Suppressed heat shock protein response in the kidney of exercise-trained diabetic rats.

Impaired expression of heat shock proteins (HSPs) and increased oxidative stress may contribute to the pathophysiology of diabetes by disrupted tissue protection. Acute exercise induces oxidative stress, whereas exercise training up-regulates endogenous antioxidant defenses and HSP expression. Although diabetic nephropathy is a major contributor to diabetic morbidity, information regarding the effect of HSPs on kidney protection is limited. This study evaluated the effects of eight-week exercise training on kidney HSP expression and markers of oxidative stress at rest and after acute exercise in rats with or without streptozotocin-induced diabetes. Induction of diabetes increased DNA-binding activity of heat shock factor-1, but decreased the expression of HSP72, HSP60, and HSP90. The inflammatory markers IL-6 and TNF-alpha were increased in the kidney tissue of diabetic animals. Both exercise training and acute exercise increased HSP72 and HSP90 protein levels only in non-diabetic rats. On the other hand, exercise training appeared to reverse the diabetes-induced histological changes together with decreased expression of TGF-beta as a key inducer of glomerulosclerosis, and decreased levels of IL-6 and TNF-alpha. Notably, HSP72 and TGF-beta were negatively correlated. In conclusion, impaired HSP defense seems to contribute to kidney injury vulnerability in diabetes and exercise training does not up-regulate kidney HSP expression despite the improvements in histopathological and inflammatory markers.

Exercise training and experimental diabetes modulate heat shock protein response in brain.

In diabetes, defense systems against cellular stress are impaired. Heat shock proteins (HSPs) function primarily as molecular chaperones. Factors that raise tissue HSP levels may slow progression of diabetes and improve diabetic complications that also affect brain tissue. This study tested the effect of an 8-week exercise training on brain HSP response in rats with or without streptozotocin-induced diabetes (SID). In untrained animals, the HSP levels were not different between SID and non-diabetic groups. Endurance training, however, increased HSP72 and HSP90 protein in non-diabetic rats, whereas SID significantly decreased the effect of training on these HSPs. At the mRNA level, HSP60, HSP90 and GRP75 were increased due to training, whereas HSP72 mRNA was only increased in exercise-trained diabetic animals. Training or diabetes had no effect on protein carbonyl content, a marker of oxidative damage. Altogether, our findings suggest that endurance training increases HSP expression in the brain, and that experimental diabetes is associated with an incomplete HSP response at the protein level.

Novel burn device for rapid, reproducible burn wound generation.

INTRODUCTION: Scarring following full thickness burns leads to significant reductions in range of motion and quality of life for burn patients. To effectively study scar development and the efficacy of anti-scarring treatments in a large animal model (female red Duroc pigs), reproducible, uniform, full-thickness, burn wounds are needed to reduce variability in observed results that occur with burn depth. Prior studies have proposed that initial temperature of the burner, contact time with skin, thermal capacity of burner material, and the amount of pressure applied to the skin need to be strictly controlled to ensure reproducibility. The purpose of this study was to develop a new burner that enables temperature and pressure to be digitally controlled and monitored in real-time throughout burn wound creation and compare it to a standard burn device. METHODS: A custom burn device was manufactured with an electrically heated burn stylus and a temperature control feedback loop via an electronic microstat. Pressure monitoring was controlled by incorporation of a digital scale into the device, which measured downward force. The standard device was comprised of a heat resistant handle with a long rod connected to the burn stylus, which was heated using a hot plate. To quantify skin surface temperature and internal stylus temperature as a function of contact time, the burners were heated to the target temperature (200±5°C) and pressed into the skin for 40s to create the thermal injuries. Time to reach target temperature and elapsed time between burns were recorded. In addition, each unit was evaluated for reproducibility within and across three independent users by generating burn wounds at contact times spanning from 5 to 40s at a constant pressure and at pressures of 1 or 3lbs with a constant contact time of 40s. Biopsies were collected for histological analysis and burn depth quantification using digital image analysis (ImageJ). RESULTS: The custom burn device maintained both its internal temperature and the skin surface temperature near target temperature throughout contact time. In contrast, the standard burner required more than 20s of contact time to raise the skin surface temperature to target due to its quickly decreasing internal temperature. The custom burner was able to create four consecutive burns in less than half the time of the standard burner. Average burn depth scaled positively with time and pressure in both burn units. However, the distribution of burn depth within each time-pressure combination in the custom device was significantly smaller than with the standard device and independent of user. CONCLUSIONS: The custom burn device's ability to continually heat the burn stylus and actively control pressure and temperature allowed for more rapid and reproducible burn wounds. Burns of tailored and repeatable depths, independent of user, provide a platform for the study of anti-scar and other wound healing therapies without the added variable of non-uniform starting injury.

