Nakashima S, Nagata K, Ueeda K, Nozawa Y
Department of Biochemistry, Gifu University School of Medicine, Japan.
Arch Biochem Biophys. 1988 Mar;261(2):375-83. doi: 10.1016/0003-9861(88)90353-0.
Addition of a guanine nucleotide analog, guanosine 5'-O-(thiotriphosphate) (GTP gamma S)(1-100 microM) induced release of [3H]arachidonic acid from [3H]arachidonate-prelabeled rabbit neutrophils permeabilized with saponin. The chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced arachidonate release was enhanced by GTP gamma S, Ca2+, or their combination. Ca2+ alone (up to 100 microM) did not effectively stimulate lipid turnover. However, the combination of fMLP plus GTP gamma S elicited greater than additional effects in the presence of resting level of free Ca2+. The addition of 100 microM of GTP gamma S reduced the Ca2+ requirement for arachidonic acid liberation induced by fMLP. Pretreatment of neutrophils with pertussis toxin resulted in the abolition of arachidonate release and diacylglycerol formation. Neomycin (1 mM) caused no significant reduction of arachidonate release. In contrast, about 40% of GTP gamma S-induced arachidonate release was inhibited by a diacylglycerol lipase inhibitor, RHC 80267 (30 microM). These observations indicate that liberation of arachidonic acid is mediated by phospholipase A2 and also by phospholipase C/diacylglycerol lipase pathways. Fluoride, which bypasses the receptor and directly activates G proteins, induced arachidonic acid release and diacylglycerol formation. The fluoride-induced arachidonate release also appeared to be mediated by these two pathways. The loss of [3H]arachidonate was seen in phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine. These data indicate that a G protein is involved between the binding of fMLP to its receptor and activation of phospholipase A2, and also that the arachidonic acid release is mediated by both phospholipase A2 and phospholipase C/diacylglycerol lipase.
添加鸟嘌呤核苷酸类似物5'-O-(硫代三磷酸)鸟苷(GTPγS)(1 - 100微摩尔)可诱导用皂角苷通透的、预先用[3H]花生四烯酸标记的兔中性粒细胞释放[3H]花生四烯酸。趋化肽N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸(fMLP)诱导的花生四烯酸释放可被GTPγS、Ca2+或它们的组合增强。单独的Ca2+(高达100微摩尔)不能有效刺激脂质周转。然而，在游离Ca2+处于静息水平时，fMLP加GTPγS的组合引发的效应大于两者单独作用之和。添加100微摩尔的GTPγS降低了fMLP诱导花生四烯酸释放所需的Ca2+浓度。用百日咳毒素预处理中性粒细胞会导致花生四烯酸释放和二酰基甘油形成的消失。新霉素(1毫摩尔)不会显著降低花生四烯酸释放。相反，二酰基甘油脂肪酶抑制剂RHC 80267(30微摩尔)可抑制约40%的GTPγS诱导的花生四烯酸释放。这些观察结果表明，花生四烯酸的释放由磷脂酶A2介导，也由磷脂酶C/二酰基甘油脂肪酶途径介导。氟化物绕过受体直接激活G蛋白，诱导花生四烯酸释放和二酰基甘油形成。氟化物诱导的花生四烯酸释放似乎也由这两条途径介导。在磷脂酰肌醇、磷脂酰胆碱和磷脂酰乙醇胺中可观察到[3H]花生四烯酸的损失。这些数据表明，在fMLP与其受体结合和磷脂酶A2激活之间涉及一种G蛋白，并且花生四烯酸的释放由磷脂酶A2和磷脂酶C/二酰基甘油脂肪酶介导。
