Inhibition of the calcium store-operated calcium entry pathway by chemotactic peptide and by phorbol ester develops gradually and independently along differentiation of HL60 cells.
N-formyl-methionyl-leucyl-phenylalanine (fMLP) inhibited transiently the entry of Ca2+ and Mn2+ induced by emptying with thapsigargin the Ca2+ stores of HL60 cells differentiated toward granulocytes. Phorbol 12,13-dibutyrate (PDB) produced a permanent inhibition of this store-operated Ca2+ entry pathway (SOCP), suggesting that inhibition was due to protein phosphorylation mediated by protein kinase C (PKC). Inhibition by PDB was prevented by the PKC inhibitors staurosporin and chelerythrine. Inhibition by fMLP was prevented by chelerythrine but only partially by staurosporin. The characteristics of the inhibition were similar to those reported in human neutrophils (Montero, M., Alvarez, J., and García-Sancho, J. (1993) J. Biol. Chem. 268, 13055-13061). Neither fMLP nor PDB inhibited significantly SOCP in undifferentiated HL60 cells. Single-cell [Ca2+]i measurements at different stages of differentiation showed that inhibition by fMLP and PDB developed independently, suggesting different inhibitory mechanisms. The simplest explanation would be that inhibition by fMLP takes place through activation of a protein kinase distinct from PKC and that the PDB-activated PKC isoform necessary to phosphorylate and inhibit SOCP is expressed only along differentiation. Additionally, inhibition by both fMLP and PDB developed gradually. At intermediate stages of differentiation, PDB was able to produce a partial and maintained inhibition and fMLP a partial and short-lived inhibition of SOCP.
Montero
M
M
Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid, Spain.
Garcia-Sancho
J
J
Alvarez
J
J
D000470:Alkaloids / Q000494:pharmacology; D053119:Benzophenanthridines; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation; D006801:Humans; D008345:Manganese / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D010617:Phenanthridines / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D019311:Staurosporine; D014407:Tumor Cells, Cultured
D000470:Alkaloids; D053119:Benzophenanthridines; D010617:Phenanthridines; D015240:Phorbol 12,13-Dibutyrate; D008345:Manganese; D009240:N-Formylmethionine Leucyl-Phenylalanine; C016299:chelerythrine; D011493:Protein Kinase C; D019311:Staurosporine; D002118:Calcium