Intracellular Zn2+ accumulation enhances suppression of synaptic activity following spreading depolarization

Spreading depolarization (SD) is a feed‐forward wave that propagates slowly throughout brain tissue and recovery from SD involves substantial metabolic demand. Presynaptic Zn²⁺ release and intracellular accumulation occurs with SD, and elevated intracellular Zn²⁺ ([Zn²⁺]_i) can impair cellular metabolism through multiple pathways. We tested here whether increased [Zn²⁺]_i could exacerbate the metabolic challenge of SD, induced by KCl, and delay recovery in acute murine hippocampal slices. [Zn²⁺]_i loading prior to SD, by transient ZnCl₂ application with the Zn²⁺ ionophore pyrithione (Zn/Pyr), delayed recovery of field excitatory post‐synaptic potentials (fEPSPs) in a concentration‐dependent manner, prolonged DC shifts, and significantly increased extracellular adenosine accumulation. These effects could be due to metabolic inhibition, occurring downstream of pyruvate utilization. Prolonged [Zn²⁺]_i accumulation prior to SD was required for effects on fEPSP recovery and consistent with this, endogenous synaptic Zn²⁺ release during SD propagation did not delay recovery from SD. The effects of exogenous [Zn²⁺]_i loading were also lost in slices preconditioned with repetitive SDs, implying a rapid adaptation. Together, these results suggest that [Zn²⁺]_i loading prior to SD can provide significant additional challenge to brain tissue, and could contribute to deleterious effects of [Zn²⁺]_i accumulation in a range of brain injury models.     

Significance of Low Nanomolar Concentration of Zn<Superscript>2+</Superscript> in Artificial Cerebrospinal Fluid

Much less attention has been paid to Zn²⁺ in artificial cerebrospinal fluid (ACSF), i.e., extracellular medium, used for in vitro slice experiments than divalent cations such as Ca²⁺. Approximately 2 mM Ca²⁺ is added to conventional ACSF from essentiality of Ca²⁺ signaling in neurons and glial cells. However, no Zn²⁺ is added to it, even though the importance of Zn²⁺ signaling in them is recognizing. On the other hand, synaptic Zn²⁺ homeostasis is changed during brain slice preparation. Therefore, it is possible that not only neuronal excitation but also synaptic plasticity such as long-term potentiation is modified in ACSF without Zn²⁺, in which original physiology might not appear. The basal (static) levels of intracellular (cytosolic) Zn²⁺ and Ca²⁺ are not significantly different between brain slices prepared with conventional ACSF without Zn²⁺ and pretreated with ACSF containing 20 nM ZnCl₂ for 1 h. In the case of mossy fiber excitation, however, presynaptic activity assessed with FM 4–64 is significantly suppressed in the stratum lucidum of brain slices pretreated with ACSF containing Zn²⁺, indicating that hippocampal excitability is enhanced in brain slices prepared with ACSF without Zn²⁺. The evidence suggests that low nanomolar concentration of Zn²⁺ is necessary for ACSF. Furthermore, exogenous Zn²⁺ has opposite effect on LTP induction between in vitro and in vivo experiments. It is required to pay attention to extracellular Zn²⁺ concentration to understand synaptic function precisely.     

Augmentation of zinc ion stimulation of lymphoid cells by calcium and lithium

Stimulation of hamster lymph node cells by optimal concentrations of ZnCl₂ (10 μM) was found to be enhanced by addition of 1–25 mM LiCl to the serum-free cultures. Maximal enhancement occurred at 10 mM Li⁺. Similar concentrations of either KCl or NaCl did not potentiate stimulation. Addition of 1 mM CaCl₂, but not 1–25 mM MgCl₂, also potentiated Zn²⁺ stimulation of lymph node cells. When the cultures were supplemented with 1 mM Ca²⁺ + 10 mM Li⁺, a synergistic potentiation of Zn²⁺ stimulation occurred. In addition, the dose response curve for Zn²⁺ was shifted such that maximal stimulation occurred at 100–250 μM Zn²⁺, a concentration of Zn²⁺ which was toxic for the unsupplemented cultures. In Ca²⁺ + Li⁺ supplemented cultures, Zn²⁺ stimulated [³H]thymidine incorporation to levels comparable to those obtained when hamster lymphoid cells were stimulated with lectins. In addition to Zn²⁺ stimulation, Ca²⁺ + Li⁺ supplemented medium also enhanced Hg²⁺ stimulation of hamster lymph node cells but did not change the dose response curve for Hg²⁺. Therefore, the observed ionic effects on Zn²⁺ stimulation of lymphocytes were unique to this mitogen, when compared to either Hg²⁺ stimulation or previously reported lectin stimulation of hamster lymphoid cells.     

