-
The Journal of Physiology Nov 20001. Persistent sodium channel activity was recorded before and during hypoxia from cell-attached and inside-out patches obtained from cultured hippocampal neurons at a...
1. Persistent sodium channel activity was recorded before and during hypoxia from cell-attached and inside-out patches obtained from cultured hippocampal neurons at a pipette potential (Vp) of +30 mV. Average mean current (IU) of these channels was very low under normoxic conditions and was similar in cell-attached and excised inside-out patches (-0.018 +/- 0.010 and -0.025 +/- 0.008 pA, respectively, n = 24). 2. Hypoxia increased the activity of persistent sodium channels in 10 cell-attached patches (IU increased from -0. 026 +/- 0.016 pA in control to -0.156 +/- 0.034 pA during hypoxia, n = 4, P = 0.013). The increased persistent sodium channel activity was most prominent at a VP between +70 and +30 mV (membrane potential, Vm = -70 to -30 mV) and could be blocked by lidocaine, TTX or R56865 (n = 5). Sodium cyanide (NaCN, 5 mM; 0.5-5 min) increased persistent sodium channel activity in cell-attached patches (n = 3) in a similar manner. 3. Hypoxia also increased sodium channel activity in inside-out patches from hippocampal neurons. Within 2-4 min of exposure to hypoxia, I had increased 9-fold to -0. 18 +/- 0.04 pA (n = 21, P = 0.001). Sodium channel activity increased further with longer exposures to hypoxia. 4. The hypoxia-induced sodium channel activity in inside-out patches could be inhibited by exposure to 10-100 microM lidocaine applied via the bath solution (I = -0.03 +/- 0.01 pA, n = 8) or by perfusion of the pipette tip with 1 microM TTX (I = -0.01 +/- 0.01 pA, n = 3). 5. The reducing agent dithiothreitol (DTT, 2-5 mM) rapidly abolished the increase in sodium channel activity caused by hypoxia in excised patches (I = -0.01 +/- 0.01 pA, n = 4). Similarly, reduced glutathione (GSH, 5-20 mM) also reversed the hypoxia-induced increase in sodium channel activity (IU = -0.02 +/- 0.02 pA, n = 5). 6. These results suggest that persistent sodium channels in neurons can sense O2 levels in excised patches of plasma membrane. Hypoxia triggers an increase in sodium channel activity. The redox reaction involved in increasing the sodium channel activity probably occurs in an auxiliary regulatory protein, co-localized in the plasma membrane.
Topics: Anesthetics, Local; Animals; Antimetabolites; Cell Hypoxia; Cells, Cultured; Hippocampus; Lidocaine; Neurons; Oxidation-Reduction; Oxygen Consumption; Patch-Clamp Techniques; Rats; Rats, Wistar; Sodium Channel Blockers; Sodium Channels; Sodium Cyanide; Tetrodotoxin
PubMed: 11080255
DOI: 10.1111/j.1469-7793.2000.00107.x -
Journal of Bacteriology Jan 1995The branched respiratory chain of Pseudomonas aeruginosa contains at least two terminal oxidases which are active under normal physiological conditions. One of these,...
The branched respiratory chain of Pseudomonas aeruginosa contains at least two terminal oxidases which are active under normal physiological conditions. One of these, cytochrome co, is a cytochrome c oxidase which is completely inhibited by concentrations of the respiratory inhibitor potassium cyanide as low as 100 microM. The second oxidase, the cyanide-insensitive oxidase, is resistant to cyanide concentrations in excess of 1 mM as well as to sodium azide. In this work, we describe the isolation and characterization of a mutant of P. aeruginosa defective in cyanide-insensitive respiration. This insertion mutant was isolated with mini-D171 (a replication-defective derivative of the P. aeruginosa phage D3112) as a mutagen and by screening the resulting tetracycline-resistant transductants for the loss of ability to grow in the presence of 1 mM sodium azide. Polarographic studies on the NADH-mediated respiration rate of the mutant indicated an approximate 50% loss of activity, and titration of this activity against increasing cyanide concentrations gave a monophasic curve clearly showing the complete loss of cyanide-insensitive respiration. The mutated gene for a mutant affected in the cyanide-insensitive, oxidase-terminated respiratory pathway has been designated cio. We have complemented the azide-sensitive phenotype of this mutant with a wild-type copy of the gene by in vivo cloning with another mini-D element, mini-D386, carried on plasmid pADD386. The complemented cio mutant regained the ability to grow on medium containing 1 mM azide, titration of its NADH oxidase activity with cyanide gave a biphasic curve similar to that of the wild-type organism, and the respiration rate returned to normal levels. Spectral analysis of the cytochrome contents of the membranes of the wild type, the cio mutant, and the complemented mutant suggests that the cio mutant is not defective in any membrane-bound cytochromes and that the complementing gene does not encode a heme protein.
