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doi: 10.1242/10.1242/jcs.00627

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Regulation of eosinophil membrane depolarization during NADPH oxidase activation

Jennifer L. Bankers-Fulbright1,*, Gerald J. Gleich3, Gail M. Kephart1, Hirohito Kita1,2 and Scott M. O'Grady4,5

1 Department of Medicine, Mayo Clinic Rochester, Rochester, MN 55905, USA
2 Department of Immunology, Mayo Clinic Rochester, Rochester, MN 55905, USA
3 Department of Dermatology, University of Utah, Salt Lake City, UT 84132, USA
4 Department of Physiology, University of Minnesota, St Paul, MN 55108, USA
5 Department of Animal Science, University of Minnesota, St Paul, MN 55108, USA



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  		Fig. 1. The time course of eosinophil depolarization correlates with activation of the proton conductance. (A) Eosinophils were loaded with 600 nM diBAC4(3) and analzyed at room temperature by confocal microscopy (magnification=630{chi}). Inset is a 3{chi} electronic magnification of the area in the box. A representative experiment is shown (n=4). (B) Eosinophils were stimulated with 800 nM PMA at room temperature. Depolarization ({blacktriangleup}; n=26) was assayed using diBAC4(3) and flow cytometry as described in Materials and Methods. Conductance values ({blacksquare}; n=6) were calculated from current measurements following a voltage step from the holding potential (–60) to +80 mV.

 


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  		Fig. 2. ZnCl2 potentiates PMA-stimulated eosinophil depolarization. Transmembrane potential was determined using diBAC4(3) and flow cytometry. Absolute membrane potential was calculated as described in Materials and Methods. Eosinophils were stimulated at room temperature with 800 nM PMA in the absence ({bullet}; n=4) or presence ({blacktriangleup}; n=4) of 250 µM ZnCl2. ZnCl2 was added immediately before stimulation. Mean values±s.e.m. are shown; where the error bars are not visible they are smaller than the symbol.

 


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  		Fig. 3. Varying extracellular pH predictably affects eosinophil depolarization. (A) Eosinophil intracellular pH was determined using BCECF/AM and calibrated as described in Materials and Methods. Buffer pH was verified and adjusted before each experiment. Unstimulated eosinophils were resuspended at 37°C at pH 6.0 (n=8), pH 6.5 (n=8), pH 7.0 (n=8) and pH 7.4 (n=8). The mean is stated above each bar (mean±s.e.m.). (B) Superoxide production was determined using the cytochrome c colorimetric assay as described in Materials and Methods. Buffer pH was verified and adjusted before each experiment. Cells were stimulated with 2 nM PMA at 37°C at pH 6.0 (n=5), pH 6.5 (n=5), pH 7.0 (n=5) and pH 7.4 (n=5). NADPH oxidase rate was calculated as described in Materials and Methods (means±s.e.m.). **P<0.01. (C) Eosinophil membrane potential was measured using diBAC4(3) and flow cytometry. Media pH was verified and adjusted before each experiment. Eosinophils were stimulated with 800 nM at room temperature at pH 6.0 ({chi}; n=3), pH 6.5 ({square}; n=4), pH 7.0 ({blacktriangleup}; n=8), pH 7.4 ({bullet}; n=8) and pH 8.0 (*; n=4). Means±s.e.m. are shown.

 


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  		Fig. 4. PKC blockers prevent PMA-induced eosinophil depolarization. Eosinophils were resuspended in diBAC4(3) and stimulated with 800 nM PMA at room temperature. Absolute transmembrane potential was determined by flow cytometry as described in Materials and Methods. Eosinophils were stimulated at room temperature with 800 nM PMA alone ({bullet}; n=9) or in the presence of 5 µM Gö6976 ({blacktriangleup}; n=3), 1 µM GF109203X ({blacktriangledown}; n=3), or 10 µM rottlerin ({blacksquare}; n=4). PKC inhibitors were added immediately before stimulation with PMA. Means±s.e.m. are shown.

 

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