Selective Inhibition of Free Apolipoprotein-Mediated Cellular Lipid Efflux by Probucol

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Abstract
We attempted to demonstrate selective modulation of lipid-free apolipoprotein-mediated cellular lipid efflux in order to test the hypothesis that it is an event independent of nonspecific physicochemical cholesterol exchange. Probucol, a unique cholesterol-lowering drug, was found to selectively suppress this pathway in vitro in mouse peritoneal macrophage. Probucol was given to the cells via the uptake of acetylated low-density lipoprotein (LDL) into which it had been incorporated. The uptake of lipoprotein-cholesteryl ester by the macrophage was the same whether the acetylated LDL was probucol-carrying or probucol-free, and probucol accumulated in the cell in parallel to cholesterol when carried by the lipoprotein. Incorporation of [35S]methionine into cell protein was unaffected by probucol accumulated in the cells. The efflux of cellular cholesterol and phospholipid mediated by lipid-free human apolipoproteins (apo) A-I, A-II, and E was all completely inhibited by probucol. Reversible binding of free apoA-I to the cellular surface was also completely blocked by probucol in this condition. On the other hand, nonspecific cholesterol exchange between LDL and macrophage was unaffected by probucol. Thus, probucol selectively inhibited apolipoprotein-mediated cellular lipid efflux by blocking specific binding of free apolipoprotein to the cell without influencing nonspecific lipid exchange. In the absence of lecithin: cholesterol acyltransferase (LCAT) reaction, apparent cellular cholesterol efflux to high-density lipoprotein (HDL) was reduced by probucol by 40−70% while the rate of cholesterol influx from HDL to the cells was unaffected, resulting in cancellation of the net cellular cholesterol efflux to HDL. However, the increase of the net cholesterol efflux to HDL by LCAT was unaffected by probucol. Net cellular cholesterol efflux to HDL in the absence of LCAT, therefore, seems to depend on an apolipoprotein-mediated mechanism.