Tilorone-induced lysosomal storage of glycosaminoglycans in cultured corneal fibroblasts: biochemical and physicochemical investigations.
Tilorone (2,7-bis[2-(diethylamino)ethoxy]-fluoren-9-one) and several other bis-basic compounds are known to induce lysosomal glycosaminoglycan (GAG) storage. The responsible pathomechanism has not been elucidated yet. The assumption of an unspecific disturbance of lysosomal proenzyme targeting due to elevation of endosomal pH is opposed by the hypothesis of formation of a complex between tilorone and GAGs within the lysosomes, which renders GAGs indigestible to glycosidases. In cultures of bovine corneal fibroblasts the amounts of intracellular GAGs [dermatan sulphate (DS), heparan sulphate (HS) and chondroitin sulphate (CS)] were quantified. The fibroblasts were exposed to tilorone (5 microM), which was found to be readily taken up by the cells and to be accumulated within acidic compartments to finally achieve millimolar concentrations. Under these conditions the GAG storage is predominantly due to the accumulation of DS; however, the DS secretion into the culture medium was not affected. The HS accumulation was much less pronounced, accounting only for 3% of total GAG storage. Ammonium chloride (10 mM), which is known to diminish lysosomal enzyme activity by interfering with the mannose 6-phosphate receptor-mediated transport, prevents both HS and DS breakdown. By means of NMR spectroscopy it was shown that tilorone itself tends to display a concentration-dependent aggregation which was enhanced in the presence of GAGs. The diethylamino groups of tilorone interact physicochemically with DS, and to a smaller extent with HS, but not with chondroitin 4-sulphate. Thus, the strength of the interaction between tilorone and the different GAGs in vitro correlates with the potency of tilorone to inhibit the breakdown of the individual GAGs in cultured bovine fibroblasts. The results support the hypothesis of a specific interaction between tilorone and particular GAGs, rendering these resistant to enzymic degradation.
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002809:Chondroitin Sulfates / Q000737:chemistry / Q000378:metabolism; D003315:Cornea / Q000166:cytology / Q000378:metabolism*; D003871:Dermatan Sulfate / Q000737:chemistry / Q000378:metabolism; D004586:Electrophoresis; D005347:Fibroblasts / Q000378:metabolism; D006025:Glycosaminoglycans / Q000032:analysis / Q000378:metabolism*; D006497:Heparan Sulfate / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D013994:Tilorone / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*
D000643:Ammonium Chloride; D002809:Chondroitin Sulfates; D003871:Dermatan Sulfate; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D013994:Tilorone