Agonist-induced Calcium Flux, Phosphoinositide Metabolism, Aggregation and Enzyme Secretion in Human Neutrophils

Formyl-methionyl-leucyl-phenylalanine (FMLP), platelet activating factor (PAF) and leukotriene B4 (LTB4) are potent activators of human neutrophils. Using human neutrophils prelabelled with the fluorescent indicator dye, Quin 2, or with [32P]-orthophosphate, we examined the effects of these stimuli on intracellular free calcium concentration, [Ca2+]i, and on various indices of phosphoinositide metabolism, including [32P]-phosphatidic acid (PtdA) formation. The concentration-dependence of the observed changed in [Ca2+]i or [32P]-PtdA were then compared to stimulus-induced aggregation and enzyme release (beta-N-acetylglucosaminidase (NAG) and lysozyme). FMLP, PAF and LTB4 caused a concentration-dependent elevation of [Ca2+]i, aggregation and enzyme release. However, unlike FMLP and PAF, LTB4 (less than or equal to 2.5 microM) did not cause significant formation of [32P]-PtdA. The concentration response curves for agonist-induced elevation of [Ca2+]i lie to the left of those for aggregation and enzyme release. FMLP and PAF also caused an elevation of [Ca2+]i at concentrations lower than those required to elicit [32P]-PtdA formation. These observations suggest that [Ca2+]i elevation per se cannot mediate human neutrophil functional responses to FMLP, PAF and LTB4. Consequently there may exist other mediator(s) that act in concert with [Ca2+]i or are triggered by [Ca2+]i elevation to promote human neutrophil activation. Both the elevation of [Ca2+]i and the formation of these putative mediator(s) in response to LTB4 apparently occur independently of inositol phospholipid hydrolysis.