Fas mediated apoptosis of human Jurkat T-cells: intracellular events and potentiation by redox-active alpha-lipoic acid.

Activation of caspases is required in Fas receptor mediated apoptosis. Maintenance of a reducing environment inside the cell has been suggested to be necessary for caspase activity during apoptosis. We explored the possibility to potentiate Fas mediated killing of tumor cells by alpha-lipoic acid (LA), a redox-active drug and nutrient that is intracellularly reduced to a potent reductant dihydrolipoic acid. Treatment of cells with 100 microM LA for 72 h markedly potentiated Fas-mediated apoptosis of leukemic Jurkat cells but not that of peripheral blood lymphocytes from healthy humans. In Jurkat, Fas activation was followed by rapid loss of cell thiols, decreased mitochondrial membrane potential, increased [Ca2+]i and increased PKC activity; all these responses were potentiated in LA pretreated cells. PKCdelta played an important role in mediating the effect of LA on Fas-mediated cell death. In response to Fas activation LA treatment potentiated caspase 3 activation by over 100%. The ability of LA to potentiate Fas mediated killing of leukemic cells was abrogated by a caspase 3 inhibitor suggesting that increased caspase 3 activity in LA-treated Fas-activated cells played an important role in potentiating cell death. This work provides first evidence showing that inducible caspase 3 activity may be pharmacologically up-regulated by reducing agents such as dihydrolipoic acid.

Increased resting and exercise-induced oxidative stress in young IDDM men.

OBJECTIVE: To assess the effect of acute physical exercise on oxidative stress and glutathione redox status and the relation to physical fitness in otherwise healthy young men with IDDM. RESEARCH DESIGN AND METHODS: Nine men with IDDM (HbA1 7.3 +/- 1.7%), ages 21-30 years, and 13 matched control subjects exercised on a bicycle ergometer for 40 min at 60% of their maximal oxygen consumption (VO2max). Oxidative stress was assessed with plasma thiobarbituric acid reactive substance (TBARS) levels (an index of lipid peroxidation) and, in response to exercise, also glutathione redox status. For glutathione redox status, blood total glutathione (TGSH) and oxidized glutathione (GSSG) were determined. Blood samples were drawn immediately before and after exercise. RESULTS: Resting plasma TBARS levels were markedly elevated in diabetic patients (2.2 +/- 0.7 vs. 0.9 +/- 0.4 mumol/l; P = 0.0002). Mean blood TGSH was higher in diabetic subjects (1,203 +/- 221 vs. 936 +/- 156 mmol/l; P = 0.002), with no significant difference in GSSG or GSSG/TGSH values. Exercise increased plasma TBARS and blood GSSG by approximately 50% in both groups. Resting plasma TBARS had a strong inverse correlation (r = -0.82; P = 0.006), and the exercise-induced percentage increase in TBARS had a strong positive correlation (r = 0.81, P = 0.008) with VO2max in diabetic subjects only. CONCLUSIONS: Glutathione redox status appears to be adequate in healthy young moderately active diabetic men. On the other hand, they demonstrated increased resting and postexercise oxidative stress as indicated by plasma TBARS. Although exercise acutely induces oxidative stress, in patients with diabetes, physical fitness may have a protective effect against oxidative stress.

