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 huma n eosinophils were studied using the permeabilized-patch voltage-clamp tech nique, 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 mu M AA. 3. AA activated diphenylene iodinium (DPI)-inhibitable inward current refle cting 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 wi th AA was profoundly enhanced: H+ current amplitude (I-H) increased 4.6 tim es, activation was 4 times faster, and the H+ conductance-voltage (g(H) - V ) relationship was shifted to substantially more negative voltages. The ele ctrophysiological effects of AA resembled those reported for PMA, except th at zn AA did not consistently slow tau (tail) (deactivation of H+ currents) . 5. The stimulation of both proton and electron currents by AA was reversibl e upon washout. Repeated exposure elicited repeated responses. The activati on of H+ currents by AA wa's dissociable from its activation of NADPH oxida se; H+ currents were enhanced at low concentrations of AA that did not elic it detectable I, or when NADPH oxidase was inhibited by DPI. 6. Most of the effects of AA on H+ currents qualitatively resemble those re ported 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.