Involvement of ion channels in human eosinophil respiratory burst

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Involvement of ion channels in human eosinophil respiratory burst Andreas Schwingshackl, MDa, Redwan Moqbel, PhD, FRCPathb, Marek Duszyk, PhDa Journal of Allergy and Clinical Immunology Volume 106, Issue 2, Pages 272-279 (August 2000) DOI: 10.1067/mai.2000.107752 Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 1 Dose-response curve of PMA-induced O2•– production in human eosinophils (1 × 106). A, Time course of O2•– production stimulated by 0 to 100 ng/mL PMA added at the time indicated by the arrow . B, Dose-response curve expressed as nmoles/liter/106 cells/min and fitted by equation 1 (see the “Methods” section) with Pmax of 9.34 nmoles/liter/106 cells/min, Kd of 4.98 ng/mL, and n of 3.5. Data are plotted as means ± SEM of 3 different experiments. Journal of Allergy and Clinical Immunology 2000 106, 272-279DOI: (10.1067/mai.2000.107752) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 2 The effects of channel blockers on O2•– production. Before PMA stimulation (10 ng/mL), cells (1 × 106) were treated with clotrimazole (100 μmol/L, n = 7), BaCl2 (5 mmol/L, n = 4), amiloride (100 μmol/L, n = 7), DIDS (50 μmol/L, n = 10), niflumic acid (100 μmol/L, n = 3), DPC (1 mmol/L, n = 5), DNDS (0.5 mmol/L, n = 3), acetazolamide (100 μmol/L, n = 5), and diltiazem (50 nmol/L, n = 3). Values are expressed as percentages of O2•– production of PMA-treated eosinophils. A significant decrease in O2•– production was observed in the presence of BaCl2, DIDS, and diltiazem (P < .05). Journal of Allergy and Clinical Immunology 2000 106, 272-279DOI: (10.1067/mai.2000.107752) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 3 The effect of Cl– ions on eosinophil O2•– production. In a high-Cl– buffer, PMA (10 ng/mL) generated 9.3 ± 0.5 nmoles/liter/106 cells/min O2•–. In the presence of DIDS (50 μmol/L), PMA-stimulated O2•– production was reduced by 22% (n = 10, P < .05). In a low-Cl– buffer, PMA generated 6.1 ± 1 nmoles/liter/106 cells/min of O2•–, and DIDS had no effect on PMA-stimulated O2•– production (6.05 ± 1.2 nmoles/liter/106 cells/min, n = 3). unst, Unstimulated. Journal of Allergy and Clinical Immunology 2000 106, 272-279DOI: (10.1067/mai.2000.107752) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 4 A, Effect of Cl– on the whole cell current in human eosinophils. B, Current-voltage relationships for the recordings shown in A . Reducing the Cl– concentration from 145.1 mmol/L to 11.2 mmol/L decreased the whole-cell current and shifted the reversal potential from –32 mV to –19 mV. The holding potential was –60 mV, and 20-mV steps ranging from –80 to 80 mV were applied every 200 ms. C, Activation of the whole-cell current by PMA (10 ng/mL). D, The effects of DIDS and ion replacement studies on eosinophil whole-cell currents. The mean values of 4 to 7 recordings obtained from 4 different individuals are shown as percentages of whole-cell currents in resting eosinophils. PMA increased the whole-cell current by 168% (P < .01). In the presence of DIDS, the whole-cell current decreased by 43% (P < .01), and PMA increased the current by 75% (P < .05). The replacement of Cl– with gluconate reduced the baseline current by 43% (P < .01), and PMA increased the current by 62% (P < .05). The replacement of K+ with Cs+ decreased the baseline current by 49% (P < .01) but had no effect on the PMA-stimulated current when compared with the PMA-induced whole-cell current in a high-Cl– solution (P > .05). Journal of Allergy and Clinical Immunology 2000 106, 272-279DOI: (10.1067/mai.2000.107752) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 5 A, Lack of CFTR mRNA expression in human eosinophils (Eos) . The human airway epithelial cell line Calu-3 was used as a positive control for CFTR expression. B, ClC mRNA expression in human eosinophils. Eosinophils expressed mRNA for ClC-3 but not for ClC-2, ClC-4, ClC-5, or ClC-6. HEK 293 cells were used as positive controls for ClC expression. The data are representative of 3 different experiments. M represents a DNA standard (100 bp DNA ladder). Journal of Allergy and Clinical Immunology 2000 106, 272-279DOI: (10.1067/mai.2000.107752) Copyright © 2000 Mosby, Inc. Terms and Conditions