Andrzej Malecki

Arachidonic Acid‐Induced Oxidative Injury to Cultured Spinal Cord Neurons

Abstract : Spinal cord trauma can cause a marked release of free fatty acids, in particular, arachidonic acid (AA), from cell membranes. Free fatty acids, and AA by itself, may lead to secondary damage to spinal cord neurons. To study this hypothesis, cultured spinal cord neurons were exposed to increasing concentrations of AA (0.01‐10 μM). AA‐induced injury to spinal cord neurons was assessed by measurements of cellular oxidative stress, intracellular calcium levels, activation of nuclear factor‐κB (NF‐κB), and cell viability. AA treatment increased cell intracellular calcium concentrations and decreased cell viability. Oxidative stress increased significantly in neurons exposed to 1 and 10 μM AA. In addition, AA treatment activated NF‐κB and decreased levels of the inhibitory subunit, IκB. It is interesting that manganese superoxide dismutase protein levels and levels of intracellular total glutathione increased in neurons exposed to this fatty acid for 24 h, consistent with a compensatory response to increased oxidative stress. These results strongly support the hypothesis that free fatty acids contribute to the tissue injury observed following spinal cord trauma.

4-Hydroxynonenal Induces Oxidative Stress and Death of Cultured Spinal Cord Neurons

Abstract: 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 μM HNE caused ∼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‐sulfophenyl)‐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.

Zinc nutrition and apoptosis of vascular endothelial cells: implications in atherosclerosis

Little is known about the requirements and function of zinc in maintaining endothelial cell integrity, especially during stressful conditions, such as the inflammatory response in cardiovascular disease. There is evidence that zinc requirements of the vascular endothelium are increased during inflammatory conditions such as atherosclerosis, where apoptotic cell death is also prevalent. Apoptosis is a morphologically distinct mechanism of programmed cell death which involves the activation of a cell-intrinsic suicide program, and there is evidence that factors such as inflammatory cytokines (e.g., tumor necrosis factor [TNF]) and pure or oxidized lipids are necessary to induce the cell death pathway. Because of its constant exposure to blood components, including prooxidants, diet-derived fats, and their derivatives, the endothelium is very susceptible to oxidative stress and to apoptotic injury mediated by blood lipid components, prooxidants, and cytokines. Thus, it is likely that the cellular lipid environment, primarily polyunsaturated fatty acids, can potentiate the overall endothelial cell injury by increasing cellular oxidative stress and cytokine release in proximity to the endothelium, which then could further induce apoptosis and disrupt endothelial barrier function. Our data suggest that zinc deficiency exacerbates the detrimental effects of specific fatty acids (e.g., linoleic acid) and inflammatory cytokines, such as TNF, on vascular endothelial functions. We propose that a major mechanism of zinc protection against disruption of endothelial cell integrity during inflammatory conditions, is by the ability of zinc to inhibit the pathways of signal transduction leading to apoptosis and especially mechanisms that lead to upregulation of caspase genes.

Piracetam and vinpocetine exert cytoprotective activity and prevent apoptosis of astrocytes in vitro in hypoxia and reoxygenation

The aim of the present study was to establish whether piracetam (2-pyrrolidon-N-acetamide; PIR) and vinpocetine (a vasoactive vinca alkaloid; VINP) are capable of protecting astrocytes against hypoxic injury. Using the model of astrocyte cell culture we observed the cells treated with PIR and VINP during and after in vitro simulated hypoxia. Cell viability was determined by Live/Dead Viability/Cytotoxicity Assay Kit, LDH release assay and MTT conversion test. Apoptotic cell death was distinguished by a method of Hoechst 33342 staining underfluorescence microscope and caspase-3 colorimetric assay. In addition the intracellular levels of ATP and phosphocreatine (PCr) were evaluated by bioluminescence method. Moreover, the effect of the drugs on the DNA synthesis was evaluated by measuring the incorporation of [3H]thymidine into DNA of astrocytes. PIR (0.01 and 1 mM) and VINP (0.1 and 10 microM) were added to the medium both during 24 h normoxia, 24 h hypoxia or 24 h reoxygenation. Administration of 1 mM PIR or 0.1 microM VINP to the cultures during hypoxia significantly decreases the number of dead and apoptotic cells. The antiapoptic effects of drugs in the above mentioned concentrations was also confirmed by their stimulation of mitochondrial function, the increase of intracellular ATP, and the inhibition of the caspase-3 activity. The prevention of apoptosis was accompanied by the increase in ATP and PCr levels and increase in the proliferation of astrocytes exposed to reoxygenation. The higher concentration of VINP (10 microM) was detrimental in hypoxic conditions. Our experiment proved the significant cytoprotective effect of 1 mM PIR and 0.1 microM VINP on astrocytes in vitro.

