The use of several types of metal cations--a new approach to the study of the metallophilia of Ranvier's nodes of nerve fibers]

Mechanisms of metal affinity of Ranvier's nodes of myelinated nerve fibers in the peripheral nerve system have been investigated. It is indicated that Fe(3+)-ions are able to reduce to Fe(2+)-ions. Ag(+)-ions compete with Fe(3+)-ions for places of adsorption in a gap [correction of chick] between myelinated segments of Ranvier's node. It is demonstrated that Fe(3+)- and Fe(2+)-ions are adsorbed in the gap [correction of chink], while Ag(+)-ions are adsorbed in the gap [correction of chink] and axon in the bulbs of the node. One can increase ferrocyanide of ferrous sediment by several times by ferricyanide of ferrous sediment as well as by ferrocyanide of silver and of metal silver ones. A possibility to use several kinds of metal ions for demonstration of cytochemical heterogeneity of nerve fibre membrane is discussed.     

Kinetic characterization of reductant dependent processes of iron mobilization from endocytic vesicles.

The reductant dependence of iron mobilization from isolated rabbit reticulocyte endosomes containing diferric transferrin is reported. The kinetic effects of acidification by a H(+)-ATPase are eliminated by incubating the endosomes at pH 6.0 in the presence of 15 microM FCCP to acidify the intravesicular milieu and to dissociate 59Fe(III) from transferrin. In the absence of reductants, iron is not released from the vesicles, and iron leakage is negligible. The second-order dependence of rate constants and amounts of 59Fe mobilized from endosomes using ascorbate, ferrocyanide, or NADH are consistent with reversible mechanisms. The estimated apparent first-order rate constant for mobilization by ascorbate is (2.7 +/- 0.4) x 10(-3) s-1 in contrast to (3.2 +/- 0.1) x 10(-4) s-1 for NADH and (3.5 +/- 0.6) x 10(-4) s-1 for ferrocyanide. These results support models where multiple reactions are involved in complex processes leading to iron transfer and membrane translocation. A type II NADH dehydrogenase (diaphorase) is present on the endosome outer membrane. The kinetics of extravesicular ferricyanide reduction indicate a bimolecular-bimolecular steady-state mechanism with substrate inhibition. Ferricyanide inhibition of 59Fe mobilization is not detected. Significant differences between mobilization and ferricyanide reduction kinetics indicate that the diaphorase is not involved in 59Fe(III) reduction. Sequential additions of NADH followed by ascorbate or vice versa indicate a minimum of two sites of 59Fe(III) residence; one site available to reducing equivalents from ascorbate and a different site available to NADH. Sequential additions using ferrocyanide and the other reductants suggest interactions among sites available for reduction. Inhibition of ascorbate-mediated mobilization by DCCD and enhancement of ferrocyanide and NADH-mediated mobilization suggest a role for a moiety with characteristics of a proton pore similar to that of the H(+)-ATPase. These data provide significant constraints on models of iron reduction, translocation, and mobilization by endocytic vesicles.     

Ferrocyanide carbon-13 NMR line broadening as a probe of electron transfer reactions

The electron transfer reaction between ferrocyanide ion and the blue copper protein, stellacyanin, has been investigated by means of 13C NMR line broadening of the inorganic oxidant. The temperature dependence of the ferrocyanide line broadening gives an activation energy for the electron transfer reaction of 17 +/- 3 kJ. The apparent rate constant decreases with increasing concentration of K4Fe(CN)6, a result which can be explained either by formation of a strong precursor ferrocyanide--stellacyanin [Cu(II)] complex or by increased formation of KFe(CN)3-6 ion pairs. The direct electron transfer between ferrocyanide and ferricyanide has also been studied by 13C NMR line broadening of the former species. The ferricyanide concentration dependence of the exchange line broadening yields a value for the apparent second-order rate constant at 25 degrees C of k = 1.65 . 10(3) M-1 . s-1, in agreement with previously reported values derived from 14N NMR and isotope exchange studies. This rate constant shows a linear dependence on the K+ concentration, independent of ionic strength, a result which confirms the importance of ion pair species such as KFe(CN)3-6 and KFe(CN)2-6 in the direct electron transfer mechanism. The general applications of the method are discussed, including the considerations which suggest that a wide range of electron transfer rates, from about 1 s-1 to 4 . 10(3) s-1, are, in principle, accessible to this technique. The potential utility of ferrocyanide 13C spin--lattice relaxation time measurements is decreasing the lower limit of this range is also discussed.     

