Zinc toxicity in various lung cell lines is mediated by glutathione and GSSG reductase activity

In a previous work, it was shown that in cells after a decrease of cellular glutathione content, toxic zinc effects, such as protein synthesis inhibition or GSSG (glutathione, oxidized form) increases, were enhanced. In this study, zinc toxicity was determined by detection of methionine incorporation as a parameter of protein synthesis and GSSG increase in various lung cell lines (A549, L2, 11Lu, 16Lu), dependent on enhanced GSSG reductase activities and changed glutathione contents. After pretreatment of cells with dl-buthionine-[R,S]-sulfoximine (BSO) for 72 h, cellular glutathione contents were decreased to 15–40% and GSSG reductase activity was increased to 120–135% in a concentration-dependent manner. In BSO pretreated cells, the IC₅₀ values of zinc for methionine incorporation inhibition were unchanged as compared to cells not pretreated. The GSSG increase in BSO pretreated cells by zinc was enhanced in L2, 11Lu, and 16Lu cells, whereas in A549 cells, the GSSG increase by zinc was enhanced only after pretreatment with the highest BSO concentration. Inhibition of GSSG reductase in alveolar epithelial cells was observed at lower zinc concentrations than needed for methionine incorporation inhibition, whereas in fibroblastlike cells, inhibition of GSSG reductase occurred at markedly higher zinc concentrations as compared to methionine incorporation inhibition. These results demonstrate that GSSG reductase is an important factor in cellular zinc susceptibility. We conclude that reduction of GSSG is reduced in zinc-exposed cells. Therefore, protection of GSH oxidation by various antioxidants as well as enhancement of GSH content are expected to be mechanisms of diminishing toxic cellular effects after exposure to zinc.     

Astrocyte control of fetal cortical neuron glutathione homeostasis: up-regulation by ethanol

Ethanol increases apoptotic neuron death in the developing brain and at least part of this may be mediated by oxidative stress. In cultured fetal rat cortical neurons, Ethanol increases levels of reactive oxygen species (ROS) within minutes of exposure and reduces total cellular glutathione (GSH) shortly thereafter. This is followed by onset of apoptotic cell death. These responses to Ethanol can be blocked by elevating neuron GSH with N-acetylcysteine or by co-culturing neurons with neonatal cortical astrocytes. We describe here mechanisms by which the astrocyte-neuron gamma-glutamyl cycle is up-regulated by Ethanol, enhancing control of neuron GSH in response to the pro-oxidant, Ethanol. Up to 6 days of Ethanol exposure had no consistent effects on activities of gamma-glutamyl cysteine ligase or glutathione synthetase, and GSH content remained unchanged (p < 0.05). However, glutathione reductase was increased with 1 and 2 day Ethanol exposures, 25% and 39% for 2.5 and 4.0 mg/mL Ethanol by 1 day, and 11% and 16% for 2.5 and 4.0 mg/mL at 2 days, respectively (p < 0.05). A 24 h exposure to 4.0 mg/mL Ethanol increased GSH efflux from astrocyte up to 517% (p < 0.05). Ethanol increased both gamma-glutamyl transpeptidase expression and activity on astrocyte within 24 h of exposure (40%, p = 0.05 with 4.0 mg/mL) and this continued for at least 4 days of Ethanol treatment. Aminopeptidase N activity on neurons increased by 62% and 55% within 1 h of Ethanol for 2.5 and 4.0 mg/mL concentration, respectively (p < 0.05), remaining elevated for 24 h of treatment. Thus, there are at least three key points of the gamma-glutamyl cycle that are up-regulated by Ethanol, the net effect being to enhance neuron GSH homeostasis, thereby protecting neurons from Ethanol-mediated oxidative stress and apoptotic death.     

