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 s ignaling pathways thought to be involved in protein kinase C (PKC)-sti mulated superoxide anion (O-2(-)) generation in all-trans retinoic aci d-treated human promyelocytic HL-60 cell line (HL-60), targeting PKC, mitogen-activated protein kinase (MAPK), MAPK kinase (MEK), protein se rine-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 agent s inhibited phorbol 12-myristate 13-acetate-stimulated O-2(-) generati on significantly in the all-trans retinoic acid-treated HL-60 cells (e xpressed as percentage of control, P < .05): 1) PKC inhibitors: stauro sporine (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 m u 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 pho spholipase 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 inh ibitors enhanced or did not significantly affect phorbol 12-myristate 13-acetate-stimulated O-2(-) generation (expressed as percentage of co ntrol): 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 2 b 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-li poxygenase inhibitor: WY 50,295 (100 mu M, 140 +/- 23%); 5) MEK inhibi tor: PD98059 (100 mu M, 94 +/- 6.7%); and 6) the PTP inhibitor: orthov anadate (100 mu M, 131 +/- 25%). Our pharmacological study suggests th at, in neutrophil-like HL-60 cells, the signaling pathways leading to PMA-stimulated O-2(-) generation appear to involve PKC, MAPK, phosphol ipase 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.