The blood antioxidant defence capacity during intermittent hypoxic training in elite swimmers.

The main objective of this study was to examine the chronic effect of simulated intermittent normobaric hypoxia on blood antioxidant defence capacity in swimmers. The study included 14 male and 14 female competitive swimmers performing part of land training under simulated intermittent normobaric hypoxia (O2 = 15.5%) or in normoxia. Land interval training took place twice per week, with a total of 8 training units during the study, performed with individualized intensity. The activities of blood antioxidant enzymes did not change significantly during the first and last training unit in the hypoxic and normoxic group. However, when comparing individual variables a significant effect of exercise was observed on GPx an CAT activities, whereas training units significantly differentiated GPx and GR activities. The oxygen conditions and gender had a significant influence on CAT activity. The total antioxidant capacity was not significantly affected. Only in male swimmers from the hypoxic group did the training significantly increase resting levels of MDA. In conclusion, training in normobaric hypoxia was not an adequate stimulus for the excessive response of the antioxidant defence system, despite increased oxidative stress in these conditions.

Macrophage migration inhibitory factor induces cell death and decreases neuronal nitric oxide expression in spinal cord neurons.

Macrophage migration inhibitory factor is a potent proinflammatory cytokine; however, its role in spinal cord injury is poorly understood. Therefore, the aim of the present study was to investigate the effects of macrophage migration inhibitory factor on spinal cord neuron survival and viability. Due to the importance of nitric oxide metabolism in these events, part of our study was also focused on the influence of recombinant macrophage migration inhibitory factor on neuronal nitric oxide expression. Exposure of cultured mouse spinal cord neurons to macrophage migration inhibitory factor markedly increased cellular oxidative stress as measured by 2',7'-dichlorofluorescein fluorescence and intracellular calcium levels. In addition, an antagonist of the inositol 1,4,5-triphosphate receptor, 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate, completely blocked the macrophage migration inhibitory factor-induced increase in intracellular calcium levels. Macrophage migration inhibitory factor treatment also decreased cell viability, increased cellular lactate dehydrogenase release, and induced chromatin condensation and aggregation in cultured spinal cord neurons. Finally, exposure to macrophage migration inhibitory factor markedly decreased expression and activity of neuronal nitric oxide, accompanied by a decrease in cellular guanosine 3'5'-cyclic monophosphate levels. The present results indicate that macrophage migration inhibitory factor can induce dysfunction of spinal cord neurons, leading to cell death through oxidative stress and intracellular calcium-dependent pathways.

Human immunodeficiency virus-1 Tat protein up-regulates interleukin-6 and interleukin-8 expression in human breast cancer cells.

OBJECTIVE: To examine whether HIV-1 Tat protein increases the metastatic potential of human breast cancer cells through induction of pro-inflammatory tumor microenvironment. METHODS: Real-time RT-PCR and ELISA were employed to determine the mRNA and protein expression of IL-6 and IL-8 in highly metastatic human breast cancer cell line, MDA-MB-231. To investigate the transcriptional regulatory mechanisms of Tat-mediated up-regulation of IL-6 and IL-8, EMSA and reporter gene assay were carried out. RESULTS: Exposure of MDA-MB-231 cells to Tat resulted in a significant and dose-dependent up-regulation of IL-6 and IL-8 mRNA and protein expression. HIV-1 Tat protein also markedly increased NF-kappaB DNA-binding activity and transactivation in MDA-MB-231 cells. Additionally, pretreatment with NF-kappaB inhibitors significantly attenuated the ability of Tat to up-regulate IL-6 and IL-8 expression. It was also found that exposure of MDA-MB-231 cells to Tat induced up-regulation of MMP-9 expression at both mRNA and protein levels. CONCLUSIONS: These results suggest that HIV-1 Tat protein up-regulates expression of IL-6 and IL-8 in human breast cancer cells by NF-kappaB-dependent pathway. These data may contribute to exploration of the new molecular mechanisms leading to novel approaches for the therapeutic drug developments specifically targeted against the inflammatory pathways of breast cancer metastasis in AIDS patients.

Methamphetamine induces AP-1 and NF-kappaB binding and transactivation in human brain endothelial cells.

