Intracellular transport of cholesterol in type C Niemann-Pick fibroblasts

The purpose of this study was to determine the capacity of Niemann-Pick type C (NPC) fibroblasts to transport cholesterol from the cell surface to intracellular membranes. This is relevant in light of the observations that NPC cells display a sluggish metabolism of LDL-derived cholesterol, a phenomenon which could be explained by a defective intracellular transport of cholesterol. Treatment of NPC cells for 4 h with 0.1 mg/ml of LDL failed to increase the incorporation of [14C]oleic acid into cholesterol [14C]oleate, an observation consistent with previous reports on this cell type (Pentchev et al. (1985) Proc. Natl. Acad. Sci. USA 82, 8247). Normal fibroblasts, however, displayed the classical upregulation (6-fold over control) of the endogenous esterification reaction in response to LDL exposure. Incubation of normal or NPC fibroblasts with sphingomyelinase (100 mU/ml; Staphylococcus aureus) led to a rapid and marked increase (9- and 10-fold for normal and NPC fibroblasts, respectively, after 4 h) in the esterification of plasma-membrane-derived [3H]cholesterol suggesting that sphingomyelin degradation forced a net transfer of cholesterol from the cell surface to the endoplasmic reticulum. The similar response in normal and mutant fibroblasts to the degradation of sphingomyelin suggests that plasma membrane cholesterol can be transported into the substrate pool of ACAT to about the same extent in these two cell types. Degradation of cell sphingomyelin in NPC fibroblasts also resulted in the movement of 20-25% of the cellular cholesterol from a cholesterol oxidase susceptible pool into oxidase-resistant pools, implying that a substantial amount of plasma membrane cholesterol was internalized after sphingomyelin degradation. This cholesterol internalization was not accompanied by an increased rate of membrane internalization, as measured by [3H]sucrose uptake. Although NPC cells showed a relative accumulation of unesterified cholesterol and a sluggish esterification of LDL-derived cholesterol when exposed to LDL, these cells responded like normal fibroblasts with regard to their capacity to transport cholesterol from the cell surface into intracellular sites in response to sphingomyelin degradation. It therefore appears that NPC cells, in contrast to the impaired intracellular movement of lipoprotein-derived cholesterol, do not display a general impairment of cholesterol transport between the cell surface and the intracellular regulatory pool of cholesterol.     

Reversible effects of sphingomyelin degradation on cholesterol distribution and metabolism in fibroblasts and transformed neuroblastoma cells.

Plasma-membrane sphingomyelin appears to be one of the major determinants of the preferential allocation of cell cholesterol into the plasma-membrane compartment, since removal of sphingomyelin leads to a dramatic redistribution of cholesterol within the cell [Slotte & Bierman (1988) Biochem. J. 250, 653-658]. In the present study we examined the long-term effects of sphingomyelin degradation on cholesterol redistribution in cells and determined the reversibility of the process. In a human lung fibroblast-cell line, removal of 80% of the sphingomyelin led to a rapid and transient up-regulation (3-fold) of acyl-CoA:cholesterol acyltransferase (ACAT) activity, and also, within 30 h, to the translocation of about 50% of the cell non-esterified cholesterol from a cholesterol oxidase-susceptible compartment (i.e. the cell surface) to oxidase-resistant compartments. At 49 h after the initial sphingomyelin degradation, the cell sphingomyelin level was back to 45% of the control level, and the direction of cell cholesterol flow was toward the cell surface, although the original distribution was not achieved. In a transformed neuroblastoma cell line (SH-SY5Y), the depletion of sphingomyelin led to a similarly rapid and transient up-regulation of ACAT activity, and to the translocation of about 25% of cell-surface cholesterol into internal membranes (within 3 h). The flow of cholesterol back to the cholesterol oxidase-susceptible pool was rapid, and a pretreatment cholesterol distribution was reached within 20-49 h. Also, the resynthesis of sphingomyelin was faster in SH-SY5Y neuroblastoma cells and reached control levels within 24 h. The findings of the present study show that the cellular redistribution of cholesterol, as induced by sphingomyelin degradation, is reversible and suggest that the normalization of cellular cholesterol distribution is linked to the re-synthesis of sphingomyelin.     

