NADPH-OXIDASE-DEPENDENT SUPEROXIDE PRODUCTION BY MYOCYTE-DERIVED H9C2CELLS - INFLUENCE OF ISCHEMIA, HEAT-SHOCK, CYCLOHEXIMIDE AND CYTOCHALASIN-D

Extracellular oxygen radicals produced by H9c2 rat heart cells in mono layer cultures during ischemia and subsequent reoxygenation were monit ored using the luminol-horseradish peroxidase-enhanced chemiluminescen ce technique. As expected, the photon count diminishes during ischemia but again rapidly attains normal values following reoxygenation. In t he presence of superoxide dismutase, this photon emission is repressed , as is also the case in the presence of diphenylene iodonium, a speci fic inhibitor of NADPH-oxidase activity. Thus, the conclusion seems ju stified that H9c2 rat heart cells in monolayer cultures produce supero xide 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 o f 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 hea t shock temperature. This inactivation is independent of protein synth esis and actin cytoskeletal structures, but the recovery of the enzyme 's activity is dependent on these entities. (C) 1997 Academic Press Li mited.