Cellular and molecular mechanisms of methamphetamine (METH)-induced neurotoxicity may involve alterations of cellular redox status and induction of inflammatory genes in endothelial cells. To study these hypotheses, molecular signaling pathways of METH-induced inflammatory responses via activation of redox-sensitive transcription factors were investigated in human brain microvascular endothelial cells (HBMEC). A dose-dependent depletion of total glutathione levels was detected in HBMEC exposed to METH. In addition, electrophoretic mobility shift assay (EMSA) showed significant increases in DNA binding activities of redox-responsive transcription factors, AP-1 and NF-kappaB, in HBMEC treated with METH. METH-mediated AP-1 or NF-kappaB activation was accompanied by induction of transactivation of AP-1 or NF-kappaB, as measured by dual luciferase assay using specific reporter plasmids. Because NF-kappaB and AP-1 are known to regulate expression of inflammatory genes, expression of the gene encoding for tumor necrosis factor-alpha (TNF-alpha) was also studied in METH-treated HBMEC. A dose-dependent overexpression of the TNF-alpha gene was observed in HBMEC treated with METH. The importance of AP-1 and NF-kappaB in METH-induced TNF-alpha gene was confirmed in functional promoter studies using constructs of the TNF-alpha promoter with mutated AP-1 or NF-kappaB sites. These results indicate that METH-induced disturbances in cellular redox status and activation of AP-1 and NF-kappaB can play critical roles in the signaling pathways leading to upregulation of inflammatory genes in human brain endothelial cells.

Cocaine activates redox-regulated transcription factors and induces TNF-alpha expression in human brain endothelial cells.

Cocaine abuse is frequently associated with cerebrovascular pathology. Although the cellular and molecular mechanisms of these alterations are not fully understood, they may involve oxidative injury or dysfunction of brain microvascular endothelial cells. To test this hypothesis, total glutathione levels, activation of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), as well as induction of the TNF-alpha gene expression were determined in human brain microvascular endothelial cells (HBMEC) exposed to cocaine. Exposure of HBMEC to cocaine resulted in a dose-dependent depletion of total glutathione levels. In addition, cocaine markedly activated redox-regulated transcription factors, NF-kappaB and AP-1. Activation of these transcription factors was accompanied by induction of AP-1- or NF-kappaB-dependent transcription, as measured by dual luciferase assay in HBMEC transfected with the AP-1- or NF-kappaB-responsive reporter constructs. Furthermore, HBMEC treatment with cocaine induced a dose-dependent expression of the tumor necrosis factor-alpha (TNF-alpha) gene. These results indicate that exposure to cocaine can trigger inflammatory pathways via activation of redox-sensitive transcription factors and induction of expression of the inflammatory genes in HBMEC. These events may contribute to the cerebrovascular insults observed in cocaine-abused patients.

High-energy diets, fatty acids and endothelial cell function: implications for atherosclerosis.

Diets high in fat and/or calories can lead to hypertriglyceridemia and postprandial lipemia and thus are considered a risk factor for the development of atherosclerosis. Plasma chylomicron levels are elevated in humans after consuming a high-fat meal, and hepatic synthesis of VLDL is increased when caloric intake is in excess of body needs. High lipoprotein lipase activity and subsequent hydrolysis of triglyceride-rich lipoproteins may be an important source of elevated concentrations of fatty acid anions in the proximity to the endothelium and hence a major risk factor for atherosclerosis. We have shown that selected fatty acids, as well as lipoprotein lipase-derived remnants of lipoproteins isolated from hypertriglyceridemic subjects, can activate vascular endothelial cells and disrupt endothelial integrity. Our studies suggest that omega-6 fatty acids, and especially linoleic acid, cause endothelial cell dysfunction most markedly as well as can potentiate TNF-mediated endothelial cell injury. We propose that high-energy diets, and especially diets rich in linoleic acid, are atherogenic by contributing to an imbalance in cellular oxidative stress/antioxidant status of the endothelium, which can lead to activation of oxidative stress-responsive transcription factors, inflammatory cytokine production and the expression of adhesion molecules. Our data also suggest that nutrients, which have antioxidant and/or membrane stabilizing properties, can protect endothelial cells. These findings contribute to the understanding of the interactive role of high fat/calorie diets and subsequent hypertriglyceridemia with inflammatory components and nutrients that exhibit antiatherogenic properties in the development of atherosclerosis. Moreover, results from our research further support the concept that high-fat/calorie diets and associated postprandial hypertriglyceridemia are significant risk factors for atherosclerosis.

Interleukin 4 induces transcription of the 15-lipoxygenase I gene in human endothelial cells.