Cholesterol oxidase susceptibility of cholesterol and 5-androsten-3 beta-ol in pure sterol monolayers and in mixed monolayers containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

This study has examined the importance of the isocaproic side chain at C-17 of cholesterol to sterol/phospholipid interactions in monolayer membranes and to the cholesterol oxidase-susceptibility of cholesterol in pure and mixed monolayers at the air/water interface. The interactions between cholesterol or 5-androsten-3 beta-ol (which lacks the C-17 side chain) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in monolayers indicated that 5-androsten-3 beta-ol was not very efficient in causing condensation of the monolayer packing of POPC. Whereas cholesterol condensed the packing of POPC at all molar fractions examined (i.e., 0.25, 0.50 and 0.75 with regard to POPC), 5-androsten-3 beta-ol caused a slight condensing effect on POPC packing only in the equimolar mixture. The mean molecular area requirement of 5-androsten-3 beta-ol (in pure sterol monolayers at different lateral surface pressures) was 2.2-6.7% less than that observed for cholesterol. The pure 5-androsten-3 beta-ol monolayer also collapsed at lower lateral surface pressures compared with the pure cholesterol monolayer (34 mN/m and 45 mN/m, respectively). The cholesterol oxidase (Streptomyces sp.) catalyzed oxidation of cholesterol or 5-androsten-3 beta-ol in pure monolayers in the air/water interface (10 mN/m) proceeded with very similar rates, indicating that the enzyme did not recognize that the C-17 side chain of 5-androsten-3 beta-ol was missing. The oxidation of cholesterol or 5-androsten-3 beta-ol in mixed POPC-containing monolayers (equimolar mixture) also revealed similar reaction rates, although the reaction was slower in the mixed monolayer compared with the pure sterol monolayer. When the oxidation of cholesterol and 5-androsten-3 beta-ol was examined by monitoring the production of H2O2 (the sterol was solubilized in 2-propanol and the assay conducted in phosphate buffer), the maximal reaction rate observed with 5-androsten-3 beta-ol was only about 41% of that measured with cholesterol. From the cholesterol oxidase point-of-view, it can be concluded that the enzyme did not recognize the C-17 side chain of cholesterol (or lack of it in 5-androsten-3 beta-ol), when the sterol was properly oriented as a monolayer at the air/water interface. However, when the substrate was presented to the enzyme in a less controlled orientation (organic solvent in water), 5-androsten-3 beta-ol may have oriented itself unfavorably compared with the orientation of cholesterol, thereby leading to slower oxidation rates.     

Cholesterol oxidase catalyzed oxidation of cholesterol in mixed lipid monolayers: effects of surface pressure and phospholipid composition on catalytic activity

The catalytic activity of cholesterol oxidase from Streptomyces sp. in mixed monolayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), N-oleoylsphingomyelin (O-SPM), and cholesterol (CHL) has been determined at lateral surface pressures between 10 and 30 mN/m. The highest cholesterol oxidase activity (determined at 37 degrees C) was observed at surface pressures around 20 mN/m in a POPC/CHL monolayer (50:50 mol %). Above and below this surface pressure, the enzyme activity decreased markedly. A similar optimal activity vs surface pressure relationship was observed also for an O-SPM/CHL monolayer (50:50 mol %). The activity of cholesterol oxidase toward cholesterol in the O-SPM/CHL monolayer was, however, less than in the corresponding POPC mixed monolayer. The surface activity of cholesterol oxidase decreased markedly when the temperature was lowered to 20 degrees C, and hardly any enzyme activity was observed in an O-SPM/CHL monolayer at 25 mN/m or above. With a monolayer containing POPC/O-SPM/CHL (42:18:40 mol %), maximal cholesterol oxidase activity was observed at the lowest surface pressure tested (i.e., 10 mN/m), and the catalytic activity decreased markedly with increasing lateral surface pressures in the monolayer. The results of this study show (i) that the activity of cholesterol oxidase in general is highly dependent on the lateral surface pressure in the substrate membranes and (ii) that sphingomyelin, by interacting tightly with cholesterol, can prevent or restrain the accessibility of cholesterol for oxidation by cholesterol oxidase.     