Electrochemical evaluation of lactate dehydrogenase immobilized in polyacrylamide gels

Lactate dehydrogenase (EC 1.1.1.27) has been immobilized in polyacrylamide gels over a platinum grid matrix. The immobilized enzyme is used to oxidize L-lactate in the presence of nicotinamide adenine dinucleotide (NAD+) and ferricyanide. The NADH produced is then chemically oxidized back to NAD+ by ferricyanide. The coupled reduction of ferricyanide ions to ferrocyanide ions results in a measurable electrochemical potential. This measurable zero-current potential is found to be Nernstian in nature and directly proportional to the logarithm values of L-lactate concentration over the range of 2 X 10(-5) to 5 X 10(-2)M. The results indicate that immobilized lactate dehydrogenase can be incorporated into a system to detect L-lactate acid in aqueous solutions.     

PHYTOPLANKTON PLASMA MEMBRANE REDOX ACTIVITY: EFFECT OF IRON LIMITATION AND INTERACTION WITH PHOTOSYNTHESIS1

Phytoplankton plasma membrane electron transport activity was determined by monitoring the reduction of the impermeant artificial electron acceptor ferricyanide in a range of diatoms. The results revealed that constitutive plasma membrane electron transport activity of marine diatoms is high compared with chlorophytes and higher plant cells. Diatom plasma membrane electron transport activity was not significantly increased by iron limitation. This lack of induction on iron limitation indicates that diatoms have an iron acquisition strategy that is distinct from chlorophytes and the dicotyledon higher plants that exhibit marked increases in plasma membrane ferricyanide reductase activity on iron limitation. The interaction of the constitutive plasma membrane electron transport with photosynthesis was also investigated. We found that 1) ferricyanide reduction at the plasma membrane was progressively inhibited in response to increasing irradiances; 2) the presence of extracellular ferricyanide, but not the reduced couple ferrocyanide, caused a marked inhibition of carbon fixation at high irradiance; and 3) extracellular electron acceptors ferricyanide and hexachloroiridate (but not ferrocyanide) induced an immediate and reversible decrease in fluorescence yields (F_o and F_m). The extent to which extracellular electron acceptors affected CO₂ fixation, F_o, and F_m was related to the level of constitutive ferricyanide reductase activity, the species with highest ferricyanide reduction rates being most sensitive. The data suggest that consumption of electrons and/or reductant at the plasma membrane by external acceptors may compete directly with CO₂ fixation for electrons, alter cytosolic‐chloroplast redox poise, and/or induce a redox‐signaling cascade that alters photosynthetic metabolism.     

Primary donor recovery kinetics in reaction centers from Rhodopseudomonas viridis. The influence of ferricyanide as a rapid oxidant of the acceptor quinones