Involvement of intracellular Ca2+ in oxidant-induced NF-kappa B activation.

In human Jurkat T cells and its subclone Wurzburg cells oxidant challenge elevated [Ca2+]i by mobilizing Ca2+ from intracellular stores. In Jurkat cells this effect was rapid and transient, but in Wurzburg cells the response was slow and sustained. H2O2-induced NF-kappaB activation in Wurzburg cells was not influenced by the presence of extracellular EGTA but was totally inhibited in cells that were loaded with esterified EGTA. In Jurkat cells that are not sensitive to H2O2-induced NF-kappaB activation, H2O2 potentiated NF-kappaB activation in the presence of sustained high [Ca2+]i following thapsigargin treatment. NF-kappaB regulatory effect of alpha-lipoate and N-acetylcysteine appeared to be, at least in part, due to their ability to stabilize elevation of [Ca2+]i following oxidant challenge. Results of this study indicate that a sustained elevated [Ca2+]i is a significant factor in oxidant-induced NF-kappaB activation.

Inward potassium transport systems in skeletal muscle derived cells are highly sensitive to oxidant exposure.

Strenuous physical exercise causes a remarkable perturbation of K+ homeostasis in skeletal muscle tissue. Potassium efflux is crucial for a number of physiological control processes; however, exercise-induced perturbation of K+ homeostasis in skeletal muscle is suggested to be implicated in the generation of muscle fatigue. Physical exercise is also known to induce oxidative stress; a possible contribution of oxygen free radicals to the development of muscle fatigue has been hypothesized. To reveal the dose-dependent effect of oxidant exposure on inward and outward K+ (86RbCl) transporting systems, skeletal muscle derived L6 cells were treated with different concentrations of tert-butylhydroperoxide (TBOOH). We document the responses of (1) the ouabain-sensitive component of K+ influx (Na+,K+ pump), (2) bumetanide-sensitive ouabain-insensitive component of K+ influx (Na+,K+, 2Cl- cotransporter), (3) ouabain- and bumetanide-insensitive component of K+ influx (passive permeability of the cell membrane to inward K+), (4) ouabain-insensitive component of K+ efflux, and (5) passive leakage component of K+ efflux following exposure of L6 cells to oxidant treatment. Even very low doses of TBOOH (25 mumol) caused powerful activation of the Na+,K+ pump. Following TBOOH treatment, activity of the Na+,K+,2Cl- cotransporter was remarkably inhibited. Such a treatment also significantly decreased the permeability of the cell membrane to inward flux of K+ (passive influx). Thus, we observed that even very low doses of oxidant had remarkable specific effects on the different components of K+ influx in the skeletal muscle derived cells. However, K+ efflux mechanisms appeared to be rather insensitive to the extracellular oxidant challenge.

Upregulation of oxidant-induced VEGF expression in cultured keratinocytes by a grape seed proanthocyanidin extract.

Angiogenesis plays a central role in wound healing. Among many known growth factors, vascular endothelial growth factor (VEGF) is believed to be the most prevalent, efficacious, and long-term signal that is known to stimulate angiogenesis in wounds. The wound site is rich in oxidants such as hydrogen peroxide mostly contributed by neutrophils and macrophages. Proanthocyanidins or condensed tannins are a group of biologically active polyphenolic bioflavonoids that are synthesized by many plants. This study provides first evidence showing that natural extracts such as grape seed proanthocyanidin extract containing 5000 ppm resveratrol (GSPE) facilitates oxidant-induced VEGF expression in keratinocytes. Using a ribonuclease protection assay (RPA), the ability of GSPE to regulate oxidant-induced changes in several angiogenesis-related genes were studied. While mRNA responses were studied using RPA, VEGF protein release from cells to the culture medium was studied using ELISA. Pretreatment of HaCaT keratinocytes with GSPE upregulated both hydrogen peroxide as well as TNF-alpha-induced VEGF expression and release. The current results suggest that GSPE may have beneficial therapeutic effects in promoting dermal wound healing and other related skin disorders.

