Dopamine inhibits the expression of proinflammatory cytokines of microglial cells through the formation of dopamine quinone in the mouse striatum.

We previously reported that dopamine (DA) attenuated lipopolysaccharide (LPS)-induced expression of proinflammatory cytokines through the formation of DA quinone (DAQ) in murine microglial cell line BV-2 and primary murine microglial cells. To reveal whether DA inhibits the expression of proinflammatory cytokines of microglial cells through the formation of DAQ in the central nervous system (CNS), in this study, we examined the effect of DAQ on LPS-induced mRNA expression of proinflammatory cytokines in C57BL/6 mouse brain under two experimental conditions: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration and l-dopa/carbidopa administration. Acute MPTP administration reduced the number of tyrosine hydroxylase-positive cells in the substantia nigra, and decreased the level of quinoprotein, an indicator of DAQ formation, in the striatum. Real-time RT-PCR analysis revealed that intraperitoneal administration of LPS increased the mRNA levels of proinflammatory cytokines, including tumor-necrosis factor-α and interleukin-1ß, in the striatum. These increases were enhanced in MPTP-treated mice. On the other hand, l-dopa/carbidopa administration increased the level of quinoprotein, attenuated the LPS-induced mRNA expression of proinflammatory cytokines, and reduced the LPS-induced increase in the number of microglial cells in the striatum. These results suggest that DA attenuate the expression of proinflammatory cytokines in microglia through the formation of DAQ in the CNS.

Noradrenaline protects neurons against H2 O2 -induced death by increasing the supply of glutathione from astrocytes via ß3 -adrenoceptor stimulation.

Oxidative stress has been implicated in a variety of neurodegenerative disorders, such as Alzheimer's and Parkinson's disease. Astrocytes play a significant role in maintaining survival of neurons by supplying antioxidants such as glutathione (GSH) to neurons. Recently, we found that noradrenaline increased the intracellular GSH concentration in astrocytes via ß3 -adrenoceptor stimulation. These observations suggest that noradrenaline protects neurons from oxidative stress-induced death by increasing the supply of GSH from astrocytes to neurons via the stimulation of ß3 -adrenoceptor in astrocytes. In the present study, we examined the protective effect of noradrenaline against H2 O2 -induced neurotoxicity using two different mixed cultures: the mixed culture of human astrocytoma U-251 MG cells and human neuroblastoma SH-SY5Y cells, and the mouse primary cerebrum mixed culture of neurons and astrocytes. H2 O2 -induced neuronal cell death was significantly attenuated by pretreatment with noradrenaline in both mixed cultures but not in single culture of SH-SY5Y cells or in mouse cerebrum neuron-rich culture. The neuroprotective effect of noradrenaline was inhibited by SR59230A, a selective ß3 -adrenoceptor antagonist, and CL316243, a selective ß3 -adrenoceptor agonist, mimicked the neuroprotective effect of noradrenaline. DL-buthionine-[S,R]-sulfoximine, a GSH synthesis inhibitor, negated the neuroprotective effect of noradrenaline in both mixed cultures. MK571, which inhibits the export of GSH from astrocytes mediated by multidrug resistance-associated protein 1, also prevented the neuroprotective effect of noradrenaline. These results suggest that noradrenaline protects neurons against H2 O2 -induced death by increasing the supply of GSH from astrocytes via ß3 -adrenoceptor stimulation.

Endogenous Apelin Is Protective Against Age-Associated Loss of Retinal Ganglion Cells in Mice.

Age-associated loss of retinal ganglion cells (RGCs) causes visual deficits, but there is not yet any therapeutic agent to prevent the loss of these cells. Herein, we report that apelin, an endogenous peptide ligand of APJ receptor, is protective against the age-related loss of RGCs in mice. The mRNA expression of apelin was reduced in the retina of old mice compared with that in young mice, whereas retinal APJ expression increased with age. Immunofluorescence staining showed that APJ was present in RGCs and their surrounding cells expressed apelin. In addition, both functional and histological analyses demonstrated that apelin deficiency accelerated the loss of RGCs associated with age in mice. These results suggest that endogenous apelin plays a protective role against the degeneration of RGCs and that the apelinergic axis may be a new target for preventing age-related visual impairment.

