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 A(2)

The purpose of this investigation was to pharmacologically probe the signaling pathways thought to be involved in protein kinase C (PKC)-stimulated superoxide anion (O-2(-)) 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 A(2), cyclooxygenase (GO) and 5-lipoxygenase with selected inhibitors. The following agents inhibited phorbol 12-myristate 13-acetate-stimulated O-2(-) 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 mu M, 3 +/- 2%); sphingosine (100 mu M, 15 +/- 7%); 2) PSP 1 and 2a inhibitors, okadaic acid (10 mu M, 35 +/- 1%); calyculin A (10 mu M, 73 +/- 1%); 3) MAPK inhibitor: SE-203580 (100 mu M, 62 +/- 1%); 4) PTP inhibitors: phenylarsine oxide (1 mu M, 12 +/- 9%); diamide (1 mM, 21 +/- 11%); and 5) secretory phospholipase A, inhibitors: manoalide (1 mu M, 24 +/- 10%); scalaradial (1 mu M, 11 +/- 4%). Exogenously added arachidonic acid-stimulated O-2(-) generation in a time- and dose-dependent manner. The following inhibitors enhanced or did not significantly affect phorbol 12-myristate 13-acetate-stimulated O-2(-) generation (expressed as percentage of control): 1) PTK inhibitors: genistein (100 mu M, 69 +/- 12%); CGP 53716 (100 mu M, 67 +/- 10%); herbimycin A (10 mu M, 67.4 +/- 1%); 2) PSP 2b inhibitors: cyclosporin A (30 mu M, 71 +/- 5%); FK506 (30 mu M, 88 +/- 7%); 3) CO inhibitor: indomethacin (100 mu M, 111 +/- 12%); 4) 5-lipoxygenase inhibitor: WY 50,295 (100 mu M, 140 +/- 23%); 5) MEK inhibitor: PD98059 (100 mu M, 94 +/- 6.7%); and 6) the PTP inhibitor: orthovanadate (100 mu M, 131 +/- 25%). Our pharmacological study suggests that, in neutrophil-like HL-60 cells, the signaling pathways leading to PMA-stimulated O-2(-) generation appear to involve PKC, MAPK, phospholipase A(2), 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 O-2(-) generation.