Golgi SNAP receptor complex 1 (GS28) has been implicated in vesicular transport between intra-Golgi networks and between endoplasmic reticulum (ER) and Golgi. Additional role(s) of GS28 within cells have not been well characterized. We observed decreased expression of GS28 in rat ischemic hippocampus. In this study, we examined the role of GS28 and its molecular mechanisms in neuronal (SK-N-SH) cell death induced by hydrogen peroxide (H2O2). GS28 siRNA-transfected cells treated with H2O2 showed a significant increase in cytotoxicity under glutathione (GSH)-depleted conditions after pretreatment with buthionine sulfoximine, which corresponded to an increase of intracellular reactive oxygen species (ROS) in the cells. Pretreatment of GS28 siRNA-transfected cells with p38 chemical inhibitor significantly inhibited cytotoxicity; we also observed that p38 was activated in the cells by immunoblot analysis. We confirmed the role of p38 MAPK in cotransfected cells with GS28 siRNA and p38 siRNA in the cell viability assay, flow cytometry, and immunoblot. Involvement of apoptotic or autophagic processes in the cells was not shown in the cell viability, flow cytometry, and immunoblot analyses. However, pretreatment of the cells with necrostatin-1 completely inhibited H2O2-induced cytotoxicity, ROS generation, and p38 activation, indicating that the cell death is necroptotic. Collectively these data imply that H2O2 induces necroptotic cell death in the GS28 siRNA-transfected cells and that the necroptotic signals are mediated by sequential activations in RIP1/p38/ROS. Taken together, these results indicate that GS28 has a protective role in H2O2-induced necroptosis via inhibition of p38 MAPK in GSH-depleted neuronal cells.
Animals ; Buthionine Sulfoximine ; Cell Death ; Cell Survival ; Endoplasmic Reticulum ; Flow Cytometry ; Glutathione ; Hippocampus ; Hydrogen ; Hydrogen Peroxide ; Imidazoles ; Indoles ; Methionine ; Neurons ; p38 Mitogen-Activated Protein Kinases ; Rats ; Reactive Oxygen Species ; RNA, Small Interfering ; SNARE Proteins

OBJECTIVETo explore the role of glial cell metabolism in the generation and regulation of central respiratory rhythm.

METHODSThe medulla oblongata slices (600-700 microm) containing the medial region of the nucleus retrofacialis (mNRF) with the hypoglossal nerve rootlets retained from 12 neonatal (0-3 days) Sprague-Dawley rats were prepared and perfused with modified Kreb's solution (MKS). Upon recording of respiratory rhythmical discharge activity (RRDA) of the rootlets of the hypoglossal nerve, the brain slices were treated with glial cell metabolism antagonist L-methionine sulfoximine (L-MSO, 50 micromol/L) for 20 min followed by application of glial cell metabolism agonist L-glutamine (L-GLN, 30 micromol/L) for 20 min, or with L-MSO for 20 min with additional L-GLN for 20 min. The changes in the RRDA of the rootlets of the hypoglossal nerve in response to the treatments were recorded.

RESULTSL-MSO prolonged the respiratory cycle (RC) and expiratory time (TE), and reduced the integral amplitude (IA) and the inspiratory time (TI) in the brain slices. L-GLN induced a significant decrease in RC and TE, but IA and TI showed no obvious variations. The effect of L-MSO on the respiratory rhythm was reversed by the application of L-GLN.

CONCLUSIONGlial cell metabolism may play an important role in the modulation of RRDA in neonatal rat brainstem.

Animals ; Animals, Newborn ; Glutamine ; pharmacology ; In Vitro Techniques ; Medulla Oblongata ; metabolism ; physiology ; Methionine Sulfoximine ; pharmacology ; Neuroglia ; metabolism ; Periodicity ; Rats ; Rats, Sprague-Dawley ; Respiration

The present study investigated the role of spinal glutamate recycling in the development of orofacial inflammatory pain or trigeminal neuropathic pain. Experiments were carried out on male Sprague-Dawley rats weighing between 230 and 280 g. Under anesthesia, a polyethylene tube was implanted in the atlanto-occipital membrane for intracisternal administration. IL-1β-induced inflammation was employed as an orofacial acute inflammatory pain model. IL-1β (10 ng) was injected subcutaneously into one vibrissal pad. We used the trigeminal neuropathic pain animal model produced by chronic constriction injury of the infraorbital nerve. DL-threo-β -benzyloxyaspartate (TBOA) or methionine sulfoximine (MSO) was administered intracisternally to block the spinal glutamate transporter and the glutamine synthetase activity in astroglia. Intracisternal administration of TBOA produced mechanical allodynia in naïve rats, but it significantly attenuated mechanical allodynia in rats with interleukin (IL)-1 β-induced inflammatory pain or trigeminal neuropathic pain. In contrast, intracisternal injection of MSO produced anti-allodynic effects in rats treated with IL-1β or with infraorbital nerve injury. Intracisternal administration of MSO did not produce mechanical allodynia in naive rats. These results suggest that blockade of glutamate recycling induced pro-nociception in naïve rats, but it paradoxically resulted in anti-nociception in rats experiencing inflammatory or neuropathic pain. Moreover, blockade of glutamate reuptake could represent a new therapeutic target for the treatment of chronic pain conditions.
Amino Acid Transport System X-AG ; Anesthesia ; Animals ; Astrocytes ; Chronic Pain ; Constriction ; Glutamate-Ammonia Ligase ; Glutamic Acid* ; Humans ; Hyperalgesia ; Inflammation ; Interleukins ; Male ; Membranes ; Methionine Sulfoximine ; Models, Animal ; Neuralgia* ; Polyethylene ; Rats* ; Rats, Sprague-Dawley ; Recycling*