Systemic Administration of an Apelin Receptor Agonist Prevents NMDA-Induced Loss of Retinal Neuronal Cells in Mice.

Glutamate excitotoxicity via N-methyl-D-aspartate (NMDA) receptors is thought to be a factor involved in the loss of retinal neuronal cells, including retinal ganglion cells, in retinal diseases such as diabetic retinopathy and acute angle closure glaucoma. Herein we report the protective effect of systemic administration of ML233, an apelin receptor agonist, against retinal neuronal cell death induced by the intravitreal injection of NMDA into mice. Intraperitoneal administration of ML233 prevented the NMDA-induced reduction in the amplitude of scotopic threshold responses (STR), which mainly reflect the activity of the retinal ganglion cells. Immunohistochemical staining showed that ML233 inhibited the NMDA-induced loss of retinal ganglion cells and amacrine cells. In addition, ML233 suppressed the breakdown of spectrin αII, a neuronal cytoskeleton protein cleaved by calpain activation, in the retina after intravitreal injection of NMDA. Intraperitoneal administration of ML233 increased the phosphorylation of Akt, a potent anti-apoptotic protein in neurons, in the retina. Furthermore, oral administration of ML233 protected against the decrease in the STR amplitudes and the loss of retinal ganglion cells caused by NMDA. These results suggest that systemic administration of ML233 protected retinal neurons from NMDA receptor-mediated excitotoxicity and that drugs activating the apelin receptor may be a new candidate for preventing the progression of these retinal diseases.

Dopamine attenuates lipopolysaccharide-induced expression of proinflammatory cytokines by inhibiting the nuclear translocation of NF-κB p65 through the formation of dopamine quinone in microglia.

Many reports have indicated that dopamine has immunomodulatory effects on peripheral immune cells. The purpose of this study was to reveal the immunomodulatory effect of dopamine on the expression of proinflammatory cytokines in microglial cells, which are the immune cells of the central nervous system. In murine microglial cell line BV-2 cells, pretreatment with dopamine for 24 h attenuated the lipopolysaccharide (LPS)-induced expression of proinflammatory cytokines such as tumor-necrosis factor-α, interleukin-1ß, and interleukin-6. Neither (5R)-8-chloro-3-methyl-5-phenyl-1,2,4,5-tetrahydro-3-benzazepin-7-ol; hydrochloride (SCH-23390) nor sulpiride, which are dopamine D1-like and D2-like receptor antagonists, respectively, affected the attenuation of LPS-induced expression of cytokines by dopamine. In addition, pretreatment with neither (-)-(6aR,12bR)-4,6,6a,7,8,12b-Hexahydro-7-methylindolo[4,3-a]phenanthridin (CY208-243) nor bromocriptine, dopamine D1-like and D2-like receptor agonists, respectively, was effective in doing so. However, N-acetylcysteine (NAC), which inhibits dopamine oxidation to dopamine quinone, did inhibit this attenuated expression. Dopamine increased the level of quinoproteins, and this increase was inhibited by NAC. Western blot and immunocytochemical analyses revealed that dopamine inhibited LPS-induced nuclear translocation of nuclear factor-kappa B (NF-κB) p65. Dopamine also attenuated the expression of cytokines and the nuclear translocation of NF-κB p65 induced by LPS in mouse microglial cells in primary culture. These results suggest that dopamine attenuated LPS-induced expression of cytokines by inhibiting the nuclear translocation of NF-κB p65 through the formation of dopamine quinone in microglial cells.

An apelin receptor antagonist prevents pathological retinal angiogenesis with ischemic retinopathy in mice.