Effect of GP120 on glutathione peroxidase activity in cortical cultures and the interaction with steroid hormones

GP120 (the protein component of the HIV viral coat) is neurotoxic and may contribute to the cell loss associated with AIDS-related dementia. Previously, it has been shown in rat cortical mixed cultures that gp120 increased the accumulation of hydrogen peroxide and superoxide, two reactive oxygen species (ROS). We now demonstrate that gp120 increased activity of the key antioxidant glutathione peroxidase (GSPx), presumably as a defensive mechanism against the increased ROS load. Both estrogen and glucocorticoids (GCs), the adrenal steroid released during stress, blunted this gp120 effect on GSPx activity. The similar effects of estrogen and of GCs are superficially surprising, given prior demonstrations that GCs exacerbated and estrogens protected against gp120 neurotoxicity. We find that these similar effects of estrogen and GCs on GSPx regulation arose, in fact, from very different routes, which are commensurate with these prior reports. Specifically, estrogen has demonstrated antioxidant properties that may prevent the ROS increase (therefore acting as a neuroprotective agent) and rendered unnecessary the compensatory GSPx increased activity. To verify this we have added H2O2 to estrogen + gp120-treated cells, and GSPx activity was increased. However, with addition of H2O2 to GCs + gp120-treated cells there was no increase in activity. GCs appeared to decrease enzyme production and or activity and therefore under insult conditions ROS levels rose in the cell resulting in increased neurotoxicity. Overexpression of GSPx enzyme via herpes vector system reversed the GCs-induced loss of enzyme and eliminated the GCs exacerbation of gp120 neurotoxicity.     

Protection by exogenous pyruvate through a mechanism related to monocarboxylate transporters against cell death induced by hydrogen peroxide in cultured rat cortical neurons

In cortical neurons cultured for 3 or 9 days in vitro (DIV), exposure to hydrogen peroxide (H(2)O(2)) led to a marked decrease in cell viability in a concentration-dependent manner at a concentration range of 10 microm to 1 mm irrespective of the duration between 6 and 24 h. However, H(2)O(2) was more potent in decreasing cellular viability in cortical neurons cultured for 9 DIV than in those for 3 DIV. Pyruvate was effective in preventing the neuronal cell death at 1 mm even when added 1-3 h after the addition of H(2)O(2). Semi-quantitative RT-PCR and western blotting analyses revealed significantly higher expression of both mRNA and protein for a particular monocarboxylate transporter (MCT) in neurons cultured for 9 DIV than in those for 3 DIV. A specific inhibitor of MCT significantly attenuated the neuroprotection by pyruvate in neurons cultured for 9 DIV, without markedly affecting that in neurons cultured for 3 DIV. These results suggest that vulnerability to H(2)O(2) may at least in part involve expression of particular MCT isoforms responsible for the bi-directional transport of pyruvate across cell surfaces in cultured rat cortical neurons.     

Influence of glutathione on zinc-mediated cellular toxicity

The effect of zinc on various pulmonary cell lines has been studied by measuring the depletion of total cellular glutathione after exposure to zinc(II) chloride at different concentrations. Total cellular glutathione (cGS) was measured at 31 ± 3 nmol/mg, 3.8 ± 0.6 nmol/mg, and 3.7 ±1.2 nmol/mg protein in A549, L2, and 11Lu cells, respectively. After treatment with buthionine sulfoximine (BSO), the cGS levels decreased by 20% in A549 cells and below 0.2 nmol/mg in L2 and 11Lu cells. Exposure of A549 cells to 25–200 μM ZnCl₂ for 4 h alone decreased the cGS content to 60–80%. There was little additional effect in BSO-pretreated cells. In L2 and 11Lu cells, the decrease of cGS was 70–85% following exposure to 15–150 μM ZnCl₂ for 2 h. If BSO was also used, the decrease in cGS was 85–95% in L2 cells and 75–85% in 11Lu cells. Exposure to 25–250 μM ZnCl₂ for 2 h diminished protein synthesis as determined by radiolabeled methionine incorporation, with half-maximum inhibition (EC₅₀) from 40–160 μM ZnCl₂. To attain similar EC₅₀ values in BSO-pretreated cells, only about half the zinc concentrations were required as compared to cells without pretreatment. The decrease of cGS was accompanied by an increased ratio of oxidized : reduced glutathione that was more pronounced in cells with low glutathione content.     

