Figure 5. Effect of paraformaldehyde on 125I-PFO* binding to human fibroblasts after treatment with SMase.
On day 0, SV-589 cells were setup in medium A at 1 × 105 cells per 60-mm dish. On day 2, cells were switched to lipoprotein-deficient medium C. On day 3, cells were treated with fresh medium C containing 50 μM compactin and 50 μM mevalonate and incubated for 16 hr at 37°C. On day 4, each monolayer was washed with ice-cold PBS at 4°C and treated with 2 ml ice-cold PBS in the presence or absence of 1% (vol/vol) paraformaldehyde as indicated. After incubation at 4°C for 45 min, the paraformaldehyde-containing medium was removed, and each monolayer was washed four times with buffer A at room temperature. (A) 125I-PFO* binding. After treatment with paraformaldehyde and washing, cells were incubated with 2 ml of fresh medium D containing 50 μM mevalonate and 50 μM compactin in the presence or absence of 100 milliunits/ml SMase as indicated. After incubation for 30 min at 37°C, the cells were washed five times as described in ‘Materials and methods’ and then incubated with 2 ml of ice-cold buffer A containing 25 μg/ml 125I-PFO* (64 × 103 cpm/μg). After 2 hr at 4°C, the total amount of cell surface binding of 125I-PFO* was determined. (B) Cholesterol esterification. After treatment with paraformaldehyde and washing, each monolayer received 2 ml of fresh medium D containing 50 μM mevalonate and 50 μM compactin in the presence or absence of 50 μg protein/ml LDL. After incubation for 4 hr, 0.2 mM sodium [14C]oleate-albumin (7733 dpm/nmol) was added to the cell and incubated for additional 2 hr. After the desired incubation, the cells were harvested and the content of cholesteryl [14C]oleate was determined. (A and B) Each bar represents the mean of triplicate incubations with the individual values shown. Paraformaldehyde treatment did not significantly affect the cellular uptake of [14C]oleate as indicated by parallel measurements of the incorporation of [14C]oleate into [14C]triglycerides. In the absence of paraformaldehyde, the amount of [14C]triglycerides formed (nmol/mg protein per hr) was 51 (no addition) and 43 (+LDL); in the presence of paraformaldehyde, the values were 57 (no addition), and 32 (+LDL). These triglyceride values represent the mean of triplicate incubations. In a separate experiment, the aforementioned mean triglyceride values were 74, 46, 57, and 66 nmol/mg per hr, respectively.