Changes in membrane properties during energy depletion-induced cell injury studied with fluorescence microscopy

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.

Peroxynitrite induces apoptosis of HL-60 cells by activation of a caspase-3 family protease

“…During peroxynitrite-induced apoptotic cell death in our experimental system, PARP appears to be inactivated due to proteolytic cleavage of PARP by a caspase-3 family protease rather than activated. Although PARP cleavage may not be a direct cause of apoptosis, proteolytic cleavage of PARP by caspase-3 may function to conserve cellular energy required for the apoptotic process (46), thereby avoiding severe depletion of ATP stores that could cause irreversible structural damage and necrosis (45). In addition, the poly(ADP-ribosy)lation can negatively regulate the Ca 2ϩ /Mg 2ϩ -dependent endonuclease implicated in internucleosomal DNA fragmentation (41).…”

Molecular Mechanisms and Genetics of Oxidative Stress in Alzheimer’s Disease

“…In various neurodegenerative disorders, including AD, a decrease in brain ATP generation is observed. Membrane fluidity changes observed upon iodoacetic acid-induced inhibition of ATP production could be rescued by treatment with anti-oxidants tirilazad and gossypol [403] suggesting that reduced ATP availability may result in oxidative stress and membrane damage. Upon aging, glucose metabolism derails progressively as a result of changes in brain insulin [404], and cortisol [405] levels.…”

Road to Alzheimer’s Disease: The Pathomechanism Underlying

“…The ensuing mitochondrial dysfunction not only leads to ROS production but also to a chronic energy crisis in the neurons. The energy depletion may also increase the catabolism of membrane cholesterol [48] , and when the reduction in cholesterol is moderate, there is a disorganization of lipid rafts that favors the contact between ␤ -secretase and amyloid precursor protein (APP), which results in increased A ␤ production [49] .…”