Activation of NADPH oxidase-related proton and electron currents in human eosinophils by arachidonic acid

1. Effects of arachidonic acid (AA) on proton and electron currents in human eosinophils were studied using the permeabilized-patch voltage-clamp technique, using an applied NH4+ gradient to control pH(i). 2. Superoxide anion (O-2(-)) release was assessed by cytochrome c reduction in human eosinophils. Significant O-2(-) release was stimulated by 5-10 muM AA. 3. AA activated diphenylene iodinium (DPI)-inhibitable inward current reflecting electron efflux through NADPH oxidase. These electron currents (I-e) were elicited in human eosinophils at AA concentrations (3-10 muM) similar to those that induced O-2(-) release. 4. The voltage-gated proton conductance (g(H)) in eosinophils stimulated with AA was profoundly enhanced: H+ current amplitude (I-H) increased 4.6 times, activation was 4 times faster, and the H+ conductance-voltage (g(H) - V) relationship was shifted to substantially more negative voltages. The electrophysiological effects of AA resembled those reported for PMA, except that zn AA did not consistently slow tau (tail) (deactivation of H+ currents). 5. The stimulation of both proton and electron currents by AA was reversible upon washout. Repeated exposure elicited repeated responses. The activation of H+ currents by AA wa's dissociable from its activation of NADPH oxidase; H+ currents were enhanced at low concentrations of AA that did not elicit detectable I, or when NADPH oxidase was inhibited by DPI. 6. Most of the effects of AA on H+ currents qualitatively resemble those reported in whole-cell studies, reflecting a more direct action than PMA. The results are compatible with AA being an immediate activator of both NADPH oxidase and proton channels in human eosinophils.