Topics: Azides; Cloning, Molecular; Cyanides; Drug Resistance, Microbial; Genetic Complementation Test; Mutagenesis, Insertional; Oxidoreductases; Oxygen Consumption; Pseudomonas aeruginosa; Sodium Azide
PubMed: 7814333
DOI: 10.1128/jb.177.2.432-438.1995 -
The New Phytologist Jan 2021In drought-stressed plants a coordinated cascade of chemical and transcriptional adjustments occurs at the same time as embolism formation. While these processes do not...
In drought-stressed plants a coordinated cascade of chemical and transcriptional adjustments occurs at the same time as embolism formation. While these processes do not affect embolism formation during stress, they may prime stems for recovery during rehydration by modifying apoplast pH and increasing sugar concentration in the xylem sap. Here we show that in vivo treatments modifying apoplastic pH (stem infiltration with a pH buffer) or reducing stem metabolic activity (infiltration with sodium vanadate and sodium cyanide; plant exposure to carbon monoxide) can reduce sugar accumulation, thus disrupting or delaying the recovery process. Application of the vanadate treatment (NaVO an inhibitor of many ATPases) completely halted recovery from drought-induced embolism for up to 24 h after re-irrigation, while partial recovery was observed in vivo in control plants using X-ray microcomputed tomography. Our results suggest that stem hydraulic recovery in poplar is a biological, energy-dependent process that coincides with accumulation of sugars in the apoplast during stress. Recovery and damage are spatially coordinated, with embolism formation occurring from the inside out and refilling from the outside in. The outside-in pattern highlights the importance of xylem proximity to the sugars within the phloem to the embolism recovery process.
Topics: Droughts; Embolism; Plant Stems; Water; X-Ray Microtomography; Xylem
PubMed: 32890423
DOI: 10.1111/nph.16912 -
Journal of Neurochemistry Oct 2019Volume-regulated anion channel (VRAC) is a glutamate-permeable channel that is activated by physiological and pathological cell swelling and promotes ischemic brain...
Volume-regulated anion channel (VRAC) is a glutamate-permeable channel that is activated by physiological and pathological cell swelling and promotes ischemic brain damage. However, because VRAC opening requires cytosolic ATP, it is not clear if and how its activity is sustained in the metabolically compromised CNS. In the present study, we used cultured astrocytes - the cell type which shows prominent swelling in stroke - to model how metabolic stress and changes in gene expression may impact VRAC function in the ischemic and post-ischemic brain. The metabolic state of primary rat astrocytes was modified with chemical inhibitors and examined using luciferin-luciferase ATP assays and a Seahorse analyzer. Swelling-activated glutamate release was quantified with the radiotracer D-[ H]aspartate. The specific contribution of VRAC to swelling-activated glutamate efflux was validated by RNAi knockdown of the essential subunit, leucine-rich repeat-containing 8A (LRRC8A); expression levels of VRAC components were measured with qRT-PCR. Using this methodology, we found that complete metabolic inhibition with the glycolysis blocker 2-deoxy-D-glucose and the mitochondrial poison sodium cyanide reduced astrocytic ATP levels by > 90% and abolished glutamate release from swollen cells (via VRAC). When only mitochondrial respiration was inhibited by cyanide or rotenone, the intracellular ATP levels and VRAC activity were largely preserved. Bypassing glycolysis by providing the mitochondrial substrates pyruvate and/or glutamine led to partial recovery of ATP levels and VRAC activity. Unexpectedly, the metabolic block of VRAC was overridden when ATP-depleted cells were exposed to extreme cell swelling (≥ 50% reduction in medium osmolarity). Twenty-four hour anoxic adaptation caused a moderate reduction in the expression levels of the VRAC component LRRC8A, but no significant changes in VRAC activity. Overall, our findings suggest that (i) astrocytic VRAC activity and metabolism can be sustained by low levels of glucose and (ii) the inhibitory influence of diminishing ATP levels and the stimulatory effect of cellular swelling are the two major factors that govern VRAC activity in the ischemic brain.
