FIGURE 5.

The effects of 4-HPR on the reversibility of inhibition of desaturase activity in rat liver microsomes or SMS-KCNR cells. A, the effects of inhibition by 4-HPR over time. Rat liver microsomes were treated with 1, 2.5, and 5 μm of 4-HPR, and the in vitro assay was performed for either 20, 60, or 120 min along with controls. Values represent the remaining activity (% to controls), and they represent the mean ± S.D. of two independent experiments performed in quadruplicate. B, the effects of dilution of 4-HPR on DES activity. Rat liver microsomes were preincubated with either 0.5 or 5 μm 4-HPR for 60 min at 37 °C. After preincubation, the microsomes were centrifuged and washed twice. The in vitro enzyme assay was performed with 100 μg of either “control microsome” or “preincubated and washed microsomes” for 60 or 120 min. Control microsomes were not washed or preincubated with 4-HPR. The values represent the remaining activity (% to controls), and they represent the mean ± S.D. of two independent experiments performed in quadruplicate. C, the effect of 4-HPR inhibition on SMS-KCNR cells. Cells were treated with 5 μm of 4-HPR for 2 h. After 2 h, cells were washed with PBS, and the medium was replaced by fresh medium. Total cell homogenates were prepared from SMS-KCNR cells after 2 h of 4-HPR treatment, and at 24, 48, and 72 h after 4-HPR treatment. Determination of DES activity was performed at these time points, using the in vitro assay with 400 μg of total cell homogenate for 20 min. Values represent the remaining activity (% to controls), and they represent the mean ± S.D. of two independent experiments performed in quadruplicate.