No abstract

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.

Exposure of cultured human astrocytoma cells to iodoacetic acid results in rapid depletion of cellular ATP and cell death. Pathophysiological changes in the injured cells, including formation of reactive oxygen species (ROS), cell viability, glutathione, pH, and cytosolic calcium, were characterized at the cellular level via fluorescence microscopy. After iodoacetic acid treatment, cellular ATP and intracellular glutathione fell sharply to undetectable levels within 2 h. ROS, as detected by the oxidation of dichlorofluorescein, appeared in 20 min and reached a maximum before the loss of membrane integrity. Cells became acidotic within 10 min. Cytosolic free calcium concentration exhibited a slow increase and then a sharp influx shortly before the rupture of the cell membrane. The addition of lipophilic antioxidants, nordihydroguaiaretic acid or the troloxamine U-78517F, eliminated the accumulation of ROS and delayed the onset of cell death without affecting other parameters observed in the early phase of the injury. We conclude that ROS is formed and may play important roles during lethal cell injury caused by energy depletion.