s-nitro-n-acetylpenicillamine and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

Mature mouse oligodendrocytes (OLs) are susceptible to death in demyelinating diseases such as multiple sclerosis and in brain injury following neurotrauma, ischemia, or stroke. To understand mechanisms leading to death of mature OLs and develop strategies for protection, we utilized cultures of mature mouse OLs to investigate the role of caspases and calpains in OL cell death mediated by different mechanisms. The agents used were (i) staurosporine, which induces apoptotic death via inhibition of protein kinases; (ii) kainate, which activates non-NMDA glutamate receptors; (iii) thapsigargin, which releases intracellular calcium stores; and (iv) SNAP, which releases active NO species and causes necrotic cell death. Inhibitors blocking primary effector caspases (including caspase 3), the FAS (death receptor)-mediated initiator caspases (including caspase 8), and stress-induced caspases (including caspase 9), were tested for their protective effects. Inhibition of caspases 3, 8, and 9 each robustly protected OLs following insult with staurosporine, thapsigargin, or kainate when added at optimal times. The time of addition of the inhibitors for maximal protection varied with the agent, from 1 h of preincubation before addition of staurosporine to 6 h after addition of kainate. Much less protection was seen for the NO generator SNAP under any condition. The role of calcium in OL death in each model was investigated by chelating extracellular Ca++ with EGTA, and by inhibiting the Ca++-activated calpain proteases. Calcium chelation did not protect against staurosporine, but decreased OL death initiated by kainate, thapsigargin, or NO. The calpain inhibitors PD150606 and calpain inhibitor I protected from cell death initiated by staurosporine, kainate, and thapsigargin, but not from cell death initiated by the NO donor SNAP.

Topics: Amino Acid Chloromethyl Ketones; Animals; Calcium; Calpain; Caspase Inhibitors; Cell Death; Cells, Cultured; Cysteine Proteinase Inhibitors; Kainic Acid; Mice; Mice, Inbred BALB C; Oligodendroglia; Penicillamine; Staurosporine; Thapsigargin

Dendritic cells (DCs) play a crucial role in the amplification of the immune response by promoting antigen presentation, T-lymphocyte proliferation, and proinflammatory cytokine and nitric oxide (NO) production. We have previously shown that the exogenous NO donor, s-nitroso-N-acetyl-penicillamine, promotes DC apoptosis by disrupting the mitochondrial membrane potential, which induces cytochrome-C release and activates caspase 3. To further elucidate the signaling pathway, we examined the expression of cellular inhibitors of apoptosis proteins (cIAPs) and poly (ADP-ribose) polymerase cleavage (PARP), a terminal event in the apoptotic cascade.. DC2.4 were exposed to 250 micromol/L s-nitroso-N-acetyl-penicillamine for various intervals. Apoptosis and necrosis were measured by terminal deoxynucleotidyl transferase nick-end labeling assay or flow cytometry with Annexin V and propidium iodide. DC2.4 were cultured with the pan-caspase inhibitor, ZVAD (100 micromol/L). cIAP, pro-caspases, and PARP expression or activation was measured by Western blot. Caspase enzyme activity was confirmed with the use of specific substrates.. NO-induced DC apoptosis correlated with the downregulation of cIAP expression. Caspase 3 and 6 were upregulated by SNAP and significantly inhibited by ZVAD. Maximal PARP cleavage occurred at 8 hours and coincided with the downregulation of cIAP and peak caspase 3 and near maximal caspase 6 activity.. NO-induced DC apoptosis is associated with the downregulation of cIAP expression, which facilitates caspase cascade activation and subsequent PARP cleavage.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Bone Marrow Cells; Caspase 3; Caspase 6; Caspases; Cell Line, Transformed; Cell Survival; Cysteine Proteinase Inhibitors; Dendritic Cells; Down-Regulation; Enzyme Activation; In Situ Nick-End Labeling; Mice; Nitric Oxide; Nitric Oxide Donors; Penicillamine; Poly(ADP-ribose) Polymerases; Up-Regulation