Hardy S J, Robinson B S, Poulos A, Harvey D P, Ferrante A, Murray A W
School of Biological Sciences, Flinders University of South Australia, Adelaide.
Eur J Biochem. 1991 Jun 15;198(3):801-6. doi: 10.1111/j.1432-1033.1991.tb16084.x.
The oxygen-dependent respiratory burst is a key neutrophil function required for the killing of bacteria. However, despite intensive investigation, the molecular events which initiate the respiratory burst remain unclear. Recent reports have suggested the agonist-induced hydrolysis of cellular phosphatidylcholine (PtdCho) by phospholipase D may be an essential requirement for initiating or mediating the respiratory burst. We have investigated the effects of the chemotactic peptide N-formylmethionylleucylphenylalanine (fMLF), the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and the polyunsaturated fatty acids arachidonic [20:4 (n-6)] and docosahexaenoic [22:6 (n-3)] acids in light of this hypothesis. Ethanol-inhibited superoxide production in response to 20:4, 22:6 and fMLF, in a dose-dependent fashion, suggesting an involvement of phospholipase D. The phosphatidic-acid phosphohydrolase inhibitor DL-propranolol completely inhibited superoxide production induced by both 20:4 and 22:6, and partially inhibited the response to TPA. In contrast, superoxide production in response to fMLF was increased by propranolol. fMLF and TPA, but not the fatty acids, stimulated phospholipase D as indicated by the accumulation of phosphatidic acid and, in the presence of ethanol, phosphatidylethanol derived from PtdCho. Extracellular Ca2+ was found to be an essential requirement for fMLF-induced superoxide production. However, responses to the fatty acids were dramatically enhanced under Ca(2+)-free conditions. Responses to TPA were independent of the extracellular Ca2+ concentration. Both fatty acids and fMLF, but not TPA, mobilised Ca2+ from intracellular stores, a response insensitive to the effects of both ethanol and propranolol. These results show that, unlike fMLF and TPA, the fatty acids do not cause hydrolysis of PtdCho by phospholipase D. However, the data indirectly suggests that the fatty acids may initiate the phospholipase-D-catalysed hydrolysis of phospholipids other than PtdCho.
氧依赖型呼吸爆发是杀灭细菌所需的关键中性粒细胞功能。然而，尽管进行了深入研究，引发呼吸爆发的分子事件仍不清楚。最近的报道表明，激动剂诱导的磷脂酶D对细胞磷脂酰胆碱（PtdCho）的水解可能是启动或介导呼吸爆发的必要条件。根据这一假设，我们研究了趋化肽N-甲酰甲硫氨酰亮氨酰苯丙氨酸（fMLF）、佛波酯12-O-十四酰佛波醇13-乙酸酯（TPA）以及多不饱和脂肪酸花生四烯酸[20:4（n-6）]和二十二碳六烯酸[22:6（n-3）]的作用。乙醇以剂量依赖的方式抑制对20:4、22:6和fMLF的超氧化物生成，提示磷脂酶D参与其中。磷脂酸磷酸水解酶抑制剂DL-普萘洛尔完全抑制20:4和22:6诱导的超氧化物生成，并部分抑制对TPA的反应。相反，普萘洛尔增加了对fMLF的超氧化物生成。如磷脂酸的积累以及在乙醇存在下由PtdCho衍生的磷脂酰乙醇所示，fMLF和TPA而非脂肪酸刺激了磷脂酶D。发现细胞外Ca2+是fMLF诱导超氧化物生成的必要条件。然而，在无Ca2+条件下，对脂肪酸的反应显著增强。对TPA的反应与细胞外Ca2+浓度无关。脂肪酸和fMLF而非TPA从细胞内储存库中动员Ca2+，该反应对乙醇和普萘洛尔的作用均不敏感。这些结果表明，与fMLF和TPA不同，脂肪酸不会通过磷脂酶D导致PtdCho水解。然而，数据间接表明脂肪酸可能启动磷脂酶D催化的除PtdCho之外的其他磷脂水解。