Zinc(II) ion mediates tamoxifen-induced autophagy and cell death in MCF-7 breast cancer cell line

Treatment of MCF-7 cells with tamoxifen induced vacuole formation and cell death. Levels of the autophagy marker, microtubule-associated protein light chain 3 (LC3)-II also increased, and GFP-LC3 accumulated in and around vacuoles in MCF-7 cells exposed to tamoxifen, indicating that autophagy is involved in tamoxifen-induced changes. Live-cell confocal microscopy with FluoZin-3 staining and transmission electron microscopy with autometallographic staining revealed that labile zinc(II) ion (Zn²⁺) accumulated in most acidic LC3(+) autophagic vacuoles (AVs). Chelation of Zn²⁺ with N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) blocked the increase in phospho-Erk and LC3-II levels, and attenuated AV formation and cell death. Conversely, the addition of ZnCl₂ markedly potentiated tamoxifen-induced extracellular signal-regulated kinase (Erk) activation, autophagy and cell death, indicating that Zn²⁺ has an important role in these events. Tamoxifen-induced death was accompanied by increased oxidative stress and lysosomal membrane permeabilization (LMP) represented as release of lysosomal cathepsins into cytosol. Treatment with the antioxidant N-acetyl-l-cysteine (NAC) blunted the increase in Zn²⁺ levels and reduced LC3-II conversion, cathepsin D release and cell death induced by tamoxifen. And cathepsin inhibitors attenuated cell death, indicating that LMP contributes to tamoxifen-induced cell death. Moreover, TPEN blocked tamoxifen-induced cathepsin D release and increase in oxidative stress. The present results indicate that Zn²⁺ contributes to tamoxifen-induced autophagic cell death via increase in oxidative stress and induction of LMP.     

The effect of zinc and magnesium on calcium uptake into the rat duodenum slices

Summary The experiments were carried out on 80 male Wistar rats, divided into four groups as follows: group 1, treated orally with ZnCl₂ at a dose of 10 mg Zn²⁺/kg for 14 days; group 2, control; group 3, MgCl₂-treated at a dose of 5 mg Mg²⁺/kg; group 4, treated with ZnCl₂ plus MgCl₂ in the same manner as groups 1 and 3. The influx of calcium into the rat duodenum slices was investigated in vitro by the method of Papworth and Patrick. Over a range of calcium concentrations (0–10 mM) the influx of this element was defined as a sum of a saturable term (active transport) and a linear term dependent on concentration (passive transport). In the zinc-treated rats only the saturable term was affected. The study of this term by Lineweaver-Burk plots showed a decrease of the half-saturation constant,K _t, while the maximal value,J _m, remained unchanged. Moreover, magnesium was shown to interact with zinc at gut level because simultaneous oral administration of Mg²⁺ and Zn²⁺ to rats protected them against the inhibition of calcium uptake observed when Zn²⁺ was given alone.     

Properties of Magnesium Chelatase in Greening Etioplasts: METAL ION SPECIFICITY AND EFFECT OF SUBSTRATE CONCENTRATIONS

Evidence was obtained by means of low temperature spectrofluorimetry and high pressure liquid chromatography for the exclusive chelation of Mg²⁺ into protoporphyrin IX by a plastid preparation extracted from greening cucumber cotyledons. Under the experimental conditions which were optimized for Mg²⁺ chelation, Zn²⁺ chelation was not detected. However, Zn²⁺ chelation was observed when ATP was omitted and ZnCl₂ was added to the incubation mixture. The observed Zn²⁺ chelation was partially nonenzymic. The requirements for the enzyme, Mg chelatase, are discussed in terms of the regulatory function of this enzyme.     

