Dual stimulation of phospholipase activity in human monocytes. Role of calcium-dependent and calcium-independent pathways in arachidonic acid release and eicosanoid formation.

Human peripheral blood monocytes, prelabeled with [3H]arachidonic acid (AA), release labeled eicosanoids in response to soluble or particulate stimuli. Treatment with 12-O-tetradecanoate phorbol-13 acetate (20 nM), calcium ionophores, A23187 (2 microM) or ionomycin (1 microM), or serum-treated zymosan (300 micrograms) resulted in production of cyclooxygenase (CO) metabolites, 6-keto-PG-F1 alpha, thromboxane-B2, PGE2, PGF2 alpha, PGD2, PGB2, 12-L-hydroxy-5,8,10-heptadecatrienoic acid; 15-lipoxygenase products, including 15-hydroxyeicosatetraenoic acid (HETE); and unmetabolized AA. Labeled 5-lipoxygenase (LO) products, 5-HETE, and leukotriene-B4 were detected only after exposure to ionophore or serum-treated zymosan. The calcium dependence of 5-LO activation was confirmed in experiments where calcium was omitted from the incubation medium, and EGTA (0.5 mM) was added, as well as by direct measurement of increased intracellular calcium in phagocytosing monocytes. Combined or sequential treatment with two stimuli increased the release of unmetabolized AA without a commensurate augmentation of labeled metabolites, indicating that release of CO and LO metabolites does not necessarily reflect the extent of phospholipase activation. Quantitation of individual eicosanoids by RIA confirmed results by using radionuclides. These studies show the following. Activation of human monocyte phospholipase may be regulated by at least two pathways, one "12-O-tetradecanoate phorbol-13 acetate-like," which is largely independent of calcium, and another which is mediated by increased intracellular Ca2+ ("ionophore-like"). "Physiologic" stimulation of monocyte arachidonate release, such as that seen accompanying phagocytosis of opsonized particles, may occur via either a calcium-sensitive or calcium-insensitive pathway or both. Calcium may regulate eicosanoid formation at the level of phospholipase or 5-LO. Free AA, CO products, and 12- or 15-LO products are ordinarily released after phagocytosis, but leukotriene-B4, 5-HETE, or other 5-LO metabolites are produced only under conditions where calcium concentrations are optimal.