naphthoquinones and pepstatin

Oxidative stress is a primary pathogenesis in the brain, which is particularly vulnerable to oxidative stress. Maintenance of astrocyte functions under oxidative stress is essential to prevent neuronal injuries and to recover neuronal functions in various pathologic conditions. Imidazoline drugs have affinities for imidazoline receptors, which are highly distributed in the brain, and have been shown to be neuroprotective. This study presented the protective effects of several imidazoline drugs against oxidative cytotoxicity, in primary cultures of astrocytes. Imidazoline drugs, such as idazoxan, guanabenz, guanfacine, BU224, and RS-45041-190, showed protective effects against naphthazarin-induced oxidative cytotoxicity, as evidenced by LDH release and Hoechst 33342/propidium iodide staining. The imidazoline drugs stabilized lysosomes and inhibited naphthazarin-induced lysosomal destabilization, as evidenced by acridine orange relocation. Guanabenz inhibited, the leakage of lysosomal cathepsin D to cytosol, the decreased mitochondrial potential, and the release of mitochondrial cytochrome c, which were induced by naphthazarin. The lysosomal destabilization by oxidative stress and other apoptotic signals and subsequent cathepsin D leakage to the cytosol can induce apoptotic changes of mitochondria and eventually cell death. Therefore, lysosomal stabilization by imidazoline drugs may be ascribed to their protective effects against oxidative cytotoxicity.

Topics: Acridine Orange; Adrenergic alpha-Antagonists; Animals; Animals, Newborn; Antineoplastic Agents; Apoptosis; Astrocytes; Cathepsin D; Cell Division; Cell Line; Cerebral Cortex; Cytochrome c Group; Fish Venoms; Guanabenz; Guanfacine; HIV Protease Inhibitors; Idazoxan; Imidazoles; Indoles; Isoindoles; L-Lactate Dehydrogenase; Ligands; Lysosomes; Membrane Potentials; Mitochondria; Naphthoquinones; Neuroglia; Oxidation-Reduction; Pepstatins; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, Drug

Apoptosis was inhibited in rat cardiomyocytes pretreated with the aspartic protease inhibitor pepstatin A and subsequently exposed to naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Cathepsin D was released from lysosomes to the cytosol upon exposure to naphthazarin, and the enzyme activity decreased simultaneously. Later, cathepsin D reappeared in granules of increased size, and enzyme activity was restored. Activation of caspase-3-like proteases was detected, and the number of cells showing apoptotic morphology increased with time. Pepstatin A pretreatment did not prevent release of cathepsin D from lysosomes but did significantly inhibit subsequent naphthazarin-induced caspase activation and apoptotic morphology. This suggests that cathepsin D exerts its apoptosis-stimulating effect upstream of caspase-3-like activation.

Topics: Animals; Apoptosis; Caspase 3; Caspases; Cathepsin D; Cells, Cultured; Enzyme Activation; Female; Fluorescent Antibody Technique; Free Radicals; Heart; In Situ Nick-End Labeling; Male; Myocardium; Naphthoquinones; Oxidative Stress; Pepstatins; Rats; Rats, Sprague-Dawley; Time Factors

Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential-sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (delta psi(m)) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of delta psi(m), and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.

Topics: Apoptosis; Cathepsin D; Cell Line; Cytochrome c Group; Fibroblasts; Flow Cytometry; Fluorescent Antibody Technique; Humans; Lysosomes; Male; Membrane Potentials; Microscopy, Electron; Mitochondria; Naphthoquinones; Oxidative Stress; Pepstatins; Protease Inhibitors