Pathological retinal angiogenesis is caused by the progression of ischemic retinal diseases and can result in retinal detachment and irreversible blindness. This neovascularization is initiated from the retinal veins and their associated capillaries and involves the overgrowth of vascular endothelial cells. Since expression of the apelin receptor (APJ) is restricted to the veins and proliferative endothelial cells during physiological retinal angiogenesis, in the present study, we investigated the effect of APJ inhibition on pathological retinal angiogenesis in a mouse model of oxygen-induced retinopathy (OIR). In vitro experiments revealed that ML221, an APJ antagonist, suppressed cultured-endothelial cell proliferation in a dose-dependent manner. Intraperitoneal administration of ML221 inhibited pathological angiogenesis but enhanced the recovery of normal vessels into the ischemic regions in the retina of the OIR model mice. ML221 did not affect the expression levels of vascular endothelial growth factor (VEGF) and its receptor (VEGFR2) in the retina. APJ was highly expressed in the endothelial cells within abnormal vessels but was only detected in small amounts in morphologically normal vessels. These results suggest that APJ inhibitors selectively prevent pathological retinal angiogenesis and that the drugs targeting APJ may be new a candidate for treating ischemic retinopathy.

Apelin protects against NMDA-induced retinal neuronal death via an APJ receptor by activating Akt and ERK1/2, and suppressing TNF-α expression in mice.

Glutamate excitotoxicity mediated by N-methyl-d-aspartate (NMDA) receptors is an important cause of retinal ganglion cell death in glaucoma. To elucidate whether apelin protects against retinal neuronal cell death, we examined protective effects of exogenous and endogenous apelin on neuronal cell death induced by intravitreal injection of NMDA in the retinas of mice. An intravitreal injection of NMDA induced neuronal cell death in both the retinal ganglion cell layer and inner nuclear layer, and reduced the amplitudes of scotopic threshold response (STR) in electroretinography studies. Both cell death and STR amplitudes decrease induced by NMDA were prevented by a co-injection of [Pyr1]-apelin-13, and were facilitated by apelin deficiency. The neuroprotective effects of [Pyr1]-apelin-13 were blocked by an apelin receptor APJ antagonist, and by inhibitors of Akt and extracellular signal-regulated kinase 1/2 signaling pathways. Additionally, an intravitreal injection of tumor necrosis factor-α (TNF-α) neutralizing antibody prevented NMDA-induced retinal neuronal cell death, and exogenous and endogenous apelin suppressed NMDA-induced upregulation of TNF-α in the retina. These results suggest that apelin protects neuronal cells against NMDA-induced death via an APJ receptor in the retina, and that apelin may have beneficial effects in the treatment of glaucoma.

Dopamine inhibits lipopolysaccharide-induced nitric oxide production through the formation of dopamine quinone in murine microglia BV-2 cells.

Dopamine (DA) has been suggested to modulate functions of glial cells including microglial cells. To reveal the regulatory role of DA in microglial function, in the present study, we investigated the effect of DA on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in murine microglial cell line BV-2. Pretreatment with DA for 24 h concentration-dependently attenuated LPS-induced NO production in BV-2 cells. The inhibitory effect of DA on LPS-induced NO production was not inhibited by SCH-23390 and sulpiride, D1-like and D2-like DA receptor antagonists, respectively. In addition, pretreatment with (-)-(6aR,12bR)-4,6,6a,7,8,12b-Hexahydro-7-methylindolo[4,3-a]phenanthridin (CY 208-243) and bromocriptine, D1-like and D2-like DA receptor agonists, respectively, did not affect the LPS-induced NO production. N-Acetylcysteine, which inhibits DA oxidation, completely inhibited the effect of DA. Tyrosinase, which catalyzes the oxidation of DA to DA quionone (DAQ), accelerated the inhibitory effect of DA on LPS-induced NO production. These results suggest that DA attenuates LPS-induced NO production through the formation of DAQ in BV-2 cells.

Noradrenaline increases intracellular glutathione in human astrocytoma U-251 MG cells by inducing glutamate-cysteine ligase protein via ß3-adrenoceptor stimulation.