Immunomodulation following zinc supplementation during chelation of lead in male rats

Influence of zinc supplementation (30 and 45 mg kg^–1, orally once for 5 days) during chelation of lead (0.3 mmol kg^–1, chelating agent, i.p., once for 5 days) on some selected variables of the immune system was investigated in male rats. Treatment with CaNa₂EDTA either alone or in combination with zinc (30 mg kg^–1) produced a significant recovery in lead induced alteration in primary antibody forming cells to T-dependent antigen and the delayed-type hypersensitivity response to bovine albumin. However, biologically significant recovery was observed only with zinc at a dose of 45 mg kg¹. It is assumed that zinc depletion during lead exposure and chelation treatment lead to harmful effects on cellular proliferation by inhibiting DNA synthesis and various enzymes during mitosis. The zinc supplementation fulfills this requirement during proliferation and clonal expansion of immunocompetent cells augmenting the immune system.     

Protective effects of 15-deoxy-Delta12,14-prostaglandin J2 against glutamate-induced cell death in primary cortical neuron cultures: induction of adaptive response and enhancement of cell tolerance primarily through up-regulation of cellular glutathione

There is increasing evidence to suggest that reactive oxygen species, including a variety of lipid oxidation products and other physiologically existing oxidative stimuli, can induce an adaptive response and enhance cell tolerance. In the present study, by using cultured cortical neurons, we investigated the effect of electrophilic lipids, such as 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and 4-hydroxy-2-nonenal (4-HNE) against the cell death induced by H(2)O(2) and glutamate. Pre-treatment with both 15d-PGJ(2) and 4-HNE at sublethal concentrations resulted in a significant protective effect against oxidative stress, and 15d-PGJ(2), in particular, exhibited a complete protective effect against glutamate-induced neuronal cell death. Pre-treatment with 15d-PGJ(2) increased the intracellular glutathione (GSH) as well as the gene expression of glutamate-cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis. 15d-PGJ(2) protected cells from glutamate-induced GSH depletion, while the inhibition of cellular GSH synthesis by buthionine sulfoximine abolished the adaptive response induced by 15d-PGJ(2). These findings indicate that at low levels, 15d-PGJ(2) acts as a potent survival mediator against glutamate-induced insults via the induction of an adaptive response primarily through the up-regulation of the intracellular GSH synthesis.     

锌离子调节皮质酮对原代培养大鼠海马神经元的损伤

探讨皮质酮对原代培养大鼠海马神经元的损伤效应及锌的调节作用。用原位染色和RT-PCR方法,分别检测神经元的损伤情况及NMDA受体三种亚基(NRl、NR2A、NR2B)mRNA的表达。皮质酮(5μmol／L)作用2,4h可明显降低海马神经元的存活率,导致神经元凋亡,并随着作用时间的延长而加重;锌离子明显影响皮质酮对海马神经元的损伤效应：同时加入皮质酮和低、中浓度Zn^2 (10、100μmol／L),可明显降低神经元凋亡率,而加入高浓度Zn^2 (250μmol／L)则加重神经元损伤。皮质酮作用24h后,海马神经元NRl、NR2BmRNA的表达水平增高,而同时加入低、中浓度Zn^2 (10、100μmol／L)的海马神经元NRl、NR2BmRNA表达水平与对照组接近;NR2AmRNA表达无明显变化。这些结果表明,锌对皮质酮所致应激损伤的调节具有双向性;NMDA受体亚基水平的变化可能是其中重要环节之一。     

