Abstract
The effects of ceramide analogs on the uptake of markers for fluid-phase (horseradish peroxidase, HRP) and receptor-mediated (low density lipoprotein, LDL) endocytosis were studied in Chinese hamster fibroblasts. N-Hexanoyl-D-erythro-sphingosine (C6-Cer) decreased the uptake of HRP in a dose-dependent manner. Internalization was inhibited > 40% with 25 microM C6-Cer, relative to controls, and was apparent within 5 min. Internalization of HRP was also inhibited by other Cer analogs and by treatment with agents that raise levels of endogenous Cer (sphingomyelinase or the glycosphingolipid synthesis inhibitor, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP)), but not by N-hexanoyl-D-erythrosphinganine (C6-dihydro-Cer) or sphingosine. Internalization of LDL was also inhibited by C6-Cer in a concentration-dependent manner, but was less pronounced than the effect on HRP internalization (10% versus 40% inhibition with 25 microM C6-Cer), suggesting that ceramide might affect fluid-phase and receptor-mediated endocytosis to different extents. C6-Cer also slowed HRP and LDL transport from endosomes to lysosomes as studied by analysis of endocytic vesicles on Percoll density gradients and induced a redistribution of endocytic organelles as determined by fluorescence microscopy of intact cells using appropriate markers. This resulted in decreased degradation of 125I-labeled LDL in the presence of C6-Cer. These results suggest that endogenous ceramide may modulate endocytosis.
Highlights
The effects ofceramide analogs on the uptake ofmarkers for fluid-phase and receptor-mediated endocytosis were studied in Chinese hamster fibroblasts, NHexanoyl-D-erythro-sphingosine (C6-Cer) decreased the uptake of HRP in a dose-dependent manner
1 The abbreviations used are: Cer, ceramide (N-acyl-D-erythro-sphingosine); C2-Cer, N-acetyl-D-erythro-sphingosine; Cs-Cer, N-hexanoylD-erythro-sphingosine; Cs-NBD-Cer, N-{6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl}-D-erythro-sphingosine; CHO-K1, Chinese hamster ovary cells; DiI, 3,3'-dioctadecylinodocarbocyanine; BSA, bovine serum albumin; FITC-dextran, fluorescein isothiocyanate-conjugated dextran; HMEM, 10 mM HEPES-buffered Eagle's minimum essential medium, pH 7.4, without indicator and with 1.3 mM CaCl2 and golipids, has been shown to act as a second messenger in a number of pathways, including those induced by tumor necrosis factor a, vitamin Da, interleukin 1/3, and interleukin 4 (reviewed in Hannun (1994), Hannun et al (1993), Kolesnick
!LM C6-Cer was the same as that prepared from cells chased in the absence of C6-Cer, suggesting that C6-Cer had little with the results shown in Fig. 2B, the effect of C6-Cer on the transport of HRP from early endosomes to lysosomes was more pronounced than that seen for low density lipoprotein (LDL) (Fig. 3C)
Summary
Vol 270, No 22, Issue of June 2, pp. 13291-13297, 1995 Printed in U.S.A. The effects ofceramide analogs on the uptake ofmarkers for fluid-phase (horseradish peroxidase, HRP) and receptor-mediated (low density lipoprotein, LDL) endocytosis were studied in Chinese hamster fibroblasts, NHexanoyl-D-erythro-sphingosine (C6-Cer) decreased the uptake of HRP in a dose-dependent manner. Ceramide (N-acyl-D-erythro-sphingosine); C2-Cer, N-acetyl-D-erythro-sphingosine; Cs-Cer, N-hexanoylD-erythro-sphingosine; Cs-NBD-Cer, N-{6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl}-D-erythro-sphingosine; CHO-K1, Chinese hamster ovary cells; DiI, 3,3'-dioctadecylinodocarbocyanine; BSA, bovine serum albumin; FITC-dextran, fluorescein isothiocyanate-conjugated dextran; HMEM, 10 mM HEPES-buffered Eagle's minimum essential medium, pH 7.4, without indicator and with 1.3 mM CaCl2 and golipids, has been shown to act as a second messenger in a number of pathways, including those induced by tumor necrosis factor a, vitamin Da, interleukin 1/3, and interleukin 4 (reviewed in Hannun (1994), Hannun et al (1993), Kolesnick (1992), and Mathias and Kolesnick (1993)) Studies from this laboratory have suggested that membrane traffic may be affected by alterations in the intracellular levels of Cer. Previously, we demonstrated that short chain analogs of Cer inhibit the movement of vesicular stomatitis virus glycoprotein through the medial and trans compartments of the Golgi apparatus and its subsequent transport to the cell surface. These findings, in conjunction with our earlier work, suggest that Cer may be a general modulator of membrane traffic
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