Glutathione (GSH) plays a critical role in protecting cells from oxidative damage. Since neurons rely on the supply of GSH from astrocytes to maintain optimal intracellular GSH concentrations, the GSH concentration of astrocytes is important for the survival of neighboring neurons against oxidative stress. The neurotransmitter noradrenaline is known to modulate the functions of astrocytes and has been suggested to have neuroprotective properties in neurodegenerative diseases. To elucidate the mechanisms underlying the neuroprotective properties of noradrenaline, in this study, we investigated the effect of noradrenaline on the concentrations of intracellular GSH in human U-251 malignant glioma (MG; astrocytoma) cells. Treatment of the cells with noradrenaline for 24h concentration-dependently increased their intracellular GSH concentration. This increase was inhibited by a non-selective ß-adrenoceptor antagonist propranolol and by a selective ß3-adrenoceptor antagonist SR59230A, but not by a non-selective α-adrenoceptor antagonist phenoxybenzamine, or by a selective ß1-adrenoceptor antagonist atenolol or by a selective ß2-adrenoceptor antagonist butoxamine. In addition, the selective ß3-adrenoceptor agonist CL316243 increased the intracellular GSH in U-251 MG cells. Treatment of the cells with noradrenaline (10µM) for 24h increased the protein level of the catalytic subunit of glutamate-cysteine ligase (GCLc), the rate-limiting enzyme of GSH synthesis; and this increase was inhibited by SR59230A. These results thus suggest that noradrenaline increased the GSH concentration in astrocytes by inducing GCLc protein in them via ß3-adrenoceptor stimulation.

[A self-improvement and participatory career development education program involving internships and volunteer training experience for pharmacy students: results verified in a follow-up survey three years after participation].

The Faculty of Pharmaceutical Sciences, Setsunan University, offers the Self-improvement and Participatory Career Development Education Program: Internship and Volunteer Training Experience for Pharmacy Students to third-year students. We previously reported that the training experience was effective in cultivating important attributes among students, such as a willingness to learn the aims of pharmacists, an awareness of their own role as healthcare workers, and a desire to reflect on their future careers and lives. A follow-up survey of the participants was carried out three years after the training experience. The questionnaire verified that the training experience affected attendance at subsequent lectures and course determination after graduation. We confirmed the relationship between the participants' degree of satisfaction with the training experience and increased motivation for attending subsequent lectures. Through the training experience, participants discovered future targets and subjects of study. In addition, they became more interested in subsequent classroom lessons and their future. The greater the participants' degree of satisfaction with their training experience, the more interest they took in practical training and future courses. The present study clarified that the training experience was effective in cultivating important attributes such as a willingness to learn and an interest in future courses. Moreover, the training positively affected the course determination after graduation.

Laser-induced choroidal neovascularization in mice attenuated by deficiency in the apelin-APJ system.

PURPOSE: To investigate the role of the apelin-APJ system in the development of choroidal neovascularization (CNV). METHODS: Experimental CNV was induced by laser photocoagulation in wild-type (WT), apelin-deficient (apelin-KO), and apelin receptor (APJ)-deficient (APJ-KO) mice. The gene expression levels of angiogenic or inflammatory factors were determined by quantitative real-time reverse transcription-polymerase chain reaction. APJ expression in CNV lesions was examined by immunohistochemistry. The sizes of the CNV lesions in the three mouse models were measured and compared histologically using isolectin B4 staining. Macrophage recruitment was measured by flow cytometric analysis. Proliferation of endothelial cells was determined using the alamar Blue assay. RESULTS: Laser photocoagulation significantly increased expression of apelin and APJ in the retina-retinal pigment epithelium (RPE) complex. APJ immunoreactive cells were found in the CNV lesions and colocalized with platelet endothelial cell adhesion molecule-1, an endothelial cell marker. The sizes of the CNV lesions in apelin-KO or APJ-KO mice decreased significantly compared with those in the WT mice. Macrophages in the RPE complex of the apelin-KO mice, in which gene expression of the inflammatory factors was almost equal to that in WT mice, were recruited as a result of laser photocoagulation to the same degree as in WT mice. In addition, apelin small and interfering RNA (siRNA) suppressed proliferation of endothelial cells independently of vascular endothelial growth factor (VEGF) receptor 2 signaling, while VEGF increased expression of apelin and APJ in human umbilical vein endothelial cells. CONCLUSIONS: The results suggested that the apelin-APJ system contributes to CNV development partially independent of the VEGF pathway.