The reticulocyte-type 15-lipoxygenase (15-LO-I) has been implicated in atherogenesis because of its capability of oxidizing low density lipoprotein. Therefore, we investigated the expression of the 15-LO-I gene in human umbilical vein endothelial cells (HUVEC). Nonactivated HUVEC did not exhibit detectable 15-LO-I mRNA. However, exposure of the cells to interleukin 4 (IL-4) induced the transcription of the 15-LO-I gene in a time- and concentration-dependent manner. Interestingly, this induction was not paralleled by a concomitant production of the functional 15-LO-I enzyme, as indicated by activity assays and immunoblotting. To gain more information about the mechanism of the induction process, we investigated IL-4-dependent activation of nuclear transcription factors for which binding sites were previously identified in the 5'-flanking region of the human 15-LO-I gene. Electrophoretic mobility shift assays revealed that IL-4 can activate signal transducer and activator of transcription 6, activator protein 2, GATA motif-binding transcription factor 1, nuclear factor 1, and SP-1 in HUVEC in a time- and concentration-dependent manner. Activation of these transcription factors was observed as early as 30 min after cytokine exposure. These data indicate that IL-4 upregulates the transcription of the 15-LO-I gene in human vascular endothelial cells, and this process may involve the activation of several nuclear transcription factors. The lack of active 15-LO-I protein in the presence of functional 15-LO-I mRNA suggests additional regulatory elements of 15-LO expression at posttranscriptional levels.

Liposome-mediated high-efficiency transfection of human endothelial cells.

Liposome-mediated transfection of endothelial cells provides a valuable experimental technique to study cellular gene expression and may also be adapted for gene therapy studies. However, the widely recognized disadvantage of liposome-mediated transfection is low efficiency. Therefore, studies were performed to optimize transfection techniques in human endothelial cells. The majority of the experiments were performed with primary cultures of human umbilical vein endothelial cells (HUVEC). In addition, selected experiments were performed using human brain microvascular endothelial cells and human dermal microvascular endothelial cells. To study transfection rates, HUVEC were transfected with the pGL3 vector, containing the luciferase reporter gene, complexed with several currently available liposomes, such as different Perfect Lipid (pFx) mixtures, DMRIE-C, or lipofectin. The optimal transfection rate was achieved in HUVEC transfected for 1.5 h with 5 microg/ml of DNA plasmid in the presence of 36 microg/ml of pFx-7. In addition, transfection with the VR-3301 vector encoding for human placental alkaline phosphatase revealed that, under the described conditions, transfection efficiency in HUVEC was approximately 32%. Transfections mediated by other liposomes were less efficient. The usefulness of the optimized transfection technique was confirmed in HUVEC transfected with NF-kappaB or AP-1-responsive constructs and stimulated with TNF or LPS. We conclude that among several currently available liposomes, pFx-7 appears to be the most suitable for transfections of cultured human endothelial cells.

The role of methyl-linoleic acid epoxide and diol metabolites in the amplified toxicity of linoleic acid and polychlorinated biphenyls to vascular endothelial cells.

Selected dietary lipids may increase the atherogenic effects of environmental chemicals, such as polychlorinated biphenyls (PCBs), by cross-amplifying mechanisms leading to dysfunction of the vascular endothelium. We have shown previously that the omega-6 parent fatty acid, linoleic acid, or 3,3',4,4'-tetrachlorobiphenyl (PCB 77), an aryl hydrocarbon (Ah) receptor agonist, independently can cause disruption of endothelial barrier function. Furthermore, cellular enrichment with linoleic acid can amplify PCB-induced endothelial cell dysfunction. We hypothesize that the amplified toxicity of linoleic acid and PCBs to endothelial cells could be mediated in part by cytotoxic epoxide metabolites of linoleic acid called leukotoxins (LTX) or their diol derivatives (LTXD). Exposure to LTXD resulted in a dose-dependent increase in albumin transfer across endothelial cell monolayers, whereas this disruption of endothelial barrier function was observed only at a high concentration of LTX. Pretreatment with the cytosolic epoxide hydrolase inhibitor 1-cyclohexyl-3-dodecyl urea partially protected against the observed LTX-induced endothelial dysfunction. Endothelial cell activation mediated by LTX and/or LTXD also enhanced nuclear translocation of the transcription factor NF-kappa B and gene expression of the inflammatory cytokine IL-6. Inhibiting cytosolic epoxide hydrolase decreased the LTX-mediated induction of both NF-kappa B and the IL-6 gene, whereas the antioxidant vitamin E did not block LTX-induced endothelial cell activation. Most importantly, inhibition of cytosolic epoxide hydrolase blocked both linoleic acid-induced cytotoxicity, as well as the additive toxicity of linoleic acid plus PCB 77 to endothelial cells. Interestingly, cellular uptake and accumulation of linoleic acid was markedly enhanced in the presence of PCB 77. These data suggest that cytotoxic epoxide metabolites of linoleic acid play a critical role in linoleic acid-induced endothelial cell dysfunction. Furthermore, the severe toxicity of PCBs in the presence of linoleic acid may be due in part to the generation of epoxide and diol metabolites. These findings have implications in understanding interactive mechanisms of how dietary fats can modulate dysfunction of the vascular endothelium mediated by certain environmental contaminants.

