The long range goal of this project is to determine the mechanisms by which cationic amphiphilic drugs (amiodarone) and aminoglycosides (gentamicin) cause phospholipid storage in the lysosomes of their target tissues leading ultimately to cell damage. The project stresses evaluation of drug-induced alterations in the intracellular processing of complex lipids. A newly-discovered class of intracellular inhibitor proteins will be characterized and the role of these protein(s) in drug-induced lipidosis will be assessed. The proposed studies have health-related implications since the drugs are used by large numbers of patients and the studies are relevant to side effects suffered by users which include: (1) nephrotoxicity (aminoglycosides) and (2) hepatic and pulmonary lipidosis and fibrosis (amiodarone).
The specific aims are to determine the biochemical basis for the phospholipidosis produced by cationic amphiphiles such as amiodarone (specific aim 1) and aminoglycosides (specific aim 2). The problem will be approached by determining the intracellular distribution of these agents in liver, lung and kidney and measuring their intralysosomal concentration using our recently published method. Lysosomal phospholipases A and C will be purified and inhibitor studies carried out to determine the mechanism(s). Drug binding to substrate will also be measured and taken into account in determination of the mechanism(s) involved in inhibition of the lipolytic enzymes. Since fibrosis is a serious clinical side effect of amiodarone studies will be undertaken with amiodarone to evaluate the relationship of phospholipidosis to fibrosis and collagen metabolism in the liver and lung of rats in vivo and in cultured fibroblasts. The third specific aim regards the characterization of the intracellular protein inhibitors of phospholipase which we recently identified during the purification of kidney phospholipase A1. The inhibitory proteins will be purified based on their ability to inhibit pure phospholipases and their involvement in drug induced lipidosis and aminoglycoside nephrotoxicity will be investigated in vitro and in vivo. The effect of glucocorticoids on these proteins will be determined since lysosomal """"""""stabilization"""""""" by glucocorticoids might involve the cortisol-dependent synthesis of phospholipase inhibitors similar to that described for """"""""lipomodulin"""""""", """"""""macrocortin"""""""" and """"""""renocortin"""""""".
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