Phenazine Methosulphate Research Articles

Bacterial mutagenicity tests of phenazine Methosulphate and three tetrazolium salts

Tetrazolium salts, in combination with phenazine methosulphate (PMS) are widely used laboratory reagents. PMS, and 3 tetrazolium salts (MTT, [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide]; TTC,[2,3,5-triphenyl tetrazolium chloride]; and NBT, [2,2′-di- p-nitrophenyl-5,5′-diphenyl-3,3′-(3,3′-dimethoxy-4,4′ were tested for mutagenicity in a range of Escherichia coli WP2 (trp −) and Salmonella typhimurium TA (his − strains in the presence rat-liver supernatant (S9). Without S9, PMS was mutagenic to E. coli uvrApKM101, S. typhimurium TA100 and TA98 giving (in the dose range 0.5–10 μg/plate) linear doseresponse curves with slopes of 2.3, 1.3 and 0.5 revertants/nmol respectively. Addition of S9 drastically reduced the mutagenicity of PMS in these bacteria. MTT, in the absence of S9, was mutagenic to E. coli uvrApKM101, S.thyphimurium TA100 and TA98, giving linear dose-response curves with slopes of 3.8, 12.1 and 1.0 respectively, at doses ranging from 0.5 to 10 or 50 μg/plate. Addition of S9 markedly reduced the mutagenicity of MTT. MTT was very weakly mutagenic in E. coli WP2 (0.22 revertants/nmol), significantly more mutagenic in WP2 uvrA (1.29 revertants/nmol), and non-mutagenic in WP2 lexA (negative slope due to toxicity), suggesting that MTT causes excisable “misrepair” DNA damage. PMS and MTT are often used in combination: however, PMS did not potentiate the mutagenicity of MTT.

Factors Controlling the Formation of Akinetes Adjacent to Heterocysts in the Cyanobacterium Cylindrospermum licheniforme Kutz.

Summary: Substances which stimulate the formation of akinetes (spores) in Cylindrospermum licheniforme Kutz. are secreted into a phosphate-free sporulation medium by filaments of that cyanobacterium. One such substance, purified from the centrifugal supernatant fluid of sporulating cultures, initiated sporulation in a phosphate-containing culture medium. Certain amino acids, particularly tryptophan, and calcium glucuronate also strongly stimulated sporulation. Acetylene and ethylene were inhibitory. No significant effect of cyclic nucleotides was observed. The addition of 12.5 to 15 % (v/v) H2 stimulated sporulation in air or under CO2/O2/Ar (0.1:19.9:80, by vol.) by up to 2.5-fold, but did not significantly affect the reduction of acetylene by intact filaments. Thus, the stimulation of sporulation by hydrogen was not mediated by an effect on the fixation of nitrogen. Hydrogen uptake in a cell-free suspension derived from whole filaments was detected manometrically, with phenazine methosulphate as electron acceptor, at a rate of 5.8 μmol H2 (mg chlorophyll a)−1 h−1. The uptake hydrogenase activity derived from isolated heterocysts accounted for 84 × 2 % of the uptake hydrogenase activity from whole filaments, whereas no activity was detected in a fraction derived from the vegetative cells. The formation of the pattern consisting of spores contiguous with heterocysts may be controlled by either, or a combination of, (i) a sporulation-stimulatory substance, if that substance is synthesized solely in heterocysts, or (ii) some substance the concentration of which may be controlled by hydrogen assimilated by heterocysts.

Nitroblue Tetrazolium (NBT) Reduction by Bacteria

All the S. albus, E. coli and P. aeruginosa strains examined reduced nitroblue tetrazolium (NBT) to dark blue formazan. The amount of formazan produced was proportional to the number of bacteria. Under the same growth conditions, an equal number of bacteria of various strains produced different amounts of formazan. However, there were statistically verified differences in the NBT-reduction between the three species examined. The NBT-reduction took place in all phases of growth but was most intense in the early logarithmic phase. NBT was found to be toxic for bacteria, and the different strains had varying sensitivity to that effect. The NBT-reaction was markedly enhanced by phenazine methosulphate (PMS). The blue colour of formazan produced from NBT has an advantage over the red colour from triphenyltetrazolium chloride (TTC) if the reaction occurs in the presence of haemoglobin often present in biological materials. With NBT and PMS, 10(6)--10(7) bacteria are needed to form detectable amounts of formazan. The NBT-reduction by bacteria may be useful for measuring the influence of bactericidal, bacteriostatic or growth-stimulating factors on bacteria.