Nicotine protects against arachidonic-acid-induced caspase activation, cytochrome c release and apoptosis of cultured spinal cord neurons.

Hydrolysis of membrane phospholipids of spinal cord neurons is one of the first events initiated in spinal cord trauma. In this process, free fatty acids, and in particular arachidonic acid, are released. Exposure of spinal cord neurons to free arachidonic acid can compromise cell survival and initiate apoptotic cell death. In order to determine potential mechanisms of apoptosis induced by arachidonic acid, activation of caspases -3, -8, and -9, as well as the release of cytochrome c into the cytoplasm were measured in cultured spinal cord neurons exposed to 10 microM of this fatty acid. In addition, because nicotine can exert a variety of neuroprotective effects, we hypothesized that it can prevent arachidonic acid induced apoptosis of spinal cord neurons. To study this hypothesis, spinal cord neurons were pretreated with nicotine (10 microM for 2 h) before arachidonic acid exposure and caspase activation as well as markers of apoptotic cell death were studied. Treatment of spinal cord neurons with arachidonic acid for up to 24 h significantly increased cytoplasmic levels of cytochrome c, induced caspase activation and induced DNA laddering, a hallmark of apoptotic cell death. Nicotine pretreatment markedly attenuated all these effects. In addition, antagonist studies suggest that the alpha7 nicotinic receptor is primarily responsible for these anti-apoptotic effects of nicotine. These results indicate that nicotine can exert potent neuroprotective effects by inhibiting arachidonic acid induced apoptotic cascades of spinal cord neurons.

IL-4-induced oxidative stress upregulates VCAM-1 gene expression in human endothelial cells.

Vascular cell adhesion molecule-1 (VCAM-1) is expressed in early stages of atherosclerosis; however, the mechanisms of its upregulation are not fully understood. In the present study, we examined the effects of interleukin-4 (IL-4) on VCAM-1 gene expression and its transcriptional regulatory mechanism in human umbilical vein endothelial cells (HUVEC). Reverse transcription-polymerase chain reaction showed that VCAM-1 mRNA was induced in IL-4-treated HUVEC in a time- and dose-dependent manner. Among known transcription factors that have binding sites in the promoter region of the VCAM-1 gene, IL-4 activated only SP-1. In contrast, nuclear factor- kappa B (NF- kappa B), activator protein-1 (AP-1) and interferon regulatory factor-1 (IRF-1), which also have consensus binding sequences in the 5'-flanking region of the human VCAM-1 gene, were not activated. The role of SP-1 in IL-4-induced VCAM-1 expression was confirmed in HUVEC transfected with a reporter construct of the VCAM-1 promoter with mutated SP-1 binding site. As IL-4 treatment of HUVEC enhanced the intracellular oxidizing potential, as indicated by an increase in 2',7'-dichlorofluorescein (DCF) fluorescence, we studied the effect of antioxidants on IL-4-induced VCAM-1 expression. Pretreatment of HUVEC with pyrrolidine dithiocarbamate (PDTC) or N-acetylcysteine (NAC) completely prevented IL-4-induced VCAM-1 expression. In addition, PDTC inhibited IL-4-related activation of SP-1. These results suggest that IL-4-induced oxidative stress upregulates the expression of VCAM-1 gene in HUVEC at transcriptional levels via activation of SP-1 transcription factor. In contrast, NF- kappa B, AP-1 or IRF-1 do not appear to be involved in the signal transduction cascade.

