Cockcroft S, Stutchfield J
Department of Experimental Pathology, School of Medicine, University College London, U.K.
Biochem J. 1989 Nov 1;263(3):715-23. doi: 10.1042/bj2630715.
The relationship between phospholipase A2 and C activation and secretion was investigated in intact human neutrophils and differentiated HL60 cells. Activation by either ATP or fMetLeuPhe leads to [3H]arachidonic acid release into the external medium from prelabelled cells. This response was inhibited when the cells were pretreated with pertussis toxin. When the [3H]arachidonic acid-labelled cells were stimulated with fMetLeuPhe, ATP or Ca2+ ionophore A23187, and the lipids analysed by t.l.c., the increase in free fatty acid was accompanied by decreases in label from phosphatidylinositol and phosphatidylcholine. Moreover, incorporation of label into triacylglycerol and to a lesser extent phosphatidylethanolamine was evident. Activation of secretion was evident with ATP and fMetLeuPhe but not with A23187. The pharmacological specificity of the ATP receptor in HL60 cells was investigated by measuring secretion of beta-glucuronidase, formation of inositol phosphatases and release of [3H]arachidonic acid. External addition of ATP, UTP, ITP, adenosine 5'-[gamma-thio]triphosphate (ATP[S]), adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p), XTP, CTP, GTP, 8-bromo-ATP and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) to intact HL60 cells stimulated inositol phosphate production, but only the first five nucleotides were effective at stimulating secretion or [3H]arachidonic acid release. In human neutrophils, addition of ATP, ITP, UTP and ATP[S] also stimulated secretion from specific and azurophilic granules, and this was accompanied by increases in cytosolic Ca2+ and in [3H]arachidonic acid release. The addition of phorbol 12-myristate 13-acetate (PMA; 1 nM) prior to the addition of either fMetLeuPhe or ATP led to inhibition of phospholipase C activity. In contrast, this had no effect on phospholipase A2 activation, whilst secretion was potentiated. Phospholipase A2 activation by either agonist was dependent on an intact cell metabolism, as was secretion. It is concluded that (1) activation of phospholipase C does not always lead to activation of phospholipase A2, (2) phospholipase A2 is coupled to the receptor independently of phospholipase C via a pertussis-toxin-sensitive G-protein and (3) for secretion to take place, the receptor has to activate both phospholipases C and A2.
在完整的人中性粒细胞和分化的HL60细胞中研究了磷脂酶A2和C的激活与分泌之间的关系。ATP或fMetLeuPhe激活可导致预先标记的细胞将[3H]花生四烯酸释放到细胞外培养基中。当细胞用百日咳毒素预处理时，这种反应受到抑制。当用fMetLeuPhe、ATP或Ca2+离子载体A23187刺激[3H]花生四烯酸标记的细胞，并通过薄层层析分析脂质时，游离脂肪酸的增加伴随着磷脂酰肌醇和磷脂酰胆碱标记的减少。此外，明显可见标记掺入三酰甘油，且在较小程度上掺入磷脂酰乙醇胺。ATP和fMetLeuPhe可明显激活分泌，但A23187不能。通过测量β-葡萄糖醛酸酶的分泌、肌醇磷酸酶的形成以及[3H]花生四烯酸的释放，研究了HL60细胞中ATP受体的药理学特异性。向完整的HL60细胞外添加ATP、UTP、ITP、腺苷5'-[γ-硫代]三磷酸（ATP[S]）、腺苷5'-[βγ-亚氨基]三磷酸（App[NH]p）、XTP、CTP、GTP、8-溴-ATP和鸟苷5'-[γ-硫代]三磷酸（GTP[S]）可刺激肌醇磷酸的产生，但只有前五种核苷酸在刺激分泌或[3H]花生四烯酸释放方面有效。在人中性粒细胞中，添加ATP、ITP、UTP和ATP[S]也可刺激特异性颗粒和嗜天青颗粒的分泌，同时伴随着胞质Ca2+和[3H]花生四烯酸释放的增加。在添加fMetLeuPhe或ATP之前添加佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA；1 nM）可导致磷脂酶C活性受到抑制。相反，这对磷脂酶A2的激活没有影响，而分泌则增强。两种激动剂对磷脂酶A2的激活均依赖于完整的细胞代谢，分泌也是如此。得出以下结论：（1）磷脂酶C的激活并不总是导致磷脂酶A2的激活；（2）磷脂酶A2通过对百日咳毒素敏感的G蛋白独立于磷脂酶C与受体偶联；（3）为了发生分泌，受体必须激活磷脂酶C和A2。
