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 O(2) release. Similar to PMN, PMN cytoplasts (PMN-CPs) produce equivalent amounts of O(2) 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 O(2) efficiently in the absence of external Ca++ when stimulated with 10(-7) mol/L fMLP, whereas PMNs release significantly less O(2) 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 O(2) from PMN-CPs.