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 p hosphatase inhibitors calyculin A or okadaic acid, the chemotactic pep tide N-formyl-methionyl-leucyl-phenylalanine (fMLP) produced a sustain ed (> 5 min) inhibition of the Ca2+ mobilization from intracellular st ores induced by platelet activating factor (PAF) or by leukotriene B-4 (LTB(4)). No effect on Ca2+ mobilization by PAF or LTB(4) was observe d 2 min after the addition of fMLP alone or only in the presence: of p hosphatase inhibitors, but a similar inhibition was produced by high ( > 50 nM) concentrations of phorbol 12,13-dibutyrate (PDB). However, in hibition 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 phosphor ylation not mediated by PKC which interferes at some point with the tr ansduction pathway leading from the plasma membrane receptors for PAF and LTB(4) to the release of Ca2+ from the stores. Protein phosphatase s 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 s maller extent. Phosphatase inhibitors also delayed by 1-2 s the start of agonist-induced rises in [Ca2+](i), and this delay was further incr eased by previous addition of any other agonist. Finally, given that b oth phosphatase inhibitors and low concentrations of PDB (2-10 nM) str ongly inhibit Ca2+ entry, we conclude that phosphorylation down-regula tes both agonist-induced Ca2+ entry and Ca2+ mobilization, but with di fferent potency.