Antioxidant regulation of phorbol ester-induced adhesion of human Jurkat T-cells to endothelial cells.

Regulation of adhesion molecule expression and function by reactive oxygen species via specific redox sensitive mechanisms have been reported. The effects of clinically safe antioxidants in the regulation of adhesion molecule expression in human endothelial cells (ECV), and adherence of human Jurkat T cells to ECV cells were investigated. The thiol antioxidant, alpha-lipoate, at clinically relevant doses down-regulated phorbol 12-myristate 13-acetate (PMA)-induced adhesion molecule expression and cell-cell adhesion. Inhibition of PMA-induced ICAM-1 and VCAM-1 expression as well as PMA-induced adhesion of Jurkat T-cells to ECV cells by alpha-lipoate was dose dependent (50-250 microM). The effect was significant for ICAM-1 (p < .01) and VCAM-1 (p < .01) expression in cells pretreated with 100 microM alpha-lipoate compared to PMA-activated untreated cells. Inhibition of PMA-induced adhesion molecule expression and cell-cell adhesion was more pronounced when a combination of antioxidants, alpha-lipoate and alpha-tocopherol, were used compared to the use of either of these antioxidant alone. The regulation of adhesion molecule expression and function by low concentration of antioxidants investigated does not appear to be NF-kappaB regulated or transcription dependent because no change in the mRNA response was observed. Protein kinase C (PKC) has been suggested to regulate PMA-induced adhesion molecule expression by post-transcriptional stabilization of adhesion molecule mRNA. Alpha-lipoate pretreatment did not influence the response of PKC activity to PMA. Oxidants are known to be involved in the regulation of cell adhesion processes. Treatment of ECV cells with PMA induced generation of intracellular oxidants. Alpha-lipoate (100 or 250 microM) treatment decreased PMA-induced generation of intracellular oxidants. The inhibitory effect of low concentration of alpha-lipaote alone or in combination with alpha-tocopherol on agonist-induced adhesion processes observed in this study may be of potential therapeutic value.

Regulation of cellular thiols in human lymphocytes by alpha-lipoic acid: a flow cytometric analysis.

Modulation of cellular thiols is an effective therapeutic strategy, particularly in the treatment of AIDS. Lipoic acid, a metabolic antioxidant, functions as a redox modulator and has proven clinically beneficial effects. It is also used as a dietary supplement. We utilized the specific capabilities of N-ethylmaleimide to block total cellular thiols, phenylarsine oxide to block vicinal dithiols, and buthionine sulfoximine to deplete cellular GSH to flow cytometrically investigate how these thiol pools are influenced by exogenous lipoate treatment. Low concentrations of lipoate and its analogue lipoamide increased Jurkat cell GSH in a dose-dependent manner between 10 (25 microM for lipoamide) to 100 microM. This was also observed in mitogenically stimulated peripheral blood lymphocytes (PBL). Studies with Jurkat cells and its Wurzburg subclone showed that lipoate dependent increase in cellular GSH was similar in CD4+ and - cells. Chronic (16 week) exposure of cells to lipoate resulted in further increase of total cellular thiols, vicinal dithiols, and GSH. High concentration (2 and 5 mM) of lipoate exhibited cell shrinkage, thiol depletion, and DNA fragmentation effects. Based on similar effects of octanoic acid, the cytotoxic effects of lipoate at high concentration could be attributed to its fatty acid structure. In certain diseases such as AIDS and cancer, elevated plasma glutamate lowers cellular GSH by inhibiting cystine uptake. Low concentrations of lipoate and lipoamide were able to bypass the adverse effect of elevated extracellular glutamate. A heterogeneity in the thiol status of PBL was observed. Lipoate, lipoamide, or N-acetylcysteine corrected the deficient thiol status of cell subpopulations. Hence, the favorable effects of low concentrations of lipoate treatment appears clinically relevant.