Reaction of labelled65ZnCl2,65ZnEDTA and65ZnDTPA with different clay-systems and some alluvial Egyptian soils

Summary The clay fraction separated from an alluvial Egyptian soil and montmorillonite clay mineral were equilibrated with CaCl₂ or NaCl solution then treated with humic acid isolated from composted clover straw to obtain different clay systems: Ca-clay, Ca-clay-HA, Na-clay, Na-clay-HA, Ca-mont and Ca-mont-HA. These clays as well as seven soil samples were reacted with different amounts of labelled⁶⁵ZnCl₂,⁶⁵ZnEDTA and⁶⁵ZnDTPA. The effectiveness of these Zn-sources for maintaining soluble Zn²⁺ ions in the equilibrium solution was the greatest for ZnDTPA and the lowest for ZnCl₂. Ca-clay provided greater Zn sorption capacity than Na-clay, and complexing the clay with humic acid depressed its capacity for Zn sorption. At the pH of the clay-systems (pH=6.5), the possibilities of Zn(OH)₂ formation were reduced especially in the presence of Zn-chelates. Reactions of⁶⁵ZnE DTA and⁶⁵ZnDTPA with the seven soils produced higher levels of soluble Zn²⁺ ions in the equilibrium solution rather than⁶⁵ZnCl₂ meanwhile ZnDTPA was more effective than ZnEDTA. The calculated Zn(OH)₂ ion product in the solution of ZnCl₂-soil systems indicated the precipitation of Zn as Zn(OH)₂. However, this was not valid in the Zn-chelates-soil systems. The results also revealed the role of soil carbonate, organic matter and soil texture as soil variables affecting Zn sorption by natural soils.     

Extracellular Zinc Ion Regulates Transient Receptor Potential Melastatin 5 (TRPM5) Channel Activation through Its Interaction with a Pore Loop Domain

The transient receptor potential melastatin 5 (TRPM5) channel is a monovalent cation channel activated by intracellular Ca²⁺. Expression of this channel is restricted to taste cells, the pancreas and brainstem, and is thought to be involved in controlling membrane potentials. Its endogenous ligands are not well characterized. Here, we show that extracellular application of Zn²⁺ inhibits TRPM5 activity. In whole-cell patch-clamp recordings, extracellular application of ZnCl₂ inhibited step-pulse-induced TRPM5 currents with 500 nm free intracellular Ca²⁺ in a dose-dependent manner (IC₅₀ = 4.3 μm at −80 mV). ZnSO₄ also inhibited TRPM5 activity. Extracellular application of ZnCl₂ inhibited TRPM5 activation at several temperatures. Furthermore, inhibition by 30 μm ZnCl₂ was impaired in TRPM5 mutants in which His at 896, and Glu at 926 and/or Glu at 939 in the outer pore loop were replaced with Gln. From these results, we conclude that extracellular Zn²⁺ inhibits TRPM5 channels, and the residues in the outer pore loop of TRPM5 are critically involved in the inhibition.     

Amyloid β-Mediated Zn2+ Influx into Dentate Granule Cells Transiently Induces a Short-Term Cognitive Deficit

We examined an idea that short-term cognition is transiently affected by a state of confusion in Zn²⁺ transport system due to a local increase in amyloid-β (Aβ) concentration. A single injection of Aβ (25 pmol) into the dentate gyrus affected dentate gyrus long-term potentiation (LTP) 1 h after the injection, but not 4 h after the injection. Simultaneously, 1-h memory of object recognition was affected when the training was performed 1 h after the injection, but not 4 h after the injection. Aβ-mediated impairments of LTP and memory were rescued in the presence of zinc chelators, suggesting that Zn²⁺ is involved in Aβ action. When Aβ was injected into the dentate gyrus, intracellular Zn²⁺ levels were increased only in the injected area in the dentate gyrus, suggesting that Aβ induces the influx of Zn²⁺ into cells in the injected area. When Aβ was added to hippocampal slices, Aβ did not increase intracellular Zn²⁺ levels in the dentate granule cell layer in ACSF without Zn²⁺, but in ACSF containing Zn²⁺. The increase in intracellular Zn²⁺ levels was inhibited in the presence of CaEDTA, an extracellular zinc chelator, but not in the presence of CNQX, an AMPA receptor antagonist. The present study indicates that Aβ-mediated Zn²⁺ influx into dentate granule cells, which may occur without AMPA receptor activation, transiently induces a short-term cognitive deficit. Extracellular Zn²⁺ may play a key role for transiently Aβ-induced cognition deficits.     

