Sources for superoxide release: Lessons from blockade of electron transport, NADPH oxidase, and anion channels in diaphragm

Isolated diaphragm releases low levels of superoxide (O-2(.-)) 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 O-2(.-) 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 O(2)(.-)release. Conversely, most blockers stimulated O-2(.-) release. As intracellular O-2(.-) generators require a mechanism for O-2(.-) transport across the membrane, anion channel blockers, probenecid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, were also tested. Neither blocker had any inhibitory effect on O-2(.-) release. These results suggest that O-2(.-) released from diaphragm is not directly dependent on mitochondrial complex activity and that it is not a reflection of passive diffusion of O-2(.-) through anion channels. Although the molecular source for extracellular O-2(.-) remains elusive, it is clearly sensitive to temperature and conditions of chemical hypoxia induced by partial or complete mitochondrial inhibition.