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

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Abstract
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 pHi. 2 Superoxide anion (O2−) release was assessed by cytochrome c reduction in human eosinophils. Significant O2− release was stimulated by 5‐10 μm AA. 3 AA activated diphenylene iodinium (DPI)‐inhibitable inward current reflecting electron efflux through NADPH oxidase. These electron currents (Ie) were elicited in human eosinophils at AA concentrations (3‐10 μm) similar to those that induced O2− release. 4 The voltage‐gated proton conductance (gH) in eosinophils stimulated with AA was profoundly enhanced: H+ current amplitude (IH) increased 4.6 times, activation was 4 times faster, and the H+ conductance‐voltage (gH‐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 τtail (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 was dissociable from its activation of NADPH oxidase; H+ currents were enhanced at low concentrations of AA that did not elicit detectable Ie 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.