Bispinck F, Fischer J, Lüllmann-Rauch R, von Witzendorff B
Department of Pharmacology, University of Kiel, Germany.
Toxicology. 1998 Jul 3;128(2):91-100. doi: 10.1016/s0300-483x(98)00052-3.
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.
几种双阳离子两亲性化合物，如免疫调节剂泰洛龙及其类似物，会损害硫酸化糖胺聚糖（GAGs）的溶酶体分解代谢。因此，它们会导致大鼠以及包括人类在内的多个物种的培养成纤维细胞中出现溶酶体GAG蓄积。体内GAG蓄积的逆转相当缓慢；在停止药物治疗后会持续数月。在本研究中，我们探究了这种缓慢逆转背后的机制。用泰洛龙（5和20微摩尔）或化合物CL-90.100（3和10微摩尔）对培养的牛角膜成纤维细胞进行4天预处理，然后在无药物培养基中进一步培养长达11天。通过生化分析细胞内GAG蓄积情况，并进行组织化学显示。通过荧光显微镜观察亚细胞药物分布（CL-90.100）。硫酸皮肤素（DS）对GAG蓄积起主要作用。用低浓度和高浓度的任何一种药物预处理后，在观察期内DS的蓄积都是不可逆的，而硫酸乙酰肝素的少量蓄积得到了解决。高浓度泰洛龙预处理引起的溶酶体酶β-己糖胺酶（E.C. 3.2.1.52）分泌增加也很容易逆转。因此，酶缺乏不能解释DS的持续蓄积。药物相关荧光在核周细胞器（可能是溶酶体）内的定位，类似于组织化学染色显示的储存GAGs的定位。在整个恢复期，荧光和阳性GAG染色在细胞内分布不变的情况下持续存在。目前的结果表明，DS蓄积的持续存在是由于在溶酶体内形成了长寿命、不可降解的DS-药物复合物。
