Prolonged use of nitric oxide donor sodium nitroprusside induces ocular hypertension in mice.

Nitric oxide (NO) donors are promising therapeutic candidates for treating intraocular hypertension (IOP) and glaucoma. This study aims to investigate the effect of prolonged use of NO donor sodium nitroprusside (SNP) on IOP. Since SNP has a short biological half-life, a nanoparticle drug delivery system (mesoporous silica nanoparticles) has been used to deliver SNP to the target tissues (trabecular meshwork and Schlemm's canal). We find that the sustained use of NO donor initially reduced IOP followed, surprisingly, by IOP elevation, which could not recover by drug withdraw but could be reversed by the antioxidant MnTMPyP application. The IOP elevation and normalization coincide with increased and reduced protein nitration in the mouse conventional outflow tissue. These findings suggest that the prolonged use of NO donor SNP may be problematic as it can cause outflow tissue damage by protein nitration. MnTMPyP is protective of the nitrative damage which could be considered to be co-applied with NO donors.

Exacerbated Headache-Related Pain in the Single Prolonged Stress Preclinical Model of Post-traumatic Stress Disorder.

Chronic headache pain is one of the most commonly reported comorbid pain conditions with post-traumatic stress disorder (PTSD) patients and resistant to effective treatment, yet no combined preclinical model of the two disorders has been reported. Here, we used a modified chronic headache pain model to investigate the contribution of single prolonged stress (SPS) model of PTSD with sodium nitroprusside (SNP)-induced hyperalgesia. Injection of SNP (2 mg/kg, i.p.) occurred every other day from day 7 to day 15 after initiation of SPS in rats. Paw withdrawal threshold (PWT) to von Frey stimuli and tail flick latencies (TFL) dramatically decreased as early as 7 days after SPS and lasted until at least day 21. Basal PWT and TFL also significantly decreased during the SNP treatment period. The lower nociceptive thresholds recovered in 6 days following the final SNP injection in SNP group, but not in SPS + SNP group. Elevated nociceptin/OFQ (N/OFQ) levels observed in cerebrospinal fluid of SPS rats were even higher in SPS + SNP group. Glial fibrillary acidic protein (GFAP) and N/OFQ peptide (NOP) receptor mRNA expression increased in dorsal root ganglia (DRG) 21 days after SPS exposure; mRNA increases in the SPS/SNP group was more pronounced than SPS or SNP alone. GFAP protein expression was upregulated in trigeminal ganglia by SPS. Our results indicate that traumatic stress exaggerated chronic SNP-induced nociceptive hypersensitivity, and that N/OFQ and activated satellite glia cells may play an important role in the interaction between both conditions.

NXY-059, a Failed Stroke Neuroprotectant, Offers No Protection to Stem Cell-Derived Human Neurons.

BACKGROUND: Developing new medicines is a complex process where understanding the reasons for both failure and success takes us forward. One gap in our understanding of most candidate stroke drugs before clinical trial is whether they have a protective effect on human tissues. NXY-059 is a spin-trap reagent hypothesized to have activity against the damaging oxidative biology which accompanies ischemic stroke. Re-examination of the preclinical in vivo dataset for this agent in the wake of the failed SAINT-II RCT highlighted the presence of a range of biases leading to overestimation of the magnitude of NXY-059's effects in laboratory animals. Therefore, NXY-059 seemed an ideal candidate to evaluate in human neural tissues to determine whether human tissue testing might improve screening efficiency. MATERIALS AND METHODS: The aim of this randomized and blinded study was to assess the effects of NXY-059 on human stem cell-derived neurons in the presence of ischemia-like injury induced by oxygen glucose deprivation or oxidative stress induced by hydrogen peroxide or sodium nitroprusside. RESULTS: In MTT assays of cell survival, lactate dehydrogenase assays of total cell death and terminal deoxynucleotidyl transferase dUTP nick end labeling staining of apoptotic-like cell death, NXY-059 at concentrations ranging from 1 µm to 1 mm was completely without activity. Conversely an antioxidant cocktail comprising 100 µm each of ascorbate, reduced glutathione, and dithiothreitol used as a positive control provided marked neuronal protection in these assays. CONCLUSION: These findings support our hypothesis that stroke drug screening in human neural tissues will be of value and provides an explanation for the failure of NXY-059 as a human stroke drug.

