Sterol carrier protein-2 expression alters sphingolipid metabolism in transfected mouse L-cell fibroblasts.

The influence of sterol carrier protein-2 (SCP-2) on the cellular metabolism of sphingolipids was examined in control mouse L-cells and stably transfected clones expressing the protein SCP-2. Three approaches were used to examine for differences; (1) compositional analysis of endogenous sphingolipid classes, (2) metabolism of NBD-ceramide, and (3) live cell labelling via endocytic uptake of BODIPY-sphingomyelin. SCP-2 over expression significantly altered the endogenous levels of both neutral and acidic sphingolipid classes. Among the neutral sphingolipids, expression of SCP-2 induced a 1.7-fold increase in the level of lactosylceramide (LacCer, p < 0.05) and a similar fold decrease in the level of the higher-order neutral glycosylceramides (p < 0.05). Among the acidic sphingolipids, SCP-2 resulted in a 5.2-fold decrease in the endogenous plasma membrane level of ganglioside GM1 (p < 0.03). Incubation of both control and transfected cell lines with NBD-ceramide resulted in the rapid establishment of a steady-state distribution of NBD-labelled sphingomyelin (NBD-SM) and glucosylceramide (NBD-GlcCer). In the SCP-2 expressing clones the conversion of NBD-Cer to NBD-GlcCer was 30% lower during incubation periods between 5 and 30 min (p < 0.025). Inspection of the cells by fluorescence microscopy after incubation with BODIPY labelled sphingomyelin (BODIPY-SM) revealed similar punctuated patterns with no distinguishable differences between the cell types. These results imply that SCP-2 plays a role in modulating enzymatic steps involved in metabolism of sphingolipid homeostasis.

Differential targeting of glucosylceramide and galactosylceramide analogues after synthesis but not during transcytosis in Madin-Darby canine kidney cells.

A short-chain analogue of galactosylceramide (6-NBD-amino-hexanoyl-galactosylceramide, C6-NBD-GalCer) was inserted into the apical or the basolateral surface of MDCK cells and transcytosis was monitored by depleting the opposite cell surface of the analogue with serum albumin. In MDCK I cells 32% of the analogue from the apical surface and 9% of the analogue from the basolateral surface transcytosed to the opposite surface per hour. These numbers were very similar to the flow of membrane as calculated from published data on the rate of fluid-phase transcytosis in these cells, demonstrating that C6-NBD-GalCer acted as a marker of bulk membrane flow. It was calculated that in MDCK I cells 155 microns membrane transcytosed per cell per hour in each direction. The fourfold higher percentage transported from the apical surface is explained by the apical to basolateral surface area ratio of 1:4. In MDCK II cells, with an apical to basolateral surface ratio of 1:1, transcytosis of C6-NBD-GalCer was 25% per hour in both directions. Similar numbers were obtained from measuring the fraction of endocytosed C6-NBD-GalCer that subsequently transcytosed. Under these conditions lipid leakage across the tight junction could be excluded, and the vesicular nature of lipid transcytosis was confirmed by the observation that the process was blocked at 17 degrees C. After insertion into one surface of MDCK II cells, the glucosylceramide analogue C6-NBD-GlcCer randomly equilibrated over the two surfaces in 8 h. C6-NBD-GalCer and -GlcCer transcytosed with identical kinetics. Thus no lipid selectivity in transcytosis was observed. Whereas the mechanism by which MDCK cells maintain the different lipid compositions of the two surface domains in the absence of lipid sorting along the transcytotic pathway is unclear, newly synthesized C6-NBD-GlcCer was preferentially delivered to the apical surface of MDCK II cells as compared with C6-NBD-GalCer.

Processing of epidermal glucosylceramides is required for optimal mammalian cutaneous permeability barrier function.

The interstices of the mammalian stratum corneum contain lipids in a system of continuous membrane bilayers critical for the epidermal permeability barrier. During the transition from inner to outer stratum corneum, the content of polar lipids including glucosylceramides, decreases while ceramide content increases. We investigated whether inhibition of glucosylceramide hydrolysis would alter epidermal permeability barrier function. Daily topical applications of bromoconduritol B epoxide (BrCBE) to intact murine skin selectively inhibited beta-glucocerebrosidase, increased glucosylceramide content of stratum corneum with ceramide content remaining largely unchanged, and caused a progressive, reversible decrease in barrier function. Histochemistry of inhibitor-treated epidermis revealed persistence of periodic acid-Schiff-positive staining in stratum corneum cell membranes, consistent with retention of hexose moieties. Electron microscopy of inhibitor-treated samples revealed no evidence of toxicity or changes in the epidermal lipid delivery system. However, immature membrane structures persisted in the intercellular spaces throughout the stratum corneum, with reappearance of mature membrane structures progressing outward from the lower stratum corneum upon termination of BrCBE. Finally, the induced barrier abnormality was not reversed by coapplications of ceramide. These data demonstrate that glucosylceramide hydrolysis is important in the formation of the epidermal permeability barrier, and suggest that accumulation of glucosylceramides in stratum corneum intercellular membrane domains leads to abnormal barrier function.

Extensive precursor-product relationship between gangliosides formed from exogenous glucosylceramide in rat liver.

Glucosylceramide, radiolabelled on the glucose residue, was administered to rats and the radioactive gangliosides formed at different time points were chemically characterized. They were identified as GM3, GM1, GD1a and GD1b, each one carrying only radioactive glucose. The time course of each individual ganglioside showed that the simpler gangliosides were formed earlier but were consumed earlier than the more complex ones, resulting in radioactivity patterns that were different at each time point. Only 30 h after injection did it resemble that of endogenous rat liver gangliosides. These results indicate that an extensive precursor-product relationship actually exists in the course of ganglioside biosynthesis.

Enhanced percutaneous penetration of flufenamic acid using lipid disperse systems containing glycosylceramides.

The usefulness of lipid disperse systems, containing soybean phosphatidylcholine (PC) and glycosylceramide (GC) as lipid components, to enhance the percutaneous penetration of flufenamic acid (FA) through rat abdominal skin was examined by both in vitro permeation and in vivo absorption studies. The penetration of FA from a simple buffer suspension (pH 3.0) containing no lipid component was poor, but was markedly enhanced when FA was incorporated in PC-dispersions. However, this enhancing effect disappeared when the PC concentration in the preparation exceeded 40 mumol/ml. Enhanced penetration of FA from PC-dispersions could also be recognized when 30% propylene glycol or 30% glycerol was used as the dispersing medium instead of the aqueous buffer solution. Addition of GC to the PC-dispersions brought further enhancement of FA penetration through the skin. The maximal effect was observed when FA was incorporated in a 10%-GC system, and the cumulative amount of FA penetrating through the skin in 24 h from this system was approximately 6-fold larger than that from the simple buffer suspension. Enhanced absorption of FA from lipid disperse systems was also confirmed by in vivo application of these preparations.