Brock T G, Nagaprakash K, Margolis D I, Smolen J E
Department of Pediatrics, University of Michigan Medical Center, Ann Arbor 48109-0684.
J Membr Biol. 1994 Aug;141(2):139-48. doi: 10.1007/BF00238247.
Degranulation involves the regulated fusion of granule membrane with plasma membrane. To study the role of lipid composition in degranulation, large unilamellar vesicles (LUVs) of increasing complexity in lipid compositions were constructed and tested for Ca(2+)-mediated lipid and contents mixing. Lipid-mixing rates of LUVs composed of phosphatidylethanolamine (PE) and phosphatidylserine (PS) were strongly decreased by the addition of either phosphatidylcholine (PC) or sphingomyelin (SM), while phosphatidylinositol (PI) had little effect. "Complex" LUVs of PC:PE:SM:PI:PS (24:27:20:16:13, designed to emulate neutrophil plasma membranes) also showed very low rates of both lipid mixing and contents mixing. The addition of cholesterol significantly lowered the Ca2+ threshold for contents mixing and increased the maximum rates of both lipid and contents mixing in a dose-dependent manner. Membrane remodeling, which occurs in neutrophil plasma membranes upon stimulation, was simulated by incorporating low levels of phosphatidic acid (PA) or a diacylglycerol (DAG) into complex LUVs containing 50% cholesterol. The addition of PA both lowered the Ca2+ threshold and increased the rate of contents mixing in a dose-dependent manner, while the DAG had no significant effect. The interaction of dissimilar LUVs was also examined. Contents-mixing rates of LUVs of two different cholesterol contents were intermediate between the rates observed for the LUVs of identical composition. Thus, cholesterol needed to be present in only one fusing partner to enhance fusion. However, for PA to stimulate fusion, it had to be present in both sets of LUVs. These results suggest that the rate of degranulation may be increased by a rise in the cholesterol level of either the inner face of the plasma membrane or the outer face of the granule membrane. Further, the production of PA can promote fusion, and hence degranulation, whereas the subsequent conversion of PA to DAG may reverse this promotional effect.
脱颗粒涉及颗粒膜与质膜的调控融合。为了研究脂质组成在脱颗粒中的作用，构建了脂质组成复杂度不断增加的大单层囊泡（LUVs），并测试其钙（Ca2+）介导的脂质和内容物混合情况。由磷脂酰乙醇胺（PE）和磷脂酰丝氨酸（PS）组成的LUVs的脂质混合速率，在添加磷脂酰胆碱（PC）或鞘磷脂（SM）后显著降低，而磷脂酰肌醇（PI）影响较小。PC:PE:SM:PI:PS比例为24:27:20:16:13的“复杂”LUVs（旨在模拟中性粒细胞质膜）的脂质混合和内容物混合速率也非常低。添加胆固醇显著降低了内容物混合的Ca2+阈值，并以剂量依赖的方式提高了脂质和内容物混合的最大速率。通过将低水平的磷脂酸（PA）或二酰基甘油（DAG）掺入含有50%胆固醇的复杂LUVs中，模拟了中性粒细胞质膜在刺激时发生的膜重塑过程。添加PA既降低了Ca2+阈值，又以剂量依赖的方式提高了内容物混合速率，而DAG没有显著影响。还研究了不同LUVs之间的相互作用。两种不同胆固醇含量的LUVs的内容物混合速率介于相同组成的LUVs所观察到的速率之间。因此，胆固醇只需存在于一个融合伙伴中就能增强融合。然而，要使PA刺激融合，它必须同时存在于两组LUVs中。这些结果表明，质膜内表面或颗粒膜外表面胆固醇水平的升高可能会增加脱颗粒速率。此外，PA的产生可以促进融合，从而促进脱颗粒，而随后PA向DAG的转化可能会逆转这种促进作用。