Protective Effect of Dimethyl Fumarate on an Oxidative Stress Model Induced by Sodium Nitroprusside in Mice.

Recent reports have shown that dimethyl fumarate (DMF) prevents brain damage induced by intracerebral hemorrhage and this beneficial effect is mediated by the nuclear erythroid 2 p45-related factor-2-antioxidant response element (Nrf2-ARE) pathway. However, the downstream mechanism underlying the activation of the Nrf2-ARE pathway is unclear. Here, we investigated the protective effect of DMF using an in vivo model of oxidative stress induced by sodium nitroprusside (SNP) and rat primary striatal cultures. Oral administration of DMF prevented SNP-induced motor dysfunction. Pre-administration of DMF (60-200 mg/kg) for 24 h dose-dependently protected against brain damage induced by the striatal injection of SNP. Next, we investigated the protective effect and mechanism of DMF against oxidative stress using rat primary striatal cell cultures. Treatment of striatal cells with DMF (10 µM) markedly prevented hydrogen peroxide-induced cytotoxicity. The protective effect of DMF against oxidative stress in vitro was inhibited by zinc protoporphyrin IX, an inhibitor of heme oxygenase-1, but not by buthionine sulfoximine, an inhibitor of glutathione synthesis. These results suggest that the activation of heme oxygenase-1 plays an important role in the protective effect of DMF.

Rutin, A Natural Flavonoid Protects PC12 Cells Against Sodium Nitroprusside-Induced Neurotoxicity Through Activating PI3K/Akt/mTOR and ERK1/2 Pathway.

Free radicals induced neural damage is implicated in CNS diseases and rutin isolated form Lonicera japonica are reported to have neuroprotective activity. Previously, we confirmed that rutin exerted neuroprotective effect against sodium nitroprusside (SNP)-induced cell death in PC12 cells. However, the neuroprotective mechanism of rutin is still not fully uncovered. Here, we found that rutin significantly decreased SNP-induced reactive oxygen species in PC12 cells. Rutin reversed the declined GSH/GSSG ratio and mitochondrial membrane potential induced by SNP. Moreover, rutin activated both the protein Akt/mTOR and the extracellular signal-regulated kinase (ERK1/2) signaling pathways and the neuroprotective effects of rutin were blocked by either the specific PI3K inhibitor LY294002 or the MAPK pathway inhibitor PD98059. In summary, these results demonstrated that the neuroprotective effects of rutin might be through activating both the PI3K/Akt/mTOR and ERK1/2 signaling pathways. Our findings support that rutin may have therapeutic potential for the treatment of CNS diseases related to NO neurotoxicity.

[Protective effect of rutin against oxidative injury in neuronal cells].

OBJECTIVE: To observe the protective effect of rutin (RUT) on neuronal cells against sodium nitroprusside (SNP) induced neurotoxicity. METHODS: PC12 cells were treated with different concentration of SNP for 24 h and MTT assay was applied to analyze the survival rate; PC12 cells were pretreated with rutin for 1 h, and then incubated for 24 h with SNP. MTT assay, morphological observation, as well as immunofluorescence were performed to evaluate both the SNP neurotoxicity and the protective effects of RUT, Western blot was used to analyzed the level of phosphorylated extra cellular regulated protein kinases (ERK1/2) after treatment with RUT, the results were also testified in primary cultured neurons. RESULTS: Results from MTT assay showed that SNP caused cell death in a concentration-dependent manner in PC12 cells. The effect of SNP was observed at 200 - 1 000 micromol/L and was significant at 800 micromol/L. 25 micromol/L rutin partly blocked the neurotoxicity of SNP by preventing PC12 cells from apoptosis. Hoechst and PI staining indicated that SNP treatment decreased the number of viable cells and induced shrinkage and aggregation of the nucleus, whereas RUT pretreatment attenuated the toxic effects of SNP, after treatment with RUT in PC12 cells, the phosphorylation of ERK1/2 was increased and peaked at 20 min. Most importantly, the protective effect of RUT on PC12 cells was confirmed on cultured neurons. CONCLUSION: RUT possesses protective effect against neuronal apoptosis induced by SNP and this effect may be partially related with ERK1/2 signaling.