No abstract available.
Buthionine Sulfoximine* ; Cell Line* ; Cisplatin* ; Humans* ; Lung Neoplasms* ; Lung* ; Stomach*

OBJECTIVES: To evaluate the effect of glutathione(GSH) on lead induced modulation of nitric oxide(NO) synthesis, and to examine how lead modulates NO production in macrophages. METHODS: This study was observed in a culture of RAW 264.7 cells, which originated from a tumor in a Balb/c mouse that was induced by the Abelson murine leukemia virus. The compounds investigated were lead chloride, N-acetyl-cystein(NAC), and Buthionine Sulfoximine(BSO). RESUJLTS: ATP synthesis in RAW 264.7 cells was unchanged by each lead concentration exposure in a dose dependent manner. The NO synthesis was decreased when exposed to lead(PbCl2) concentration 0.5 micro M. The presence of 300 micro M NAC, used as a pretreatment in the culture medium, caused the recovery of the lead induced decrease in NO synthesis, but in the presence of 300 micro M BSO as a pretreatment, there was no recoverey. Pretreatment with NAC and BSO had no affect on ATP synthesis at any of the lead concentrations used. CONCLUSIONS: These results indicated that GSH has a protective effect toward lead toxicity, and suggested that the inhibition of NO production in macrophage due to lead toxicity may be related to cofactors of iNOS (inducible nitric oxide synthase)
Abelson murine leukemia virus ; Acetylcysteine ; Adenosine Triphosphate ; Animals ; Buthionine Sulfoximine ; Glutathione* ; Macrophages ; Mice ; Nitric Oxide

OBJECTIVES: To evaluate the effect of glutathione(GSH) on lead induced modulation of nitric oxide(NO) synthesis, and to examine how lead modulates NO production in macrophages. METHODS: This study was observed in a culture of RAW 264.7 cells, which originated from a tumor in a Balb/c mouse that was induced by the Abelson murine leukemia virus. The compounds investigated were lead chloride, N-acetyl-cystein(NAC), and Buthionine Sulfoximine(BSO). RESUJLTS: ATP synthesis in RAW 264.7 cells was unchanged by each lead concentration exposure in a dose dependent manner. The NO synthesis was decreased when exposed to lead(PbCl2) concentration 0.5 micro M. The presence of 300 micro M NAC, used as a pretreatment in the culture medium, caused the recovery of the lead induced decrease in NO synthesis, but in the presence of 300 micro M BSO as a pretreatment, there was no recoverey. Pretreatment with NAC and BSO had no affect on ATP synthesis at any of the lead concentrations used. CONCLUSIONS: These results indicated that GSH has a protective effect toward lead toxicity, and suggested that the inhibition of NO production in macrophage due to lead toxicity may be related to cofactors of iNOS (inducible nitric oxide synthase)
Abelson murine leukemia virus ; Acetylcysteine ; Adenosine Triphosphate ; Animals ; Buthionine Sulfoximine ; Glutathione* ; Macrophages ; Mice ; Nitric Oxide

PURPOSE: Cycloporine A (CsA) is a immunosuppressive agent most widely used in organ transplanted patients for preventing immunorejection, but it has some side effects, including hypertension, nephrotoxicity, hepatotoxicity and diabetes. The mechanism of toxicity of CsA was not completely understood, but the reactive oxygen species has been proposed to be involved in the reaction of toxicity of CsA. The purpose of this study is to determine the effects of glutathione, as a physiological antioxidant on CsA induced cytotoxicity in rat insulinoma (RINm5F) cells. METHODS: RINm5F cells were incubated in the presence of CsA (105~108 M) and buthionine sulfoximine (BSO), as a inhibitor of r-glutamyl cysteine synthetase, was added to cultured media (RPMI1640). Twenty four hours of incubation with CsA and BSO, viable cells were determined by MTT method. RESULTS: CsA decreased cell viability in dose response in cultured RINm5F cells and significantly decreased according to redusing glutathione. CONCLUSION: These results suggested that CsA may induce the diabetes and glutathione have some roles in the pathogenesis of CsA-induced diabetes.
Animals ; Buthionine Sulfoximine ; Cell Survival ; Cyclosporine* ; Cysteine ; Glutathione* ; Humans ; Hypertension ; Insulinoma* ; Ligases ; Rats ; Reactive Oxygen Species