Pine bark extract pycnogenol downregulates IFN-gamma-induced adhesion of T cells to human keratinocytes by inhibiting inducible ICAM-1 expression.

Expression of intercellular adhesion molecule-1 (ICAM-1) is necessary for leukocyte/keratinocyte interactions. Upregulation of ICAM-1 expression in keratinocytes has been observed in several inflammatory dermatoses, such as psoriasis, atopic dermatitis, and lupus erythematosus. Inflammatory cytokines, such as interferon-gamma (IFN-gamma), upregulate ICAM-1 expression in keratinocytes. Because of potent antioxidant and anti-inflammatory properties of the French maritime pine bark extract, Pycnogenol (Horphag Research, Geneva, Switzerland), its effects were investigated on the interaction of T cells with keratinocytes after activation with IFN-gamma and the molecular mechanisms involved in such interactions. Studies were performed using a human keratinocyte cell line, HaCaT. Cell adhesion in the presence of IFN-gamma was studied using a coculture assay. Treatment of HaCaT cells with 20 U/ml IFN-gamma for 24 h markedly induced adherence of Jurkat T cells to HaCaT cells. PYC pretreatment (50 microg/ml, 12 h) significantly inhibited IFN-gamma induced adherence of T cells to HaCaT cells (p < .01). ICAM-1 plays a major role in the IFN-gamma-induced adherence of T cells to keratinocytes. Thus, the effect of PYC on IFN-gamma-induced ICAM-1 expression was investigated as well. Pretreatment of HaCaT cells with PYC significantly inhibited IFN-gamma-induced expression of ICAM-1 expression in HaCaT cells. The downregulation of inducible ICAM-1 expression by PYC was both dose and time dependent. A 50 microg/ml dose of PYC and a 12 h pretreatment time (i.e., before activation with IFN-gamma) provided maximal (approximately 70%) inhibition of inducible ICAM-1 expression in HaCaT cells. Gamma-activated sequence present on the ICAM-1 gene confers IFN-gamma responsiveness in selected cells of epithelial origin (e.g., keratinocytes) that are known to express ICAM-1 on activation with IFN-gamma. Gel-shift assays revealed that PYC inhibits IFN-gamma-mediated activation of Stat1, thus suggesting a transcriptional regulation of inducible ICAM-1 expression by PYC. These results indicate the therapeutic potential of PYC in patients with inflammatory skin disorders.

Neuroprotective effects of alpha-lipoic acid and its positively charged amide analogue.

Elevated levels of extracellular glutamate have been linked to reactive oxygen species mediated neuronal damage and brain disorders. Lipoic acid is a potent antioxidant that has previously been shown to exhibit neuroprotection in clinical studies. A new positively charged water soluble lipoic acid amide analog, 2-(N,N-dimethylamine) ethylamido lipoate HCl (LA-plus), with a better cellular reduction and retention of the reduced form was developed. This novel antioxidant was tested for protection against glutamate induced cytotoxicity in a HT4 neuronal cell line. Glutamate treatment for 12 h resulted in significant release of LDH from cells to the medium suggesting cytotoxicity. Measurement of intracellular peroxides showed marked (up to 200%) increase after 6 h of glutamate treatment. Compared to lipoic acid, LA-plus was more effective in (1) protecting cells against glutamate induced cytotoxicity, (2) preventing glutamate induced loss of intracellular GSH, and (3) disallowing increase of intracellular peroxide level following the glutamate challenge. The protective effect of LA-plus was found to be independent of its stereochemistry. The protective function of this antioxidant was synergistically enhanced by selenium. These results demonstrate that LA-plus is a potent protector of neuronal cells against glutamate-induced cytotoxicity and associated oxidative stress.

Redox-modulating gene therapies for human diseases.

