NADPH-oxidase-dependent superoxide production by myocyte-derived H9c2 cells: influence of ischemia, heat shock, cycloheximide and cytochalasin D.
Extracellular oxygen radicals produced by H9c2 rat heart cells in monolayer cultures during ischemia and subsequent reoxygenation were monitored using the luminol-horseradish peroxidase-enhanced chemiluminescence technique. As expected, the photon count diminishes during ischemia but again rapidly attains normal values following reoxygenation. In the presence of superoxide dismutase, this photon emission is repressed, as is also the case in the presence of diphenylene iodonium, a specific inhibitor of NADPH-oxidase activity. Thus, the conclusion seems justified that H9c2 rat heart cells in monolayer cultures produce superoxide radicals extracellularly due to an NADPH oxidase-like action. In order to characterize this extracellular superoxide-generating system, we determined its sensitivity to increased temperatures, inhibition of protein synthesis and perturbations of cytoskeletal structures. Heat shocks result in a delayed inactivation of the NADPH oxidase activity followed by recovery, the kinetics of which depend on the imposed heat shock temperature. This inactivation is independent of protein synthesis and actin cytoskeletal structures, but the recovery of the enzyme's activity is dependent on these entities.
Souren
J E
JE
Department of Molecular Cell Biology, Utrecht University, Padualaan 8, Utrecht, 3584 CH, The Netherlands.
Van Der Mast
C
C
Van Wijk
R
R
D000818:Animals; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D018869:Heat-Shock Response; D017202:Myocardial Ischemia / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D013481:Superoxides / Q000378:metabolism*
D019384:Nucleic Acid Synthesis Inhibitors; D011500:Protein Synthesis Inhibitors; D013481:Superoxides; D015638:Cytochalasin D; D003513:Cycloheximide; D019255:NADPH Oxidases; D010100:Oxygen