Sources for Superoxide Release: Lessons from Blockade of Electron Transport,NADPH Oxidase,and Anion Channels in Diaphragm

Abstract

Isolated diaphragm releases low levels of superoxide (O₂ ^•-) at rest and much higher levels during heat stress. The molecular source is unknown. The hypothesis was tested that heat stress stimulates mitochondrial complex activity or NADPH oxidases, resulting in increased O₂ ^•- release. The mitochondria within intact rat diaphragm were inhibited at complex I (amobarbital or rotenone) or complex I and II (rotenone plus thenoyltrifluoroacetone). NADPH oxidases were blocked by diphenyliodonium. None of these treatments inhibited O₂ ^•- release. Conversely, most blockers stimulated O₂ ^•- release. As intracellular O₂ ^•- generators require a mechanism for O₂ ^•- transport across the membrane, anion channel blockers, probenecid and 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid, were also tested. Neither blocker had any inhibitory effect on O₂ ^•- release. These results suggest that O₂ ^•- released from diaphragm is not directly dependent on mitochondrial complex activity and that it is not a reflection of passive diffusion of O₂ ^•- through anion channels. Although the molecular source for extracellular O₂ ^•- remains elusive, it is clearly sensitive to temperature and conditions of "chemical hypoxia" induced by partial or complete mitochondrial inhibition.