Indomethacin Inhibits the Cellular Receptor-Dependent Processing of Low Density Lipoproteins; Cyclic Nucleotides Have No Effect *

Abstract

Low density lipoproteins (LDL) are taken up by cells via specific adsorptive endocytosis, lipoproteins are then disassembled in lysosomes and cholesterol is delivered to microsomes where it regulates intracellular cholesterol metabolism. A number of agents affect the LDL pathway. For example, N-ethylamaleimide inhibits the internalization of LDL receptors; colchicine, a microtubule-disaggregating agent, inhibits the fusion of pinosomes with lysosomes; and chloroquine inhibits the lysosomal degradation of LDL. Because prostaglandins and cyclic nucleotides affect many receptor-related cell functions, the effects of inhibitors of prostaglandin cyclooxygenase on the LDL pathway of cultured human cells whose LDL receptors were expressed, suppressed or deficient were examined. The effects of cyclic nucleotides and phosphodiesterase inhibitors were also examined. Heparin-elutable surface binding, heparin-resistant intracellular accumulation and lysosomal degradation of [125I]-LDL were assessed in the presence of 0-420 .mu.M indomethacin, 0-34 .mu.M meclofenamate and 0-200 .mu.M aspirin. In the presence of LDL receptors, indomethacin (280 .mu.M) inhibited [125I]LDL accumulation and degradation by .apprx. 50% in fibroblasts by .apprx. 35% in smooth muscle cells and by .apprx. 35% in endothelial cells. Meclofenamate inhibited [125I]LDL accumulation and degradation up to 40% in fibroblasts and 15% in aortic smooth muscle cells. Aspirin, even at maximum doses, had no inhibitory effects. Cells with decreased receptor activity were not inhibited by these drugs. Indomethacin did not affect cellular protein mass or protein synthesis and its effects on the LDL pathway were reversible, suggesting that it did not act by inhibiting energy-dependent processes. Indomethacin did not affect the number of functioning [125I]LDL receptors. Rather, it affected the tightness of binding of [125I]LDL to receptors and thereby inhibited the internalization of [125I]LDL. The lack of effect of aspirin and the need for larger doses of indomethacin and meclofenamate than are needed for the inhibition of cyclooxygenase (.apprx. 10 .mu.M) suggest that the inhibition of LDL internalization occurred via a cyclooxygenase-independent pathway. The effects of cyclic nucleotides (cAMP and cGMP) and their stable derivatives (dibutyryl and 8-bromo) were evaluated in experiments which contained 0.1-2 mM nucleotides or the same concentrations of phosphodiesterase inhibitors. Fibroblasts, incubated for 4-16 h in the presence of these reagents, bound and processed the same amounts of [125I]LDL as did cells incubated in the absence of these agents. Combinations of cyclic nucleotides and phosphodiesterase inhibitors diminished the processing of [125I]LDL by 15-30%. Cyclic nucleotides may not play important roles in modulating the LDL pathway of cultured fibroblasts.