Nucleotide-, chemotactic peptide- and phorbol ester-induced exocytosis in HL-60 leukemic cells.
Undifferentiated and differentiated HL-60 leukemic cells possess 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 beta-glucuronidase release in HL-60 cells. In dibutyryl cyclic AMP (Bt2cAMP)-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-[3-thio]triphosphate (ATP[gamma S]), and UTP increased cytosolic Ca2+ from 100 nM up to 1.2 microM with EC50 values of 4 nM, 1 microM and 100 nM, respectively. In these cells, ATP[gamma S] induced exocytosis with an EC50 of 4 microM 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 beta-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 beta-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 beta-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.(ABSTRACT TRUNCATED AT 400 WORDS)
Wenzel-Seifert
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K
Abteilung für Allgemeine Medizin und Nephrologie, Universitätsklinikum Steglitz, Berlin, Germany.
Seifert
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R
D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D003571:Cytochalasin B / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects* / Q000502:physiology; D005966:Glucuronidase / Q000378:metabolism; D006801:Humans; D007938:Leukemia / Q000473:pathology / Q000503:physiopathology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009711:Nucleotides / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000473:pathology / Q000502:physiology*
D009711:Nucleotides; D010703:Phorbol Esters; D013481:Superoxides; D003571:Cytochalasin B; D009240:N-Formylmethionine Leucyl-Phenylalanine; D003994:Bucladesine; D005966:Glucuronidase; D002118:Calcium