (iPLA(2)) in GEC injury. iPLA(2)beta short and iPLA(2)gamma were expressed in cultured rat GEC and normal rat glomeruli. To determine whether iPLA(2) is involved in complement-mediated arachidonic acid (AA) release, GEC were stably transfected with iPLA(2)gamma or iPLA(2)beta cDNAs (GEC-iPLA(2)gamma; GEC-iPLA(2)beta). Compared with control cells (GEC-Neo), GEC-iPLA(2)gamma and GEC-iPLA(2)beta demonstrated greater expression of iPLA(2) proteins and activities. Complement-mediated release of [(3)H]AA was augmented significantly in GEC-iPLA(2)gamma compared with GEC-Neo, and the augmented [(3)H]AA release was inhibited by the iPLA(2)-directed inhibitor bromoenol lactone (BEL). For comparison, overexpression of iPLA(2)gamma also amplified [(3)H]AA release after incubation of GEC with H(2)O(2), or chemical anoxia followed by reexposure to glucose (in vitro ischemia-reperfusion injury). In parallel with release of [(3)H]AA, complement-mediated production of prostaglandin E(2) was amplified in GEC-iPLA(2)gamma. Complement-mediated cytotoxicity was attenuated significantly in GEC-iPLA(2)gamma compared with GEC-Neo, and the cytoprotective effect of iPLA(2)gamma was reversed by BEL, and in part by indomethacin. Overexpression of iPLA(2)beta did not amplify complement-dependent [(3)H]AA release, but nonetheless attenuated complement-mediated cytotoxicity. Thus iPLA(2)gamma may be involved in complement-mediated release of AA. Expression of iPLA(2)gamma or iPLA(2)beta induces cytoprotection against complement-dependent GEC injury. Modulation of iPLA(2) activity may prove to be a novel approach to reducing GEC injury.