Topics: Adenosine Triphosphate; Animals; Astrocytes; Cell Size; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Glucose; Glutamic Acid; Ischemia; Male; Rats; Rats, Sprague-Dawley
PubMed: 31032919
DOI: 10.1111/jnc.14711 -
RSC Advances Nov 2018The co-metabolic degradation kinetics, microbial growth kinetics and electricity generation capacity were researched of strain MC-1 in a MFC (microbial fuel cell). The...
The co-metabolic degradation kinetics, microbial growth kinetics and electricity generation capacity were researched of strain MC-1 in a MFC (microbial fuel cell). The results show that Haldane and Aiba models suit the growth kinetics of a single substrate (sodium acetate) MFC with 0.995 correlation coefficient. Moreover, the Haldane model was appropriate to describe the growth kinetics of a single substrate (sodium cyanide) MFC with 0.986 correlation coefficient. The growth kinetics of a mixed substrate MFC can be explained well by the SKIP model with correlation coefficient 0.995. Second order and three-half order models were found to suitably describe the cyanide degradation process. The maximum output voltage of MFC and the cyanide degradation efficiency were significantly enhanced by using sodium acetate and cyanide as mixed substrates. Also, the trend of electricity production is related to the growth cycle of microorganisms in a MFC.
PubMed: 35558197
DOI: 10.1039/c8ra08775j -
Journal of Neuroinflammation Apr 2010Activated complement system is known to mediate neuroinflammation and neurodegeneration following exposure to hypoxic-ischemic insults. Therefore, inhibition of the...
BACKGROUND
Activated complement system is known to mediate neuroinflammation and neurodegeneration following exposure to hypoxic-ischemic insults. Therefore, inhibition of the complement activation cascade may represent a potential therapeutic strategy for the management of ischemic brain injury. Decay-accelerating factor (DAF, also known as CD55) inhibits complement activation by suppressing the function of C3/C5 convertases, thereby limiting local generation or deposition of C3a/C5a and membrane attack complex (MAC or C5b-9) production. The present study investigates the ability of DAF to protect primary cultured neuronal cells subjected to sodium cyanide (NaCN)-induced hypoxia from degeneration and apoptosis.
METHODS
Cultured primary cortical neurons from embryonic Sprague-Dawley rats were assigned one of four groups: control, DAF treatment alone, hypoxic, or hypoxic treated with DAF. Hypoxic cultures were exposed to NaCN for 1 hour, rinsed, followed by 24 hour exposure to 200 ng/ml of recombinant human DAF in normal medium. Human DAF was used in the present study and it has been shown to effectively regulate complement activation in rats. Neuronal cell function, morphology and viability were investigated by measuring plateau depolarization potential, counting the number dendritic spines, and observing TUNEL and MTT assays. Complement C3, C3a, C3a receptor (R) production, C3a-C3aR interaction and MAC formation were assessed along with the generation of activated caspase-9, activated caspase-3, and activated Src.
RESULTS
When compared to controls, hypoxic cells had fewer dendritic spines, reduced plateau depolarization accompanied by increased apoptotic activity and accumulation of MAC, as well as up-regulation of C3, C3a and C3aR, enhancement of C3a-C3aR engagement, and elevated caspase and Src activity. Treatment of hypoxic cells with 200 ng/ml of recombinant human DAF resulted in attenuation of neuronal apoptosis and exerted significant protection against neuronal dendritic spine loss and plateau depolarization reduction. Furthermore, treatment with DAF resulted in decreased accumulation of C3a, MAC, C3a-C3aR interaction, caspase-9, activated caspase-3, and pTyr416-Src (activated Src) tyrosine kinase.
