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Journal of Lipid Research, Vol 32, 329-339, Copyright © 1991 by Lipid Research, Inc.
Y Lange
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.
ARTICLES
Disposition of intracellular cholesterol in human fibroblasts
Department of Pathology, Rush-Presbyterian-St.Luke's Medical Center, Chicago, IL 60612-3864.
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