In reaction centers from Rhodopseudomonas viridis that contain a single quinone, the decay of the photo-oxidized primary donor, P+, was found to be biphasic when the bound, donor cytochromes were chemically oxidized by ferricyanide. The ratio of the two phases was dependent on pH with an apparent pK of 7.6. A fast phase, which dominated at high pH (t1/2 = 1 ms at pH 9.5), corresponded to the expected charge recombination of P+ and the primary acceptor QA-. A much slower phase dominated at low pH and was shown to arise from a slow reduction of P+ by ferrocyanide in reaction centers where QA- has been rapidly oxidized by ferricyanide. The rate of QA- oxidation was linear with respect to ferricyanide activity and was strongly pH-dependent. The second-order rate constant, corrected for the activity coefficient of ferricyanide, approached a maximum of 2 X 10(8) M-1 X s-1 at low pH, but decreased steadily as the pH was raised above a pK of 5.8, indicating that a protonated state of the reaction center was involved. The slow reduction of P+ by ferrocyanide was also second-order, with a maximum rate constant at low pH of 8 X 10(5) M-1 X s-1 corrected for the activity coefficient of ferrocyanide. This rate also decreased at higher pH, with a pK of 7.4, indicating that ferrocyanide also was most reactive with a protonated form of the reaction center. The oxidation of QA- by ferricyanide was unaffected by the presence of o-phenanthroline, implying that access to QA- was not via the QB-binding site. In reaction centers supplemented with ubiquinone, oxidation of reduced secondary quinone, QB-, by ferricyanide was observed but was substantially slower than that for QA-. It is suggested that Q-B may be oxidized via QA so that the rate is modulated by the equilibrium constant for QA-QB in equilibrium with QAQB-.     

Catalytic effect of ferricyanide between myoglobin and luminol and effect of temperature.

Specific catalytic oxidation of oxymyoglobin (MbO(2)) and luminol by ferricyanide was studied in a flow-injection system. MbO(2) in different redox states (ferric and ferrous) was oxidized to Mb(Fe(III)) by ferricyanide, and then specific binding of the ferrocyanide anion to Mb(Fe(III)) to the His 119 (GH1) region accelerated the electron transfer between Mb(Fe(III)) and luminol, which produced a chemiluminescence (CL) signal at 425 nm. The increased CL emission was correlated with the myoglobin concentration in the range 0.16-7.5 microg/mL. Thermogravimetry and differential scanning calorimetry were used to investigate the temperature effects on this reaction. The results showed that the CL intensity in the presence of myoglobin changed considerably with heating in the range 15-50 degrees C, and the maximal CL intensity was observed at 40 degrees C, corresponding to the glass transition temperature of myoglobin. The effect of different ligands and interferences were also studied.     

Thansplasmalemma redox reactions and ion transport in photosynthetic and heterotrophic plant cells

Extracellular ferricyanide reduction, NADH and ferrocyanide oxidation were investigated by spectrophotometrical method on photosynthetic freshwater plants ( Elodea canadensis Rich., Vallisneria spiralis L., Nitella flexilis L.) and heterotrophic tissues (roots of Triticum vulgare L., Hordeum vulgare L., Zea mays L., Pisum sativum L., Avena sativa L., Allium sativa L., Allium cepa L.). All species had ferricyanide reductase activity. The roots of land plants also carried out extracellular oxidation of NADH and ferrocyanide in contrast to leaves of the freshwater plants. External NADH stimulated ferricyanide reductase activity, but only with those objects that had external NADH oxidase activity. In all species ferricyanide decreased the membrane potential (MP), decreased the membrane resistance measured at a fixed current and inhibited K⁺ influx measured by flame photometry. The factors affecting ferricyanide reductase activity also influenced the inhibitory effect of ferricyanide on the MP and K⁺ transport. These results demonstrate a connection between transport, electrogenic and redox functions of the plasmalemma.     

Potentiometric determination of coenzyme-dependent oxidoreductase enzymes with recycling of the coenzyme

By enzymatically establishing a rapid (essentially equilibrium) coupling of a redox coenzyme such as NAD with the components of the ferrocyanide–ferricyanide half-cell (e.g., using excess diaphorase) the half-cell potential can be used to monitor another enzymatic reaction involving the same coenzyme. This approach provides a general, rapid potentiometric method of assaying coenzyme-dependent oxidoreductase enzymes. We show that these assay systems can be designed for multiple turnover of coenzyme (in our case NAD) during a single assay thereby amplifying the rate of electromotive force (emf) change with a concomitant increase in sensitivity of enzyme assay. This allows the use of small concentrations of coenzyme and extension of the range of enzyme concentrations that may be assayed.     