Melatonin receptor agonist-induced reduction of SNP-released nitric oxide and cGMP production in isolated human non-pigmented ciliary epithelial cells.

The present study was designed to determine the effects of melatonin and its receptor agonists on SNP-released nitric oxide (NO) and cGMP production in aqueous humor producing cells of the ciliary body because these effects may play a role in melatonin receptor-mediated regulation of intraocular pressure (IOP). NO release protocols were carried out using human non-pigmented ciliary epithelial (hNPCE) cells treated in dye free DMEM containing l-arginine (10(-3) M). The cGMP experimental protocols were performed using dye free DMEM containing 3-isobutyl-1-methylxanthine (IBMX, 10(-4) M). The effects of varying concentrations (10(-13), 10(-11), 10(-9), 10(-7), and 10(-5) M) of melatonin, 5-MCA-NAT (putative MT(3) agonist), N-butanoyl-2-(2-methoxy-6H-isoindolo[2, 1-a]indol-11-yl)ethanamine (IIK7; selective MT(2) agonist) or S-27633-1 (selective MT(1) agonist) on sodium nitroprusside (SNP)-released NO or cGMP production were determined in separate experiments. NO and cGMP levels were measured using a colorimetric assay or enzyme immunoassay (EIA), respectively. Melatonin receptor selectivity was evaluated using luzindole (LUZ; nonselective MT(1)/MT(2) antagonist) or 4-phenyl-2-propionamidotetralin (4P-PDOT; selective MT(2) antagonist). Melatonin, 5-MCA-NAT, and IIK7 all caused concentration-dependent reduction of SNP-released NO and cGMP production. The inhibitory actions of melatonin, 5-MCA-NAT and IIK7 were either completely blocked at 10(-13), 10(-11), and 10(-9) M concentrations of the agonists or partially at 10(-7) and 10(-5) M in the presence of luzindole or 4P-PDOT. Results from this study suggest that melatonin and its analogs, 5-MCA-NAT and IIK7 inhibit SNP-released NO and cGMP production via activation of MT(2) receptors in human NPCE cells. These actions may play a role in melatonin agonist-induced regulation of aqueous humor secretion and IOP.

Neuroprotective effect of 2-(4-methoxyphenyl)ethyl-2-acetamido-2-deoxy-ß-D-pyranoside against sodium nitroprusside-induced neurotoxicity in HT22 cells.

2-(4-Methoxyphenyl) ethyl-2-acetamido-2-deoxy-ß-D-pyranoside (GlcNAc-Sal), the salidroside analog was synthesized and shown to inhibit hypoglycemia and serum limitation induced apoptosis in PC12 cells. This study investigated the protective effects of GlcNAc-Sal on sodium nitroprusside (SNP)-induced cytotoxicity in HT22 cells. Cell viability tests and Hoechst 33342 staining comfirmed that GlcNAc-Sal pretreatment attenuated SNP-stimulated apoptotic cell death in HT22 cells in a concentration-dependent manner. The measurements of reactive oxygen species (ROS), nitric oxide (NO) production and apoptosis-related gene and protein expression suggest that the protection of GlcNAc-Sal, shown in this study, might be mediated by inhibiting intracellular ROS and NO production, and regulating apoptosis-related gene and protein expression during SNP stimulation. Perhaps, this study might contribute to the development of GlcNAc-Sal as an agent for preventing and/or treating a variety of NO-induced brain diseases.

Protective effect of luteolin on an oxidative-stress model induced by microinjection of sodium nitroprusside in mice.

Accumulating lines of evidence showed that luteolin, a polyphenolic compound, has potent neuroprotective effects. The purpose of this study was to examine whether luteolin can protect against sodium nitroprusside (SNP)-induced oxidative damage in mouse brain. Intrastriatal co-injection of luteolin (3 - 30 nmol) with SNP (10 nmol) dose-dependently protected against brain damage and motor dysfunction. Oral administrations of luteolin (600 - 1200 mg/kg) dose-dependently protected against brain damage and motor dysfunction induced by striatal injection of SNP. Furthermore, luteolin (30 - 100 µM) concentration dependently protected against Fe(2+)-induced lipid peroxidation in mouse brain homogenate. Luteolin (1 - 100 µg/ml) showed potent DPPH radical scavenging ability, when compared with ascorbic acid and glutathione. Finally, a ferrozine assay showed that luteolin (30 - 100 µg/ml) has Fe(2+)-chelating ability, but this was weaker than that of ethylenediaminetetraacetic acid. These results suggest that intrastriatal or oral administration of luteolin protected mice brain from SNP-induced oxidative damage by scavenging and chelating effects.

