Regional Loss of the Mitochondrial Membrane Potential in the Hepatocyte Is Rapidly Followed by Externalization of Phosphatidylserines at That Specific Site during Apoptosis

Abstract

The spatio-temporal relationship between a decrease in the mitochondrial membrane potential (MMP) and externalization of phosphatidylserines (PS) during induction of apoptosis was investigated in single freshly isolated hepatocytes. Apoptosis was induced in the hepatocytes in three different ways: attack by activated Natural Killer cells, exposure to ATP, or exposure to the inhibitor of protein synthesis cycloheximide. Fluorescence microscopy showed staining of externalized PS at those areas where the staining for MMP was lost whereas in other areas the mitochondria remained intact for longer periods of time, indicating coupling between local loss of MMP and local PS exposure. To discriminate whether the decrease in MMP itself or a decrease in ATP induced PS externalization, hepatocytes were treated with rotenone, which resulted in a rapid collapse of cellular ATP but left the MMP intact for a much longer period. Addition of fructose prevented the decrease of ATP to ϳ30% and also delayed the collapse of the MMP. This indicates that ATP was needed for the maintenance of the MMP probably via reverse action of the ATP synthase. In a subsequent study hepatocytes were incubated with Natural Killer cells for induction of apoptosis followed by addition of rotenone to deplete ATP. Under these conditions the PS staining co-localized with mitochondrial MMP indicating that PS externalization does not require a collapse in MMP. Moreover, exposure of PS was evenly distributed over the whole plasma membrane. In conclusion, we propose that after an apoptotic stimulus some mitochondria start to loose their MMP, which results in cessation of ATP production and perhaps even consumption of ATP. This results in an overall decrease in cellular ATP. ATPconsuming enzyme reactions most distal from still intact mitochondria will be most sensitive to such a decrease. Apparently the translocase that keeps phosphatidylserines inward-oriented is such a sensitive enzyme.

Nitric oxide protects osteoblasts from oxidative stress‐induced apoptotic insults via a mitochondria‐dependent mechanism

“…Depolarization of the mitochondrial membrane potential can enhance the release of apoptotic factors from mitochondria to the cytoplasm and induce cell apoptosis. 13,14 Pretreatment of NO completely ameliorated the oxidative stress-caused decreases in the mitochondrial membrane potential. Thus, recovery of the mitochondrial membrane potential is an important reason explaining NO's protection against oxidative stress-induced osteoblast apoptosis.…”

“…Increases in the synthesis or translocation of the proapoptotic Bax protein can trigger depolarization of the mitochondrial membrane potential, enhancing the release of apoptotic factors such as cytochrome c, and ultimately leading to cell apoptosis. [13][14][15] Being an effector for death regulation, NO has been shown to decrease bone mineral density through induction of osteoblast apoptosis in inflammationinduced osteoporosis. 16 Our previous studies showed that NO can activate mitochondrial apoptotic signals that induce osteoblast apoptosis.…”

Temporal Alteration of Connexin43 Localization during Ultraviolet Light-induced Apoptosis

“…The apoptotic process involves a series of biochemical steps and structural changes including mitochondrial membrane potential (MMP) depolarization, chromatin condensation, nuclear fragmentation, cytoskeletal reorganization, cell shrinkage, and membrane blebbing [3,16]. Caspases have been implicated as mediators of apoptosis in a variety of mammalian cell types [11,18].…”

Apoptotic insults to human chondrocytes induced by sodium nitroprusside are involved in sequential events, including cytoskeletal remodeling, phosphorylation of mitogen‐activated protein kinase kinase kinase‐1/c‐Jun N‐terminal kinase, and Bax‐Mitochondria‐Mediated caspase activation

“…Intracellular ATP levels participate in regulation of cell apoptosis and necrosis. 23,37 Therefore, NO may decrease cellular ATP levels through suppression of the mitochondrial membrane potential and complex I enzyme activity in human chondrocytes and induces cell insults.…”

“…16,22 Increases in the synthesis or translocation of Bax, a proapoptotic protein, can trigger depolarization of the mitochondrial membrane potential, enhancing the release of cytochrome c, and ultimately leading to cell apoptosis. 23 Phosphorylation of MAPKs by MEKK1 has been reported to activate the Bax-caspase protease pathway and plays a pivotal role in high glucose-induced apoptosis of human endothelial cells. 24 However, the roles of the cytoskeleton and MEKK1/JNK in NO-induced insults to chondrocytes need to be evaluated.…”