Evidence for the extracellular reduction of ferricyanide by rat liver. A trans-plasma membrane redox system

1. Reduction of ferricyanide by the isolated perfused rat liver and by isolated rat hepatocytes was studied. 2. Ferricyanide was reduced to ferrocyanide by the perfused liver at a linear rate of 0.22mumol/min per g of liver. Ferricyanide was not taken up by the liver and the perfusate concentration of ferricyanide+ferrocyanide remained constant throughout the perfusion. Perfusate samples from livers perfused without ferricyanide did not reduce ferricyanide. 3. Isolated hepatocytes reduced ferricyanide in a biphasic manner. The initial rate of 2.3mumol/min per g of cells proceeded for approx. 3min and derived from low-affinity sites (apparent K(m)>1.3mm). The secondary rate of 0.29mumol/min per g of cells was maintained for the remainder of the incubation and derived from higher affinity sites (apparent K(m)0.13mm). Disruption of the cells resulted in an increase in the low-affinity rate and a decrease in the high-affinity rate. 4. Ferrocyanide was oxidized by isolated hepatocytes but not by perfused liver. The apparent K(m) for ferrocyanide oxidation by hepatocytes was 1.3mm. 5. Oxidized cytochrome c was reduced by isolated hepatocytes in the presence of 1mm-KCN but at a rate less than that of the reduction of ferricyanide. 6. Properties of the ferricyanide-reducing activities of intact hepatocytes and the perfused liver were examined. The low-affinity rate, present only in cell and broken cell preparations, was inhibited by 1mum-rotenone and 0.5mm-ferrocyanide, and stimulated by 0.1mm-KCN. The mitochondrial substrate, succinate, also stimulated this rate. The perfused liver showed only a high-affinity activity for ferricyanide reduction. This activity was also present in liver cells and was unaffected by rotenone, antimycin A, KCN, NaN(3), or p-hydroxymercuribenzoate but was inhibited by 2.6mm-CaCl(2), 2-heptyl-4-hydroxyquinoline-N-oxide and ferrocyanide. Overall, these results are consistent with the occurrence of a trans-plasma membrane redox system of liver that reduces extracellular ferricyanide to ferrocyanide. The reduction process shows properties which are similar to that of the NADH:ferricyanide oxidoreductase found in isolated liver plasma membranes but different from that of mitochondria.     

Ferrous iron-dependent volatilization of mercury by the plasma membrane of Thiobacillus ferrooxidans

Of 100 strains of iron-oxidizing bacteria isolated, Thiobacillus ferrooxidans SUG 2-2 was the most resistant to mercury toxicity and could grow in an Fe(2+) medium (pH 2.5) supplemented with 6 microM Hg(2+). In contrast, T. ferrooxidans AP19-3, a mercury-sensitive T. ferrooxidans strain, could not grow with 0.7 microM Hg(2+). When incubated for 3 h in a salt solution (pH 2.5) with 0.7 microM Hg(2+), resting cells of resistant and sensitive strains volatilized approximately 20 and 1.7%, respectively, of the total mercury added. The amount of mercury volatilized by resistant cells, but not by sensitive cells, increased to 62% when Fe(2+) was added. The optimum pH and temperature for mercury volatilization activity were 2.3 and 30 degrees C, respectively. Sodium cyanide, sodium molybdate, sodium tungstate, and silver nitrate strongly inhibited the Fe(2+)-dependent mercury volatilization activity of T. ferrooxidans. When incubated in a salt solution (pH 3.8) with 0.7 microM Hg(2+) and 1 mM Fe(2+), plasma membranes prepared from resistant cells volatilized 48% of the total mercury added after 5 days of incubation. However, the membrane did not have mercury reductase activity with NADPH as an electron donor. Fe(2+)-dependent mercury volatilization activity was not observed with plasma membranes pretreated with 2 mM sodium cyanide. Rusticyanin from resistant cells activated iron oxidation activity of the plasma membrane and activated the Fe(2+)-dependent mercury volatilization activity of the plasma membrane.     