Neurotrophic and neurotoxic effects of zinc on neonatal cortical neurons

Although zinc exerts direct neurotoxic action, this metal is also essential for the activity of numerous biological systems and zinc deficiency has been associated with various pathologies. We investigated the cellular responses and neuronal viability following exposure to different concentrations of zinc in primary cultures of neonatal rat cortical neurons. Higher concentrations of zinc (0.15 and 0.2 mM) triggered excessive zinc influx, glutathione depletion and ATP loss leading to necrotic neuronal death. In contrast, lower concentrations of zinc (0.05 and 0.1 mM) attenuated serum-deprivation induced apoptotic neuronal death. The antiapoptotic action of low amounts of zinc was found both in mixed cultures and neuron-enriched cultures indicating the independence of glial mediator. Neurotrophic action was not accompanied by significant alteration in those cellular responses but required chelatable zinc. The N-methyl-D-aspartate (NMDA) antagonist, MK-801, mimicked the beneficial effect of zinc in protecting neuronal death. Moreover, both MK-801 and zinc eliminated NMDA-induced neuronal injury. The results suggest that zinc is an intrinsic factor for neuron survival and exogenous zinc, in low amounts, is an active neuroprotectant against serum deprivation in part through the antagonism of NMDA receptor activation.     

Peroxidase-mediated conjugation of glutathione to unsaturated phenylpropanoids. Evidence against glutathione S-transferase involvement

A number of plant species are thought to possess a glutathione S-transferase enzyme (GST: EC 2.5.1.18) that will conjugate glutathione (GSH) to trans -cinnamic acid (CA) and para -coumaric acid (4-CA). However, we present evidence that this activity is mediated by peroxidase enzymes and not GSTs. The N-terminal amino acid sequence of the GSH-conjugating enzyme purified from etiolated corn shoots exhibited a strong degree of homology to cytosolic ascorbate peroxidase enzymes (APX: EC 1.11.1.11) from a number of plant species. The GSH-conjugating and APX activities of corn could not be separated during chromatography on hydrophobic-interaction. anion-exchange, and gel filtration columns. Spectral analysis of the enzyme revealed that the protein had a Soret band at 405 nm. When the enzyme was reduced with dithionite, the peak was shifted to 423 nm with an additional peak at 554 nm. The spectrum of the dithionite-reduced enzyme in the presence of 0.1 m M KCN exhibited peaks at 430, 534 and 563 nm. These spectra are consistent with the presence of a heme moiety. The GSH-conjugating and APX activities of the enzyme were both inhibited by KCN. NaN₃, p -chloromercuribenzoate ( p CMB), and iodoacetate. The APX specific activity of the enzyme was 1.5-fold greater than the GSH-conjugating specific activity with 4-CA. In addition to the corn enzyme, a pea recombinant APX (rAPX) and horseradish peroxidase (HRP; EC 1.11.1.7) were also able to conjugate GSH to CA and 4-CA. The peroxidase enzymes may generate thiyl free radicals of GSH that react with the alkyl double bond of CA and 4-CA resulting in the formation of a GSH conjugate.     

Metabolomic analysis of spontaneous neutrophil apoptosis reveals the potential involvement of glutathione depletion

Spontaneous apoptosis of neutrophils plays a key role in maintaining immune homeostasis and resolving inflammation. However, the mechanism triggering this apoptosis remains obscure. In the present study, we performed a global metabolomics analysis of neutrophils undergoing spontaneous apoptosis by using hydrophilic interaction chromatography ultra-high-performance liquid chromatography-tandem quadrupole/time-of-flight mass spectrometry and found 23 metabolites and 42 related pathways that were altered in these cells. Among them, glutathione, which is known to be involved in apoptosis, was particularly interesting. We found that L-pyroglutamic acid, glutamate, and their glutathione-mediated embolic pathways were all changed. Our findings confirmed the glutathione levels decreased in apoptotic neutrophils. Exogenous glutathione and LPS treatment delayed neutrophil apoptosis and decreased the levels of pro-apoptotic protein caspase-3. γ-glutamylcyclotransferase, 5-oxoprolinase, and ChaC1, which participated in glutathione degradation, were all activated. At the same time, the down-regulation of ATP production suggested the activity of glutathione biosynthesis may be attenuated even if glutamate-cysteine ligase and glutathione synthase, which are two ATP-dependent enzymes participating in glutathione biosynthesis, were enhanced. To our knowledge, this is the first report highlighting a global metabolomics analysis using hydrophilic interaction chromatography ultra-high-performance liquid chromatography-tandem quadrupole/time-of-flight mass spectrometry and the potential involvement of glutathione depletion in spontaneous apoptosis of neutrophils demonstrating that LPS could delay this process.     

