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American Journal of Pathology, Vol 132, 479-488, Copyright © 1988 by American Society for Investigative Pathology

REGULAR ARTICLES

ATP and microfilaments in cellular oxidant injury

DB Hinshaw, BC Armstrong, JM Burger, TF Beals and PA Hyslop
Department of Surgery, University of Michigan, Ann Arbor.

Oxidant injury produces dramatic changes in cytoskeletal organization and cell shape. ATP synthetic pathways are major targets of oxidant injury resulting in rapid depletion of cellular ATP following oxidant exposure. The relation of ATP depletion to the changes in microfilament organization seen following H2O2 exposure were examined in the P388D1 cell line. Three hours of glucose depletion alone resulted in a decline in cellular ATP levels to less than 10% of controls, which was comparable to ATP levels in cells 30 to 60 minutes after exposure to 5 mM H2O2 in the presence of glucose. Adherent cells stained with rhodamine phalloidin, a probe specific for polymerized (F) actin, revealed a progressive shortening of microfilaments into globular aggregates within cells depleted of glucose over 3 hours, a pattern similar to earlier observations of H2O2-injured cells after 1 hour. The changes in cellular ATP associated with glucose depletion or H2O2 exposure were then correlated with G actin content measured by the DNAse 1 inhibition assay. No real differences in G actin content as a percentage of total actin were seen in P388D1 cells following 3 hours of glucose depletion or 30 to 60 minutes after exposure to 5 mM H2O2. But 2 to 3 hours after exposure to H2O2 there was a progressive decrease in G actin as a percentage of total actin within the cells. Transmission electron microscopy of cells depleted of glucose for 3 h or 1 hour after exposure to H2O2 revealed the presence of side-to-side aggregates or bundles of microfilaments within the cells. These observations suggest that declining levels of ATP either from metabolic inhibition or H2O2 injury are correlated with the fragmentation and shortening of microfilaments into aggregates. No net change in monomeric or polymeric actin was necessary for this to occur. However, at later time points after H2O2 exposure some actin assembly did occur.

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