Activation of inositol phospholipid breakdown in HL60 cells by P2-purinergic receptors for extracellular ATP. Evidence for mediation by both pertussis toxin-sensitive and pertussis toxin-insensitive mechanisms.
The mechanisms whereby P2-purinergic receptors for extracellular ATP are coupled to the inositol phospholipid-signaling system were studied in the HL60 human promyelocytic leukemia cell line. Brief pretreatment of either undifferentiated or differentiated HL60 cells with various activators of protein kinase C Ca2+/phospholipid-dependent enzyme (e.g. phorbol myristate acetate) produced a 50-fold decrease in the potency of extracellular ATP to induce mobilization of intracellular Ca2+. The ATP-induced increase in rate of inositol trisphosphate (InsP3) accumulation in these 4-beta-phorbol 12-myristate-13-acetate-treated cells was characterized by a 40% decrease in the maximal rate of InsP3 accumulation. Incubation of the cells with NaF also induced mobilization of the same Ca2+ stores released in response to extracellular ATP; this provided indirect evidence that the transmembrane signaling actions of P2-purinergic receptors may be mediated by GTP-binding regulatory proteins. This latter possibility was further supported by the finding that treatment of either undifferentiated or differentiated HL60 cells with pertussis toxin produced a significant, but partial, inhibition of ATP-induced signaling actions. These included: 1) a 60-70% decrease in the maximum rate of InsP3 accumulation, and 2) a 1.5 log unit increase in the half-maximally effective [ATP] required for mobilization of intracellular Ca2+. In cells treated with both pertussis toxin and 4-beta-phorbol 12-myristate-13-acetate, there was an 80% decrease in maximal rate of ATP-induced InsP3 accumulation and near-complete inhibition of ATP-induced Ca2+ mobilization. Significantly, the residual, pertussis toxin-insensitive portion of ATP-induced signaling was observed in the same samples of differentiated HL60 cells wherein pertussis toxin treatment produced complete abolition of InsP3 accumulation and Ca2+ mobilization in response to occupation of chemotactic peptide receptors. These results indicate that the activation of inositol phospholipid breakdown by P2-purinergic receptors in HL60 cells may be mediated by both pertussis toxin-sensitive and toxin-insensitive mechanisms; this suggests that these myeloid progenitor cells may express two distinct types of GTP-binding proteins coupled to phospholipase C.
Dubyak
G R
GR
Department of Physiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
Cowen
D S
DS
Meuller
L M
LM
D000255:Adenosine Triphosphate / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D037342:Pertussis Toxin; D010716:Phosphatidylinositols / Q000378:metabolism*; D011983:Receptors, Purinergic / Q000187:drug effects / Q000502:physiology*; D012965:Sodium Chloride / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology; D013403:Sugar Phosphates / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D011983:Receptors, Purinergic; D013403:Sugar Phosphates; D010566:Virulence Factors, Bordetella; D012965:Sodium Chloride; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000255:Adenosine Triphosphate; D012969:Sodium Fluoride; D037342:Pertussis Toxin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium