Counteracting effects of NADPH oxidase and the Na+/Ca2+ 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(+)/Ca(2+) exchanger in regulating membrane repolarisation and store-operated uptake of Ca(2+) by FMLP (1 microM)-activated human neutrophils. Diphenyleneiodonium chloride (DPI, 5-10 microM) and KB-R7943 (2.5-10 microM), inhibitors of NADPH oxidase and the reverse mode of the Na(+)/Ca(2+) exchanger respectively, were used as pharmacological probes. Transmembrane fluxes of Ca(2+), K(+) and Na(+) were determined radiometrically, while alterations in membrane potential and cytosolic Ca(2+) 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 Ca(2+), while KB-R7943 effectively antagonised these processes. SKF 96365 (10 microM), an antagonist of store-operated Ca(2+) channels, abolished the influx of Ca(2+) into FMLP-activated neutrophils, but had no effects on membrane repolarisation, suggesting that the Na(+)/Ca(2+) exchanger is primarily involved in mediating membrane repolarisation, thereby facilitating uptake of Ca(2+) via store-operated channels. These observations are compatible with prominent negative and positive regulatory roles for NADPH oxidase and the Na(+)/Ca(2+) exchanger respectively in regulating the rates of membrane repolarisation and store-operated uptake of Ca(2+) by chemoattractant-activated neutrophils.