Formation of functional synaptic connections between cultured cortical neurons from agrin‐deficient mice

Numerous studies suggest that the extracellular matrix protein agrin directs the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). Strong support for this hypothesis comes from the observation that the high density of acetylcholine receptors (AChR) normally present at the neuromuscular junction fails to form in muscle of embryonic agrin mutant mice. Agrin is expressed by many populations of neurons in the central nervous system (CNS), suggesting that this molecule may also play a role in neuron–neuron synapse formation. To test this hypothesis, we examined synapse formation between cultured cortical neurons isolated from agrin‐deficient mouse embryos. Our data show that glutamate receptors accumulate at synaptic sites on agrin‐deficient neurons. Moreover, electrophysiological analysis demonstrates that functional glutamatergic and gamma‐aminobutyric acid (GABA)ergic synapses form between mutant neurons. The frequency and amplitude of miniature postsynaptic glutamatergic and GABAergic currents are similar in mutant and age‐matched wild‐type neurons during the first 3 weeks in culture. These results demonstrate that neuron‐specific agrin is not required for formation and early development of functional synaptic contacts between CNS neurons, and suggest that mechanisms of interneuronal synaptogenesis are distinct from those regulating synapse formation at the neuromuscular junction. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 547–557, 1999     

Differential effects of ethanol on the expression of cyclo-oxygenase in cultured cortical astrocytes and neurons

The developing central nervous system is a primary target of ethanol toxicity. The teratogenic effect of ethanol may result from its action on prostaglandins. Prostaglandins are generated through the release of arachidonic acid (AA) by the action of cytosolic phospholipase A(2) (cPLA(2)) on membrane-bound phospholipids and the catalytic conversion of AA to prostaglandin E(2) (PGE(2)) by cyclo-oxygenase (COX). COX is expressed in two isoforms, constitutive COX1 and inducible COX2. Cultured astrocytes and neurons from immature cerebral cortex were used as in vitro models to investigate the effect of ethanol on PGE(2) synthesis. In both cell types, neither the activity nor the expression of cPLA(2) was affected by ethanol. PGE(2) was synthesized by astrocytes and neurons. Ethanol (200-400 mg/dL for 24 h) significantly increased PGE(2) production in both cell types and the ethanol-induced increase in PGE(2) accumulation in astrocytes was significantly greater than in neurons. These increases resulted from the effects of ethanol on COX. Overall COX activity was up-regulated by ethanol in astrocytes and neurons, and indomethacin, a nonselective blocker for COX, eliminated the ethanol-induced increases of COX activity in both cell types. Increased COX activity in astrocytes resulted from an increase in COX2 expression. NS-398, a selective COX2 blocker, completely inhibited ethanol-induced alterations in COX activity. In neurons, however, ethanol had a direct effect on COX activity in the absence of a change in COX expression. NS-398 only partially blocked ethanol-induced increases in neuronal COX activity. Thus, astrocytes are a primary target of ethanol and ethanol-induced increases in glial PGE(2) synthesis are mediated by COX, principally COX2. Ethanol toxicity may be mediated through PGE(2) in immature cortical cells.     

