ER stress upregulated PGE2/IFNγ-induced IL-6 expression and down-regulated iNOS expression in glial cells.

The disruption of endoplasmic reticulum (ER) function can lead to neurodegenerative disorders, in which inflammation has also been implicated. We investigated the possible correlation between ER stress and immune function using glial cells. We demonstrated that ER stress synergistically enhanced prostaglandin (PG) E2 + interferon (IFN) γ-induced interleukin (IL)-6 production. This effect was mediated through cAMP. Immune-activated glial cells produced inducible nitric oxide synthase (iNOS). Interestingly, ER stress inhibited PGE2 + IFNγ-induced iNOS expression. Similar results were obtained when cells were treated with dbcAMP + IFNγ. Thus, cAMP has a dual effect on immune reactions; cAMP up-regulated IL-6 expression, but down-regulated iNOS expression under ER stress. Therefore, our results suggest a link between ER stress and immune reactions in neurodegenerative diseases.

ER stress-mediated regulation of immune function under glucose-deprived condition in glial cells: up- and down-regulation of PGE2 + IFNγ-induced IL-6 and iNOS expressions.

Glucose metabolism plays central role in maintaining brain function. Under ischemic condition, where glucose levels were reduced, glial cells induce pro-inflammatory cytokine production. In the present study, we found prostaglandin (PG) E2+interferon (IFN) γ-induced interleukin (IL)-6 production was enhanced under glucose-deprived condition in the primary cultured glial cells. On the other hand, to our surprise, we found that PGE2+IFNγ-induced iNOS expression was attenuated under glucose-deprived condition. These dual effects would be mediated through endoplasmic reticulum (ER) stress, because we observed (1) up-regulation of GRP78 and CHOP under glucose-deprived condition, which was inhibited by chemical chaperon TMAO, and (2) treatment with TMAO inhibited IL-6 production under glucose-deprived condition. By activating theses responses glial cells may protect neurons because we observed increased neuronal cell viability in the immune-activated glial cell conditioned medium. Overall, our results suggest a link between ER stress and immune reactions under glucose-deprived condition in the glial cells.

Excitonic coupling in linear and trefoil trimer perylenediimide molecules probed by single-molecule spectroscopy.

Perylenediimide (PDI) molecules are promising building blocks for photophysical studies of electronic interactions within multichromophore arrays. Such PDI arrays are important materials for fabrication of molecular nanodevices such as organic light-emitting diodes, organic semiconductors, and biosensors because of their high photostability, chemical and physical inertness, electron affinity, and high tinctorial strength over the entire visible spectrum. In this work, PDIs have been organized into linear (L3) and trefoil (T3) trimer molecules and investigated by single-molecule fluorescence microscopy to probe the relationship between molecular structures and interchromophoric electronic interactions. We found a broad distribution of coupling strengths in both L3 and T3 and hence strong/weak coupling between PDI units by monitoring spectral peak shifts in single-molecule fluorescence spectra upon sequential photobleaching of each constituent chromophore. In addition, we used a wide-field defocused imaging technique to resolve heterogeneities in molecular structures of L3 and T3 embedded in a PMMA polymer matrix. A systematic comparison between the two sets of experimental results allowed us to infer the correlation between intermolecular interactions and molecular structures. Our results show control of the PDI intermolecular interactions using suitable multichromophoric structures.

Methylglyoxal has deleterious effects on thioredoxin in human aortic endothelial cells.

Methylglyoxal (MG), a precursor of advanced glycation end products (AGEs), is elevated in diabetic patient's plasma. Some studies have demonstrated that MG induces oxidative stress and apoptosis. Thioredoxin (Trx) is a cytoprotective protein with anti-oxidative and anti-apoptosis functions. In this study, we examined the effects of MG on Trx in human aortic endothelial cells (HAECs). MG increased oxidized-hydroethidine fluorescence intensity, suggesting intracellular accumulation of reactive oxygen species. Flow cytometric analyses with annexin-V/propidium iodide double staining revealed that cells incubated with MG displayed features characteristic of apoptosis. The condensation of chromatin, the release of cytochrome c into cytosol, and the collapse of mitochondrial membrane potential by MG were observed. The exposure to MG decreased Trx protein levels through transcription regulation. MG induced the oxidative damage of peroxiredoxin, a Trx-dependent peroxidase. These results suggest that MG has deleterious effects on Trx in HAECs, which may be contribute to oxidative stress and apoptosis.