Intracellular flavonoids as electron donors for extracellular ferricyanide reduction in human erythrocytes.

Reduction of extracellular ferricyanide [Fe(CN)(6)](-3) to ferrocyanide by intact cells reflects the activity of a trans-plasma membrane oxidoreductase that, in human red blood cells, utilizes ascorbic acid as an electron donor. We herein report that the flavonoids quercetin and myricetin, while inhibiting dehydroascorbic acid uptake-and thus the erythrocyte ascorbic acid content-effectively stimulate the extracellular reduction of ferricyanide. Other flavonoids such as rutin, acacetin, apigenin, and genistein do not show the same effect. The notion that quercetin or myricetin may serve as an intracellular donor for a trans-plasma membrane oxidoreductase is supported by the following lines of evidence: (i) they afford direct reduction of ferricyanide; (ii) extracellular reduction of ferricyanide was not mediated by direct effects of the flavonoids released by the cells and was abolished by the sulphydryl reagent parachloromercuribenzenesulfonic acid (pCMBS); (iii) the intracellular concentrations of quercetin or myricetin well correlate with increases in ferricyanide reduction; (iv) the intracellular concentration of the flavonoids dramatically declines after ferricyanide exposure. Taken together, the results presented in this study demonstrate that myricetin and quercetin, which accumulate in large amounts in red blood cells, act as intracellular substrates of a pCMBS-sensitive trans-plasma membrane oxidoreductase. This may represent a novel mechanism whereby these flavonoids exert beneficial effects under oxidative stress conditions.     

High-throughput screening for cellobiose dehydrogenases by Prussian Blue in situ formation

Extracellular fungal flavocytochrome cellobiose dehydrogenase (CDH) is a promising enzyme for both bioelectronics and lignocellulose bioconversion. A selective high-throughput screening assay for CDH in the presence of various fungal oxidoreductases was developed. It is based on Prussian Blue (PB) in situ formation in the presence of cellobiose (<0.25 mM), ferric acetate, and ferricyanide. CDH induces PB formation via both reduction of ferricyanide to ferrocyanide reacting with an excess of Fe3? (pathway 1) and reduction of ferric ions to Fe2? reacting with the excess of ferricyanide (pathway 2). Basidiomycetous and ascomycetous CDH formed PB optimally at pH 3.5 and 4.5, respectively. In contrast to the holoenzyme CDH, its FAD-containing dehydrogenase domain lacking the cytochrome domain formed PB only via pathway 1 and was less active than the parent enzyme. The assay can be applied on active growing cultures on agar plates or on fungal culture supernatants in 96-well plates under aerobic conditions. Neither other carbohydrate oxidoreductases (pyranose dehydrogenase, FAD-dependent glucose dehydrogenase, glucose oxidase) nor laccase interfered with CDH activity in this assay. Applicability of the developed assay for the selection of new ascomycetous CDH producers as well as possibility of the controlled synthesis of new PB nanocomposites by CDH are discussed.     

Oxidation of sperm whale oxymyoglobin, catalyzed by ferrocyanide ions: kinetics and mechanisms

Specific catalytic oxidation of sperm whale oxymyoglobin by small amounts of potassium ferri- and ferrocyanide, from 1 to 20% in relation to the protein concentration, was studied. The mechanism of catalysis was shown to involve specific binding of the ferrocyanide anion to the protein. The influence of pH and ionic strength of the medium, the [Fe(CN)6]4- concentration and of chemical modification of Mb histidines by bromoacetate, as well as the effect of the Mb complexing with redox-inactive zinc ion on the rate of reaction was examined. The zinc ion forms a stable complex with His 119(GH1) on the Mb surface at the equimolar Zn2+ concentration. The kinetic scheme of the reaction was analyzed, and the equilibrium and kinetic parameters were obtained. It was first shown that the strong oxidant such as potassium ferricyanide is able to react with the same protein by two distinct mechanisms: (i) a simple outer sphere electron transfer over the heme edge and (ii) electron transfer after the specific binding of [Fe(CN)6]4- to oxyMb in the His 119(GH1) region, thus catalyzing the protein oxidation.     

