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

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 NH 4 + 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 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 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 AA did not consistently slow r 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.

Arachidonic acid triggers an oxidative burst in leukocytes

“…For this reason, significant results can be obtained with cell lines that are usually considered devoid of superoxide-generating systems (9,10), such as T lymphocytes (Jurkat) and undifferentiated HL-60 cells, as shown here. "Professional" superoxide-generating cells, such as macrophages and PMN-differentiated HL-60 cells, showed the greatest capacity for NBT reduction, but the production of superoxide was also clearly demonstrated in lymphocytes, even without cell stimulation.…”

“…It seems that AA might act as a detergent, but its effect must involve other mechanisms as well, as Many theories have been proposed to explain AA stimulation of the oxidative burst. In the PMA-stimulated NADPH system, it could cause dissociation of inhibitors of components of the NADPH complex, promote an ideal membrane environment for enzyme function, promote the phosphorylation or translocation of p47 phox (15,23,25), stimulate the NADPH oxidase proton pump (10,26), activate or promote PKC translocation (12,23,(27)(28)(29), stimulate the cell, mimicking, when in the micellar state, particulate stimuli (14,22), or stimulating the cell via the generation of eicosanoids (6). Regarding the mitochondrial-derived superoxide production, AA could act by its known mitochondrial uncoupling ability (carrying protons across the inner mitochondrial membrane), stimulating the opening of the permeability transition pore or acting as a prooxidant, and affecting electron transport (10,30,31).…”

Regulation of eosinophil membrane depolarization during NADPH oxidase activation

“…Although originally thought to be part of the NADPH oxidase complex, the proton channel now appears to be a distinct protein that is, nevertheless, concomitantly regulated by PKC (DeCoursey et al, 2001a). Stimuli such as arachidonic acid and PMA, which activate the proton channels in neutrophils and eosinophils, also stimulate electron transport through the oxidase (DeCoursey et al, 2000;DeCoursey et al, 2001b;Cherny et al, 2001). Although PMAstimulated NADPH oxidase activity can be blocked with the PKCδ selective inhibitor, rottlerin, the proton conductance is insensitive to PKCδ inhibition (Bankers-Fulbright, 2001).…”

“…This is consistent with recent results obtained in neutrophils (Jankowski and Grinstein, 1999). Although several groups have described the presence of proton channels in eosinophils (Gordienko et al, 1996;Schrenzel et al, 1996;Bánfi et al, 1999;Bankers-Fulbright et al, 2001;Cherny et al, 2001;DeCoursey et al, 2001a;DeCoursey et al, 2001b), the membrane potential determined by current clamp measurements indicated that depolarization following activation of NADPH oxidase was not sufficient to activate the proton channel (Gordienko et al, 1996;Tare et al, 1998;Bánfi et al, 1999). Eosinophil resting membrane potential has been reported to be between -60 and -80 mV (Gordienko et al, 1996;Tare et al, 1998).…”

“…The NADPH oxidase and its associated proton channel are often activated by the same stimuli (DeCoursey et al, 2000;DeCoursey et al, 2001b;Cherny et al, 2001). We have previously shown that superoxide production by PMAstimulated eosinophils is inhibited by the selective PKCδ Journal of Cell Science 116 (15) Fig.…”