Nitric oxide and peroxynitrite induce cellular death in bovine chromaffin cells: evidence for a mixed necrotic and apoptotic mechanism with caspases activation.
Treatment of chromaffin cells with nitric oxide (NO) donors (SNP and SNAP) and peroxynitrite produces a time- and dose-dependent necrotic and apoptotic cell death. Necrotic cell death was characterized by both an increase in lactate dehydrogenase and ATP release and changes in nuclei and cell morphology (as seen with fluorescence microscopy analysis with propidium iodide and Hoechst 33342). Apoptotic cell death was characterized by nuclear fragmentation and presence of apoptotic cell bodies, by a decrease in DNA content, and by an increase in DNA fragmentation. Treatment of chromaffin cells with lipopolysaccharide (LPS) or cytokines (interferon-gamma, tumor necrosis factor-alpha) resulted only in apoptotic cell death. Apoptotic effects of NO-inducing compounds were specifically reversed, depending on the stimuli, by the NO scavenger carboxy-PTIO (CPTio) or by the NOS inhibitors L-NMA and thiocitrulline. NO-induced apoptotic death in chromaffin cells was concomitant to a cell cycle arrest in G0G1 phase and a decrease in the number of chromaffin cells in the G2M and S phases of cell cycle. All NO-producing compounds were able to induce activation of caspase 3 and cytochrome c release, and specific inhibitors of caspase 3 and 9, such as Ac-DEVD-CHO (CPP32) and Ac-Z-LEHD-FMK, respectively, prevented NO-induced apoptosis in chromaffin cells. These results suggest that chromaffin cells could be good models for investigating the molecular basis of degeneration in diseases showing death of catecholaminergic neurons, phenomenon in which NO plays an important role.
Vicente
S
S
Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Complutense University of Madrid, Madrid, Spain.
Pérez-Rodríguez
R
R
Oliván
A M
AM
Martínez Palacián
A
A
González
M P
MP
Oset-Gasque
M J
MJ
D000704:Analysis of Variance; D000818:Animals; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002417:Cattle; D002453:Cell Cycle / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects*; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D005455:Fluorescent Antibody Technique / Q000379:methods; D016166:Free Radical Scavengers / Q000494:pharmacology*; D006360:Heat-Shock Proteins / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009336:Necrosis / Q000139:chemically induced / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D013481:Superoxides / Q000378:metabolism
D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D006360:Heat-Shock Proteins; D020030:Nitric Oxide Donors; D010446:Peptide Fragments; C110881:S-nitro-N-acetylpenicillamine; D013481:Superoxides; C063820:SNP nonapeptide; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D045304:Cytochromes c; D007770:L-Lactate Dehydrogenase; D053148:Caspase 3; D020169:Caspases; D010396:Penicillamine