The effect of PI3K inhibitors and cytochalasin D on LDL uptake and net cholesterol accumulation in LDLR −/− macrophages.

Abstract

<p>LDLR−/− bone marrow-derived macrophages were pretreated 1 h with either drug vehicle, 50 µM LY294002, 100 nm wortmannin, or 4 µg/ml cytochalasin D. For cholesterol accumulation, macrophages then were incubated with 1 mg/ml LDL and inhibitor for 24 h. For ¹²⁵I-LDL uptake, macrophages then were incubated 5 h with 200 µg/ml ¹²⁵I-LDL and inhibitor. All incubations were performed in serum-free medium containing 50 ng/ml M-CSF. The percent inhibition of net cholesterol accumulation compares macrophages treated with LDL alone and LDL with inhibitor after basal cholesterol values were subtracted from each. The percent inhibition of ¹²⁵I-LDL uptake compares macrophages treated with ¹²⁵I-LDL alone and ¹²⁵I-LDL with inhibitor. ** = <i>p</i><0.01. *** = <i>p</i><0.001. All inhibitors showed almost complete inhibition of macropinosome formation as assessed by phase-microscopy. For LY294002 and wortmannin experiments, control macrophages incubated with LDL or ¹²⁵I-LDL alone showed net cholesterol accumulation and ¹²⁵I-LDL uptake values of 226±8 nmole/mg cell protein and 2.7±0.1 µg/mg cell protein, respectively. For cytochalasin D experiments, control macrophages incubated with LDL or ¹²⁵I-LDL alone showed net cholesterol accumulation and ¹²⁵I-LDL uptake values of 230±2 nmole/mg cell protein and 3.4±0.1 µg/mg cell protein, respectively. The range of cell-associated and degraded ¹²⁵I-LDL for all treatments was 19–33% and 67–81%, respectively.</p