benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and Ischemia

Abstract Deposition of ubiquitinated protein aggregates is a hallmark of neurodegeneration in both acute neural injuries, such as stroke, and chronic conditions, such as Parkinson's disease, but the underlying mechanisms are poorly understood. In the present study, we examined the role of Zn2+ in ischemia-induced impairment of the ubiquitin-proteasome system in the CA1 region of rat hippocampus after transient global ischemia. We found that scavenging endogenous Zn2+ reduced ischemia-induced ubiquitin conjugation and free ubiquitin depletion. Furthermore, exposure to zinc chloride increased ubiquitination and inhibited proteasomal enzyme activity in cultured hippocampal neurons in a concentration- and time-dependent manner. Further studies of the underlying mechanisms showed that Zn(2+)-induced ubiquitination required p38 activation. These findings indicate that alterations in Zn2+ homeostasis impair the protein degradation pathway.

Topics: Actins; Animals; CA1 Region, Hippocampal; Cells, Cultured; Chelating Agents; Coumarins; Disease Models, Animal; Dose-Response Relationship, Drug; Edetic Acid; Embryo, Mammalian; Enzyme Inhibitors; Fluorescent Dyes; Green Fluorescent Proteins; Imidazoles; Ischemia; Leupeptins; Male; Microtubule-Associated Proteins; Neurons; Oligopeptides; p38 Mitogen-Activated Protein Kinases; Proteasome Endopeptidase Complex; Pyrimidines; Rats; Rats, Sprague-Dawley; Statistics, Nonparametric; Time Factors; Transfection; Ubiquitin; Zinc

A previous exposure to a non-harmful ischemic insult (preconditioning) protects the brain against subsequent harmful ischemia (ischemic tolerance). In contrast to delayed gene-mediated ischemic tolerance, little is known about the molecular mechanisms that regulate rapid ischemic tolerance, which occurs within 1 h following preconditioning. Here we have investigated the degradation of the pro-apoptotic Bcl-2 family member Bim as a mechanism of rapid ischemic tolerance. Bim protein levels were reduced 1 h following preconditioning and occurred concurrent with an increase in Bim ubiquitination. Ubiquitinated proteins are degraded by the proteasome, and inhibition of the proteasome with MG132 (a proteasome inhibitor) prevented Bim degradation and blocked rapid ischemic tolerance. Inhibition of p42/p44 mitogen-activated protein kinase activation by U0126 reduced Bim ubiquitination and Bim degradation and blocked rapid ischemic tolerance. Finally, inhibition of Bim expression using antisense oligonucleotides also reduced cell death following ischemic challenge. Our results suggest that following preconditioning ischemia, Bim is rapidly degraded by the ubiquitin-proteasome system, resulting in rapid ischemic tolerance. This suggests that the rapid degradation of cell death-promoting proteins by the ubiquitin-proteasome pathway may represent a novel therapeutic strategy to reduce cell damage following neuropathological insults, e.g. stroke.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Butadienes; Cell Death; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p21; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Ischemia; Ischemic Preconditioning; Leupeptins; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurons; Nitriles; Oligonucleotides, Antisense; Phosphorylation; Propidium; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Time Factors; Ubiquitin

The objectives of this study were to 1) determine the time-course of T-lymphocyte adhesion to monolayers of human umbilical vein endothelial cell (HUVEC) that were exposed to 60 min of anoxia followed by 24 h of reoxygenation, and 2) define the mechanisms responsible for the hyperadhesivity of postanoxic HUVEC to human T-lymphocytes.. Human peripheral blood mononuclear leukocytes were isolated from heparinized peripheral blood. T-lymphocytes were obtained by negative selection using a MACS column. HUVEC monolayers were exposed to anoxia/reoxygenation (A/R), and then reacted with 51Cr -labeled T-lymphocytes in adhesion assays.. A/R leads to an increased adhesion of T-lymphocytes to HUVEC monolayers, with peak responses occurring at 8 h after reoxygenation. This adhesion response was largely attributed to the CD4+ T-cell subset. The hyperadhesivity of A/R-exposed HUVEC was inhibited by monoclonal antibodies directed against either LFA-1, VLA-4, ICAM-1, or VCAM-1, indicating a contribution of these adhesion molecules and their ligands. Moreover, T-cell hyperadhesivity was attenuated by anti- IL-8. consistent with a role for this chemokine in the adhesion response. Protein synthesis inhibitors (actinomycin D and cycloheximide) as well as chemical inhibitors of (and binding ds-oligonucleotides to) NFkappaB and AP-1 significantly attenuated the A/R-induced T-lymphocyte adhesion responses. The kinetics of VCAM-1 on post-anoxic HUVEC correlated with the T-lymphocyte adhesion response.. A/R elicits a T-lymphocyte-endothelial cell adhesion response that involves transcription-dependent surface expression of VCAM-1.

Topics: Antibodies, Monoclonal; Benzamides; Cell Adhesion; Cell Adhesion Molecules; Cell Hypoxia; Cells, Cultured; Cysteine Endopeptidases; Endothelium, Vascular; Humans; Interleukin-8; Ischemia; Kinetics; Leupeptins; Multienzyme Complexes; NF-kappa B; Oxygen; Proteasome Endopeptidase Complex; Protein Synthesis Inhibitors; Reperfusion Injury; T-Lymphocyte Subsets; Thionucleotides; Time Factors; Transcription Factor AP-1; Tumor Necrosis Factor-alpha; Umbilical Veins; Vascular Cell Adhesion Molecule-1