NUCLEOTIDE-INDUCED, CHEMOTACTIC PEPTIDE-INDUCED AND PHORBOL ESTER-INDUCED EXOCYTOSIS IN HL-60 LEUKEMIC-CELLS

Undifferentiated and differentiated HL-60 leukemic cells posses nucleotide receptors which functionally couple to phospholipase C via pertussis toxin-sensitive guanine nucleotide-binding proteins (G-proteins). We investigated the role of extracellular nucleotides in the regulation of B-glucuronidase release in HL-60 cells. In dibutyryl cyclic AMP (Bt2cAMP)-differentiated HL-60 cells, the chemotactic peptide, N-formyl-L-methionyl-L-leucy-L-phenylalanine (fMet-Leu-Phe), the phosphorothioate analogue of ATP, adenosine 5'-O-[3-thio]triphosphate (ATP[gamma-S]), and UTP increased cytosolic Ca2+ from 100 nM up to 1.2 mu-M with EC50 values of 4 nM, 1 mu-M and 100 nM, respectively. In these cells, ATP[gamma-S] induced exocytosis with an EC50 of 4 mu-M and an effectiveness amounting to 50-70% of that of fMet-Leu-Phe. ATP, ITP, UTP, CTP, and uridine 5'-O-[2-thio]diphosphate activated exocytosis as well. Phorbol myristate acetate (PMA) induced exocytosis with an EC50 of 115 ng/ml and an effectiveness similar to that of ATP[gamma-S]. Cytochalasin B (CB) differently potentiated exocytosis induced by ATP[gamma-S], fMet-Leu-Phe and PMA. Treatment of Bt2cAMP-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 Ca2+ and B-glucuronidase release induced by fMet-Leu-Phe but only partially inhibited the effects of ATP[gamma-S] and UTP. fMet-Leu-Phe at a non-stimulatory concentration (1 nM) potentiated ATP[gamma-S]-induced B-glucuronidase release in the presence but not in the absence of CB. In contrast, ATP[gamma-S] and fMet-Leu-Phe synergistically activated superoxide formation in the absence of CB. PMA potentiated superoxide formation induced by ATP[gamma-S] or fMet-Leu-Phe and did not affect exocytosis induced by ATP[gamma-S] or fMet-Leu-Phe. In undifferentiated HL-60 cells, fMet-Leu-Phe, ATP[gamma-S], UTP and PMA did not induce B-glucuronidase release. fMet-Leu-Phe did not increase cytosolic Ca2+ in undifferentiated HL-60 cells, whereas ATP[gamma-S] and UTP were similarly potent and effective as in Bt2cAMP-differentiated cells. In differentiated HL-60 cells, fMet-Leu-Phe induced aggregation, and ATP[gamma-S] 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. (III) Specific signal transduction components for exocytosis are expressed during myeloid differentiation which are apparently different from G-proteins, phospholipase C, protein kinase C, and the Ca2+-mobilizing system. (IV) Exocytosis and superoxide formation are independently regulated.