Baseline levels of reactive oxygen species (ROS) are generated as an integral component of cellular function. Under certain conditions, e.g., the presence of an elevated concentration of transition metal (Fe/Cu) ions, drug metabolism, or ischemia-reperfusion, ROS generation is exaggerated to an extent that overwhelms cellular antioxidant defenses and results in oxidative stress. Oxidative stress has been characterized by the assessment of oxidative damage to cellular components, e.g., protein, lipid, and nucleic acid. More recent studies have determined that at a concentration much below that required for inflicting oxidative damage, ROS may serve as cellular second messengers through the regulation of numerous signal transduction pathways. For this reason, much of the current medical focus in this area has been directed toward the understanding of redox-driven physiological and pathophysiological processes in the cell. The goal of such research is to formulate effective strategies for manipulating the cellular redox environment in a manner that is beneficial for restoring normal cell functions in the setting of disease.

Mitochondrial control of inducible nitric oxide production in stimulated RAW 264.7 macrophages.

Addition of glucose to activated RAW 264.7 macrophages or addition of mitochondrial electron-transfer chain inhibitors enhanced the cellular nitric oxide production. An additive effect of rotenone or antimycin A and glucose on enhancing nitric oxide production was shown. Uncoupling the mitochondria by a chemical uncoupler decreased nitric oxide production. The mitochondria membrane potential was found to be important for cell viability. Although nitric oxide is the physiological inhibitor of mitochondrial respiration, this study indicates that mitochondria were not inhibited in the activated macrophages. Furthermore, a role of mitochondria in the rapid regulation of nitric oxide synthesis by the inducible nitric oxide synthase has been demonstrated.

High expression of human 15-lipoxygenase induces NF-kappaB-mediated expression of vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and T-cell adhesion on human endothelial cells.

Expression of 15-lipoxygenase (15-LO) is induced over 100-fold in early fatty streak lesions. 15-LO activity leads to the production of specific lipid hydroperoxides, which can have major effects on the expression of proinflammatory genes involved in atherogenesis. We have used retrovirus-mediated gene transfer to achieve stable high expression of 15-LO in human endothelial ECV304 cells. These cells were used to study the effects of 15-LO on the expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), activation of nuclear factor kappa B (NF-kappaB), and T-cell adhesion on endothelial cells. NF-kappaB activation was greatly potentiated by increased 15-LO activity in the stably transduced cells, and both VCAM-1 and ICAM-1 were significantly induced in these cells in response to tumor necrosis factor-alpha (TNF-alpha) and phorbol 12-myristate 13-acetate (PMA) stimulation, as studied by flow cytometry. The induction of ICAM-1 was sensitive to antioxidants in a dose-dependent manner. The adherence of Jurkat T cells on the 15-LO-expressing endothelial cells was markedly induced after PMA stimulation. These results indicate that 15-LO activity may be involved in the early pathogenesis of atherosclerosis by inducing VCAM-1 and ICAM-1 expression and by increasing T-cell adhesion on the endothelium.

Physical exercise induces activation of NF-kappaB in human peripheral blood lymphocytes.

Current understanding of nuclear factor-kappaB (NF-kappaB) activation is derived mostly from in vitro studies, and in vivo human data are limited. This study provides first evidence showing that physical exercise (80% maximal O2 consumption, 1 h) may trigger NF-kappaB activation, as determined by electrophoretic mobility shift assay, in peripheral blood lymphocytes of physically fit young men. Supershift assay showed that the NF-kappaB protein complex contained the transcriptionally active p65 protein. Plasma levels of NF-kappaB-directed gene products such as tumor necrosis factor-alpha and interleukin-2 receptor confirmed that physical exercise caused NF-kappaB transactivation. Exercise-induced NF-kappaB activation in lymphocytes was associated with elevated levels of lipid peroxidation by-products in the plasma.

Thiol homeostasis and supplements in physical exercise.

