Pörn M I, Slotte J P
Department of Biochemistry and Pharmacy, Abo Akademi University, Turku, Finland.
Biochem J. 1995 May 15;308 ( Pt 1)(Pt 1):269-74. doi: 10.1042/bj3080269.
The distribution of cellular unesterified cholesterol was studied in fibroblasts, which had been depleted of plasma membrane sphingomyelin by exposure to exogenous sphingomyelinase. This treatment has previously been shown to induce an increase in cholesterol esterification, a decrease in the biosynthesis of cholesterol, and a decreased susceptibility of cell cholesterol to oxidation with cholesterol oxidase. When the cellular localization of cholesterol was studied with fluorescent filipin staining, sphingomyelin depletion did not cause any visible changes in the filipin-cholesterol staining pattern, suggesting that the major part of cellular cholesterol was retained in the plasma membrane after sphingomyelinase treatment. After the oxidation of cell-surface cholesterol with cholesterol oxidase, the plasma membrane was no longer stained by filipin, but the plasma membrane cholesterol of sphingomyelin-depleted cells appeared to be resistant to oxidation with cholesterol oxidase when sphingomyelinase was used as an oxidation-promoting agent. However, the use of hypotonic buffer or phosphatidylcholine-specific phospholipase C together with cholesterol oxidase resulted in a complete oxidation of the cell-surface cholesterol in sphingomyelin-depleted cells, as evidenced by the filipin-cholesterol staining pattern. Similar results were obtained when [3H]cholesterol-labelled fibroblasts were used for determination of the susceptibility to cholesterol oxidation. The kinetics of [3H]cholesterol oxidation in sphingomyelin-depleted cells with cholesterol oxidase in hypotonic buffer indicated that approximately 85% of the cellular cholesterol still resided in the plasma membrane after sphingomyelin depletion. These results are contradictory to earlier reports on sphingomyelinase-induced changes in cellular cholesterol distribution and suggest that minor changes in the kinetics of cholesterol transport from the plasma membrane to the endoplasmic reticulum may be responsible for the sphingomyelinase-induced changes in the rates of cholesterol metabolism. Whereas the use of phospholipases to promote the oxidation of cholesterol in some instances might lead to misinterpretations, the use of hypotonic buffer together with cholesterol oxidase proved to be a more reliable method for the determination of cellular cholesterol distribution.
研究了在外源性鞘磷脂酶作用下耗尽质膜鞘磷脂的成纤维细胞中细胞未酯化胆固醇的分布情况。此前已证明这种处理会导致胆固醇酯化增加、胆固醇生物合成减少以及细胞胆固醇对胆固醇氧化酶氧化的敏感性降低。当用荧光制霉菌素染色研究胆固醇的细胞定位时，鞘磷脂耗尽并未导致制霉菌素 - 胆固醇染色模式出现任何可见变化，这表明在鞘磷脂酶处理后，细胞胆固醇的主要部分仍保留在质膜中。在用胆固醇氧化酶氧化细胞表面胆固醇后，质膜不再被制霉菌素染色，但当使用鞘磷脂酶作为氧化促进剂时，鞘磷脂耗尽细胞的质膜胆固醇似乎对胆固醇氧化酶的氧化具有抗性。然而，使用低渗缓冲液或磷脂酰胆碱特异性磷脂酶C与胆固醇氧化酶一起使用时，鞘磷脂耗尽细胞的细胞表面胆固醇会完全氧化，这由制霉菌素 - 胆固醇染色模式证明。当使用[3H]胆固醇标记的成纤维细胞来测定胆固醇氧化敏感性时，也获得了类似的结果。在低渗缓冲液中用胆固醇氧化酶氧化鞘磷脂耗尽细胞中[3H]胆固醇的动力学表明，鞘磷脂耗尽后约85%的细胞胆固醇仍存在于质膜中。这些结果与早期关于鞘磷脂酶诱导细胞胆固醇分布变化的报道相矛盾，并表明从质膜到内质网的胆固醇转运动力学的微小变化可能是鞘磷脂酶诱导胆固醇代谢速率变化的原因。虽然在某些情况下使用磷脂酶促进胆固醇氧化可能会导致误解，但使用低渗缓冲液与胆固醇氧化酶一起使用被证明是一种更可靠的测定细胞胆固醇分布的方法。