Protection of cells from nitric oxide-mediated apoptotic death by glutathione C60 derivative.

The influence of the glutathione C60 derivative on the cytotoxicity of a highly reactive free radical NO (nitric oxide) has been investigated. Consistent with its cytoprotective abilities, the derivative scavenges ROS (reactive oxygen species) and RNS (reactive nitrogen species) both in vitro and under cell-free conditions. Moreover, the glutathione C60 derivative protected PC12 cells from the cytotoxic effect of the NO-releasing compound, SNP (sodium nitroprusside). Addition of glutathione C60 derivative alone did not induce apoptosis and necrosis. The results suggest that the glutathione C60 derivative has the potential to prevent NO-mediated cell death without evident toxicity.

Differential response of three cell types to dual stress of nitric oxide and radiation.

The perception of toxicity to nitric oxide (NO) and irradiation (IR) by three different cell types has been studied. The three cell types are the macrophage like RAW264.7 cells, EL4 lymphoma cells, and splenocytes, which represent the different components of a tumor. These three cell types respond differently to NO donors (SNP and SNAP) and radiation treatment. The macrophages were found to be most radio-resistant and insensitive to NO donors. The innate resistance of the macrophages was not due to its antioxidant defense system since there was no significant activation of the enzymes (superoxide dismutases, catalase, and glutathione peroxidase) in RAW264.7 cells after NO donor and irradiation. But the cell cycle arrest of the three cell types was different from each other. The EL4 cells were found to arrest in the G2/M phase while the macrophages were found arrested in the G1 phase of the cell cycle. Such specific killing of the tumor cell in response to NO donor while sparing the macrophages can be of immense importance to radiotherapy.

Evidence for the nitric oxide pathway as a potential mode of action in fenoldopam-induced vascular injury.

Fenoldopam, a dopaminergic DA1 agonist, induces vasodilatation via nitric oxide (NO), and this may be associated with mesenteric arterial injury. NO is produced from the enzymatic action of nitric oxide synthase (NOS), which is regulated by the shear-stress mediating protein caveolin-1. Profound vasodilatation and accompanied decreased shear are early events that could initiate vascular injury. Therefore, it is of interest to determine the role of caveolin-1 and the NO pathway in fenoldopam-induced vascular injury. At sites of fenoldopam-induced mesenteric arterial injury, decreased caveolin-1 expression and apoptosis were prominent immunohistochemical findings. An additional finding at these sites of injury were loss and/or reduced expression of caveolin-1 regulated structural proteins, connexin-43, (gap junction) ZO-1, and claudin (tight junctions). Because functional loss of caveolin-1 is associated with increased NOS activity and vasodilatation via NO, studies were conducted to show a NO donor produced vascular lesions in the mesenteric arteries morphologically similar to those induced by fenoldopam. Moreover, the incidence and severity of fenoldopam-induced vascular injury were reduced when an NOS inhibitor or a scavenger of NO-generated free radicals were coadministered with fenoldopam. Collectively, these data suggest that caveolin-1 and its regulated NO pathway may play an important role in vasodilatory drug-induced vascular injury.

Fe(II) and sodium nitroprusside induce oxidative stress: a comparative study of diphenyl diselenide and diphenyl ditelluride with their napthyl analog.