Inhibition of apelin expression switches endothelial cells from proliferative to mature state in pathological retinal angiogenesis.

The recruitment of mural cells such as pericytes to patent vessels with an endothelial lumen is a key factor for the maturation of blood vessels and the prevention of hemorrhage in pathological angiogenesis. To date, our understanding of the specific trigger underlying the transition from cell growth to the maturation phase remains incomplete. Since rapid endothelial cell growth causes pericyte loss, we hypothesized that suppression of endothelial growth factors would both promote pericyte recruitment, in addition to inhibiting pathological angiogenesis. Here, we demonstrate that targeted knockdown of apelin in endothelial cells using siRNA induced the expression of monocyte chemoattractant protein-1 (MCP-1) through activation of Smad3, via suppression of the PI3K/Akt pathway. The conditioned medium of endothelial cells treated with apelin siRNA enhanced the migration of vascular smooth muscle cells, through MCP-1 and its receptor pathway. Moreover, in vivo delivery of siRNA targeting apelin, which causes exuberant endothelial cell proliferation and pathological angiogenesis through its receptor APJ, led to increased pericyte coverage and suppressed pathological angiogenesis in an oxygen-induced retinopathy model. These data demonstrate that apelin is not only a potent endothelial growth factor, but also restricts pericyte recruitment, establishing a new connection between endothelial cell proliferation signaling and a trigger of mural recruitment.

Apelin deficiency accelerates the progression of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motor neurons. Recent studies have implicated that chronic hypoxia and insufficient vascular endothelial growth factor (VEGF)-dependent neuroprotection may lead to the degeneration of motor neurons in ALS. Expression of apelin, an endogenous ligand for the G protein-coupled receptor APJ, is regulated by hypoxia. In addition, recent reports suggest that apelin protects neurons against glutamate-induced excitotoxicity. Here, we examined whether apelin is an endogenous neuroprotective factor using SOD1(G93A) mouse model of ALS. In mouse CNS tissues, the highest expressions of both apelin and APJ mRNAs were detected in spinal cord. APJ immunoreactivity was observed in neuronal cell bodies located in gray matter of spinal cord. Although apelin mRNA expression in the spinal cord of wild-type mice was not changed from 4 to 18 weeks age, that of SOD1(G93A) mice was reduced along with the paralytic phenotype. In addition, double mutant apelin-deficient and SOD1(G93A) displayed the disease phenotypes earlier than SOD1(G93A) littermates. Immunohistochemical observation revealed that the number of motor neurons was decreased and microglia were activated in the spinal cord of the double mutant mice, indicating that apelin deficiency pathologically accelerated the progression of ALS. Furthermore, we showed that apelin enhanced the protective effect of VEGF on H(2)O(2)-induced neuronal death in primary neurons. These results suggest that apelin/APJ system in the spinal cord has a neuroprotective effect against the pathogenesis of ALS.

Caspase-4 directly activates caspase-9 in endoplasmic reticulum stress-induced apoptosis in SH-SY5Y cells.