We examined the neurotoxic effects of 3 glutathione (GSH) depletors, buthionine sulfoximine (BSO), diethyl maleate (DEM) and phorone, under the presence of trolox, cycloheximide (CHX), pyrrolidine dithiocarbamate (PDTC) or MK-801 in primary mouse cortical cell cultures. All three depletors induced neuronal death in dose and exposure time dependent manner, and decreased total cellular GSH contents. The patterns of the neuronal death and the GSH decrements were dependent on the individual agents. DEM (200 micrometer) induced rapid and irreversible decrement of the GSH. BSO (1 mM) also decreased the GSH irreversibly but the rate of decrement was more progressive than that of DEM. Phorone (1 mM) reduced the GSH content to 40% by 4 hr exposure, that is comparable to the decrement of BSO, but the GSH recovered and reached over the control value by 36 hr exposure. BSO showed a minimal neurotoxicity (0-10%) at the end of 24 hr exposure, but marked neuronal cell death at the end of 48 hr exposure. The BSO (1 mM)-induced neurotoxicity was markedly inhibited by trolox or CHX and partially attenuated by MK-801. DEM induced dose-dependent cytotoxicity at the end of 24 hr exposure. Over the doses of 400 micrometer, glial toxicity also appeared. DEM (200 micrometer)-induced neurotoxicity was markedly inhibited by trolox or PDTC. Phorone (1 mM) induced moderate neurotoxicity (40%) at the end of 48 hr exposure. Only CHX showed significant inhibitory effect on the phorone-induced neurotoxicity. These results suggest that the GSH depletors induce neuronal injury via different mechanisms and that GSH depletors should be carefully employed in the researches of neuronal oxidative injuries.
Animals ; Buthionine Sulfoximine ; Cell Culture Techniques* ; Cell Death ; Cycloheximide ; Dizocilpine Maleate ; Glutathione* ; Mice* ; Neurons

This study was performed to evaluate in vitro cytotoxicity of chemotherapeutic agents in established human retinoblastoma cell line, Y79 and to study the possibility of enhancing the cytotoxicity of chemotherapeutic agents by administration of buthionine sulfoximine(BSO) which lowers the intracellular glutathione(GSH) level. IC50 defined as the concentration which inhibits the cell survival rates to 50% compared with control group was used to evaluate cytotoxicity. Intracellular level after 13.50 adminstration were measured and compared with the level prior to administration of BSO. Doxorubicin, cisplatin, and melphalan showed significant decrease of IC50 by administration of BSO(p<0.01). But vincristine did not show significant decrease of IC50 by administration of BSO(p>0.05). Intracellular GSH level prior to the administration of BSO was 0.931nM/mg protein. After the administration of BSO, they were lowered to 0.095nM/mg protein in both BSO concentrations. Results listed above suggest that cytotoxicity of doxorubicin, cisplatin, and melphalan can be enhanced by ESO. This effect may be mediated by decreased intracellualr level of GSH by BSO.
Buthionine Sulfoximine* ; Cell Line* ; Cell Survival ; Cisplatin ; Doxorubicin ; Drug Therapy ; Glutathione ; Humans ; Inhibitory Concentration 50 ; Melphalan ; Retinoblastoma* ; Vincristine

OBJECTIVE: The purpose of this study is to determine the effects of glutathione on cisplatin-induced cytotoxicity of human cervical carcinoma cell lines (SiHa: squamous cell carcinoma cell, CaSki: epidermoid metastatic carcinoma cell). METHODS: Human cervical carcinoma cells (SiHa, CaSki) were incubated with culture media (RPMI1640) in the presence of cisplatin and/or buthionine sulfoximine (BSO), as a inhibitor of gamma-glutamyl- cysteine synthetase, and/or glutathione (GSH) and/or 2-oxo 4-thiazolidine carboxylic acid (OTC). The viable cells were examined by using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and was determined by spectrophotometer at 570 nm. RESULTS: The incubation of cervical cells with cisplatin resulted in an decreasing cells viability by dose response. The MTT reduction rate were not different by BSO (5 mM) treatment in cervical cell lines. The viable cells were increased significantly by glutathione (5 mM) or OTC (5 mM) in cisplatin-treated cell lines. CONCLUSION: gamma-glutamylcysteine synthetase inhibitor had no effect on cisplatin toxicity. GSH and OTC had effect on cisplatin cytotoxicity. So, These result suggested that cervical cancer line cells were more susceptive to protective effects of glutathione and OTC than BSO on cisplatin induced-toxicity.
Buthionine Sulfoximine ; Carcinoma, Squamous Cell ; Cell Line* ; Cisplatin ; Culture Media ; Cysteine ; Glutathione* ; Humans* ; Ligases ; Uterine Cervical Neoplasms*