Best Katrina B, Ohran Allison J, Hawes Andrea C, Hazlett Theodore L, Gratton Enrico, Judd Allan M, Bell John D
Department of Physiology and Developmental Biology, Brigham Young University, Provo, Utah 84602, USA.
Biochemistry. 2002 Nov 26;41(47):13982-8. doi: 10.1021/bi026796r.
Normally, cell membranes resist hydrolysis by secretory phospholipase A(2). However, upon elevation of intracellular calcium, the cells become susceptible. Previous investigations demonstrated a possible relationship between changes in lipid order caused by increased calcium and susceptibility to phospholipase A(2). To further explore this relationship, we used temperature as an experimental means of manipulating membrane physical properties. We then compared the response of human erythrocytes to calcium ionophore at various temperatures in the range of 20-50 degrees C using fluorescence spectroscopy and two-photon fluorescence microscopy. The steady state fluorescence emission of the environment-sensitive probe, laurdan, revealed that erythrocyte membrane order decreases systematically with temperature throughout this range, especially between 28 and 45 degrees C. Furthermore, the ability of calcium ionophore to induce increased membrane order and susceptibility to phospholipase A(2) depended similarly on temperature. Both responses to calcium influx were enhanced as membrane fluidity increased. Analysis of the spatial distribution of laurdan fluorescence at several temperatures indicated that the ordering effect of intracellular calcium on fluid membranes generates an increase in the number of fluid-solid boundaries. Hydrolysis of the membrane appeared to progress outward from these boundaries. We conclude that phospholipase A(2) prefers to hydrolyze lipids in fluid regions of human erythrocyte membranes, but primarily when those regions coexist with domains of ordered lipids.
正常情况下，细胞膜可抵抗分泌型磷脂酶A2的水解作用。然而，细胞内钙水平升高时，细胞就变得易受影响。先前的研究表明，钙增加引起的脂质有序性变化与对磷脂酶A2的敏感性之间可能存在关联。为了进一步探究这种关系，我们使用温度作为操纵膜物理性质的实验手段。然后，我们利用荧光光谱法和双光子荧光显微镜，比较了人红细胞在20至50摄氏度范围内不同温度下对钙离子载体的反应。环境敏感探针劳丹的稳态荧光发射显示，在整个这个温度范围内，尤其是在28至45摄氏度之间，红细胞膜的有序性随温度系统性降低。此外，钙离子载体诱导膜有序性增加以及对磷脂酶A2敏感性增加的能力同样取决于温度。随着膜流动性增加，对钙内流的这两种反应均增强。对几个温度下劳丹荧光空间分布的分析表明，细胞内钙对流体膜的有序化作用会使流体 - 固体边界的数量增加。膜的水解似乎从这些边界向外推进。我们得出结论，磷脂酶A2更倾向于水解人红细胞膜流体区域中的脂质，但主要是当这些区域与有序脂质区域共存时。
