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Intracellular localization of beta-glucuronidase in fibroblasts after direct transfer from macrophages.

ABSTRACT: The subcellular distribution of beta-glucuronidase acquired by deficient human fibroblasts during co-culture with peritoneal macrophages was compared with that taken up by receptor-mediated endocytosis. Labelled enzyme taken up via receptors was located initially in a low-density endosomal fraction and was transferred to lysosomes within a few minutes. The beta-glucuronidase acquired during 24 h of co-culture was present almost entirely within lysosomes and had a distribution profile identical with that of endogenous beta-hexosaminidase. Monensin prevented transfer of radiolabelled enzyme from endosomes to lysosomes and had a similar effect on the distribution of enzyme acquired by direct transfer, causing beta-glucuronidase to accumulate within endosomes. When the temperature was lowered from 37 degrees C to 19 degrees C, the rate of transfer of enzyme from endosomes to lysosomes was decreased during both direct transfer and indirect receptor-mediated endocytosis. These results show that a lysosomal enzyme acquired by direct transfer during cell-to-cell contact follows a similar intracellular route and has a similar distribution to that of enzymes taken up via cell-surface receptors.

SUBMITTER: Dean MF

PROVIDER: S-EPMC1135414 | biostudies-other | 1988 Dec

REPOSITORIES: biostudies-other

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Project description:IntroductionFlavonoids are converted to inactive metabolites like glucuronides in the gut, and circulate mainly as glucuronides in blood stream, resulting in low concentrations of active aglycones in plasma. It is therefore unclear how oral flavonoids exert their effects in tissues. We recently reported the plasma pharmacokinetics of some flavonoids and suggested the possibility that the absorbed flavonoids modified macrophage functions leading to enhance bacterial clearance. We aimed to confirm their pharmacological profiles focusing on tissue macrophages.MethodsPseudoinfection was induced by intradermal injection of FITC-conjugated and killed Staphylococcus aureus into the ears of mice treated with or without genistein 7-O-glucuronide (GEN7G, 1 mg/kg, i.v.). FACS analysis was performed on single cell suspensions dispersed enzymatically from the skin lesions at 6 h post pseudoinfection to evaluate phagocytic activities of monocytes/macrophages (CD11b+ Ly6G- ) and neutrophils (CD11b+ Ly6G+ ). Phagocytosis of the FITC-conjugated bacteria by four glucuronides including GEN7G was evaluated in cultures of mouse macrophages.ResultsAfter GEN7G injection, genistein was identified in the inflamed ears as well as GEN7G, and the phagocytic activity of CD11b+ Ly6G- cells was increased. GEN7G was converted to genistein by incubation with macrophage-related β-glucuronidase. Macrophage culture assays revealed that GEN7G increased phagocytosis, and the action was dampened by a β-glucuronidase inhibitor. Binding of aglycones to estrogen receptors (ERs), putative receptors of flavonoid aglycones, correlated to biological activities, and glucuronidation reduced the binding to ERs. An ER antagonist suppressed the increase of macrophage function by GEN7G, whereas estradiol enhanced phagocytosis as well.ConclusionsThis study suggests a molecular mechanism by which oral flavonoids are carried as glucuronides and activated to aglycones by β-glucuronidase in tissue macrophages, and contributes to the pharmacological study of glucuronides.

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Intracellular localization of beta-glucuronidase in fibroblasts after direct transfer from macrophages.

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