Neutrophil cytoplasts: Relationships of superoxide release and calcium pools

Abstract

Activation of human polymorphonuclear neutrophils (PMNs) by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) leads to a transient increase in intracellular level of ionized calcium and an alteration of the plasma membrane permeability. Calcium has been proposed as a second messenger for activation of the PMN. Modulation of intracellular pools of calcium is of importance in the regulation of PMN activation. We have studied the changes in membrane-bound and cytoplasmic calcium in PMN and PMN devoid of granules and nucleus by quantifying changes in chlorotetracycline (CTC) and Quin 2 fluorescence and comparing their relation to O2·¯ release. Similar to PMN, PMN cytoplasts (PMN-CPs) produce equivalent amounts of O2·¯ in response to 10-7 mol/L fMLP. The decrease in CTC fluorescence following fMLP stimulation is not significantly different in PMN-CP (-9.9% ± 3.7%) from that observed in PMN (-12.7% ± 2.33%), suggesting that the trigger pool of Ca++ is present in PMN-CPs. Although PMNs show a net increase in free Ca++ as measured by Quin 2. PMN-CPs display a lower sustained rise, which is totally abolished in the absence of external Ca++. PMN-CPs release O2·¯ efficiently in the absence of external Ca++ when stimulated with 10-7 mol/L fMLP, whereas PMNs release significantly less O2·¯, under the same conditions. Our results suggest that a rapid rise in free Ca++, as monitored by Quin 2 fluorescence, is not required for expression of full activation of the oxidase system and release of O2·¯ from PMN-CPs.