PROTEIN-KINASE C-DEPENDENT REGULATION OF SULFIDOPEPTIDE LEUKOTRIENE BIOSYNTHESIS AND LEUKOTRIENE C-4 SYNTHASE IN NEUTROPHILIC HL-60 CELLS

In response to calcium ionophore (A23187) stimulation, human granulocy te/macrophage colony-stimulating factor-primed, dimethylsulfoxide-diff erentiated HL-60 cells (which resemble mature granulocytes) synthesize d leukotrienes (LTs) LTA(4), LTB(4), LTC(4), and LTD(4). The synthesis of the sulfidopeptide LTs, LTC(4) and LTD(4), was specifically inhibi ted in cells incubated in the presence of both A23187 and phorbol-12-m yristate-13-acetate (PMA), an activator of protein kinase C (PKC). In contrast, neither the synthesis of LTB(4), a product of the nonpeptide branch of the LT pathway, nor the formation of LTA(4), the precursor for both branches of the LT biosynthetic pathway, was significantly af fected by the presence of PMA during A23187 stimulation. The inhibitio n by PMA of LTC(4) production in A23187-stimulated HL-60 cells was dos e dependent, with an IC50 value of approximately 3.5 nM. The PKC inhib itor staurosporine completely reversed the inhibition by PMA of LTC(4) production in A23187-stimulated cells, in a dose-dependent fashion, w ith an IC50 value of approximately 30 nM. Bisindolylmaleimide, another PKC inhibitor, was also able to prevent PMA-mediated inhibition of LT C(4) formation, whereas inhibitors of protein kinase A, tyrosine kinas es, or the respiratory-burst oxidase were not. Measurement of LTC(4) s ynthase enzymatic activity in cells challenged with A23187 and PMA in the presence or absence of staurosporine demonstrated that the activit y of the LTC(4) synthase enzyme was inhibited in cells costimulated wi th A23187 and PMA and that inhibition could also be completely prevent ed by the presence of staurosporine. Because PMA is known to activate PKC, and staurosporine and bisindolylmaleimide are inhibitors of PKC, these results suggest that LTC(4) synthase in HL-60 cells may be phosp horegulated.