CHEMOTACTIC PEPTIDE DOWN-REGULATION OF CALCIUM MOBILIZATION INDUCED BY PLATELET-ACTIVATING-FACTOR AND BY LEUKOTRIENE B-4 IN HUMAN NEUTROPHILS IS UNCOVERED BY PROTEIN PHOSPHATASE INHIBITORS

When human neutrophils were incubated in the presence of the protein phosphatase inhibitors calyculin A or okadaic acid, the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) produced a sustained (> 5 min) inhibition of the Ca2+ mobilization from intracellular stores induced by platelet activating factor (PAF) or by leukotriene B-4 (LTB(4)). No effect on Ca2+ mobilization by PAF or LTB(4) was observed 2 min after the addition of fMLP alone or only in the presence: of phosphatase inhibitors, but a similar inhibition was produced by high (> 50 nM) concentrations of phorbol 12,13-dibutyrate (PDB). However, inhibition by PDB was sensitive to the protein kinase C (PKC) inhibitors staurosporin and Ro 31-8220, while inhibition by fMLP and calyculin A was not. These results suggest that fMLP induces a transient phosphorylation not mediated by PKC which interferes at some point with the transduction pathway leading from the plasma membrane receptors for PAF and LTB(4) to the release of Ca2+ from the stores. Protein phosphatases 1 and/or 2A revert the inhibition effected by fMLP within less than 2 min. PAF and LTB(4) were also able to activate this mechanism to a smaller extent. Phosphatase inhibitors also delayed by 1-2 s the start of agonist-induced rises in [Ca2+](i), and this delay was further increased by previous addition of any other agonist. Finally, given that both phosphatase inhibitors and low concentrations of PDB (2-10 nM) strongly inhibit Ca2+ entry, we conclude that phosphorylation down-regulates both agonist-induced Ca2+ entry and Ca2+ mobilization, but with different potency.