Figure 1.
Representative time courses for the oxidation of intact fibroblast cholesterol.
Replicate flasks were preincubated for 3°C with DME containing 5% LPDS without (○) or with 1.3 mM HPCD complexed with 0.16 mM cholesterol (•). The control and enriched cells, containing 29 and 53 µg cholesterol/mg cell protein, respectively, were fixed, washed, incubated with cholesterol oxidase at 37°C and aliquots extracted at the designated intervals for analysis, as described in Methods and ref. [8].
Figure 2.
Influence of cell cholesterol on plasma membrane and intracellular cholesterol.
To increase cell cholesterol, flasks were incubated for 8°C with DME containing 5% LPDS plus 2.2 mM HPCD bearing 0.25 mM cholesterol (•, ○) or for 3 h with 1.3 mM HPCD bearing 0.08 mM cholesterol (▴, Δ). In addition, cell cholesterol was reduced by 7% by incubating a flask for 30 min at 37°C with DME containing 5% LPDS plus 4.3 mM HPCD (♦, ◊). The cells were fixed and the kinetics of oxidation analyzed as in Figure 1. Intracellular (Panel A) and plasma membrane cholesterol (Panel B) are plotted versus the relative total cell cholesterol in each of 10 experiments, and the plots were provided with linear least squares best fits. The means (∇) and standard deviation bars at the left of each panel represent the matched untreated control cells for these experiments.
Figure 3.
Response of endoplasmic reticulum cholesterol to a jump in PM cholesterol.
Replicate flasks were preincubated for 6°C in PBS with or without 3 mM HPCD bearing 0.33 mM cholesterol. The cells were then quickly rinsed with PBS at room temperature, overlaid with PBS and chased at 37°C. At the designated times of chase, the cells in each flask were harvested and homogenized and total cell cholesterol, cell protein, and unesterified ER cholesterol quantified as described [9]. Ordinate values indicate the ratio of enriched to control cell ER cholesterol. The symbols designate two separate experiments. The zero time points were unenriched controls. Control cells had 27.0 and 26.7 µg cholesterol/mg protein; the multiple enriched flasks in the two time courses had averages of 42.6±4.2 SD (n = 3) and 48.5±2.2 SD (n = 4) µg cholesterol/mg protein.
Figure 4.
Gradient distribution of cholesterol in homogenates of control versus cholesterol-enriched fibroblasts.
Replicate flasks of fibroblasts were incubated at 37°C for 3.5 h in LPDS in the absence (•) or presence (○) of 1.2 mM HPCD bearing 0.12 mM cholesterol. The control and enriched cells contained 42 and 70 µg cholesterol/mg protein, respectively. The cells were washed, dissociated, homogenized and equivalent amounts of homogenate (measured as protein) spun to equilibrium on sucrose gradients [53]. The gradient fractions were extracted and analyzed for cholesterol (CH) and phospholipid (PL). Panel A, nmol cholesterol/fraction. Panel B, cholesterol/phospholipid, mole/mole, per fraction (left axis) and the ratios of the mole ratios in enriched versus control in fractions bearing significant amounts of material (right axis, no symbols). Abscissa, sucrose concentration as weight/volume. A representative experiment.
Figure 5.
Simulation of the distribution of complexed and uncomplexed cholesterol in plasma membranes and intracellular membranes as a function of total cell cholesterol.
CH/PL denotes moles cholesterol/mole phospholipid. Total plasma membrane cholesterol (◊); plasma membrane cholesterol complexes (•); total intracellular cholesterol (▴); uncomplexed intracellular cholesterol (▪); complexed intracellular cholesterol (▿); uncomplexed plasma membrane cholesterol (○). The cross designates typical literature values for the CH/PL of unmodified intact fibroblasts and for the CH/PL of their plasma membranes. See the text for a discussion of the values employed.