Properties of Pseudomonas AM1 primary-amine dehydrogenase immobilized on agarose

1. 1. The primary-amine dehydrogenase of Pseudomonas AM1 (primary-amine:(acceptor) oxidoreductase (deaminating), EC 1.4.99.-) was purified by an improved method and covalently attached to cyanogen bromide-activated Sepharose 4B. The immobilized enzyme showed very little change in its sensitivity to heat and to inhibition by semicarbazide as compared with the soluble enzyme, but had enhanced stability at 0°C. The pH optimum of the imobilized enzyme remained unchanged at pH 7.4. 2. 2. A new type of spectrophotometric assay is described in which sedimentation of the immobilized enzyme in the cuvette is prevented by increasing the viscosity by the presence of 10% (w/w) polyethylene glycol ( M r 20 000 ). Detailed kinetic analysis using this assay showed only insignificant differences in the K m values for n- butylamine and phenazine methosulphate between the soluble and Agarose-bound enzymes. The results are compared with those for other oxidoreductase enzymes immobilized on Sepharose.

Stimulation of monovalent cation fluxes by electron donors in the human red cell membrane.

When human red cells are incubated at 37 degrees C with the artificial electron donor system ascorbate + phenazine methosulphate the fluxes of Rb+ (K+) through the cell membrane are increased. The effect of this donor system is much stronger in energy-depleted than in normal cells. The same effects are produced by HS-glutathione, NADH or NADPH loaded into resealed ghosts, but these electron donors were ineffective when added to the incubation medium. The Rb+ (K+) fluxes induced by electron donors resemble closely those induced by an increase of intracellular Ca2+ (Gardos effect). The electron donors require the presence of intracellular Ca2+ to be effective, but at levels that do not stimulate by themselves the fluxes of K+. Flavoenzyme inhibitors (atebrin and chlorpromazine), oligomycin and quinine prevented the effects of both electron donors and Ca2+ alone; antimycin, upcouplers and ethacrynic acid inhibited them partially; ouabain, furosemide, and rotenone had no effect. The results could be explained if the effect of electron donors is to bring about a change in the redox state of some membrane component(s) that makes intracellular Ca2+ more effective to elicit rapid K+ movements. Plasma membrane oxidoreductase activities could be engaged in this change.

The terminal oxidase of Photobacterium phosphoreum. A novel cytochrome

The terminal oxidase of Photobacterium phosphoreum has been purified to the electrophoretically homogeneous state and some of its properties have been studied. The enzyme catalyses oxidation of ascorbate in the presence of phenazine methosulphate or N, N, N′, N′-tetramethyl- p-phenylenediamine. The reaction is inhibited by cyanide. Nitrite at comparatively high concentrations inhibits the enzyme, but the enzyme does not catalyse nitrite reduction with ascorbate plus the electron mediator as the electron donor. The enzyme shows the absorption peaks at 632, 565, 534 and 436 nm in the reduced form. It has two kinds of haems: protohaem and haem d. Namely, the enzyme is a ‘cytochrome bd’-type oxidase; a novel cytochrome.

The influence of energy-transfer inhibitors on proton permeability and photophosphorylation in normal and preilluminated Rhodospirillum rubrum chromatophores

(1) Chromatophores were preilluminated in the presence of phenazine methosulphate or diaminodurene, and without phosphorylation substrates; next they were transferred to fresh medium and assayed for light-induced proton uptake, light-induced 9-aminoacridin fluorescence quenching, and photophosphorylation. (2) Preillumination in the presence of phenazine methosulphate or diaminodurene causes an inhibition of the photophosphorylation rate. The presence of ADP + MgCl 2 + phosphate, or ADP + MgCl 2 + arsenate during preillumination provides full protection against this effect. (3) Preilluminated chromatophores are leaky for protons. The leak is expressed as an accelerated dark decay, and a diminished extent of succinate-supported, light-induced proton uptake. The extent of light-induced 9-aminoacridin fluorescence quenching is also diminished. (4) The proton leak can be closed by oligomycin and by dicyclohexyl carbodiimide (at concentrations similar to those used to inhibit photophosphorylation), but not by aurovertin. Closure of the proton leak results in partial restoration of the photophosphorylation rate. (5) The inhibition of phosphorylation by oligomycin or dicyclohexyl carbodiimide is time-dependent. In untreated chromatophores, the time-dependence is determined by the extent of membrane energization. In preilluminated chromatophores, the time-dependence is determined in addition by the extent to which the proton leaks have been closed. The reasons for this are briefly discussed.

