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.