The effect of possible inhibitors of fluid-phase pinocytosis on LDL uptake and net cholesterol accumulation in wild-type M-CSF-differentiated macrophages.

Abstract

<p>Wild-type bone marrow-derived macrophages were pretreated 1 h with drug vehicle or the indicated drug. For cholesterol accumulation, macrophages then were incubated with 1 mg/ml LDL and inhibitor for 6 h. For ¹²⁵I-LDL uptake, macrophages then were incubated with 200 µg/ml ¹²⁵I-LDL and inhibitor for 6 h. 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 (except the experiment that compares macrophages treated with LDL and dynamin peptide inhibitor with LDL and control peptide) 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. Control values for macrophages incubated with LDL alone or ¹²⁵I-LDL alone are indicated in parentheses. For cholesterol accumulation, control values are expressed as nmol net cholesterol accumulation/mg cell protein. For ¹²⁵I-LDL uptake, control values are expressed as µg ¹²⁵I-LDL uptake/mg cell protein. The range of cell-associated and degraded ¹²⁵I-LDL for all treatments except bafilomycin A1-treated macrophages was 15–19% and 82–86%, respectively. Cell-associated and degraded ¹²⁵I-LDL for bafilomycin A1-treated macrophages was 91% and 9%, respectively. * = <i>p</i><0.05. ** = <i>p</i><0.01. *** = <i>p</i><0.001. ND = not determined. “+” indicates a decrease in the number of macropinosomes and “−” indicates no effect on macropinosomes. “−” in percent inhibition columns indicates stimulation rather than inhibition.</p