Reconstituting the barrier properties of a water-tight epithelial membrane by design of leaflet-specific liposomes
Reconstituting the barrier properties of a water-tight epithelial membrane by design of leaflet-specific liposomes
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DOI：
10.1074/jbc.m003494200
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发表时间：
2000-09-29
影响因子：
4.8
通讯作者：
Zeidel, ML
中科院分区：
文献类型：
--
作者：
Hill, WG;Zeidel, ML
为了确定脂质成分和双层不对称对屏障功能至关重要的方面，我们检查了脂质体的通透性，脂质体模拟屏障上皮细胞顶膜的单个小叶，Madin-Darby犬肾脏1型细胞。利用已发表的脂类组合物，我们制备了含有磷脂酰胆碱、鞘磷脂、糖鞘脂脂和胆固醇的表面脂质体，以及含有磷脂酰乙醇胺、磷脂酰丝氨酸和胆固醇的细胞质脂质体。细胞内脂质体对水(P-f)、溶质和氨氮的渗透渗透率是表面脂质体的18-90倍(P-f(Ex)=2.4+/-0.4×10(-4)cm/S，P-f(Cy)=4.4+/-0.3×10(-3)cm/S)；P-甘油(EX)=2.5+/-0.3×10(-8)cm/S，P-甘油(Cy)=2.2+/-0.02×10(-6)cm/S；P-NH3(EX)=0.13+/-0.4×10(-4)cm/S，P-NH3(Cy)=7.9+/-1.0×10(-3)cm/S。而表面脂质体的表观质子渗透率是胞浆脂质体的4倍(PH+(Ex)=1.1+/-0.1×10(-2)cm/S，PH+(Cy)=2.7+/-0.6×10(-3)cm/S)。通过加入单叶通透性，我们计算了4.6x10(-4)cm/S的Madin-Darby犬肾尖膜的理论P-f，与实验值吻合较好。在不含鞘糖脂或鞘磷脂的表面脂质体中，透过率是对照组的2-7倍，表明这两种物质都具有屏障作用。去除胆固醇导致通透性增加40-280倍。Fire的结论是：1)我们已经重建了屏障膜的生物物理性质，2)屏障存在于表面小叶中，3)鞘磷脂和神经鞘糖脂都在降低膜通透性方面发挥作用，但胆固醇需要绝对的物质来介导这种作用，4)这些结果进一步证实了这样的假设，即每个小叶都提供了独立的渗透阻力，以及5)鞘磷脂/胆固醇相互作用促进了质子渗透。
To define aspects of lipid composition and bilayer asymmetry critical to barrier function, we examined the permeabilities of liposomes that model individual leaflets of the apical membrane of a barrier epithelium, Madin-Darby canine kidney type 1 cells. Using published Lipid compositions we prepared exofacial liposomes containing phosphatidylcholine, sphingomyelin, glycosphingolipids, and cholesterol; and cytoplasmic liposomes containing phosphatidylethanolamine, phosphatidylserine, and cholesterol. The osmotic permeability of cytoplasmic liposomes to water (P-f), solutes, and NH3 was 18-90-fold higher than for the exofacial liposomes (P-f(ex) = 2.4 +/- 0.4 x 10(-4) cm/s, P-f(cy) = 4.4 +/- 0.3 x 10(-3) cm/s; P-glycerol(ex) = 2.5 +/- 0.3 x 10(-8) cm/s, P-glycerol(cy) = 2.2 +/- 0.02 x 10(-6) cm/s; P-NH3(ex) = 0.13 +/- 0.4 x 10(-4) cm/s, P-NH3(cy) = 7.9 +/- 1.0 x 10(-3) cm/s). By contrast, the apparent proton permeability of exofacial liposomes was 4-fold higher than cytoplasmic liposomes (PH+(ex) = 1.1 +/- 0.1 x 10(-2) cm/s, PH+(cy) = 2.7 +/- 0.6 x 10(-3) cm/s). By adding single leaflet permeabilities, we calculated a theoretical P-f for a Madin-Darby canine kidney apical membrane of 4.6 x 10(-4) cm/s, which compares favorably with experimentally determined values. In exofacial liposomes lacking glycosphingolipids or sphingomyelin, permeabilities were 2-7-fold higher, indicating that both species play a role in barrier function. Removal of cholesterol resulted in 40-280-fold increases in permeability. Fire conclude: 1) that we have reconstituted the biophysical properties of a barrier membrane, 2) that the barrier resides in the exofacial leaflet, 3) that both sphingomyelin and glycosphingolipids play a role in reducing membrane permeability but that there is an ab solute requirement for cholesterol to mediate this effect, 4) that these results further validate the hypothesis that each leaflet offers an independent resistance to permeation, and 5) that proton permeation was enhanced by sphingolipid/cholesterol interactions.