Thiols are a class of organic sulfur derivatives (mercaptans) characterized by the presence of sulfhydryl residues. In biological systems, thiols have numerous functions, including a central role in coordinating the antioxidant defense network. Physical exercise may induce oxidative stress. In humans, a consistent marker of exercise-induced oxidative stress is blood glutathione oxidation. Physical training programs have specific effects on tissue glutathione metabolism that depend on the work program and the type of tissue. Experimental studies show that glutathione metabolism in several tissues sensitively responds to an exhaustive bout of exercise. Study of glutathione-deficient animals clearly indicates the central importance of having adequate tissue glutathione to protect against exercise-induced oxidative stress. Among the various thiol supplements studied, N-acetyl-L-cysteine and alpha-lipoic acid hold the most promise. These agents may have antioxidant effects at the biochemical level but are also known to influence redox-sensitive cell signaling.

Nuclear factor kappa B activity in response to oxidants and antioxidants.

The goal of this chapter was to review the current protocols that are available to measure the activation of NF-kappa B. The methods discussed all have their pitfalls when used in isolation. To obtain meaningful information, nuclear translocation and transcriptional activation should be studied in conjunction. Study of NF-kappa B regulated protein expression is the most physiologically relevant approach to monitoring the transcription regulatory effect of NF-kappa B. Because of the limitations of transcriptional analysis in primary cell cultures or tissues, incorporation of multiple approaches is recommended when the involvement of NF-kappa B in a disease process is evaluated.

Flow cytometric determination of cellular thiols.

Several biochemical techniques are based on chromatography or electrophoresis for the determination of thiols from biological samples. These techniques are indispensable for the accurate and sensitive detection of specific thiols. Flow cytometric determination of cellular thiols is a powerful technique that is perhaps best suited for clinical application, particularly for cells in blood or other body fluids. Information can be obtained from a small sample amount with a relatively little and quick sample treatment. This technique offers an unique advantage to study the thiol status of a subset of cells because data are collected from individual cells. Multiparameter flow cytometry allows the study of different subsets of immunotyped cells. A major drawback of the flow cytometric method is the lack of specificity for the determination of distinct thiols. The reaction between MBB and thiols is not specific for any particular intracellular thiol, although almost all of the entire thiol-reacted bimane emission is specific for thiols in general. This limitation can be partly overcome by the treatment of cells with known thiol regulatory agents as described in the section on the differential assessment of cellular thiols.

Cellular and mitochondrial changes in glutamate-induced HT4 neuronal cell death.

Elevated levels of extracellular glutamate are neurotoxic. The cytotoxic property of extracellular glutamate is known to mediate two primary mechanisms, excitotoxicity and excitotoxicity-independent processes. The excitotoxicity-independent pathway was investigated in the current study in a mouse hippocampal-derived HT4 cell line. Exposure of HT4 cells to glutamate for 12h induced loss of cell viability preceded by rapid loss of intracellular reduced glutathione followed by accumulation of intracellular reactive oxygen species, elevation of intracellular Ca(2+), progressive loss of mitochondrial membrane potential swelling and loss of mitochondrial outer membrane integrity. Glutamate-induced loss of DNA integrity has been detected. The antioxidants alpha-tocopherol and trolox, mitochondrial calcium uniporter inhibitor Ruthenium Red and protein synthesis inhibitor cycloheximide all showed protection against glutamate-induced toxicity. None of the protective agents except for alpha-tocopherol controlled the glutamate-induced reactive oxygen species build-up. However, these cell death regulators prevented the glutamate-induced mitochondrial damage and regulated glutamate-induced increase in intracellular Ca(2+). Carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone, a mitochondrial uncoupler, partially protected against glutamate-induced cell death and mitochondrial damage, while the mitochondrial ribosomal inhibitor chloramphenicol and extracellular Ca(2+) chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid did not protect the cells against glutamate treatment. The results of this study demonstrated that mitochondrial dysfunction was a key event in the excitotoxicity-independent component of neuronal cell death. Reactive oxygen species accumulation and glutathione depletion were prominent in glutamate-treated cells; however, these events were not direct mediators of cell death.