Linoleic acid-induced VCAM-1 expression in human microvascular endothelial cells is mediated by the NF-kappa B-dependent pathway.

Vascular cell adhesion molecule-1 (VCAM-1) has been reported to play an important role in cancer metastasis via the adhesive interaction between tumor cells and endothelial cells. In this study, we examined the effects of linoleic acid on VCAM-1 expression and its transcriptional regulatory mechanism in human microvascular endothelial cells (HMEC-1). Time- and dose-dependent increases of VCAM-1 mRNA levels were observed in linoleic acid-treated HMEC-1 as detected by reverse transcriptase-polymerase chain reaction. Flow cytometry analysis showed a significant and dose-dependent upregulation of VCAM-1 expression in HMEC-1 stimulated with linoleic acid compared with controls. To clarify the transcriptional regulatory pathway, we investigated the role of nuclear factor-kappa B (NF-kappa B) in the expression of VCAM-1 by linoleic acid in HMEC-1. Nuclear extracts from HMEC-1 stimulated with linoleic acid showed a dose-dependent increase in binding activity to the NF-kappa B consensus sequences. These effects were preventable by cotreatment with inhibitors of NF-kappa B activity, such as sodium salicylate, aspirin, or pyrrolidine dithiocarbamate. In addition, pretreatment with NF-kappa B inhibitors markedly suppressed the ability of linoleic acid to induce VCAM-1 gene expression. The role of NF-kappa B in linoleic acid-induced VCAM-1 expression was confirmed by functional promoter studies in HMEC-1 transfected with reporter constructs of the VCAM-1 promoter with or without mutated NF-kappa B binding site. These results indicate that linoleic acid upregulates VCAM-1 expression in HMEC-1 through the NF-kappa B-dependent pathway.

IL-4 induces apoptosis of endothelial cells through the caspase-3-dependent pathway.

The present study was designed to investigate the hypothesis that interleukin-4 (IL-4) can induce apoptosis of human endothelial cells and to study regulatory pathways of this process. Indeed, DNA ladder assay and flow cytometry study showed that IL-4 can induce apoptosis of endothelial cells in a time- and dose-dependent manner. In addition, IL-4 markedly increased activity of caspase-3, and inhibition of this enzyme suppressed IL-4-induced apoptosis in a dose-dependent manner. These results provide the first evidence that IL-4 can induce apoptosis of human endothelial cells. In addition, the data indicate that the caspase-3-dependent pathway is critically involved in this process.

Fatty acid-mediated activation of vascular endothelial cells.

Vascular endothelial cell activation and dysfunction are critical early events in atherosclerosis. Selected dietary lipids (eg, fatty acids) may be atherogenic by activating endothelial cells and by potentiating an inflammatory response. Due to their prooxidant property, unsaturated fatty acids may play a critical role in endothelial cell activation and injury. To test this hypothesis, porcine endothelial cells were exposed to 18-carbon fatty acids differing in the degree of unsaturation, ie, 90 micromol/L stearic (18:0), oleic (18:1n-9), linoleic (18:2n-6), or linolenic acid (18:3n-3) for 6 to 24 hours and/or tumor necrosis factor alpha ([TNF-alpha] 500 U/L) for up to 3 hours. Compared with control cultures, treatment with 18:0 and 18:2 decreased glutathione levels, suggesting an increase in cellular oxidative stress. Both 18:2 and 18:0 activated the transcription factor nuclear factor kappaB (NF-kappaB) the most and 18:1 the least. This NF-kappaB-dependent transcription was confirmed in endothelial cells by luciferase reporter gene assay. The fatty acid-mediated activation of NF-kappaB was blocked by preenrichment of the cultures with 25 micromol/L vitamin E. All fatty acids except 18:1 and 18:3 increased transendothelial albumin transfer, and 18:2 caused the most marked disruption of endothelial integrity. Preenrichment of endothelial cells with 18:2 followed by exposure to TNF-alpha resulted in a 100% increase in interleukin-6 (IL-6) production compared with TNF-alpha exposure alone. In contrast, cellular preenrichment with 18:0, 18:1, or 18:3 had no effect on TNF-alpha-mediated production of IL-6. Cellular release of radiolabeled arachidonic acid (20:4) was markedly increased only by cell exposure to 18:2 and 18:3, and the release of 20:4 appeared to be mainly from the phosphatidylethanolamine fraction. These data suggest that oleic acid does not activate endothelial cells. Furthermore, linoleic acid and other omega-6 fatty acids appear to be the most proinflammatory and possibly atherogenic fatty acids.