Ferrocyanide as electron donor to cytochrome aa3. Cytochrome c requirement for oxygen uptake

1. In the absence of cytochrome c, ferrocyanide or ferrous sulphate reduces cytochrome c oxidase (EC 1.9.3.1), but no continuous oxygen uptake ensues, as it does with N, N, N′, N′-Tetramethyl- p-phenylenediamine or reduced phenazine methosulphate as reductants, unless a substoichiometric amount of cytochrome c or an excess of clupein is present. Cytochrome c cannot be replaced by porphyrin cytochrome c. 2. Cytochrome c, porphyrin cytochrome c and clupein all stimulate the reduction of cytochrome aa 3 by ferrocyanide. 3. A model is proposed to explain these findings in which a high-affinity site for cytochrome c on the oxidase regulates the access of hydrophilic electron donors to a low-affinity site, and reduction via the high-affinity site is required for continuous oxygen uptake. 4. Furthermore, it is shown that upon reaction of oxidase with ferrocyanide, cyano-oxidase is formed.

Stimulation of glucose transport in rat adipocytes by calcium

Glucose transport in rat adipocytes was studied by monitoring the conversion of [1-14C]-glucose to 14CO2 in a system where glucose transport was made rate-limiting by increasing the flux through the pentose phosphate pathway with phenazine methosulphate, an agent which rapidly reoxidizes NADPH. Calcium increased both basal and insulin-stimulated apparent rates of glucose transport by approximately 40%. The maximum velocity of the apparent rate of glucose transport was increased by extracellular calcium both in the presence or absence of insulin. There was no change in the glucose concentration required for half-maximal rates of 14CO2 production. Calcium also enhanced the stimulation of apparent rates of glucose transport by insulin when examined over a range of hormone concentrations. Adipocyte cAMP concentrations were significantly lowered by calcium under conditions which led to increased apparent rates of glucose transport. In contrast, cobalt and nickel, antagonists of calcium action, elevated adipocyte cAMP levels and inhibited apparent rates of glucose transport. Agents which inhibit transmembrane calcium flux (verapamil, tetracaine, and procaine) inhibited apparent rates of glucose transport despite a reduction in adipocyte cAMP concentration.On the basis of the above data we suggest that calcium may increase apparent rates of glucose transport in rat adipocytes both by lowering intracellular cAMP concentration and by a further mechanism independent of changes in the level of cAMP. These results are consistent with the hypothesis that glucose transport in rat adipocytes may be controlled, in part, by a cAMP-induced phosphorylation mechanism.

Enhancement of rat brain tryptophan metabolism by chronic ethanol administration and possible involvement of decreased liver tryptophan pyrrolase activity

1. Chronic ethanol administration enhances rat brain 5-hydroxytryptamine synthesis by increasing the availability of circulating tryptophan to the brain. This increased availability is not insulin-mediated or lipolysis-dependent. 2. Under these conditions, tryptophan accumulates in the liver and apo-(tryptophan pyrrolase) activity is completely abolished, but could be restored by administration of regenerators of liver NAD+ and/or NADP+. 3. All four regenerators used (fructose, Methylene Blue, phenazine methosulphate and sodium pyruvate) prevented the ethanol-induced increase in liver tryptophan concentration and the increased availability of tryptophan to the brain. 4. It is suggested that the enhancement of brain tryptophan metabolism by chronic ethanol administration is caused by the decreased hepatic tryptophan pyrrolase activity. The results are briefly discussed in relation to previous work with ethanol. 5. Fructose enhances the conversion of tryptophan into 5-hydroxyindol-3-ylacetic acid in brains of ethanol-treated rats, whereas Methylene Blue inhibits this conversion in both control and ethanol-treated animals.

