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

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 NH 4 + gradient to control pH i . Superoxide anion (O 2 − ) release was assessed by cytochrome c reduction in human eosinophils. Significant O 2 − release was stimulated by 5–10 μ m AA. 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 μ m ) similar to those that induced O 2 − release. 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 AA did not consistently slow τ tail (deactivation of H + currents). 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 was dissociable from its activation of NADPH oxidase; H + currents were enhanced at low concentrations of AA that did not elicit detectable I e or when NADPH oxidase was inhibited by DPI. 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.