Chemotactic peptide down-regulation of calcium mobilization induced by platelet-activating factor and by leukotriene B4 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 B4 (LTB4). No effect on Ca2+ mobilization by PAF or LTB4 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 LTB4 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 LTB4 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.
Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid, Spain.
M
M
Montero
J
J
Garcia-Sancho
J
J
Alverez
D000470:Alkaloids / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015536:Down-Regulation; D004988:Ethers, Cyclic / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007975:Leukotriene B4 / Q000494:pharmacology*; D008387:Marine Toxins; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D019319:Okadaic Acid; D010080:Oxazoles / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D015398:Signal Transduction / Q000187:drug effects; D019311:Staurosporine
D000470:Alkaloids; D004988:Ethers, Cyclic; D007211:Indoles; D008387:Marine Toxins; D010080:Oxazoles; D010972:Platelet Activating Factor; D007975:Leukotriene B4; D019319:Okadaic Acid; D015240:Phorbol 12,13-Dibutyrate; D009240:N-Formylmethionine Leucyl-Phenylalanine; C059041:calyculin A; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D019311:Staurosporine; D002118:Calcium; C064758:Ro 31-8220