Here, we compare the influence of molecular structural modifications of diphenyl diselenide (DPDS) and diphenyl ditelluride (DPDT) with their naphthalene analogs, 1-dinapthyl diselenide (1-NapSe)2, 2-dinapthyl diselenide (2-NapSe)2, 1-dinapthyl distelluride (1-NapTe)2, and 2-dinapthyl ditelluride (2-NapTe)2. Fe(II)-induced hepatic thiobarbituric acid reactive species (TBARS) was in the order [(2-NapTe)2] > [(2-NapSe)2] > [(DPDS)] > [(1-NapSe)2] > [(1-NapTe)2]> [(DPDT)]. For sodium nitroprusside (SNP)-induced hepatic TBARS, the order was [(2-NapTe)2] > [(DPDT)] > [(1-NapSe)2] > [(2-NapSe)2] > [(1-NapTe)2] > [(DPDS)]. For Fe(II) and SNP-induced renal TBARS, the orders were [(2-NapTe)2] > [(1-NapTe)2] = [(DPDT)] > [(1-NapSe)2] > [(2-NapSe)2] > [(DPDS)] and [(2-NapTe)2] > [(1-NapTe)2] > [(1-NapSe)2] > [(2-NapSe)2] > [(DPDS)] > [(DPDS)], respectively. The present investigation shows that DPDS was less potent and the change in the organic moiety from an aryl to napthyl group dramatically changed the potency of diselenides. These results suggest that minor changes in the organic moiety of aromatic diselenides can profoundly modify their antioxidant properties. In view of the fact that the pharmacological properties of organochalcogens are linked, at least in part, to their antioxidant properties, it becomes important to explore the pharmacological properties of dinaphtyl diselenides and ditellurides.

Inhibition of key enzymes linked to type 2 diabetes and sodium nitroprusside-induced lipid peroxidation in rat pancreas by water extractable phytochemicals from some tropical spices.

CONTEXT: Spices have been used as food adjuncts and in folklore for ages. Inhibition of key enzymes (α-amylase and α-glucosidase) involved in the digestion of starch and protection against free radicals and lipid peroxidation in pancreas could be part of the therapeutic approach towards the management of hyperglycemia and dietary phenolics have shown promising potentials. OBJECTIVE: This study investigated and compared the inhibitory properties of aqueous extracts of some tropical spices: Xylopia aethiopica [Dun.] A. Rich (Annonaceae), Monodora myristica (Gaertn.) Dunal (Annonaceae), Syzygium aromaticum [L.] Merr. et Perry (Myrtaceae), Piper guineense Schumach. et Thonn (Piperaceae), Aframomum danielli K. Schum (Zingiberaceae) and Aframomum melegueta (Rosc.) K. Schum (Zingiberaceae) against α-amylase, α-glucosidase, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and sodium nitroprusside (SNP)-induced lipid peroxidation in rat pancreas--in vitro using different spectrophotometric method. MATERIALS AND METHODS: Aqueous extract of the spices was prepared and the ability of the spice extracts to inhibit α-amylase, α-glucosidase, DPPH radicals and SNP-induced lipid peroxidation in rat pancreas--in vitro was investigated using various spectrophotometric methods. RESULT: All the spice extracts inhibited α-amylase (IC(50) = 2.81-4.83 mg/mL), α-glucosidase (IC(50) = 2.02-3.52 mg/mL), DPPH radicals (EC(50) = 15.47-17.38 mg/mL) and SNP-induced lipid peroxidation (14.17-94.38%), with the highest α-amylase & α-glucosidase inhibitory actions and DPPH radical scavenging ability exhibited by X. aethiopica, A. danielli and S. aromaticum, respectively. Also, the spices possess high total phenol (0.88-1.3 mg/mL) and flavonoid (0.24-0.52 mg/mL) contents with A. melegueta having the highest total phenolic and flavonoid contents. DISCUSSION AND CONCLUSION: The inhibitory effects of the spice extracts on α-amylase, α-glucosidase, DPPH radicals and SNP-induced lipid peroxidation in pancreas (in vitro) could be attributed to the presence of biologically active phytochemicals such as phenolics and some non-phenolic constituents of the spices. Furthermore, these spices may exert their anti-diabetic properties through the mechanism of enzyme inhibition, free radicals scavenging ability and prevention of lipid peroxidation.

Antioxidant properties of Taraxacum officinale fruit extract are involved in the protective effect against cellular death induced by sodium nitroprusside in brain of rats.