The present study investigated the function of caspase-4 in endoplasmic reticulum (ER) stress-induced apoptosis in human neuronal cell line SH-SY5Y. Tunicamycin, which is known to induce ER stress, activated both caspase-9 and caspase-4, and the activation of caspase-4 preceded that of caspase-9. The caspase-4 inhibitor LEVD-CHO suppressed both the apoptosis and caspase-9 activation. In addition, human recombinant active caspase-4 cleaved wild type and D330A mutant substituted Asp-330 for alanine of human recombinant procaspase-9, but did not cleave D315A mutant substituted Asp-315 for alanine. These results suggest that caspase-4 directly activates caspase-9 by the processing of procaspase-9 at Asp-315 in ER stress-induced neuronal apoptosis.

Caspase-4 Directly Activates Caspase-9 in Endoplasmic Reticulum Stress-Induced Apoptosis in SH-SY5Y Cells.

The present study investigated the function of caspase-4 in endoplasmic reticulum (ER) stress-induced apoptosis in human neuronal cell line SH-SY5Y. Tunicamycin, which is known to induce ER stress, activated both caspase-9 and caspase-4, and the activation of caspase-4 preceded that of caspase-9. The caspase-4 inhibitor LEVD-CHO suppressed both the apoptosis and caspase-9 activation. In addition, human recombinant active caspase-4 cleaved wild type and D330A mutant substituted Asp-330 for alanine of human recombinant procaspase-9, but did not cleave D315A mutant substituted Asp-315 for alanine. These results suggest that caspase-4 directly activates caspase-9 by the processing of procaspase-9 at Asp-315 in ER stress-induced neuronal apoptosis.

Apelin is a crucial factor for hypoxia-induced retinal angiogenesis.

OBJECTIVE: To investigate the role of endogenous apelin in pathological retinal angiogenesis. METHODS AND RESULTS: The progression of ischemic retinal diseases, such as diabetic retinopathy, is closely associated with pathological retinal angiogenesis, mainly induced by vascular endothelial growth factor (VEGF) and erythropoietin. Although antiangiogenic therapies using anti-VEGF drugs are effective in treating retinal neovascularization, they show a transient efficacy and cause general adverse effects. New therapeutic target molecules are needed to resolve these issues. It was recently demonstrated that the apelin/APJ system, a newly deorphanized G protein-coupled receptor system, is involved in physiological retinal vascularization. Retinal angiography and mRNA expression were examined during hypoxia-induced retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Compared with age-matched control mice, retinal apelin expression was dramatically increased during the hypoxic phase in oxygen-induced retinopathy model mice. APJ was colocalized in proliferative cells, which were probably endothelial cells of the ectopic vessels in the vitreous body. Apelin deficiency hardly induced hypoxia-induced retinal angiogenesis despite the upregulation of VEGF and erythropoietin mRNA in oxygen-induced retinopathy model mice. Apelin small and interfering RNA suppressed the proliferation of endothelial cells independent of the VEGF/VEGF receptor 2 signaling pathway. CONCLUSIONS: These results suggest that apelin is a prerequisite factor for hypoxia-induced retinal angiogenesis.

Nitric oxide inhibits lipopolysaccharide-induced inducible nitric oxide synthase expression and its own production through the cGMP signaling pathway in murine microglia BV-2 cells.

The present study examined the effect of the nitric oxide (NO) donor NOC18 on lipopolysaccharide (LPS)-induced NO production to investigate a regulation mechanism of NO production by microglial cells. LPS increased the levels of NO and inducible NO synthase (iNOS) protein in BV-2 murine microglial cells in a concentration-dependent manner. Pretreatment with NOC18 for 24 h concentration-dependently attenuated the LPS-induced iNOS protein expression and NO production. The inhibitory effect of NOC18 on LPS-induced NO production was partially blocked by LY83583, a soluble guanylate cyclase inhibitor. Pretreatment with dibutyryl guanosine-3',5'-cyclic monophosphate (DBcGMP), a cell-permeable cGMP analogue, for 24 h attenuated partially LPS-induced iNOS protein expression and NO production. Furthermore, the effects of LPS on iNOS and NO production were inhibited by the c-Jun N-terminal kinase (JNK) inhibitor SP600125, and LPS-induced phosphorylation of JNK and c-Jun was inhibited by NOC18 and DBcGMP. These results suggest that NO production by microglial cells is controlled by a negative feedback mechanism via the NO/cGMP signaling pathway.

