Lysosomal glycosaminoglycan storage as induced by dicationic amphiphilic drugs: investigation into the mechanisms underlying the slow reversibility.
Several dicationic amphiphilic compounds, such as the immunomodulator tilorone and analogues, impair the lysosomal catabolism of sulphated glycosaminoglycans (GAGs). Thereby they cause lysosomal GAG storage in rats and in cultured fibroblasts of several species including man. The GAG storage is rather slowly reversible in vivo; it persists for months after discontinuance of drug treatment. In the present study, we investigated the mechanisms underlying the slow reversibility. Cultured bovine corneal fibroblasts were pretreated for 4 days with tilorone (5 and 20 microM) or with compound CL-90.100 (3 and 10 microM) and further cultured in drug-free medium for periods up to 11 days. The intracellular GAG storage was analysed biochemically and demonstrated histochemically. The subcellular drug distribution (CL-90.100) was demonstrated by fluorescence microscopy. Dermatan sulphate (DS) provided the predominant contribution towards the GAG storage. After pretreatments with the low, as well as the high concentrations of either drug, the storage of DS was irreversible during the period of observation, whereas the minor storage of heparan sulphate was resolved. The enhanced secretion of the lysosomal enzyme beta-hexosaminidase (E.C. 3.2.1.52) caused by pretreatment with the high concentration of tilorone was also readily reversible. Thus, enzyme deprivation could not be the explanation for the sustained DS storage. The localization of the drug-related fluorescence within perinuclear cell organelles, presumably lysosomes, resembled that of the stored GAGs as visualized by histochemical staining. Both, the fluorescence and the positive GAG staining persisted with unchanged intracellular distribution throughout the recovery period. The present results suggest that the persistence of the DS storage is due to the formation of long-lived, non-degradable DS-drug complexes within the lysosomes.
Department of Pharmacology, University of Kiel, Germany.
F
F
Bispinck
J
J
Fischer
R
R
Lüllmann-Rauch
B
B
von Witzendorff
D000166:Acridines / Q000633:toxicity; D000276:Adjuvants, Immunologic / Q000633:toxicity*; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003871:Dermatan Sulfate / Q000737:chemistry / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts; D006025:Glycosaminoglycans / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D006497:Heparan Sulfate / Q000737:chemistry / Q000494:pharmacology; D007150:Immunohistochemistry; D008247:Lysosomes / Q000201:enzymology / Q000378:metabolism*; D013994:Tilorone / Q000737:chemistry / Q000633:toxicity*; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D000166:Acridines; D000276:Adjuvants, Immunologic; D003871:Dermatan Sulfate; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D013994:Tilorone; D001619:beta-N-Acetylhexosaminidases; C075219:3,6-bis(2-(diethylamino)ethoxy)acridine