Zinc reverses glycine-dependent inactivation of NMDARs in cultured rat hippocampal neurons

In the presence of glutamate and co-agonists, e.g., glycine, the N-methyl-D-aspartate receptor (NMDAR) plays an important role in physiological and pathophysiological brain processes. Previous studies indicate glycine could inhibit NMDAR responses induced by high concentration of NMDA in hippocampal neurons. The mechanism underlying this inhibitory impact, however, has been unclear. In this study, the whole-cell patch-clamp recording and Ca²⁺ imaging with Fluo-3/AM under laser scanning confocal microscope were used to analyze the possible involvement of NMDAR subunits in this effect. We found that the peak current of NMDARs and Ca²⁺ influx induced by high concentration of NMDA were reduced by treatment of glycine (0.03?C10 ??mol L^?1) in a dose-dependent manner, and that the glycine-dependent inhibition of NMDAR responses, which were induced at 300 ??mol L^?1 NMDA, was reversed by ZnCl₂ through the blocking of the NR2A subunit of NMDARs, but was less influenced by ifenprodil, a NR2B inhibitor. Our results suggest that the glycine-dependent inactivation of NMDARs is potentially modulated by the regulatory subunit NR2A.     

Elevated zinc induces endothelial apoptosis via disruption of glutathione metabolism: role of the ADP translocator

Zinc is the second-most abundant transition metal within cells and an essential micronutrient. Although adequate zinc is essential for cellular function, intracellular free zinc (Zn²⁺) is tightly controlled, as sustained increases in free Zn²⁺ levels can directly contribute to apoptotic endothelial cell death. Moreover, exposure of endothelial cells to acute nitrosative and/or oxidative stress induces a rapid rise of Zn²⁺ with mitochondrial dysfunction and the initiation of apoptosis. This apoptotic induction can be mimicked through addition of exogenous ZnCl₂ and mitigated by zinc-chelation strategies, indicating Zn²⁺-dependent mechanisms in this process. However, the molecular mechanisms of Zn²⁺-mediated mitochondrial dysfunction are unknown. Here we report that free Zn²⁺ disrupts cellular redox status through inhibition of glutathione reductase, and induces apoptosis by redox-mediated inhibition of the mitochondrial adenine nucleotide transporter (ANT). Inhibition of ANT causes increased mitochondrial oxidation, loss of ADP uptake, mitochondrial translocation of bax, and apoptosis. Interestingly, pre-incubation with glutathione ethyl ester protects endothelial cells from these observed effects. We conclude that key mechanisms of Zn²⁺-mediated apoptotic induction include disruption of cellular glutathione homeostasis leading to ANT inhibition and decreases in mitochondrial ATP synthesis. These pathways could represent novel therapeutic targets during acute oxidative or nitrosative stress in cells and tissues.     