Rate of transmembrane electron transfer in chromaffin-vesicle ghosts

The chromaffin vesicle of the adrenal medulla contains a transmembrane electron carrier that may provide reducing equivalents for dopamine beta-hydroxylase in vivo. This electron-transfer system can be assayed by trapping ascorbic acid inside resealed membrane vesicles (ghosts), adding an external electron acceptor such as ferricytochrome c or ferricyanide, and following the reduction of these acceptors spectrophotometrically. Cytochrome c reduction is more rapid at high pH and is proportional to the amount of chromaffin-vesicle ghosts, at least at low ghost concentrations. At pH 7.0, ghosts loaded with 100 mM ascorbic acid reduce 60 microM cytochrome c at a rate of 0.035 +/- 0.010 mu equiv min-1 (mg of protein)-1 and 200 microM ferricyanide at a rate of 2.3 +/- 0.3 mu equiv min-1 (mg of protein)-1. The rate of cytochrome c reduction is accelerated to 0.105 +/- 0.021 mu equiv min-1 (mg of protein)-1 when cytochrome c is pretreated with equimolar ferrocyanide. Pretreatment of cytochrome c with ferricyanide also causes a rapid rate of reduction, but only after an initial delay. The ferrocyanide-stimulated rate of cytochrome c reduction is further accelerated by the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), probably because FCCP dissipates the membrane potential generated by electron transfer. These rates of electron transfer are sufficient to account for electron transfer to dopamine beta-hydroxylase in vivo and are consistent with the mediation of electron transfer by cytochrome b-561.     

Automated colorimetric determination of cellulase activity in fermentation samples using a ferricyanide-molybdoarsenic acid reagent

An automated method for the determination of cellulase activity has been developed. The enzyme incubation with the cellulose substrate is stopped at alkaline pH, after which the reaction mixture is dialyzed. The concentration of reducing sugar in the dialyzer recipient is determined by means of the colorimetric ferricyanide-molybdoarsenic acid reaction. In this reaction the ferrocyanide formed by the ferricyanide reaction is coupled with molybdoarsenic acid during formation of a molybdenum-blue color. An optimization of the analytical conditions and the reagent concentrations has been carried out. The result of the automation is an analysis with high precision (mean = 36.4; SD = 0.7%) and accuracy as well as high rate of analysis. The sensitivity is 5 mg of glucose/liter.     

A possible relationship between the effect of factors on photoactivation of photosystem II depleted of functional Mn and cytochrome b559

The photoassembly of the Mn₄CaO₅ cluster in Mn-depleted photosystem II preparations (photoactivation) was studied under the influence of oxidants, reductants and pH. New data on the effect of these factors on the photoactivation yield are presented. The presence of the oxidant, ferricyanide, negatively affected the photoactivation yield over the entire concentration range studied (0–1 mM). In contrast to ferricyanide, the addition of the reductant, ferrocyanide, up to 1 mM resulted in an increase in the photoactivation yield. Other reductants either did not significantly affect (diphenylcarbazide) or suppressed (ascorbate) the photoactivation yield. The effect of ferrocyanide on photoactivation were found to be similar dichlorophenolindophenol. Investigation of the photoactivation yield as a function of pH revealed that the maximum yield was observed at pH 6.5 in the presence of ferrocyanide and DCPIP, and at pH 5.5 without additives. In addition, the photoactivation yield at pH 5.5 was the same without and with the addition of ferrocyanide or dichlorophenolindophenol. Although ferricyanide suppressed the photoactivation, the photoactivation yield increased in the presence of ferricyanide by shifting the pH to the acidic region. The samples contained approximately 25 % of the HP cyt b₅₅₉, which was in the reduced state, as the absorbance at 559 nm was decreased upon addition of ferricyanide and subsequent addition of ferrocyanide returned the spectrum to the baseline. A possible relationship between the effect of factors on the photoactivation and the involvement of cyt b₅₅₉ in the protection of PSII from oxidative damage on the donor side is discussed.     

