Counteracting effects of NADPH oxidase and the Na+/Ca 2+ exchanger on membrane repolarisation and store-operated uptake of Ca2+ by chemoattractant-activated human neutrophils

This study was designed to investigate the possible involvement of NADPH oxidase and the Na+/Ca2+ exchanger in regulating membrane repolarisation and store-operated uptake of Ca2+ by FMLP (1 muM)-activated human neutrophils. Diphenyleneiodonium. chloride (DPI, 5-10 muM) and KB-R7943 (2.5-10 muM), inhibitors of NADPH oxidase and the reverse mode of the Na+/Ca2+ exchanger respectively, were used as pharmacological probes. Transmembrane fluxes of Ca2+, K+ and Na+ were determined radio metrically, while alterations in membrane potential and cytosolic Ca2+ were evaluated using spectrofluorimetric procedures. DPI, added to the cells at the time of maximum FMLP-activated membrane depolarisation, accelerated the rates of both membrane repolarisation and influx of Ca2+, while KB-R7943 effectively antagonised these processes. SKF 96365 (10 muM), an antagonist of store-operated Ca2+ channels, abolished the influx of Ca2+ into FMLP-activated neutrophils, but had no effects on membrane repolarisation, suggesting that the Na+/Ca2+ exchanger is primarily involved in mediating membrane repolarisation, thereby facilitating uptake of Ca2+ via store-operated channels. These observations are compatible with prominent negative and positive regulatory roles for NADPH oxidase and the Na+/Ca2+ exchanger respectively in regulating the rates of membrane repolarisation and store-operated uptake of Ca2+ by chemoattractant-activated neutrophils. (C) 2004 Elsevier Inc. All rights reserved.