Stimulation of glucose transport in rat adipocytes by insulin, adenosine, nicotinic acid and hydrogen peroxide. Role of adenosine 3′:5′-cyclic monophosphate

Glucose transport into adipocytes of the rat was measured by monitoring the conversion of [1-(14)C]glucose into (14)CO(2). Glucose transport was made rate-limiting by increasing the flux through the pentose phosphate pathway with phenazine methosulphate, an agent that rapidly reoxidizes NADPH. Under these conditions, the observed rate of glucose disappearance from the incubation medium was about 20% higher than the rate of conversion of the C-1 of glucose into (14)CO(2). Apparent rates of glucose transport were significantly increased by insulin, H(2)O(2), adenosine and nicotinic acid. Stimulation of the apparent rate of glucose transport by insulin was dependent on adipocyte concentration, the hormone being most effective at relatively high cell concentrations. Adenosine and nicotinic acid further enhanced the maximum stimulation of glucose transport by insulin. Potentiation of insulin action by adenosine was more pronounced at lower cell concentrations. At relatively high cell concentrations the stimulatory action of insulin was markedly decreased by adenosine deaminase. Stimulation of apparent rates of glucose transport by the compounds noted above were antagonized by agents that increased intracellular cyclic AMP concentrations (theophylline and isoprenaline) and by dibutyryl cyclic AMP. Intracellular concentrations of cyclic AMP were significantly lowered when adipocytes were incubated with insulin, H(2)O(2), adenosine or nicotinic acid. These effects were observed under basal conditions or when intracellular cyclic AMP concentrations were elevated by theophylline or isoprenaline. On the basis of the above data, we suggest that insulin, H(2)O(2), adenosine and nicotinic acid may all stimulate glucose transport in rat adipocytes by lowering the intracellular cyclic AMP concentration. These data therefore support the hypothesis that cyclic AMP inhibits glucose transport in rat adipocytes.

Site of Interaction between Phenazine Methosulphate and the Respiratory Chain of Bacillus subtilis

Membrane vesicles from Bacillus subtilis W23 and from the menaquinone‐deficient Bacillus subtilis aroD were incubated with phenazine methosulphate and excess soluble phenazine methosulphate was subsequently removed by washing (preincubated vesicles). Phenazine methosulphate which remains attached to the membrane is accessible from the outer surface of the membrane because it can be reduced chemically by NADH or ascorbate and the reduced forms can mediate electrons to horse heart cytochrome c in the external medium of the membrane vesicles.The oxidation rates of ascorbate in preincubated vesicles of B. subtilis W23 and B. subtilis aroD and of NADH in preincubated vesicles of B. subtilis aroD increased with increasing amounts of phenazine methosulphate attached per mg membrane protein. Electrons from membrane‐attached reduced phenazine methosulphate are essentially all mediated to the terminal part of the respiratory chain before cytochrome a601 because the oxidation of reduced phenazine methosulphate is almost completely inhibited by cyanide and only to a small extent by 2‐heptyl‐4‐hydroxy‐quinoline‐N‐oxide while NADH oxidase is strongly inhibited by both compounds. Furthermore, reduced phenazine methosulphate reduces completely cytochrome a601 and not all cytochromes b560 and c553.Active transport of amino acids is at least ten‐fold more effectively stimulated by NADH or ascorbate oxidation via phenazine methosulphate than by NADH oxidation via NADH dehydrogenase. Possible explanations are discussed.

Kinetics of the xanthine dehydrogenase reaction from Panstrongylus megistus using coupled electron acceptors in the assay system

The kinetics of xanthine dehydrogenase reaction from Panstrongylus megistus were studied using a system in which phenazine methosulphate (PMS) was associated with tetrazolium salts. The Lineweaver-Burk plots showed that K m ap for triphenyl tetrazolium chloride (TTC) was 4.4 × 10 4 M and K m ap(PMS) was 1.8 × 10 −5 M for both xanthine and hypoxanthine as electron donors. Both acceptors were able to increase the initial maximum velocity of the reaction but only phenazine methosulphate increased K m ap . Using saturating concentrations of both acceptors good linearity was obtained with time and with enzyme concentration. With standard conditions the K m ap s for xanthine and hypoxanthine were found to be 2.3 × 10 −4 and 4.0 × 10 −4 M, respectively. It was found that p- iodonitrotetrazolium violet (INT) could replace triphenyl tetrazolium chloride and the effectiveness of the association of acceptors was discussed.