CONTEXT: Taraxacum officinale Weber (Asteraceae), known as dandelion, is used for medicinal purposes due to its choleretic, diuretic, antitumor, antioxidant, antiinflammatory, and hepatoprotective properties. OBJECTIVE: We sought to investigate the protective activity of T. officinale fruit extract against sodium nitroprusside (SNP)-induced decreased cellular viability and increased lipid peroxidation in the cortex, hippocampus, and striatum of rats in vitro. To explain the mechanism of the extract's antioxidant activity, its putative scavenger activities against NO, DPPH·, OH·, and H(2)O(2) were determined. METHODS: Slices of cortex, hippocampus, and striatum were treated with 50 µM SNP and T. officinale fruit ethanolic extract (1-20 µg/mL) to determine cellular viability by MTT reduction assay. Lipid peroxidation was measure in cortical, hippocampal and striatal slices incubates with SNP (5 µM) and T. officinale fruit extract (1-20 µg/mL). We also determined the scavenger activities of T. officinale fruit extract against NO·, DPPH·, OH·, and H(2)O(2), as well as its iron chelating capacity. RESULTS: The extract (1, 5, 10, and 20 µg/mL) protected against SNP-induced decreases in cellular viability and increases in lipid peroxidation in the cortex, hippocampus, and striatum of rats. The extract had scavenger activity against DPPH· and NO· at low concentrations and was able to protect against H(2)O(2) and Fe(2+)-induced deoxyribose oxidation. CONCLUSION: T. officinale fruit extract has antioxidant activity and protects brain slices against SNP-induced cellular death. Possible mechanisms of action include its scavenger activities against reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are attributed to the presence of phenolic compounds in the extract.

Neonatal glucocorticoid overexposure alters cardiovascular function in young adult horses in a sex-linked manner.

Prenatal glucocorticoid overexposure has been shown to programme adult cardiovascular function in a range of species, but much less is known about the long-term effects of neonatal glucocorticoid overexposure. In horses, prenatal maturation of the hypothalamus-pituitary-adrenal axis and the normal prepartum surge in fetal cortisol occur late in gestation compared to other precocious species. Cortisol levels continue to rise in the hours after birth of full-term foals and increase further in the subsequent days in premature, dysmature and maladapted foals. Thus, this study examined the adult cardiovascular consequences of neonatal cortisol overexposure induced by adrenocorticotropic hormone administration to full-term male and female pony foals. After catheterisation at 2-3 years of age, basal arterial blood pressures (BP) and heart rate were measured together with the responses to phenylephrine (PE) and sodium nitroprusside (SNP). These data were used to assess cardiac baroreflex sensitivity. Neonatal cortisol overexposure reduced both the pressor and bradycardic responses to PE in the young adult males, but not females. It also enhanced the initial hypotensive response to SNP, slowed recovery of BP after infusion and reduced the gain of the cardiac baroreflex in the females, but not males. Basal diastolic pressure and cardiac baroreflex sensitivity also differed with sex, irrespective of neonatal treatment. The results show that there is a window of susceptibility for glucocorticoid programming during the immediate neonatal period that alters cardiovascular function in young adult horses in a sex-linked manner.

Sodium nitroprusside induces autophagic cell death in glutathione-depleted osteoblasts.

Previous studies reported that high levels of nitric oxide (NO) induce apoptotic cell death in osteoblasts. We examined molecular mechanisms of cytotoxic injury induced by sodium nitroprusside (SNP), a NO donor, in both glutathione (GSH)-depleted and control U2-OS osteoblasts. Cell viability was reduced by much lower effective concentrations of SNP in GSH-depleted cells compared to normal cells. The data suggest that the level of intracellular GSH is critical in SNP-induced cell death processes of osteoblasts. The level of oxidative stress due to SNP treatments doubled in GSH-depleted cells when measured with fluorochrome H2DCFDA. Pretreatment with the NO scavenger PTIO preserved the viability of cells treated with SNP. Viability of cells treated with SNP was recovered by pretreatment with Wortmannin, an autophagy inhibitor, but not by pretreatment with zVAD-fmk, a pan-specific caspase inhibitor. Large increases of LC3-II were shown by immunoblot analysis of the SNP-treated cells, and the increase was blocked by pretreatment with PTIO or Wortmannin; this implies that under GSH-depleted conditions SNP induces different molecular signaling that lead to autophagic cell death. The ultrastructural morphology of SNP-treated cells in transmission electron microscopy showed numerous autophagic vacuoles. These data suggest NO produces oxidative stress and cellular damage that culminate in autophagic cell death of GSH-depleted osteoblasts.