Effects of lipids and antioxidants on PCB-mediated dysfunction of vascular endothelial cells (EC).

Our findings suggest that exposure to specific environmental contaminants can trigger diseases of the vasculature, e.g., cardiovascular disease. In addition, high-fat diets may potentiate and diets high in antioxidant nutrients may protect against PCB-mediated endothelial cell dysfunction. Our data give an insight into the potential use of vitamin E and related antioxidants to limit PCB-mediated cell injury. These studies are significant for providing new insights into potential nutrition interventions in diseases that can be induced by the toxicity of PCBs and other halogenated compounds.

Cellular glutathione status modulates polychlorinated biphenyl-induced stress response and apoptosis in vascular endothelial cells.

Exposure to environmental contaminants, such as polychlorinated biphenyls (PCBs), may severely compromise normal function of vascular endothelial cells (EC). We have previously shown that PCB 77 (3,3',4,4'-tetrachlorobiphenyl), an arylhydrocarbon receptor (AhR) agonist, can induce oxidative stress in cultured EC. We now show that PCB 77 can activate EC and induce a cellular stress response that is reflected by the activation of c-Jun N-terminal/stress-activated protein kinases (JNK/SAPK). Our data also suggest that this PCB 77-mediated stress response can be modulated by the intracellular glutathione content. EC treated with buthionine-sulphoximine (BSO), an inhibitor of glutathione synthesis, further enhanced PCB-induced JNK/SAPK activity. This stress response was sustained only in the presence of BSO plus PCB 77. Media supplementation with the glutathione precursor N-acetyl-cysteine (NAC) reduced PCB 77-induced JNK/SAPK. Intracellular glutathione also may be implicated in PCB-induced EC apoptosis. Individual treatment with PCB, BSO, or linoleic acid induced activation of caspase 3. Compared to PCB 77 alone, annexin V activity was further amplified during combined treatment with BSO and PCB 77. DNA fragmentation was mostly observed when cells were treated with both BSO and PCB 77. The caspase 3-specific inhibitor DEVD-CHO protected cells against PCB 77/BSO-mediated apoptosis and inhibited the caspase activity without affecting JNK/SAPK activation or cellular glutathione levels. These results suggest that AhR ligands, such as PCB 77, cause vascular EC dysfunction by modulating intracellular glutathione, which subsequently leads to activation of stress-specific kinases. Furthermore, inhibition of glutathione synthesis by BSO can further potentiate the PCB 77-induced stress response and ultimately lead to apoptotic cell death.

4-Hydroxynonenal induces oxidative stress and death of cultured spinal cord neurons.

Primary spinal cord trauma can trigger a cascade of secondary processes leading to delayed and amplified injury to spinal cord neurons. Release of fatty acids, in particular arachidonic acid, from cell membranes is believed to contribute significantly to these events. Mechanisms of fatty acid-induced injury to spinal cord neurons may include lipid peroxidation. One of the major biologically active products of arachidonic acid peroxidation is 4-hydroxynonenal (HNE). The levels of HNE-protein conjugates in cultured spinal cord neurons increased in a dose-dependent manner after a 24-h exposure to arachidonic acid. To study cellular effects of HNE, spinal cord neurons were treated with different doses of HNE, and cellular oxidative stress, intracellular calcium, and cell viability were determined. A 3-h exposure to 10 microM HNE caused approximately 80% increase in oxidative stress and 30% elevation of intracellular calcium. Exposure of spinal cord neurons to HNE caused a dramatic loss of cellular viability, indicated by a dose-dependent decrease in MTS [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-s ulfophenyl)- 2H-tetrazolium, inner salt] conversion. The cytotoxic effect of HNE was diminished by pretreating neurons with ebselen or N-acetylcysteine. These data support the hypothesis that formation of HNE may be responsible, at least in part, for the cytotoxic effects of membrane-released arachidonic acid to spinal cord neurons.

Nicotine attenuates arachidonic acid-induced neurotoxicity in cultured spinal cord neurons.