A quantitative histochemical study of the optimal incubation conditions for certain oxidative enzymes in hamster cheek pouch epithelium

The assay conditions of three oxidative enzymes, lactate, succinate and glucose-6-phosphate dehydrogenases, were examined histochemically and their activities quantified by scanning and integrating microdensitometry. Optimal concentrations of substrate, co-enzyme, tetrazolium salt and of the intermediate acceptor, phenazine methosulphate, were determined for each enzyme. When studied at pH 7.5, non-specific reactions in the keratinized layer were absent and control activities in the absence of either substrate or co-enzyme were low in the other epithelial layers.

A quantitative cytochemical study of glucose-6-phosphate dehydrogenase and delta 5-3 beta-hydroxysteroid dehydrogenase activity in the membrana granulosa of the ovulable type of follicle of the rat.

During the last four days of follicular development prior to ovulation, the activities of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta OHD) and glucose-6-phosphate dehydrogenase (G-6-PD) were quantified in cryostat sections of the rat ovary. The product of the enzyme reactions were measured using a scanning and integrating microdensitometer. The enzyme activity was measured in the peripheral region, the antral region and the cumulus of the membrana granulosa (MG) of these follicles on the morning of each of the four days of the estrous cycle. G-6-PD activity was measured in the presence and absence of an intermediate hydrogen acceptor, phenazine methosulphate, to provide a measure of the quantity of Type I and Type II Hydrogen (H) generated: Type I H is considered to be related to hydroxylating reactions such as those of steroids and Type II H to other general biosynthetic activities of cells. In all three regions of the MG of follicles of the ovulable type, 3 beta OHD activity was lowest in estrus and diestrus-1, increased on diestrus-2 and peaked in proestrus. In estrus and diestrus-1, the level of 3 beta OHD activity in the three regions was comparable. However, by diestrus-2, and even more conspicuously in proestrus, enzyme activity was significantly greater in the peripheral region than in the antral region or in the cumulus. During the same period, the level of enzyme activity remained comparable in the last two regions. Throughout the estrous cycle, both Type I and Type II H generation from G-6-PD was greatest in the peripheral region, less in the antral region and least in the cumulus. In the eripheral region, Type I H generation increased progressively after diestrus-1, to reach a maximum in prestrus. In the antral region, Type I H generation increased between diestrus-1 and diestrus-2 and then remained unchanged through proestrus. In the cumulus, Type I H generation remained at levels seen in estrus throughout the remainder of the cycle. Generation of Type II H, in the peripheral region was constant throughout the estrous cycle. In contrast, in the antral region and cumulus, Type II H generation was greater in diestrus-1 and diestrus-2 than on either proestrus or estrus.

Studies on the reduction of nitroblue tetrazolium chloride mediated through the action of NADH and phenazine methosulphate

The general features of the reduction of nitroblue tetrazolium chloride (NBT) by NADH and phenazine methosulphate (PMS) have been studied under aerobic and anaerobic conditions. Under aerobic conditions the reduction appears to be mediated through the intermediate formation of the superoxide anion radical O 2 −; this reaction is strongly inhibited by superoxide dismutase and by a number of O 2 − scavengers such as propyl gallate, (+)-catechin, manganous ions, reduced glutathione and benzoquinone. Cupric ions inhibited the overall reaction by reoxidising reduced PMS. Under anaerobic conditions, superoxide dismutase had only a small inhibitory action and, with the exception of cupric ions, the other substances mentioned above were ineffective as inhibitors. The data presented show that the use of NBT to detect the presence of O 2 − is fraught with difficulties due to an equally rapid reduction of NBT by NADH and PMS under anaerobic conditions.