Comparative studies of zinc metabolism in cultured chinese hamster cells with differing metallothionein-induction capacities

Previous work in our laboratory led to the isolation of a cadmium (Cd)-resistant variant (Cd^r2C10) of the line CHO Chinese Hamster cell having a 10-fold greater resistance to the cytotoxic action of Cd²⁺ compared with the CHO cell. This resistance was attributed to an increased capacity of the Cd²⁺-resistant Cd^r2C10 subline to induce synthesis of the Cd²⁺- and Zn²⁺-binding protein(s), metallothionein(s) (MT). Evidence that Cd²⁺ behaves as an analog of the essential trace metal, Zn²⁺, especially as an inducer of MT synthesis, suggested that the Cd^r and CHO cell types could be employed to investigate cellular Zn²⁺ metabolism. In the present study, measurements were made to compare CHO and Cd^r cell types for (a) growth as a function of the level of ZnCl₂ added to the culture medium, (b) uptake and subcellular distribution of Zn²⁺, and (c) capacity to induce MT synthesis. The results of these measurements indicated that (a) both CHO and Cd^r cell types grew normally (T _d≊16–18 h) during exposures to Zn²⁺ at levels up to 100 μM added to the growth medium, but displayed abrupt growth inhibition at higher Zn²⁺ levels, (b) Cd^r cells incorporate fourfold more Zn²⁺ during a 24-h exposure to the maximal subtoxic level of Zn²⁺ and (c) the CHO cell lacks the capacity to induce MT synethesis while the Cd^r cell is proficient in this response during exposure to the maximal subtoxic Zn²⁺ level. These findings suggest that (a) the CHO and Cd^r cell systems will be useful in further studies of cellular Zn²⁺ metabolism, especially in comparisons of Zn²⁺ metabolism in the presence and absence of induction of the Zn²⁺-sequestering MT and (b) a relationship exists between cellular capacity to induce MT synthesis and capacity for cellular Zn²⁺ uptake.     

Quantitative analysis and functional evaluation of zinc ion in the <Emphasis Type="SmallCaps">d</Emphasis>-hydantoinase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> YZ-26

d-Hydantoinase (HDT) is a metal-dependent enzyme that is widely used in industrial bioconversion to d-amino acids as valuable intermediates in the fields of food, pharmaceutical industry and agriculture. In this report, we prepared apo-HDT (metal-removed HDT) and Zn²⁺-HDT (Zn²⁺-added HDT) in vitro from a recombinant HDT (re-HDT) expressed in E. coli. The Zn²⁺-HDT and re-HDT contain 2.17 and 0.95 mol Zn²⁺ per mol subunit, respectively, and they have comparable enzymatic activities. In contrast, the apo-HDT only retains 0.04 mol Zn²⁺ per mol subunit with less than 10% activity, compared with the re-HDT. When the apo-HDT was reconstituted with ZnCl₂, the enzymatic activity recovery was about 75%. Moreover, the fluorescence intensity, circular dichroism spectra and thermo-stability of the apo-HDT and Zn²⁺-HDT are quite different from those of the re-HDT. These data suggest that the re-HDT may have two Zn²⁺-binding sites, one is an intrinsic or tight-binding site (zinc-α) essential for its activity and the other is a vacant or loose-binding site (zinc-β) possibly non-essential for the activity.     

Electrical rhythmicity and spread of action potentials in longitudinal muscle of guinea pig distal colon

Using simultaneous intracellular recordings, we have characterized 1) electrical activity in the longitudinal muscle (LM) of isolated segments of guinea pig distal colon free to contract spontaneously and 2) extent of propagation of spontaneous action potentials around the circumference of the colon. In all animals, rhythmical spontaneous depolarizations (SDs) were recorded that are usually associated with the generation of action potentials. Recordings from pairs of LM cells, separated by 100 microm in the circumferential axis, revealed that each action potential was phase locked at the two electrodes (mean propagation velocity: 3 mm/s). However, at an increased electrode separation distance of 1 mm circumferentially, action potentials and SDs became increasingly uncoordinated at the two recording sites. No SDs or action potentials ever propagated from one circumferential edge to the other (i.e., 13 mm apart). When LM strips were separated from the myenteric plexus and circular muscle, rhythmically firing SDs and action potentials were still recorded. Atropine (1 microM) or tetrodotoxin (1 microM) either reduced the frequency of SDs or temporarily abolished activity, whereas nifedipine (1 microM) always abolished SDs and action potentials. Kit-positive interstitial cells of Cajal were present at the level of the myenteric plexus and circular and longitudinal muscle. In summary, SDs and action potentials in LM propagate over discrete localized zones, usually <1 mm around the circumference of the colon. Furthermore, in contrast to the classic slow wave, rhythmic depolarizations in LM appear to be generated by an intrinsic property of the smooth muscle itself and are critically dependent on opening of L-type Ca(2+) channels.     

