Ethanol-induced cell death by lipid peroxidation in PC12 cells.

Free radical generation is hypothesized to be the cause of alcohol-induced tissue injury. Using fluorescent cis-parinaric acid and TBARS, lipid peroxidation was shown to be increased in the presence of trace amounts of free ferrous ion in PC12 cells. This increase in lipid peroxidation was enhanced by ethanol in a dose dependent manner and also correlated with loss of cell viability, as measured by increased release of lactate dehydrogenase (LDH). Resveratrol, a potent antioxidant, had a protective effect against lipid peroxidation and cell death. These findings strongly suggest that ethanol-induced tissue injury and cell death is a free radical mediated process, and may be important in alcohol-related premature aging and other degenerative diseases.

Enhanced (Na+K)-ATPase activity and expression in mouse brain after chronic ethanol administration.

It is the general hypothesis that the primary mode of action of ethanol is the alteration of membrane structure and function including the conformation of receptors and ion channels essential for neurotransmission and signal transduction. However, the issue of whether ethanol affects (Na+K)-ATPase under physiological conditions remains unsettled. In this study, adult mice were treated with a daily dose of 5 g/kg of ethanol for 28 days. The RNA was isolated from brain and the (Na+K)-ATPase mRNA level was determined using Northern blot analysis. We have found an increased expression of (Na+K)-ATPase alpha-subunit in the chronically treated alcohol group as compared with that of controls. This result was further substantiated by increased protein phosphorylation as well as increased specific activity of this enzyme in the synaptosomal plasma membrane after chronic ethanol administration. Thus we have demonstrated that ethanol may directly affect (Na+K)-ATPase in vivo, leading to the increased synthesis of this enzyme through adaptive mechanisms.

Enhanced lipid peroxidation by extracellular ATP in PC12 cells.

Recently we have demonstrated that extracellular ATP acts as an excitatory neurotransmitter and enhances cell death in the presence of ferrous ions. By using a newly developed cis-parinaric acid fluorescence technique, we demonstrated that ATP, in a dose dependent manner, enhanced the increased membrane lipid peroxidation in PC12 cells when cells were incubated with micromolar FeCl2/DTP. P2 purinoceptor agonists, alpha,beta-methylene ATP and 2-methylthio-ATP, induced PC12 cell lipid peroxidation, but to a lesser extent than ATP. ATP-induced Ca(2+) influx via P2 purinoceptor activation significantly increased the intracellular Ca(2+)concentration, which may have triggered a free radical generating cascade(s), and led to membrane lipid peroxidation and cell death. Since oxidative stress has been implicated in certain neurodegenerative diseases such as aging, extracellular ATP may contribute to neuronal cell death by an oxidative mechanism involving lipid peroxidation.

Effects of extracellular ATP on Fe(2+)-induced cytotoxicity in PC-12 cells.

Extracellular ATP is known to cause a variety of changes, including the alteration of ion fluxes, cell growth, and other physiological activities. Recently, it has been suggested that ATP acts as an excitatory synaptic transmitter, which may produce a Ca2+ influx via the activation of a P2y purinoceptor. Rat pheochromocytoma (PC-12) cells are known to resemble rat sensory neurons and to possess a P2y purinoceptor. In this study, we demonstrated that extracellular ATP dose-dependently increased PC-12 cell death in the presence of ferrous ions. Voltage-sensitive calcium channel blockers and calpain and xanthine oxidase inhibitors were found to be effective at protecting PC-12 cells from Fe2+/ATP-induced lipid peroxidation and cell death. These results suggest that xanthine oxidase activation induced by calpains and subsequent free radical formation may be responsible for Fe2+/ATP-induced neuronal cell death.

Effects of focal cerebral ischemia on expression and activity of inositol 1,4,5-trisphosphate 3-kinase in rat cortex.

Results from this study clearly indicate that Ins(1,4,5)P3 3-kinase is a target enzyme of cerebral ischemia insult. This enzyme is responsible for removal of Ins(1,4,5)P3 which, in turn, plays an important role in the maintenance of intracellular Ca2+ homeostasis. Not only did a time-dependent decrease in enzyme activity occur due to the focal cerebral ischemic insult, but there was also a second phase for the decline in enzyme activity around 6 h after the insult. Examination of the mRNA for the 3-kinase in frozen brain sections suggested an increase in message at a time (around 8 h) prior to development of tissue infarct. Since the initial decline in enzyme activity during ligation correlated well with the time for development of an infarct, assay of this enzyme could be used as a biochemical marker of cerebral ischemic insult.