ALTERATIONS IN PHOSPHATIDYLCHOLINE METABOLISM OF STRETCH-INJURED CULTURED RAT ASTROCYTES

The primary objective of this study was to determine the influence of stretch-induced cell injury on the metabolism of cellular phosphatidyl choline (PC). Neonatal rat astrocytes were grown to confluency in Sila stic-bottomed tissue culture wells in medium that was usually suppleme nted with 10 mu M unlabeled arachidonate. Cell injury was produced by stretching (5-10 mm) the Silastic membrane with a 50-ms pulse of compr essed air. Stretch-induced cell injury increased the incorporation of [H-3]choline into PC in an incubation time- and stretch magnitude-depe ndent 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 pos tinjury. Stretch-induced cell injury also increased the activity of se veral enzymes involved in the hydrolysis [phospholipase A(2) (EC 3.1.1 .4) and C (PLC; EC 3.1.4.3)] and biosynthesis [phosphocholine cytidyly ltransferase (PCT; EC 2.7.7.15)] of PC, Stretch-induced increases in P C biosynthesis and PCT activity correlated well (r = 0.983) and were s ignificantly reduced by pretreating (1 h) the cells with an iron chela tor (deferoxamine) or scavengers of reactive oxygen species such as su peroxide dismutase and catalase, The stretch-dependent increase in PC biosynthesis was also reduced by antioxidants (vitamin E, vitamin E su ccinate, vitamin E phosphate, melatonin, and n-acetylcysteine), Arachi donate-enriched cells were more susceptible to stretch-induced injury because lactate dehydrogenase release and PC biosynthesis were signifi cantly less in non-arachidonate-enriched cells. In summary, the data s uggest that stretch-induced cell injury is (a) a result of an increase in the cellular level of hydroxyl radicals produced by an iron-cataly zed Haber-Weiss reaction, (b) due in part to the interaction of oxyrad icals with the polyunsaturated fatty acids of cellular phospholipids s uch as PC, and (c) reversible as long as the cell's membrane repair fu nctions (PC hydrolysis and biosynthesis) are sufficient to repair inju red 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-indu ced injury may supplement or be a reasonable alternative to some in vi vo models of brain injury for determining the mechanisms by which trau matic cell injury results in cell dysfunction.