Irreversible zinc ion inhibition of (Na+ -K+)-adenosinetriphosphatase, Na+ -phosphorylation, and K+-p-nitrophenylphosphatase of Electrophorus electricus electroplax.

Zinc ion in micromolar concentrations is an irreversible inhibitor of Electrophorus electricus electroplax microsomal (Na⁺-K⁺)-ATPase. The rate of inhibition is dependent on [ZnCl₂] and the extent of inhibition varies with the ratio of ZnCl₂ to microsomal protein. The same kinetics are observed for inhibition of K⁺ -p-nitrophenylphosphatase and steady-state levels of Na⁺ -dependent enzyme phosphorylation. The observations suggest that a Zn²⁺ -sensitive conformational restraint is important to both kinase and phosphatase activities. The fact that inhibition is irreversible has implications for models seeking to relate zinc effects in tissue to inhibition of (Na⁺-K⁺)-ATPase.     

A metallothionein-like protein of rice (rgMT) functions in E. coli and its gene expression is induced by abiotic stresses

A metallothionein-like (rgMT) gene was isolated from a rice (Oryza sativa L.) root cDNA library that was prepared from plants grown under NaHCO₃ stress. The rgMT gene expression was induced in rice leaves and roots under several abiotic stresses from salts (NaCl and NaHCO₃), drought (PEG) and metals (CuCl_2, ZnCl₂, CdCl₂). The results suggested that the rgMT gene was expressed in response to environmental stresses. The rgMT gene was expressed in Escherichia coli, and the final yield of the purified rgMT protein was 4.8 mg g⁻¹ dry cells. Tolerance of E. coli expressing GST-rgMT fusion protein to Cu²⁺, Zn²⁺ and Cd²⁺ was enhanced, and cells dry weight increased 0.04 mg, 0.17 mg and 0.07 mg in 1 ml culture treated with either CuCl₂, ZnCl₂ or CdCl₂, respectively, compared with control after 6 h culture.     

Two mechanisms for spreading depression in the chicken retina

Two mechanisms have been proposed to explain spreading depression (SD): one based on a release of glutamate (Van Harreveld, 1959), and the other on a release of potassium (Grafstein, 1956) from neuronal elements. Both glutamate and KCl cause transparency changes in the retina, comparable to those occurring in this tissue during SD. The glutamate effect is inhibited by MgCl₂ (10 mM), in contrast to the transparency change due to KCl which is not affected by Mg⁺⁺. Also SD is usually inhibited by MgCl₂ which suggests that such SDs are based on a glutamate release. Impairment of the tissue metabolism promotes SDs which are insensitive to MgCl₂. The resulting failure of the mechanisms that transport K⁺ and glutamate which leak out of the intracellular compartment back into the cells and fibers, seems to be involved in the generation of Mg⁺⁺ insensitive SDs. This may facilitate either K-based SDs or glutamate-based SDs since the inhibitory effect of Mg⁺⁺ is counteracted by an enhanced glutamate concentration. Both proposed mechanisms for SD seem to be possible under special circumstances.     

Zinc biosorption by a zinc-resistant bacterium, Brevibacterium sp. strain HZM-1

A zinc-resistant bacterium, Brevibacterium sp. strain HZM-1 which shows a high Zn²⁺-adsorbing capacity, was isolated from the soil of an abandoned zinc mine. Kinetic analyses showed that Zn²⁺ binding to HZM-1 cells follows Langmuir isotherm kinetics with a maximum metal capacity of 0.64 mmol/g dry cells and an apparent metal dissociation constant of 0.34 mM. The observed metal-binding capacity was one of the highest values among those reported for known microbial Zn²⁺ biosorbents. The cells could also adsorb heavy metal ions such as Cu²⁺. HZM-1 cells could remove relatively low levels of the Zn²⁺ ion (0.1 mM), even in the presence of large excess amounts (total concentration, 10 mM) of alkali and alkali earth metal ions. Bound Zn²⁺ ions could be efficiently desorbed by treating the cells with 10 mM HCl or 10 mM EDTA, and the Zn²⁺-adsorbing capacity of the cells was fully restored by treatment of the desorbed cells with 0.1 M NaOH. Thus, HZM-1 cells can serve as an excellent biosorbent for removal of Zn²⁺ from natural environments. The cells could grow in the presence of significant concentrations of ZnCl₂ (at least up to 15 mM) and thus is potentially applicable to in situ bioremediation of Zn²⁺-contaminated aqueous systems. Received: 1 February 2000 / Received revision: 31 March 2000 / Accepted: 1 May 2000     

