The dependence on Ca2+ of phosphatidylinositol breakdown and enzyme secretion in rabbit neutrophils stimulated by formylmethionyl-leucylphenylalanine or ionomycin

Abstract

The breakdown of [3H]phosphatidylinositol was measured in rabbit neutrophils prelabeled with [3H]glycerol by a pulse-chase procedure. To define a possible causal relationship between phosphatidylinositol breakdown and enzyme secretion in these cells, the characteristics of both these processes induced by either the receptor-directed agonist formylmethionylleucylphenylalanine (fMet-Leu-Phe) or the Ca2+-ionophore ionomycin were compared. The dependence on fMet-Leu-Phe concentration of phosphatidylinositol breakdown and secretion is identical (half-maximal at 0.3 nM). This is 30-fold less than that required for half-maximal occupation of receptors. Both secretion and breakdown of phosphatidylinositol due to fMet-Leu-Phe are modulated by extracellular Ca2+. The sensitivity to Ca2+ of both processes is enhanced by pretreatment to deplete cell Ca2+. The concentration of Ca2+ required to cause half-maximal effects of both processes in Ca2+-depleted cells on stimulation with 1 nM-fMet-Leu-Phe is 100 .mu.M. Ionomycin-stimulated secretion and breakdown of phosphatidylinositol are completely dependent on extracellular Ca2+ over similar concentration ranges. Both secretion and phosphatidylinositol breakdown due to fMet-Leu-Phe approach completion by 10 s. With ionomycin these processes are slower, terminating by 2 min. In the presence of [32P]Pi, labeling of [32P]phosphatidic acid reaches a maximum 15 min after stimulation with either fMet-Leu-Phe or ionomycin. This precedes the labeling of [32P]phosphatidylinositol and shows the expected precursor-product relationship. In rabbit neutrophils, a rise in cytosol Ca2+ is both sufficient and necessary to cause secretion and phosphatidylinositol breakdown. In cells depleted of Ca2+, the occupation of receptors by fMet-Leu-Phe is without effect on these 2 processes.