Protection against 3,4-methylenedioxymethamphetamine-induced neurodegeneration produced by glutathione depletion in rats is mediated by attenuation of hyperthermia

3,4-Methylenedioxymethamphetamine (MDMA) administration produces neurotoxic degeneration of serotonin terminals in rat brain. These effects occur only after systemic administration and not after central injection, suggesting that peripheral metabolism, possibly hepatic, is required for toxicity. Glutathione is one of the principal cellular defence mechanisms, but conjugation with glutathione can, on some occasions, increase the reactivity of certain molecules. Previous studies have shown that central administration of glutathione adducts of a MDMA metabolite produces a neurotoxicity profile similar to that of systemic MDMA. In the present study, depletion of peripheral (hepatic) glutathione by 43% with dl-buthionine-(S,R)-sulfoximine (an inhibitor of glutathione synthesis) did not attenuate MDMA-induced neurotoxicity as indicated by the 34% loss of [(3) H]paroxetine binding to the serotonin uptake sites in Dark Agouti rats treated with the inhibitor. However, a more profound depletion (92%) of glutathione by diethylmaleate (direct conjugation) administration significantly reduced the serotonergic neurotoxicity produced by MDMA. This depletion protocol also attenuated the hyperthermic response to MDMA. A combination protocol utilising both buthionine-(S,R)-sulfoximine and diethylmaleate that did not alter the hyperthermic response of the rats given MDMA also failed to attenuate the neurotoxicity. These findings indicate that glutathione depletion does not offer specific protection against MDMA-induced serotonin neurotoxicity in Dark Agouti rats.     

A study on the inhibition of rat myocardium glutathione peroxidase and glutathione reductase by moniliformin

The yeasts of patients with oral cancer has been studied before and during Xr-therapy. Gram and PAS smears revealed an increase of yeast-like structures, during treatment, from 56% to 66% of the cases. Before radiotherapy oral yeasts were isolated from 56% of the patients with cancer represented by Candida albicans (30%); C. tropicalis (12%); C. glabrata and C. krusei (4%), besides six other different species (2%). During radiotherapy yeasts were isolated in 72% of the cases, as follow: C. albicans (36%); C. tropicalis (16%); Rhodotorula rubra (6%); C. kefyr; C. krusei and Pichia farinosa (4%), besides other nine species (2%). C. albicans serotype A represented 93% of the isolated samples, before treatment and 88,8% during Xr-therapy.     

Intracellular reduction of selenite into glutathione peroxidase. Evidence for involvement of NADPH and not glutathione as the reductant

Selenium (Se) in selenite is present in an oxidized state, and must be reduced for it to be incorporated as selenocysteine into selenoenzymes such as glutathione peroxidase (GPx). In vitro, Se, as in selenite, can be reduced utilizing glutathione (GSH) and glutathione reductase (GRed). We determined the effects of decreasing GSH levels, inhibiting GRed activity, and decreasing cellular NADPH on the selenite-dependent rate of GPx synthesis in cultured cells: PC3, CHO, and the E89 glucose-6-phosphate dehydrogenase (G-6-PD)-deficient cell line. A novel statistical analysis method was developed (using Box Cox transformed regression and a bootstrap method) in order to assess the effects of these manipulations singly and in combinations. Buthionine sulfoximine (BSO) was used to decrease GSH levels, 1,3 bis-(2 chloroethyl)-1-nitrosourea (BCNU) was used to inhibit GRed activity and methylene blue (MB) was used to decrease cellular NADPH levels. This statistical method evaluates the effects of BSO, BCNU, MB and selenite alone and in combinations on GPx activity. Decreasing the GSH level (< 5% of control) did not have an effect on the selenite-dependent rate of GPx synthesis in PC3 or CHO cells, but did have a small inhibitory effect on the rate of GPx synthesis in E89 cells. Inhibiting GRed activity was also associated with either no effect (CHO, E89) or a small effect (PC3) on GPx activity. In contrast, decreasing NADPH levels in cells treated with MB was associated with a large decrease in the selenite-dependent rate of GPx synthesis to 36, 34 and 25% of control in PC3, CHO, and E89 cells, respectively. The effects of BSO plus BCNU were not synergistic in any of the cell lines. The effects of BSO plus MB were synergistic in G-6-PD-deficient E89 cells, but not in PC3 or CHO cells. We therefore conclude that under normal culture conditions, NADPH, and not glutathione, is the primary reductant of Se in selenite to forms that are eventually incorporated into GPx. For cells with abnormal ability to generate NADPH, lowering the GSH levels had a small effect on selenite-dependent GPx synthesis. GRed activity is not required for the selenite-dependent synthesis of GPx.     

