Johnson M M, Vaughn B, Triggiani M, Swan D D, Fonteh A N, Chilton F H
Department of Medicine, Mayo Clinic Jacksonville, Jacksonville, Florida, USA.
Am J Respir Cell Mol Biol. 1999 Aug;21(2):253-8. doi: 10.1165/ajrcmb.21.2.3489.
Increasing evidence suggests that the subcellular and glycerolipid localization of esterified arachidonic acid (AA) is a key factor in regulating its availability to lipases. The goal of the current study was to determine the potential of AA stored in triglycerides (TG) to serve as a substrate for lipases and 5-lipoxygenase during neutrophil (polymorphonuclear leukocytes, PMN) activation. PMN containing high concentrations of AA in TG were generated by culturing PMN in vitro with high concentrations of exogenous AA (eAA) for 12 h. Cellular AA increased 2- and 4-fold in PMNs incubated with 5 and 20 microM AA, respectively, and this increase was almost exclusively observed in neutral lipids (NL). Further analysis revealed that 88% of the AA in the NL fraction was associated with TG. Subsequent experiments were designed to determine whether this AA in TG could be mobilized and metabolized to eicosanoids during cell activation. TG pools of AA were increased as previously described and then PMN were stimulated with ionophore, A23187. In contrast to the 43-fold increase in TG AA after eAA loading (20 microM), free AA increased by only 1.9-fold after cell stimulation. Similarly, leukotriene (LT)B(4) production increased only 2-fold after loading TG with large quantities of AA. The magnitude of increase in free AA released and in LTB(4) formation was similar to the magnitude of increase in AA mass in phospholipase (PL), suggesting that PL, and not TG, served as the source of released AA and subsequent product generation. To confirm that AA in TG did not serve as a source for eicosanoid production, cellular pools of AA were differentially labeled with [(14)C]AA and [(3)H]AA, and the [(3)H]AA-to-[(14)C]AA ratio of LTB(4) and 20-hydroxyl LTB(4) produced during cell stimulation was measured. The [(3)H]AA/[(14)C]AA ratios of LTs were markedly different from the ratios in TG, thus providing further evidence that AA pools in TG are not a major source of AA for LT generation.
越来越多的证据表明，酯化花生四烯酸（AA）的亚细胞定位和甘油脂质定位是调节其对脂肪酶可用性的关键因素。本研究的目的是确定储存在甘油三酯（TG）中的AA在中性粒细胞（多形核白细胞，PMN）激活过程中作为脂肪酶和5-脂氧合酶底物的潜力。通过在体外将PMN与高浓度的外源性AA（eAA）培养12小时，生成了TG中含有高浓度AA的PMN。与分别用5和20μM AA孵育的PMN相比，细胞内AA分别增加了2倍和4倍，并且这种增加几乎完全出现在中性脂质（NL）中。进一步分析表明，NL部分中88%的AA与TG相关。随后的实验旨在确定在细胞激活过程中，TG中的这种AA是否可以被动员并代谢为类花生酸。如前所述，AA的TG池增加，然后用离子载体A23187刺激PMN。与eAA加载（20μM）后TG AA增加43倍相比，细胞刺激后游离AA仅增加1.9倍。同样，在TG中加载大量AA后，白三烯（LT）B4的产生仅增加2倍。释放的游离AA和LTB4形成的增加幅度与磷脂酶（PL）中AA质量的增加幅度相似，这表明PL而非TG是释放的AA和后续产物生成的来源。为了证实TG中的AA不是类花生酸产生的来源，用[¹⁴C]AA和[³H]AA对细胞内的AA池进行差异标记，并测量细胞刺激过程中产生的LTB4和20-羟基LTB4的[³H]AA/[¹⁴C]AA比值。LTs的[³H]AA/[¹⁴C]AA比值与TG中的比值明显不同，从而进一步证明TG中的AA池不是LT生成的主要AA来源。