Characterization of Inhibitory GABA-A Receptor Activation during Spreading Depolarization in Brain Slice

Spreading depolarization (SD) is a slowly propagating wave of near complete depolarizations of neurons and glia. Previous studies have reported large GABA releases during SD, but there is limited understanding of how GABA release and receptor activation are regulated and influence the propagating SD wavefront, as well as an excitatory phase immediately following the passage of SD. The present study characterized GABA-A type receptor (GABA_AR) currents during SD generated by KCl microinjection in acute hippocampal slices from adult mice. Spontaneous GABA_AR-mediated currents (sIPSCs) were initially enhanced, and were followed by a large outward current at the wavefront. sIPSC were then transiently supressed during the late SD phase, resulting in a significant reduction of the sIPSC/sEPSC ratio. The large outward current generated during SD was eliminated by the GABA_AR antagonist gabazine, but the channel potentiator/agonist propofol failed to potentiate the current, likely because of a ceiling effect. Extracellular Cl⁻ decreases recorded during SD were reduced by the antagonist but were not increased by the potentiator. Together with effects of GABA_AR modulators on SD propagation rate, these results demonstrate a significant inhibitory role of the initial GABA_AR activation and suggest that intracellular Cl⁻ loading is insufficient to generate excitatory GABA_AR responses during SD propagation. These results provide a mechanistic explanation for facilitating effects of GABA_AR antagonists, and the lack of inhibitory effect of GABA_AR potentiators on SD propagation. In addition, selective suppression of GABA transmission in the late SD period and the lack of effect of GABA_A modulators on the duration of SD suggests that GABA modulation may not be effective approach to protect neurons during the vulnerable phase of SD.     

Update on biochemical properties of recombinant Pseudomonas diminuta phosphotriesterase

Phosphotriesterase from Pseudomonas diminuta (PTE; EC 3.1.8.1) hydrolyzes organophosphate insecticides and chemical warfare agents. The two zinc cations in the active center can be substituted. Co²⁺-containing PTE is the most efficient but least stable isoform. Gel filtration showed that PTE is monomeric at the submicromolar concentrations used in kinetic assays. The analysis of the recombinant enzyme by X-ray fluorescence spectrometry and CCT-ICP-MS, confirms that recombinant Zn-PTE contains only Zn²⁺ whereas Co-PTE has Zn²⁺ and Co²⁺ in equimolar amount, with Co²⁺ most likely in the reported labile β-site. We noted that recombinant PTE is unstable at low concentrations and must be stabilized by a protein environment. We tested the effect of excess of various metal cofactors on PTE-catalyzed hydrolysis of paraoxon. We notably observed that ZnCl₂ induces a non-competitive partial inhibition of Zn²⁺- and Co²⁺-PTE at pH 8.5 (apparent Ki=155 μM and 52 μM, respectively). Inhibition results from interactions with colloidal Zn(OH)₂ formed in alkaline buffer that alters the catalytic machinery. NiCl₂ caused a similar effect at higher concentrations (apparent Ki=3 mM). We observed that mutating His123, a surface residue close to an alleged allosteric site, dramatically altered the bacterial expression yield of Co²⁺-PTE, Ki for Zn(OH)₂ inhibition, k_cat (up to 60 fold) for paraoxon hydrolysis, but not K_M. Issues addressed in this work are important for future biotechnological developments of PTE as a detoxifying enzyme.