Cationic amphiphilic drugs such as chloroquine, 4,4'-bis (diethylaminoethoxy)-alpha, beta-diethyldiphenylethane (DH), chlorphentermine and others can cause an iatrogenic lipid storage disease in man or animals. In the past three years, our exeriments have provided several new insights into the molecular basis for this interesting drug toxicity. In the liver of rats treated with chloroquine of DH, the excess phospholipid is found exclusively in lysosomes, while other subcellular membranes such as mitochondria and microsomes have a normal phospholipid content. Chloroquine and DH also concentrate to a great degree in lysosomes. In our studies of phospholipid degradation, we discovered an active phopholipase C in rat liver lysosomes, the first description of this enzyme outside of bacteria. We also found that chloroquine and DH are effective inhibitors of lysosomal phospholipid A and C. Thus, it appears that the cationic amphiphilic drugs cause phospholipid storage by concentrating in lysosomes and inhibiting phospholipases (directly, and indirectly, by raising pH). This renewal project proposes to examine further the molecular and metabolic basis for drug-induced lipidosis in rats and in tissue culture cells. The enzyme involved in the synthesis of bis(monoacylglycero)-phosphate, a phospholipid which is greatly elevated in this disease, will be purified from liver lysosomes as will lysosomal phospholipases A and C. The effects of cationic drugs on these three enzymes will be examined in detail. Using cultured cells, we will determine the source of the stored phospholipid, external (lipoprotein) vs. internal (autophagy). The role of biliary phospholipid excretion in this disorder will be studied. Drug concentrations in lysosomes will be determined throughout the course of the experimental lipidosis both in rat liver and in cultured cells using an advanced methodology, free-flow electrophoresis.
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