Involvement of Hsp70, a stress protein, in the resistance of long-term culture of PC12 cells against sodium nitroprusside (SNP)-induced cell death.

Sodium nitroprusside (SNP)-treated PC12 cell line is being used in our laboratory as a cell model of nitric oxide (NO)-mediated damage for in vitro evaluation of potential neuroprotective compounds, thus cell response to SNP must be standardized to gain reproducible data. The NO-donor SNP has been shown to induce cell death at high concentrations in undifferentiated PC12 cells. Differences were found in sensitivity to SNP between cells from short- and long-term cultured cells. After 24-h exposure to 100-500 microM SNP, a decrease of cell viability was observed in both short- (17, 21 and 23rd passages) and long-term cultures (46, 49 and 50th passages), with IC(50) values of 312.72 and 462.90 microM, respectively. In cells from early passages, SNP-induced cell death was accompanied by significant increases of LDH leakage, nitrite production, malondialdehyde (MDA) levels, catalase (CAT) activity, cleavage of poly(ADP-ribose)polymerase (PARP) and caspase-3 activation in comparison with those from late passages. Furthermore, untreated and SNP-treated cells from long-term cultures displayed an increase of the stress protein Hsp70 levels when compared with those from short-term cultures. Up-regulated levels of Hsp70 may be associated with cell survival. Therefore, cells may acquire a certain resistance to SNP-induced toxicity associated with an increase in cell passage-dependent Hsp70. The protein Hsp70 might modulate the cellular response to the toxic insult by increasing CAT and GSH-Px activities and decreasing caspase-3 activation. Finally, it is crucial for the standardization of this cell model of neurotoxicity, at least in part, the use of PC12 cells in an optimum and reliable range of passages.

Regulatory protein Yap1 is involved in response of yeast Saccharomyces cerevisiae to nitrosative stress.

The goal of this work was to investigate the possible involvement of protein transcription factor Yap1 in regulation of activity of antioxidant enzymes superoxide dismutase and catalase during yeast response to nitrosative stress. It was found that the inactivation of the YAP1 gene, encoding Yap1p, cancelled the activation of superoxide dismutase and catalase by NO-donors. Then, using chimeric protein Yap1-GFP, we found the accumulation of Yap1p in the nucleus in response to nitrosative stress. Therefore, we conclude that these results in combination with previous data clearly demonstrate the involvement of Yap1p in upregulation of superoxide dismutase and catalase in yeast cells in response to nitrosative stress.

PACAP38 protects rat cortical neurons against the neurotoxicity evoked by sodium nitroprusside and thrombin.

Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 is a multifunctional anti-inflammatory and anti-apoptotic neuropeptide widely distributed in the nervous system. The objective of this study is to determine whether PACAP38 is neuroprotective against sodium nitroprusside (SNP) and thrombin, two mechanistically distinct neurotoxic agents. Treatment of primary cortical neuronal cultures with 1 mM SNP for 4 h causes neuronal cell death that is significantly reduced by 100 nM PACAP38. PACAP38 down-regulates SNP-induced cell cycle protein (cyclin E) expression and up-regulates p57(KIP2), a cyclin-dependent kinase inhibitor as well as the anti-apoptotic protein Bcl-2. Similarly, neuronal death induced by 100 nM thrombin or the thrombin receptor activating peptide (TRAP 6) is reduced by PACAP38 treatment. Thrombin-stimulated cell cycle protein (cdk4) expression is decreased by PACAP38 while PACAP38 inhibits thrombin-mediated reduction of p57(KIP2). However, the decrease in Bcl-2 evoked by thrombin is not affected by PACAP38. Finally, both SNP and thrombin (or TRAP) increase caspase 3 activity, an effect that is decreased by PACAP38. These data show that PACAP38 supports neuronal survival in vitro suppressing cell cycle progression and enhancing anti-apoptotic proteins. Our results support the possibility that PACAP could be a useful therapeutic agent for reducing neuronal cell death in neurodegenerative diseases.