CONCLUSION
DAF was found to reduce neuronal cell death and apoptosis in NaCN induced hypoxia. This effect is attributed to the ability of DAF to limit complement activation and inhibit the activity of Src and caspases 9 and 3. This study supports the inhibiting of complement as a neuroprotective strategy against CNS ischemia/reperfusion injury.
Topics: Animals; Apoptosis; Blotting, Western; CD55 Antigens; Caspase 3; Caspases; Cell Hypoxia; Cell Survival; Cells, Cultured; Cerebral Cortex; Complement C3a; Complement System Proteins; Dendrites; Fluorescent Antibody Technique; Immunohistochemistry; In Situ Nick-End Labeling; Neurons; Neuroprotective Agents; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptors, Complement; src-Family Kinases
PubMed: 20380727
DOI: 10.1186/1742-2094-7-24 -
Journal of Medical Toxicology :... Dec 2018Cyanide (CN) poisoning is a serious chemical threat from accidental or intentional exposures. Current CN exposure treatments, including direct binding agents,...
INTRODUCTION
Cyanide (CN) poisoning is a serious chemical threat from accidental or intentional exposures. Current CN exposure treatments, including direct binding agents, methemoglobin donors, and sulfur donors, have several limitations. Dimethyl trisulfide (DMTS) is capable of reacting with CN to form the less toxic thiocyanate with high efficiency, even without the sulfurtransferase rhodanese. We investigated a soluble DMTS formulation with the potential to provide a continuous supply of substrate for CN detoxification which could be delivered via intramuscular (IM) injection in a mass casualty situation. We also used non-invasive technology, diffuse optical spectroscopy (DOS), to monitor physiologic changes associated with CN exposure and reversal.
METHODS
Thirty-six New Zealand white rabbits were infused with a lethal dose of sodium cyanide solution (20 mg/60 ml normal saline). Animals were divided into three groups and treated with saline, low dose (20 mg), or high dose (150 mg) of DMTS intramuscularly. DOS continuously assessed changes in tissue hemoglobin concentrations and cytochrome c oxidase redox state status throughout the experiment.
RESULTS
IM injection of DMTS increased the survival in lethal CN poisoning. DOS demonstrated that high-dose DMTS (150 mg) reversed the effects of CN exposure on cytochrome c oxidase, while low dose (20 mg) did not fully reverse effects, even in surviving animals.
CONCLUSIONS
This study demonstrated potential efficacy for the novel approach of supplying substrate for non-rhodanese mediated sulfur transferase pathways for CN detoxification via intramuscular injection in a moderate size animal model and showed that DOS was useful for optimizing the DMTS treatment.
Topics: Animals; Antidotes; Carbon Dioxide; Dose-Response Relationship, Drug; Electron Transport Complex IV; Hemoglobins; Oxidation-Reduction; Oxygen Consumption; Rabbits; Sodium Cyanide; Spectrum Analysis; Sulfides; Survival Analysis
PubMed: 30094773
DOI: 10.1007/s13181-018-0680-6 -
Journal of Neurology, Neurosurgery, and... Feb 1976Sodium cyanide was given to rats by intravenous infusion at a rate that would avert apnoea (the first sign of overdosage) in the majority. There was full physiological...
Sodium cyanide was given to rats by intravenous infusion at a rate that would avert apnoea (the first sign of overdosage) in the majority. There was full physiological monitoring in a group under anaesthesia and more limited monitoring in an unanaesthetized group. White matter was damaged in six animals and grey matter additionally in only one. It was concluded that cyanide can damage neurones only through the medium of secondary effects on circulation and respiration.
