The changes in membrane structural properties occurring during the process of
ATP depletion-induced cell injury in adherent human
astrocytoma cells (UC-11 MG) were studied with two epifluorescence techniques: 1) steady-state fluorescence anisotropy (r) to examine microstructural changes in the membrane
phospholipids and 2) fluorescence redistribution after photobleaching (FRAP) to examine membrane fluidity changes. A new method for r measurement was established that provides the unique advantage of simultaneously monitoring both vertical and horizontal polarized fluorescence emissions needed for the calculation of r. In this study, r in the
astrocytoma cells labeled with 1-(4-trimethylammonium phenyl)-6-phenyl-1,3,5-hexatriene
p-toluenesulfonate was shown to remain stable for up to 90 min. However, when the cells were treated with 75 microM
iodoacetic acid (IAA), a metabolic inhibitor that induces rapid depletion of cellular
ATP, r continually decreased, indicating a decrease in
membrane lipid order and perturbation of the bilayer structure. This decrease in r could be prevented by the pretreatment of cells with lipophilic
antioxidants such as
tirilazad or
gossypol.
Tirilazad itself caused a significant increase in r, suggesting that
tirilazad intercalates into the membrane bilayer and profoundly increases the
lipid order in uninjured cells.
Gossypol, however, did not exhibit this property. Further investigations into these phenomena with FRAP confirmed the r results and indicated that membrane fluidity increased while its structure became less rigid during the process of
ATP-induced cell injury. In addition, lipophilic
antioxidants prevented the membrane structural aberrations induced by IAA. Experimental results suggest that different mechanisms of cytoprotective action may exist for
tirilazad and the
antioxidant gossypol.
Gossypol appears to prevent or delay the observed cell injury entirely because of its
antioxidant action, whereas
tirilazad's protection is mediated not only via its
antioxidant activity, but also by its ability to increase
cell membrane lipid order.