Phospholipase C activity was measured in 1000 X G centrifuged cellular fractions isolated from cerebral cortical homogenates obtained from either control cats or cats subjected to experimental fluid-percussion brain injury. Phospholipase C activity was determined directly by measuring the Ca++-dependent conversion of membrane-bound, labeled phosphatidate to diacylglycerol or indirectly by measuring the diacylglycerol-dependent (brain diacylglycerol content) formation of phosphatidylcholine in the presence of labeled cytidine diphosphate (CDP) choline. Phospholipase C activity determined by either method was about two time greater in cell fractions isolated from animals subjected to brain injury than in controls (p less than 0.01). The brain injury-induced rise in phospholipase C activity may be responsible, at least in part, for generating diacylglycerol that may be a source of free arachidonic acid that stimulates prostaglandin synthesis. These changes may account for the rise in brain prostaglandin levels that has been demonstrated earlier to occur after this type of brain injury.

The influence of dietary fat and alcohol on hepatic microsomal levels of cytochromes P-450 2E1, 2B, and 4A; phospholipases A and C; and UDP-glucuronosyltransferase was studied in the intragastric feeding rat model for alcoholic liver injury. Eight groups of animals were evaluated. Control and ethanol fed rats received either saturated fat or corn oil and were killed after 2 weeks and 1 month of feeding. All animals were pair-fed by continuous infusion of liquid diet through permanently implanted gastric cannulas. Alcoholic liver injury developed only in the corn oil-ethanol-fed groups and was manifest by 1 month. Livers were subjected to the following analyses: pathologic evaluation of liver injury; levels of cytochromes P-450 2E1, 2B, and 4A protein and mRNA; aniline hydroxylase activity; and phospholipase A and C and UDP-glucuronosyltransferase activities. Ethanol-induced increases in cytochromes P-450 2E1 and 2B protein determined by Western blotting were greatest in the corn oil-ethanol-fed group, which developed pathologic changes in the liver. Cytochromes P-450 2E1 and 2B1 mRNA levels were unaffected, suggesting that posttranscriptional mechanisms are responsible for the increase in the corresponding P-450 proteins. In contrast, cytochrome P-450 4A levels were higher in the saturated fat-ethanol groups compared with the corn oil-ethanol groups. Phospholipase A and phospholipase C levels were higher in the corn oil-ethanol groups compared with pair-fed dextrose controls and the saturated fat-ethanol groups. UDP-glucuronosyltransferase levels declined with time in the ethanol-fed groups.(ABSTRACT TRUNCATED AT 250 WORDS)

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The primary objective of this study was to determine the influence of stretch-induced cell injury on the metabolism of cellular phosphatidylcholine (PC). Neonatal rat astrocytes were grown to confluency in Silasticbottomed tissue culture wells in medium that was usually supplemented with 10~M unlabeled arachidonate. Cell injury was produced by stretching (5-10 mm) the Silastic membrane with a 50-ms pulse of compressed air. Stretch-induced cell injury increased the incorporation of [ 3H]cholineinto PC in an incubation time-and stretch magnitude-dependent manner. PC biosynthesis was increased three-to fourfold between 1.5 and 4.5 h after injury and returned to control levels by 24 h postinjury. Stretch-induced cell injury also increased the activity of several enzymes involved in the hydrolysis [phospholipase A 2 (EC 3.1.1.4) and C (PLC; EC 3.1.4.3)] and biosynthesis [phosphocholine cytidylyltransferase (PCT; EC 2.7.7.15)] of PC. Stretch-induced increases in PC biosynthesis and PCT activity correlated well (r = 0.983) and were significantly reduced by pretreating (1 h) the cells with an iron chelator (deferoxamine) or scavengers of reactive oxygen species such as superoxide dismutase and catalase. The stretch-dependent increase in PC biosynthesis was also reduced by antioxidants (vitamin E, vitamin E succinate, vitamin E phosphate, melatonin, and n -acetylcysteine). Arachidonate-enriched cells were more susceptible to stretch-induced injury because lactate dehydrogenase release and PC biosynthesis were significantly less in non-arachidonate-enriched cells. In summary, the data suggest that stretch-induced cell injury is (a) a result of an increase in the cellular level of hydroxyl radicals produced by an iron-catalyzed Haber-Weiss reaction, (b) due in part to the interaction of oxyradicals with the polyunsaturated fatty acids of cellular phospholipids such as PC, and (c) reversible as long as the cell's membrane repair functions (PC hydrolysis and biosynthesis) are sufficient to repair injured membranes. These results suggest that stretch-induced cell injury in vitro may mimic in part experimental traumatic brain injury in vivo because alterations in cellular PC biosynthesis and PLC activity are similar in both models. Therefore, this in vitro model of stretch-induced injury may supplement or be a reasonable alternative to some in vivo models of brain injury for determining the mechanisms by which traumatic cell injury results in cell dysfunction. Key Words: Cultured astrocytes-Stretch-induced cell injury-Phosphatidylcholine biosynthesis-Phospholipase activity-Phosphocholine cytidylyltransferase activity-Oxyradical scavengers-Antioxidants.

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