ERK 1/2 signaling pathway is involved in nicotine‐mediated neuroprotection in spinal cord neurons

Evidence indicates that agonists of neuronal nicotinic receptors (nAChRs), including nicotine, can induce neuroprotective and anti‐apoptotic effects in the CNS. To study these mechanisms, the present study focused on nicotine‐mediated modulation of the extracellular regulated kinase 1 and 2 (ERK1/2) pathway in cultured spinal cord neurons. Exposure to nicotine (0.1–10 µM) for as short as 1 min markedly upregulated levels of phosphorylated ERK1/2 (pERK1/2) and increased total ERK1/2 activity. Inhibition studies with mecamylamine and α‐bungarotoxin revealed that these effects were mediated by the α7 nicotinic receptor. In addition, pre‐exposure to U0126, a specific inhibitor of the ERK1/2 signaling, prevented nicotine‐mediated anti‐apoptotic effects. To indicate if treatment with nicotine also can activate ERK1/2 in vivo, a moderate spinal cord injury (SCI) was induced in rats using a weight‐drop device and nicotine was injected 2 h post‐trauma. Consistent with in vitro data, nicotine increased levels of pERK1/2 in this animal model of spinal cord trauma. Results of the present study indicate that the ERK1/2 pathway is involved in anti‐apoptotic effects of nicotine in spinal cord neurons and may be involved in therapeutic effects of nicotine in spinal cord trauma. J. Cell. Biochem. 100: 279–292, 2007.

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.

Arachidonic acid increases choline acetyltransferase activity in spinal cord neurons through a protein kinase C-mediated mechanism

Arachidonic acid (AA) plays an important role as a signaling factor in the CNS. Therefore, exposure to AA may affect cholinergic neurons in the spinal cord. To test this hypothesis, mRNA expression and activity of choline acetyltransferase (ChAT) was measured in cultured spinal cord neurons treated with increasing concentrations (0.1–10 µm) of AA. Exposure to AA increased mRNA levels and activity of ChAT in dose‐ and time‐dependent manners. The most marked effect of AA on ChAT expression was observed in spinal cord neurons treated with 10 µm AA for 1 h. To study the mechanisms associated with these effects, ChAT mRNA levels and activity were measured in cultured spinal cord neurons exposed to AA and inhibitors of protein kinase C (PKC), such as 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine dichloride (H‐7) and chelerythrine. Inhibition of PKC completely prevented an AA‐induced increase in ChAT expression. In addition, exposure of spinal cord neurons to phorbol‐12‐myristate‐13‐acetate (PMA), an activator of PKC, mimicked AA‐induced stimulation of ChAT activity. The AA‐mediated increase in ChAT mRNA levels and activity was also prevented by treatments with EGTA, indicating the role of calcium metabolism in induction of this enzyme. In contrast, treatments with 7‐nitroindazole (7‐NI, a specific inhibitor of neuronal nitric oxide synthase), sodium vanadate (NaV, a non‐specific inhibitor of phosphatases), and N‐acetyl‐cysteine (NAC, an antioxidant) had no effect on AA‐induced changes in ChAT activity. The protein synthesis inhibitor cycloheximide completely blocked AA‐mediated increase in ChAT activity. These results indicate that the AA‐evoked increase in ChAT activity in spinal cord neurons is mediated by PKC, presumably at the transcriptional level.