Disposition of intracellular cholesterol in human fibroblasts.
Disposition of intracellular cholesterol in human fibroblasts.
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发表时间：
1991-02
影响因子：
6.5
通讯作者：
Yvonne Lange
中科院分区：
文献类型：
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作者：
Yvonne Lange
我们在培养的人成纤维细胞中检测了未酯化胆固醇的细胞内分布。用胆固醇氧化酶处理完整细胞，选择性地将细胞表面胆固醇转化为胆甾酮。匀浆等离心表明胆甾酮的浮力密度峰值为1.13 g/cm3。未氧化的总胆固醇约10%分布在两个大小大致相等的峰上：一个尖峰约为1.09 g/cm3，一个宽峰以1.18 g/cm3为中心。当完整细胞与外源性[3H]胆固醇一起孵育时，放射性标记物在37℃下以温度依赖的方式进入非氧化池，半时间为3小时。该标记物最初与非氧化胆固醇的致密峰相关，但与浮力峰无关。40 h后，浮力峰也被标记；两个峰的比活性都略低于表面胆甾酮。未氧化胆固醇的浮力密度与高尔基体、内质网或溶酶体的标记物不一致。然而，摄入的两种胞饮标志物，钙黄蛋白和辣根过氧化物酶，与未氧化胆固醇的密集峰同步。在无血清脂蛋白的细胞中，未氧化胆固醇池的大小比喂养细胞中更大，这表明细胞内胆固醇不需要归因于摄入的固醇。当洛伐他汀抑制脂蛋白剥夺细胞中的胆固醇生物合成时，不可氧化胆固醇的质量并未减少。此外，新合成的抗胆固醇氧化酶的放射性标记胆固醇不随细胞内胆固醇质量在蔗糖密度梯度上迁移。新合成的胆固醇约占未氧化甾醇总量的10%。这些数据表明，大多数细胞内胆固醇不是新合成的。我们得出结论：a)大约90%的成纤维细胞胆固醇与细胞表面有关；B)细胞内胆固醇的大部分，约占总胆固醇的10%，来自内化（内吞）质膜；c)最近合成的胆固醇，大约占总量的1%，在一个离散的细胞器中。
We have examined the intracellular distribution of unesterified cholesterol in cultured human fibroblasts. Intact cells were treated with cholesterol oxidase to selectively transform cell surface cholesterol to cholestenone. Isopycnic centrifugation of homogenates showed that the cholestenone had a peak buoyant density of 1.13 g/cm3. The approximately 10% of total cholesterol which remained unoxidized was distributed in two peaks of roughly equal size: a sharp peak at approximately 1.09 g/cm3 and a broad peak centered at 1.18 g/cm3. When intact cells were incubated with exogenous [3H]cholesterol, the radiolabel entered the nonoxidizable pool in a temperature-dependent fashion with a half time of 3 h at 37 degrees C. This label initially was associated with the dense but not the buoyant peak of nonoxidized cholesterol. After 40 h, the buoyant peak also became labeled; both peaks then had a specific activity slightly less than the surface cholestenone. The buoyant density of the unoxidized cholesterol did not coincide with markers for the Golgi apparatus, endoplasmic reticulum, or lysosomes. However, two ingested markers of pinocytosis, calcein and horseradish peroxidase, comigrated with the dense peak of unoxidized cholesterol. That the size of the unoxidized cholesterol pool was greater in cells deprived of serum lipoproteins than in fed cells suggested that none of the intracellular cholesterol need be ascribed to ingested sterols. The mass of unoxidizable cholesterol was not diminished when cholesterol biosynthesis was inhibited by lovastatin in lipoprotein-deprived cells. Furthermore, the newly synthesized radiolabeled cholesterol resistant to cholesterol oxidase did not migrate with intracellular cholesterol mass on sucrose density gradients. The newly synthesized cholesterol amounted to about 10% of the total unoxidized sterol. These data indicate that most of the intracellular cholesterol was not newly synthesized. We conclude that a) approximately 90% of fibroblast cholesterol is associated with the cell surface; b) the bulk of intracellular cholesterol, approximately 10% of total, is derived from internalized (endocytic) plasma membrane; and c) the most recently synthesized cholesterol, approximately 1% of the total, is in a discrete organelle.