Kargman S, Ali A, Vaillancourt J P, Evans J F, Nicholson D W
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada.
Mol Pharmacol. 1994 May;45(5):1043-9.
In response to calcium ionophore (A23187) stimulation, human granulocyte/macrophage colony-stimulating factor-primed, dimethylsulfoxide-differentiated HL-60 cells (which resemble mature granulocytes) synthesized leukotrienes (LTs) LTA4, LTB4, LTC4, and LTD4. The synthesis of the sulfidopeptide LTs, LTC4 and LTD4, was specifically inhibited in cells incubated in the presence of both A23187 and phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC). In contrast, neither the synthesis of LTB4, a product of the nonpeptide branch of the LT pathway, nor the formation of LTA4, the precursor for both branches of the LT biosynthetic pathway, was significantly affected by the presence of PMA during A23187 stimulation. The inhibition by PMA of LTC4 production in A23187-stimulated HL-60 cells was dose dependent, with an IC50 value of approximately 3.5 nM. The PKC inhibitor staurosporine completely reversed the inhibition by PMA of LTC4 production in A23187-stimulated cells, in a dose-dependent fashion, with an IC50 value of approximately 30 nM. Bisindolylmaleimide, another PKC inhibitor, was also able to prevent PMA-mediated inhibition of LTC4 formation, whereas inhibitors of protein kinase A, tyrosine kinases, or the respiratory-burst oxidase were not. Measurement of LTC4 synthase enzymatic activity in cells challenged with A23187 and PMA in the presence or absence of staurosporine demonstrated that the activity of the LTC4 synthase enzyme was inhibited in cells costimulated with A23187 and PMA and that inhibition could also be completely prevented by the presence of staurosporine. Because PMA is known to activate PKC, and staurosporine and bisindolylmaleimide are inhibitors of PKC, these results suggest that LTC4 synthase in HL-60 cells may be phosphoregulated.
在钙离子载体（A23187）刺激下，经人粒细胞/巨噬细胞集落刺激因子预处理、二甲基亚砜分化的HL-60细胞（类似于成熟粒细胞）可合成白三烯（LTs）LTA4、LTB4、LTC4和LTD4。在同时存在A23187和佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA，一种蛋白激酶C（PKC）激活剂）的情况下培养的细胞中，硫肽白三烯LTC4和LTD4的合成受到特异性抑制。相比之下，在A23187刺激过程中，PMA的存在对LT途径非肽分支的产物LTB4的合成以及LT生物合成途径两个分支的前体LTA4的形成均无显著影响。PMA对A23187刺激的HL-60细胞中LTC4产生的抑制作用呈剂量依赖性，IC50值约为3.5 nM。PKC抑制剂星形孢菌素以剂量依赖性方式完全逆转了PMA对A23187刺激细胞中LTC4产生的抑制作用，IC50值约为30 nM。另一种PKC抑制剂双吲哚马来酰胺也能够阻止PMA介导的LTC4形成的抑制作用，而蛋白激酶A抑制剂、酪氨酸激酶抑制剂或呼吸爆发氧化酶抑制剂则不能。在有或没有星形孢菌素的情况下，对用A23187和PMA刺激的细胞中LTC4合酶的酶活性进行测定，结果表明，在A23187和PMA共同刺激的细胞中，LTC4合酶的活性受到抑制，并且星形孢菌素的存在也能完全阻止这种抑制作用。由于已知PMA可激活PKC，而星形孢菌素和双吲哚马来酰胺是PKC抑制剂，因此这些结果表明HL-60细胞中的LTC4合酶可能受到磷酸化调节。