Topics: Animals; Apnea; Blood Pressure; Brain; Carbon Dioxide; Corpus Callosum; Cyanides; Electroencephalography; Female; Heart Arrest; Heart Rate; Hydrogen-Ion Concentration; Male; Myelin Sheath; Oxygen; Rats; Respiration; Seizures; Tetany; Thalamus
PubMed: 4588
DOI: 10.1136/jnnp.39.2.129 -
The Biochemical Journal Nov 19691. The methods for the assay of choline acetyltransferase were based on the reaction between labelled acetyl-CoA and unlabelled choline to give labelled acetylcholine....
1. The methods for the assay of choline acetyltransferase were based on the reaction between labelled acetyl-CoA and unlabelled choline to give labelled acetylcholine. 2. Both synthetic acetyl-CoA and acetyl-CoA formed from sodium [1-(14)C]acetate or sodium [(3)H]acetate by incubation with CoA, ATP, Mg(2+) and extract from acetone-dried pigeon liver were used. 3. [1-(14)C]Acetylcholine was isolated by extraction with ketonic sodium tetraphenylboron. 4. [(3)H]Acetylcholine was precipitated with sodium tetraphenylboron to remove a ketone-soluble contaminant in sodium [(3)H]acetate and then extracted with ketonic sodium tetraphenylboron. 5. The values of choline acetyltransferase activity obtained in the presence of sodium cyanide or EDTA and synthetic acetyl-CoA were similar to those obtained with acetyl-CoA synthesized in situ. 6. The assay of acetylcholinesterase was based on the formation of labelled acetate from labelled acetylcholine. The labelled acetylcholine could be quantitatively removed from the acetate by extraction with ketonic sodium tetraphenylboron. 7. The methods were tested with samples from central and peripheral nervous tissues and purified enzymes. 8. The blank values for choline acetyltransferase and acetylcholinesterase corresponded to the activities in 20ng. and 5ng. of brain tissue respectively.
Topics: Acetates; Acetylcholine; Acetylcholinesterase; Acyltransferases; Animals; Boron Compounds; Brain; Carbon Isotopes; Cats; Choline; Coenzyme A; Cyanides; Diaphragm; Edetic Acid; Guinea Pigs; Nervous System; Rats; Sciatic Nerve; Tritium
PubMed: 4982085
DOI: 10.1042/bj1150465 -
Biochimica Et Biophysica Acta Apr 2000The effect of antimycin, myxothiazol, 2-heptyl-4-hydroxyquinoline-N-oxide, stigmatellin and cyanide on respiration, ATP synthesis, cytochrome c reductase, and membrane...
The effect of antimycin, myxothiazol, 2-heptyl-4-hydroxyquinoline-N-oxide, stigmatellin and cyanide on respiration, ATP synthesis, cytochrome c reductase, and membrane potential in mitochondria isolated from dark-grown Euglena cells was determined. With L-lactate as substrate, ATP synthesis was partially inhibited by antimycin, but the other four inhibitors completely abolished the process. Cyanide also inhibited the antimycin-resistant ATP synthesis. Membrane potential was collapsed (<60 mV) by cyanide and stigmatellin. However, in the presence of antimycin, a H(+)60 mV) that sufficed to drive ATP synthesis remained. Cytochrome c reductase, with L-lactate as donor, was diminished by antimycin and myxothiazol. Cytochrome bc(1) complex activity was fully inhibited by antimycin, but it was resistant to myxothiazol. Stigmatellin inhibited both L-lactate-dependent cytochrome c reductase and cytochrome bc(1) complex activities. Respiration was partially inhibited by the five inhibitors. The cyanide-resistant respiration was strongly inhibited by diphenylamine, n-propyl-gallate, salicylhydroxamic acid and disulfiram. Based on these results, a model of the respiratory chain of Euglena mitochondria is proposed, in which a quinol-cytochrome c oxidoreductase resistant to antimycin, and a quinol oxidase resistant to antimycin and cyanide are included.
Topics: Adenosine Triphosphate; Animals; Antimycin A; Cell Respiration; Enzyme Activation; Euglena; Lactic Acid; Methacrylates; Mitochondria; NADH Dehydrogenase; Oxidative Phosphorylation; Polyenes; Sodium Cyanide; Thiazoles
PubMed: 10773165
DOI: 10.1016/s0005-2728(00)00102-x