Effect of the chelation of metal cation on the antioxidant activity of chondroitin sulfates

The antioxidant potencies of chondroitin sulfates (CSs) from shark cartilage, salmon cartilage, bovine trachea, and porcine intestinal mucosa were compared by three representative methods for the measurement of the antioxidant activity; DPPH radical scavenging activity, superoxide radical scavenging activity, and hydroxyl radical scavenging activity. CSs from salmon cartilage and bovine trachea showed higher potency in comparison with CSs from shark cartilage and porcine intestinal mucosa. Next, CS from salmon cartilage chelating with Ca²⁺, Mg²⁺, Mn²⁺, or Zn²⁺ were prepared, and their antioxidant potencies were compared. CS chelating with Ca²⁺ or Mg²⁺ ions showed rather decreased DPPH radical scavenging activity in comparison with CS of H⁺ form. In contrast, CS chelating with Ca²⁺ or Mg²⁺ ion showed remarkably enhanced superoxide radical scavenging activity than CS of H⁺ or Na⁺ form. Moreover, CS chelating with divalent metal ions, Ca²⁺, Mg²⁺, Mn²⁺, or Zn²⁺, showed noticeably higher hydroxyl radical scavenging activity than CS of H⁺ or Na⁺ form. The present results revealed that the scavenging activities of, at least, superoxide radical and hydroxyl radical were enhanced by the chelation with divalent metal ions.     

Activity-dependent regulation of HCN1 protein in cortical neurons

Homeostasis of neuronal activity is crucial to neuronal physiology. In dendrites, hyperpolarization-activated cyclic nucleotide-gated channel (HCN) 1 is considered to play critical roles in this process. While electrophysiological studies have demonstrated the dynamic modulation of I_h current mediated by HCN1 proteins, little is known about the underlying molecular and cellular mechanisms. In this study, we utilized cortical cultured neurons and biochemical methods to identify molecular and cellular mechanisms that mediate the physiological regulation of HCN1 channel functions in cortical neurons. Pharmacological manipulations of neuronal activity resulted in changes in the expression level of HCN1. In addition, the surface expression of HCN1 was dynamically regulated by neuronal activity. Both of these changes led to functional modulations of HCN1 channels. Our study suggests that coordinated changes in protein expression and surface expression of HCN1 serve as the key regulatory mechanisms controlling the function of endogenous HCN1 protein in cortical neurons.     

Interference of plasmatic reduced glutathione and hemolysis on glutathione disulfide levels in human blood

The blood reduced glutathione (GSH)/GSH disulfide (GSSG) ratio is an index of the oxidant/antioxidant balance of the whole body. Nevertheless, data indicating GSH and GSSG physiological levels are still widely divergent, especially those on GSSG, probably due to its low concentration. Standardization in methodological protocols and sample manipulation could help to minimize these discrepancies. Therefore, we have investigated how plasma reduced GSH, which is rapidly oxidized after blood withdrawal, could alter the blood GSSG measurement if the sample is not suitably processed. We have observed that an increase in plasma GSH concentration, due to red blood cell hemolysis, is responsible for a significant overestimation of blood GSSG level. Our results show that, before performing blood GSSG determination, thiols have to be rapidly blocked, to avoid possible pitfalls in GSSG measurement, in particular when hemolysis is present.