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

V. V. Cherny, L. M. Henderson *, W. Xu †, L. L. Thomas † and T. E. DeCoursey

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 (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 (I_e) were elicited in human eosinophils at AA concentrations (3-10 µM) similar to those that induced O₂^- 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 AA did not consistently slow _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 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.
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.

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