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 µ m 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 µ m 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 α7 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.
Age is strongly correlated to the onset of atherosclerotic lesion formation in humans. This may be associated with an age-related increase in the susceptibility of the vascular endothelium to oxidative injury. Such injury may result in altered endothelial function as a barrier to plasma components, such as cholesterol-rich lipoprotein remnants. To investigate this hypothesis, the relationship between endothelial cell culture age, susceptibility to oxidative injury and protection against this injury by the nutrient/antioxidant vitamin E on endothelial barrier function (transfer of albumin across endothelial monolayers) was examined. An acute 24 h exposure to 30 microM linoleic acid hydroperoxide resulted in increased albumin transfer at all cell passages tested (up to passage 50). Pre-enrichment of cells with 25 microM vitamin E always protected endothelial cells against oxidized fatty acid-induced cell injury, independent of cell age. In comparison, patterns of total cell protein and DNA were not markedly influenced by experimental treatments, although age-related declines in total DNA were noted. These data suggest that the possible correlation between age and the onset of atherosclerosis may be in part related to a decrease in endothelial barrier function due to oxidative stress, permitting more blood components to enter the arterial wall. Furthermore, vitamin E may protect endothelial cells against oxidant-mediated vascular injury.
Abstract Four methylation methods were evaluated for use in the gas chromatographic (GC) quantitation of conjugated linoleic acid (CLA) isomers, which are potential anticarcinogen. The methods were (1) sodium methoxide in methanol (NaOMe-MeOH), (2) American Oil Chemists' Society (AOCS) procedure Ce 2-66, which involves methanolic sodium hydroxide followed by boron trifluoride in methanol, (3) tetramethylguanidine in methanol (TMG-MeOH), and (4) direct transesterification with methanolbenzene- acetyl chloride (DAC). Purified methyl esters of isomerized linoleic acid containing 86% CLA isomers were methylated and analyzed by GC. The AOCS and DAC methods resulted in 3 and 50% losses in cis-9,trans-11-octadecadienoic acid (9c, 111 CLA isomer) and trans-10,cis-12 octadecadienoic acid (10t, 12c CLA isomers), respectively. Compared with the control, the AOCS and DAC methods increased the yield of the trans,trans CLA isomers (trans-9,trans-11- and trans-10, trans-12-octadecadienoic acid) by 1.07-fold and a 10-fold, respectively. A non-CLA artifact that eluted close to CLA peaks was formed during methylation by the AOCS and DAC methods. Thus, the DAC and AOCS methods are not suitable for quantitation of CLA isomers. The NaOMe-MeOH and TMG-MeOH methods, however, are suitable for quantitation of CLA isomers in fats containing low concentrations of free fatty acids.
A spectrum of cholesterol oxidation derivatives (oxysterols) is generated in food products exposed to heat or radiation in the presence of oxygen. One of these derivatives (cholestan-3β,5α,6β-triol) was shown to compromise the selective barrier function of cultured vascular endothelial cell monolayers, an action that may initiate atherosclerotic lesion formation. This study sought to investigate the relationship of cholesterol synthesis inhibition by several naturally occurring oxysterols to depression of vascular endothelial cell monolayer barrier function, determined as an increase in albumin transfer across cultured endothelial monolayers. All oxysterols tested caused a variable time- and dose-dependent elevation in trans-endothelial albumin transfer, and they were also able to inhibit cholesterol biosynthesis to varying degrees. Pure cholesterol was without effect on both counts. The correlation between the increase in albumin transfer related to oxysterol exposure and the ability of oxysterols to suppress cholesterol biosynthesis was, however, poor. Moreover, mevinolin, a water-soluble competitive inhibitor of cholesterol synthesis, reduced the rate of cholesterol synthesis to 0.9% of control but did not significantly increase albumin transfer. Cholestan-3β,5α,6β-triol caused a 660% elevation in albumin transfer while cholesterol synthesis remained at 11% of control. We conclude that changes in endothelial barrier function caused by exposure to the oxysterols examined, but not pure cholesterol, are probably related to factors other than the well-known action of cholesterol biosynthesis inhibition. These findings may have implications in the development of atherosclerosis.
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