Nitric oxide/cGMP signaling pathway protects RAW264 cells against nitric oxide-induced apoptosis by inhibiting the activation of p38 mitogen-activated protein kinase.

Nitric oxide (NO) induces apoptosis in various cells lines, while activation of the NO/cGMP signaling pathway prevents apoptosis induced by diverse stimuli, including NO. Here, we report the cytoprotective mechanisms of the NO/cGMP signaling pathway against NO-induced apoptosis in a mouse macrophage-like cell line, RAW264. Treatment with sodium nitroprusside (SNP), an NO donor, at a high-toxic concentration (4 mM) stimulated the N-terminal conformational change of Bax and its translocation to mitochondria followed by cytochrome c release and nuclear fragmentation in RAW264 cells. These changes of Bax were attenuated by pretreatment with SNP at a low-nontoxic concentration (100 microM) or dibutyryl cGMP (DBcGMP), a cell-permeable cGMP analogue. SB203580, a p38 mitogen-activated protein kinase (MAP kinase) inhibitor, blocked the effects of 4 mM SNP on Bax translocation and cell viability. Treatment with 4 mM SNP activated p38 MAP kinase and this effect was prevented by pretreatment with 100 microM SNP or DBcGMP. These findings suggest that the NO/cGMP signaling pathway inhibits NO-induced apoptosis of macrophages by suppressing the p38 MAP kinase activation, which results in N-terminal conformational change of Bax and its translocation to mitochondria.

Involvement of endoplasmic reticulum stress on the cell death induced by 6-hydroxydopamine in human neuroblastoma SH-SY5Y cells.

Endoplasmic reticulum (ER) dysfunction is known to activate the unfolded protein response, which is characterized by the activation of two divergent processes, i.e., suppression of the initiation process in global protein synthesis and expression of glucose-regulated protein 78 (Bip/Grp78) and the C/EBP homologous transcription factor CHOP/Gadd153. In this study, we examined the expression of CHOP/Gadd153 and Bip/Grp78 in human neuroblastoma SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA), which is used to prepare animal models of Parkinson's disease. 6-OHDA treatment induced cell death, in a concentration-dependent manner, which was inhibited by co-treatment with an antioxidant N-acetylcysteine. 6-OHDA was also effective in decreasing proteasome activity and in increasing the levels of high molecular ubiquitin-conjugated proteins. Furthermore, 6-OHDA induced a marked increase in the expression of both CHOP/Gadd153 and Bip/Grp78. This increase was prevented by N-acetylcysteine. Taken together, our data indicate that ER dysfunction is at least in part involved in the mechanisms underlying cell death induced by 6-OHDA in SH-SY5Y cells.

Nitric oxide protects macrophages from hydrogen peroxide-induced apoptosis by inducing the formation of catalase.

We investigated the cytoprotective effect of NO on H2O2-induced cell death in mouse macrophage-like cell line RAW264. H2O2-treated cells showed apoptotic features, such as activation of caspase-9 and caspase-3, nuclear fragmentation, and DNA fragmentation. These apoptotic features were significantly inhibited by pretreatment for 24 h with NO donors, sodium nitroprusside and 1-hydroxy-2-oxo-3,3-bis-(2-aminoethyl)-1-triazene, at a low nontoxic concentration. The cytoprotective effect of NO was abrogated by the catalase inhibitor 3-amino-1,2,4-triazole but was not affected by a glutathione synthesis inhibitor, L-buthionine-(S,R)-sulfoximine. NO donors increased the level of catalase and its activity in a concentration-dependent manner. Cycloheximide, a protein synthesis inhibitor, inhibited both the NO-induced increase in the catalase level and the cytoprotective effect of NO. These results indicate that NO at a low concentration protects macrophages from H2O2-induced apoptosis by inducing the production of catalase.