title: Pharmacological targeting of signaling pathways in protein kinase C-stimulated superoxide generation in neutrophil-like HL-60 cells: effect of phorbol ester, arachidonic acid and inhibitors of kinase(s), phosphatase(s) and phospholipase A2.
abstract: The purpose of this investigation was to pharmacologically probe the signaling pathways thought to be involved in protein kinase C (PKC)-stimulated superoxide anion (O2-) generation in all-trans retinoic acid-treated human promyelocytic HL-60 cell line (HL-60), targeting PKC, mitogen-activated protein kinase (MAPK), MAPK kinase (MEK), protein serine-threonine phosphatase(s) (PSP), protein tyrosine kinase(s) (PTK) and phosphatase(s) (PTP), secretory phospholipase A2, cyclooxygenase (CO) and 5-lipoxygenase with selected inhibitors. The following agents inhibited phorbol 12-myristate 13-acetate-stimulated O2- generation significantly in the all-trans retinoic acid-treated HL-60 cells (expressed as percentage of control, P < .05): 1) PKC inhibitors: staurosporine (100 nM, 3 +/- 1%); Ro 31-8220 (1 microM, 3 +/- 2%); sphingosine (100 microM, 15 +/- 7%); 2) PSP 1 and 2a inhibitors, okadaic acid (10 microM, 35 +/- 1%); calyculin A (10 microM, 73 +/- 1%); 3) MAPK inhibitor: SB-203580 (100 microM, 62 +/- 1%); 4) PTP inhibitors: phenylarsine oxide (1 microM, 12 +/- 9%); diamide (1 mM, 21 +/- 11%); and 5) secretory phospholipase A2 inhibitors: manoalide (1 microM, 24 +/- 10%); scalaradial (1 microM, 11 +/- 4%). Exogenously added arachidonic acid-stimulated O2- generation in a time- and dose-dependent manner. The following inhibitors enhanced or did not significantly affect phorbol 12-myristate 13-acetate-stimulated O2- generation (expressed as percentage of control): 1) PTK inhibitors: genistein (100 microM, 69 +/- 12%); CGP 53716 (100 microM, 67 +/- 10%); herbimycin A (10 microM, 67.4 +/- 1%); 2) PSP 2b inhibitors: cyclosporin A (30 microM, 71 +/- 5%); FK506 (30 microM, 88 +/- 7%); 3) CO inhibitor: indomethacin (100 microM, 111 +/- 12%); 4) 5-lipoxygenase inhibitor: WY 50,295 (100 microM, 140 +/- 23%); 5) MEK inhibitor: PD98059 (100 microM, 94 +/- 6.7%); and 6) the PTP inhibitor: orthovanadate (100 microM, 131 +/- 25%). Our pharmacological study suggests that, in neutrophil-like HL-60 cells, the signaling pathways leading to PMA-stimulated O2- generation appear to involve PKC, MAPK, phospholipase A2, arachidonic acid, PSP 1 and 2a and PTP. Furthermore, PTK, MEK, CO, 5-lipoxygenase and PSP 2b do not appear to participate in the modulation of PKC-stimulated O2- generation.
authors.affiliation: Department of Pharmacology, Chicago College of Osteopathic Medicine, Downers Grove, Illinois, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Mayer
authors.forename: S
authors.initials: S
authors.lastname: Brenic
authors.forename: K B
authors.initials: KB
authors.lastname: Glaser
mesh_terms: D016718:Arachidonic Acid / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D018922:HL-60 Cells / Q000378:metabolism*; D006801:Humans; D020929:Mitogen-Activated Protein Kinase Kinases; D009504:Neutrophils / Q000378:metabolism*; D010741:Phospholipases A / Q000502:physiology; D054467:Phospholipases A2; D011493:Protein Kinase C / Q000502:physiology*; D011494:Protein Kinases / Q000502:physiology; D017027:Protein Tyrosine Phosphatases / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D013481:Superoxides / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014212:Tretinoin / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D013481:Superoxides; D016718:Arachidonic Acid; D014212:Tretinoin; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D017027:Protein Tyrosine Phosphatases; D013755:Tetradecanoylphorbol Acetate