Relaxation in Thin Polymer Films Mapped across the Film Thickness by Astigmatic Single-Molecule Imaging.

We have studied relaxation processes in thin supported films of poly(methyl acrylate) at the temperature corresponding to 13 K above the glass transition by monitoring the reorientation of single perylenediimide molecules doped into the films. The axial position of the dye molecules across the thickness of the film was determined with a resolution of 12 nm by analyzing astigmatic fluorescence images. The average relaxation times of the rotating molecules do not depend on the overall thickness of the film between 20 and 110 nm. The relaxation times also do not show any dependence on the axial position within the films for the film thickness between 70 and 110 nm. In addition to the rotating molecules we observed a fraction of spatially diffusing molecules and completely immobile molecules. These molecules indicate the presence of thin (<5 nm) high-mobility surface layer and low-mobility layer at the interface with the substrate.

Multi-beam single-molecule defocused fluorescence imaging reveals local anisotropic nature of polymer thin films.

We developed a method to determine full three-dimensional orientation distribution of individual molecules based on wide-field defocused fluorescence imaging. Excitation efficiencies of out-of-plane oriented molecules were improved dramatically by illuminating molecules with multiple laser beams. Our high throughput approach allowed us to obtain unbiased statistical distributions of orientations of doped molecules in spin-coated polymer thin films. We found thickness- and glass transition temperature-dependent distributions of the molecular orientations which reflect local chain orientations and relaxation in the polymer thin films.

Buthionine sulfoximine promotes methylglyoxal-induced apoptotic cell death and oxidative stress in endothelial cells.

Methylglyoxal (MG), a reactive dicarbonyl produced during glucose metabolism, is found at high levels in the blood of diabetic patients. MG induces oxidative stress and apoptosis. There is evidence that MG causes glutathione (GSH) depletion. However, it remains unknown whether GSH plays a protective role against the cytotoxic effect of MG. We examined the effect of DL-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) biosynthesis, on the viability of bovine aortic endothelial cells (BAECs) exposed to MG. BAECs pretreated with BSO showed reduced ability to survive MG exposure. Flow cytometric analyses with annexin V and propidium iodide double staining revealed that BAECs exposed to MG after BSO pretreatment displayed features characteristic of apoptosis. Caspase-3 activation induced by MG was increased by BSO. Moreover, measurement of protein carbonyl levels showed that BSO promoted MG-induced oxidative stress. Taken together, these findings suggest that the depletion of GSH via BSO pretreatment promoted MG-induced apoptotic cell death and oxidative stress in BAECs.

A unique modulator of endoplasmic reticulum stress-signalling pathways: the novel pharmacological properties of amiloride in glial cells.

BACKGROUND AND PURPOSE: Stress on the endoplasmic reticulum (ER) can trigger rescuer responses such as the unfolded protein response (UPR). However, pharmacological modulators of these ER-regulated stress responses are not well understood. In the present study, we found that amiloride, a potassium-sparing diuretic, has unique properties relating to such stress. EXPERIMENTAL APPROACH: We treated mouse primary cultured glial cells with amiloride, in the absence and presence of the ER stress-inducing reagents tunicamycin (Tm) or dithiothreitol, and measured UPR and ER stress-induced cell death. IRE1alpha phosphorylation, eIF2alpha phosphorylation, X-box binding protein 1 (XBP1) splicing, glucose regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) expression by reverse transcription-polymerase chain reaction and Western blotting were used to assess UPR and lactate dehydrogenase activity was determined to measure ER stress-induced cell death. KEY RESULTS: Amiloride completely inhibited ER stress-induced activation of IRE1alpha, an ER-localized stress sensor protein, splicing of XBP1, and subsequent expression of GRP78 at the mRNA and protein levels. ER stress induces the phosphorylation of eIF2alpha, leading to the expression of CHOP or an attenuation of translation in cells. Surprisingly, treatment with amiloride alone markedly promoted the phosphorylation but actually inhibited ER stress-induced CHOP expression. Finally, we found that amiloride (200 microM) synergistically enhanced ER stress-induced cell death, which was mediated through caspases. On the other hand, a low dose of amiloride (20 microM) significantly prevented Tm-induced cell death. CONCLUSIONS AND IMPLICATIONS: These results suggest that amiloride can modulate UPR. They also suggest amiloride to be an important pharmacological agent and provide basic information for understanding and preventing ER stress-related diseases.

Methylglyoxal causes dysfunction of thioredoxin and thioredoxin reductase in endothelial cells.

Methylglyoxal (MG), a reactive dicarbonyl produced during glucose metabolism, induces oxidative stress and apoptosis. Under hyperglycemic conditions, the abnormal accumulation of MG is related to the development of diabetic complications. We examined the effects of MG on thioredoxin (Trx) and glutaredoxin (Grx) systems, two thiol-disulfide oxidoreductase systems that protect against oxidative damage of proteins, in bovine aortic endothelial cells (BAECs). The levels of protein carbonyls as markers of protein oxidation increased in BAECs exposed to MG at 5 mM, resulting in the loss of cell viability. Western blot analysis demonstrated that Trx protein level decreased when BAECs were exposed to 5 mM MG. MG also inactivated Trx reductase, which maintains Trx in the reduced/active state. Moreover, peroxiredoxin, which is dependent on Trx and Trx reductase to maintain its reduced state, was oxidized by 5 mM MG. No significant difference in the levels of Trx, Trx reductase, or peroxiredoxin was observed in BAECs exposed to MG at 1 mM; this concentration had little effect on protein carbonyl formation and cell viability. MG failed to decrease Grx activity, indicating that Trx is more susceptible to MG than Grx. Taken together, these findings suggest that MG causes dysfunction of the Trx system, including Trx and Trx reductase, in BAECs.

[methylglyoxal-induced superoxide anion production in endothelial cells].

Methylglyoxal (MG), a highly reactive dicarbonyl compound, is a metabolic by-product of glycolysis. MG is often detected at high levels in the blood of diabetic patients. We examined whether MG was capable of inducing reactive oxygen species (ROS) production in bovine aortic endothelial cells (BAECs). The viability of BAECs decreased with time on treatment with 5 mM MG, and was almost completely lost at 24 h. In contrast, MG at 1 mM had little influence on BAEC viability up to 24 h, but induced the elevation of intracellular glutathione content at 24 h. Exposure of BAECs to MG caused a dose-dependent increase in oxidized-hydroethidine fluorescence intensity, indicating ROS production. In addition, aconitase inactivation, which is an indicator of intracellular superoxide, was observed in MG-treated cells. Finally, we found that MG at 5 mM increased the fluorescence intensity of BES-So, a specific probe for superoxide. Together, the results suggest that MG induces superoxide production in endothelial cells, and that the accumulation of ROS may be linked to cytotoxic effects.

Multidrug-resistance-associated protein plays a protective role in menadione-induced oxidative stress in endothelial cells.

AIMS: Menadione, a redox-cycling quinone known to cause oxidative stress, binds to reduced glutathione (GSH) to form glutathione S-conjugate. Glutathione S-conjugates efflux is often mediated by multidrug-resistance-associated protein (MRP). We investigated the effect of a transporter inhibitor, MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), on menadione-induced oxidative stress in bovine aortic endothelial cells (BAECs). MAIN METHODS: BAECs were treated with menadione and MK571, and cell viability was measured. Modulation of intracellular GSH levels was performed with buthionine sulfoximine and GSH ethyl ester treatments. Intracellular superoxide was estimated by dihydroethidium oxidation using fluorescence microscopy or flow cytometry. Expression of MRP was determined by flow cytometry using phycoerythrin-conjugated anti-MRP monoclonal antibody. KEY FINDINGS: Intracellular GSH depletion by buthionine sulfoximine promoted the loss of viability of BAECs exposed to menadione. Exogenous GSH, which does not permeate the cell membrane, or GSH ethyl ester protected BAECs against the loss of viability induced by menadione. The results suggest that GSH binds to menadione outside the cells as well as inside. Pretreatment of BAECs with MK571 dramatically increased intracellular levels of superoxide generated from menadione, indicating that menadione may accumulate in the intracellular milieu. Finally, we found that MK571 aggravated menadione-induced toxicity in BAECs and that MRP levels were increased in menadione-treated cells. SIGNIFICANCE: We conclude that MRP plays a vital role in protecting BAECs against menadione-induced oxidative stress, presumably due to its ability to transport glutathione S-conjugate.