Evidence for two types of electron transfer processes through Photosystem II

Inhibition of electron flow from H₂O to methylviologen by 3-(34 dichlorophenyl)-1,1 dimethyl urea (DCMU), yields a biphasic curve — an initial high sensitivity phase and a subsequent low sensitivity phase. The two phases of electron flow have a different pH dependence and differ in the light intensity required for saturation.Preincubation of chloroplasts with ferricyanide causes an inhibition of the high sensitivity phase, but has no effect on the low sensitivity phase. The extent of inhibition increases as the redox potential during preincubation becomes more positive. Tris-treatment, contrary to preincubation with ferricyanide, affects, to a much greater extent, the low sensitivity phase.Trypsin digestion of chloroplasts is known to block electron flow between Q _A and Q _B, allowing electron flow to ferricyanide, in a DCMU insensitive reaction. We have found that in trypsinated chloroplasts, electron flow becomes progressively inhibited by DCMU with increase in pH, and that DCMU acts as a competitive inhibitor with respect to [H⁺]. The sensitivity to DCMU rises when a more negative redox potential is maintained during trypsin treatment. Under these conditions, only the high sensitivity, but not the low sensitivity phase is inhibited by DCMU.The above results indicate the existence of two types of electron transport chains. One type, in which electron flow is more sensitive to DCMU contains, presumably Fe in a Q _A Fe complex and is affected by its oxidation state, i.e., when Fe is reduced, it allows electron flow to Q _B in a DCMU sensitive step; and a second type, in which electron transport is less sensitive to DCMU, where Fe is either absent or, if present in its oxidized state, is inaccessible to reducing agents.Abbreviations DCMU 3-(34 dichlorophenyl)-1, 1 Dimethyl urea - MV methyl viologen - PS II Photosystem II - Tris tris (hydroxymethyl)aminomethane     

The iron component of sodium nitroprusside blocks NMDA-induced glutamate accumulation and intracellular Ca2+ elevation

These studies were designed to compare the effects of nitric oxide (NO) generating compounds with those of several iron containing, compounds which do not generate NO on glutamate receptor function. Stimulation of primary cultures of cerebellar granule cells with N-methyl-D-aspartate (NMDA) or kainate results in the elevation of intracellular calcium ([Ca²⁺]_i) and cGMP and the release of glutamate. The iron containing compounds, sodium nitroprusside (SNP), potassium ferrocyanide (K₄Fe(CN)₆) and potassium ferricyanide (K₃Fe(CN)₆) decrease the NMDA-induced release of glutamate. SNP is the only compound of the above 3 agents which generates NO. A non-iron, NO generating compound, S-nitroso-N-acetylpenicillamin (SNAP), has no effect on the NMDA-induced glutamate release. Potassium ferrocyanide (Fe II), but not potassium ferricyanide (Fe III), blocks NMDA-induced cGMP elevations after 3 min exposure times. This contrasts with the NO generating compounds (both SNP and SNAP) which elevate cGMP levels. Furthermore, both potassium ferrocyanide (Fe II) and SNP (Fe II) suppress the elevation of [Ca²⁺]_i induced by NMDA but neither potassium ferricyanide (Fe III) nor SNAP are effective in this regard. These effects are also independent of cyanide as another Fe II compound, ferrous sulfate (FeSO₄) is also able to suppress NMDA-induced elevations of [Ca²⁺]_i SNP was unable to suppress kainate receptor functions. Collectively, these results indicate that Fe II, independently of NO, has effects on NMDA receptor function.