Arachidonic acid release from cellular membranes due to spinal cord trauma may be one of the principal destructive events that can lead to progressive injury to spinal cord tissue. Exposure to arachidonic acid can compromise neuronal survival and viability. Because nicotine is known to be a neuroprotective agent, we propose that it can prevent arachidonic acid-induced neurotoxicity. To study this hypothesis, effects of nicotine on mitochondrial function, cellular energy content and apoptotic cell death were measured in cultured spinal cord neurons treated with arachidonic acid. Nicotine attenuated arachidonic acid-induced compromised cell viability and cellular ATP levels in spinal cord neurons. Nicotine exerted these protective effects when used at the concentration of 10 microM and only after a 2-h pre-treatment before a co-exposure to arachidonic acid. Antagonists of nicotinic receptors, such as alpha-bungarotoxin or mecamylamine, only partially reversed these neuroprotective effects of nicotine. In addition, nicotine prevented arachidonic acid-induced activation of caspase-3 activity and apoptotic cell death. These results indicate that nicotine pre-treatment can exert a protective effect against arachidonic acid-induced injury to spinal cord neurons.

Nicotine attenuates arachidonic acid-induced overexpression of nitric oxide synthase in cultured spinal cord neurons.

Primary spinal cord trauma can initiate a cascade of pathophysiologic events which markedly contribute to the expansion and amplification of the primary insult. The detailed mechanisms of these secondary neurochemical reactions are largely unknown; however, they involve membrane lipid derangements with the release of free fatty acids, in particular, arachidonic acid (AA). AA can induce several injury effects on spinal cord neurons. We hypothesize that upregulation of nitric oxide synthase (NOS) is among the most important mechanisms of arachidonic-acid-induced neuronal dysfunction and that nicotine can attenuate this effect. To study these hypotheses, spinal cord neurons were exposed to AA and/or nicotine, and several markers of neuronal nitric oxide synthase (nNOS) metabolism were measured. In addition, cotreatments with either inhibitors of nicotinic receptors or inhibitors of specific NOS isoforms were employed. Treatment with AA markedly increased activity of nNOS, as well as mRNA and protein levels of this enzyme. Changes in nNOS expression were accompanied by an increase in cellular cGMP and medium nitrite levels. Pretreatment with nicotine decreased AA-induced overexpression of nNOS and elevation of nitrite levels. In addition, it appeared that these nicotine effects could be partially modulated both by the alpha7 nicotinic receptors or by nonreceptor mechanisms. Alternatively, the observed changes could also be mediated by an alternate nicotinic receptor mechanism which is not blocked by alpha-bungarotoxin or mecamylamine. Results of the present study indicate that exposure to AA can lead to induction of nNOS in cultured spinal cord neurons. In addition, nicotine can exert a neuroprotective effect by attenuation of AA-induced upregulation of nNOS metabolism. These data may have therapeutic implications for the treatment of acute spinal cord trauma.

Zinc protects against apoptosis of endothelial cells induced by linoleic acid and tumor necrosis factor alpha.

BACKGROUND: Zinc requirements of the vascular endothelium may be increased in inflammatory conditions, ie, atherosclerosis, in which apoptotic cell death is prevalent. OBJECTIVE: We hypothesized that zinc deficiency may potentiate disruption of endothelial cell integrity mediated by fatty acids and inflammatory cytokines by enhancing pathways that lead to apoptosis and up-regulation of caspase genes. DESIGN: Endothelial cells were maintained in low-serum medium or grown in culture media containing selected chelators, ie, diethylenetriaminepentaacetate or N,N,N', N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), with or without zinc supplementation. Subsequently, cells were treated with linoleic acid, tumor necrosis factor alpha (TNF-alpha), or both. We studied the effect of zinc deficiency and supplementation on the induction of apoptosis by measuring caspase-3 activity, cell binding of annexin V, and DNA fragmentation. RESULTS: Our results indicated that linoleic acid and TNF-alpha independently, but more markedly in concert, up-regulated caspase-3 activity and induced annexin V binding and DNA fragmentation. Zinc deficiency, especially when induced by TPEN, dramatically increased apoptotic cell death induced by cytokines and lipids compared with control cultures. Supplementation of low-serum- or chelator-treated endothelial cells with physiologic amounts of zinc caused a marked attenuation of apoptosis induced by linoleic acid and TNF-alpha. Morphologic changes of cells observed during zinc deficiency were prevented by zinc supplementation. Media supplementation with other divalent cations (eg, calcium and magnesium) did not mimic the protective role of zinc against apoptosis. CONCLUSIONS: Our data indicate that zinc is vital to vascular endothelial cell integrity, possibly by regulating signaling events to inhibit apoptotic cell death.