Characterization and partial purification of the reversible hydrogenase of Anabaena cylindrica

Three types of hydrogenases are recognized in the literature; a reversible hydrogenase which has been highly purified and characterized from saccharolytic bacteria [ 11, an uptake hydrogenase which is seemingly incapable of evolving hydrogen and differs from the reversible hydrogenase in being saturated at low pH2 [2], and the ATP-driven hydrogen evolution catalyzed by nitrogenase in the absence of other reducible substrates [3]. Species of cyanobacteria such as Anabaena cylindrica contain all three types of hydrogenase under some conditions [4]. Hydrogen evolution by nitrogenase in this organism has been studied in some detail [5-71. The presence of an uptake hydrogenase, similar to the Azofobacter hydrogenase, has been demonstrated [&lo]. The presence of reversible hydrogenase activity can be directly shown with cultures of A. cylindrica that lack nitrogenase activity either through growth in the presence of tungsten instead of molybdenum, or through ammonium repression [ 11 ,12 1. Hydrogen is produced by isolated heterocysts or vegetative cell fragments supplied with artificial electron donors [ 131. In [ 14,151 crude extracts with hydrogenase activity were prepared from nitrate-grown cultures of A. cylindrica incubated anaerobically in the dark under an atmosphere of hydrogen. It was found that phenazine methosulphate, methylene blue, dichlorophenol-indophenol and toluidine blue were effective as electron acceptors, but the results suggested that redox coupling between the hydrogenase and ferredoxin had a very low efficiency. When crude extracts

Some properties and subcellular localization of xanthine dehydrogenase in pea leaves

Summary In cell-free extracts of pea leaves, the oxidation of hypoxanthine to xanthine and uric acid was catalyzed by a NAD + -dependent xanthine dehydrogenase. Considerable activity was also observed with NADP + as electron acceptor. However, little activity was detected with oxygen (air) alone. Comparative tests in the presence of a variety of artificial electron acceptors showed high activity for phenazine methosulphate, but only low activity for methylene blue, nitro blue tetrazolium, 2,6-dichlorophenol indophenol, and K 3 Fe(CN) 6 . It was not possible to convert the xanthine dehydrogenase to an oxidase form. The xanthine dehydrogenase activity was inhibited by KCN and allopurinol, known inhibitors of xanthine oxidase. Inhibition by salicylhydroxamic acid indicated the presence of non-heme iron. Xanthine dehydrogenase of pea leaves was not associated with any major subcellular particulate fraction (mitochondria, chloroplasts, peroxisomes, microsomes) but appeared to be a soluble enzyme of the cytoplasm.

Control of glucose transport in adipose tissue of the rat: role of insulin, ATP, and intracellular metabolites.

The purpose of this study was to elucidate some of the mechanisms of control of the glucose transport step in adipose tissue. Glucose transport was studied by monitoring the conversion of [1-14C]glucose to 14CO2 in a system where glucose transport was made rate limiting by increasing the flux through the pentose phosphate pathway with phenazine methosulphate, an agent which results in rapid rates of reoxidation of NADPH. The maximum velocity for the apparent rate of glucose transport was increased significantly by insulin. There was no change in the glucose concentration required for half-maximal rates of 14CO2 production. Glucose transport was also monitored by directly measuring the rate of glucose uptake. Glucose uptake was increased by phenazine methosulphate. The intracellular glucose-6-phosphate concentration was decreased by phenazine methosulphate. These two agents, insulin and phenazine methosulphate, seemed to act by independent mechanisms as their optimal effects on glucose uptake were additive. The apparent rate of glucose transport was decreased by ATP which resulted in a decrease in maximal velocity but did not affect the affinity for glucose. This effect of ATP was seen in the presence of absence of insulin.

Alcohol, lactate and glutamate sensors based on oxidoreductases with regeneration of nicotinamide adenine dinucleotide

Flowthrough enzyme electrodes are reported for determinations of alcohol, lactate and glutamate. Oxidoreductases mixed with immobilized NAD + cofactor are held between a suitable platinum electrode and a semipermeable membrane. The coenzyme is readily regenerated either directly by electrochemical oxidation or by using phenazine methosulphate (PMS +) as intermediate. Continuous flow conditions are used. The sensitivity obtained with the alcohol dehydrogenase electrode was 50, 620 or 810 nA mol -1 of ethanol, respectively, when regeneration was done electrochemically or with 0.1 or 0.5 mM PMS +. The sensitivities for the lactate and glutamate sensors in the presence of 0.5 mM PMS +, were 14 and 50 nA mmol -1 for D,L-lactate and L-glutamate, respectively. The calibration curves were linear for concentrations up to 0.5, 1.5 and 100 mM of glutamate, lactate and ethanol, respectively. The sensitivity of the alcohol and lactate sensors decreased by 50–55% within 60 h and that of the glutamate sensor within 6 h.