Nucleotide-, Chemotactic Peptide- and Phorbol Ester-Induced Exocytosis in HL-60 Leukemic Cells

Abstract

Undifferentiated and differentiated HL-60 leukemic cells possess nucleotide receptors which functionally couple to phospholipase C via pertussis toxin-sensitive guanine nucleotidebinding proteins (G-proteins).We investigated the role of extracellular nucleotides in the regulation of ϟ-glucuronidase release in HL-60 cells.In dibutyryl cyclic AMP (Bt 2cAMP)differentiated HL-60 cells, the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), the phosphorothioate analogue of ATP, adenosine 5’-O-[3thio]triphosphate (ATP[γS]), and UTP increased cytosolic Ca 2+ from 100 nM up to 1.2 μM with EC 50 values of 4 nM, 1 μM and 100 nM, respectively.In these cells, ATP[γS] induced exocytosis with an EC 50 of 4 μM and an effectiveness amounting to 50–70 % of that of fMetLeu-Phe.ATP, ITP, UTP, CTP, and uridine 5’-O-[2-thio]diphosphate activated exocytosis as well.Phorbol myristate acetate (PMA) induced exocytosis with an EC 50 of 115 ng/ml and an effectiveness similar to that of ATP[γS].Cytochalasin B (CB) differently potentiated exocytosis induced by ATP[γS], fMet-Leu-Phe and PMA.Treatment of Bt 2cAMP-differentiated HL-60 cells with pertussis toxin (500 ng/ml) for 24 h resulted in ADP-ribosylation of more than 97.5 % of the G-proteins.Under these conditions, pertussis toxin almost completely inhibited the increase in cytosolic Ca 2+ and (β-glucuronidase release induced by fMet-Leu-Phe but only partially inhibited the effects of ATP[γS] and UTP.fMet-Leu-Phe at a nonstimulatory concentration (1 nM) potentiated ATP[γS]-induced β-glucuronidase release in the presence but not in the absence of CB.In contrast, ATP[γS] and fMet-Leu-Phe synergistically activated superoxide formation in the absence of CB.PMA potentiated superoxide formation induced by ATP[γS] or fMet-Leu-Phe and did not affect exocytosis induced by ATP[γS] or fMet-Leu-Phe.In undifferentiated HL-60 cells, fMet-Leu-Phe, ATP[γS], UTP and PMA did not induce β-glucuronidase release.fMet-Leu-Phe did not increase cytosolic Ca 2+ in undifferentiated HL-60 cells, whereas ATP[γS] and UTP were similarly potent and effective as in Bt 2cAMP-differentiated cells.In differentiated HL-60 cells, fMet-Leu-Phe induced aggregation, and ATP[γS] induced a transient shape change.Our results show (I) that exocytosis in HL-60 cells does not obligatorily depend on CB.(II) Purine and pyrimidine nucleotides activate exocytosis via pertussis toxin-sensitive and -insensitive signal transduction pathways.