Hepatocellular uptake of 3H-dihydromicrocystin-LR, a cyclic peptide toxin

The cellular uptake of microcystin-LR, a cyclic heptapeptide hepatotoxin from the cyanobacterium Microcystis aeruginosa, was studied by means of a radiolabelled derivative of the toxin. 3H-dihydromicrocystin-LR. The uptake of 3H-dihydromicrocystin-LR was shown to be specific for freshly isolated rat hepatocytes whereas the uptake in the human hepatocarcinoma cell line Hep G2 as well as the mouse fibroblast cell line NIH-3T3, and the human neuroblastoma cell line SH-SY5Y, was negligible. By means of a surface barostat technique it was shown that the membrane penetrating capacity (surface activity) of microcystin-LR was low, indicating that the toxin requires an active uptake mechanism. The hepatocellular uptake of microcystin-LR could be inhibited in the presence of bile acid transport inhibitors such as antamanide (5 microM), sulfobromophthalein (50 microM) and rifampicin (30 microM). The uptake was also reduced in a concentration dependent manner when the hepatocytes were incubated in the presence the bile salts cholate and taurocholate. A complete inhibition of the hepatocellular uptake was achieved by 100 microM of either bile salt. The overall results indicate that the uptake of microcystin-LR is through the multispecific transport system for bile acids. This mechanism of cell entry would explain the previously observed cell specificity and organotropism of microcystin-LR.     

Rapid turn-over of plasma membrane sphingomyelin and cholesterol in baby hamster kidney cells after exposure to sphingomyelinase

Plasma membrane sphingomyelin in baby hamster kidney (BHK-21) cells was hydrolyzed with sphingomyelinase (Staphylococcus aureus) and the effects on membrane cholesterol translocation and the properties of membrane bound adenylate cyclase and Na+/K(+)-ATPase were determined. Exposure of confluent BHK-21 cells to 0.1 U/ml of sphingomyelinase led to the degradation (at 37 degrees C) of about 60% of cell sphingomyelin. No simultaneous hydrolysis of phosphatidylcholine occurred. The hydrolysis of sphingomyelin subsequently led to the translocation (within 40 min) of about 50-60% of cell [3H]cholesterol from a cholesterol oxidase susceptible pool to an oxidase resistant compartment. The translocation of [3H]cholesterol from the cell surface to intracellular membranes was accompanied by a paralleled increase in [3H]cholesterol ester formation. When cells were first exposed to sphingomyelinase (to degrade sphingomyelin) and then incubated without the enzyme in serum-free media, the mass of cell sphingomyelin decreased initially (by 60%), but then began to increase and reached control levels within 3-4 h. The rapid re-synthesis of sphingomyelin was accompanied by an equally rapid normalization of cell [3H]cholesterol distribution. The re-formation of cell sphingomyelin also led to a decreased content of cellular [3H]cholesterol esters, indicating that unesterified [3H]cholesterol was pulled out of the cholesterol ester cycle and transported to the cell surface. Exposure of BHK-21 cells to sphingomyelinase further led to a dramatically decreased activity of ouabain-sensitive Na+/K(+)-ATPase, whereas forskolin-stimulated adenylate cyclase activity was not affected. The activity of Na+/K(+)-ATPase returned to normal in parallel with the normalization of cell sphingomyelin mass and cholesterol distribution. We conclude that sphingomyelin has profound effects on the steady-state distribution of cell cholesterol, and that manipulations of cell sphingomyelin levels directly and reversibly affects the apparent distribution of cholesterol. Changes in the lipid composition of the plasma membrane also appears to selectively affect important metabolic reactions in that compartment.     

Effects of Sphingomyelin Headgroup Size on Interactions with Ceramide

Sphingomyelins (SMs) and ceramides are known to interact favorably in bilayer membranes. Because ceramide lacks a headgroup that could shield its hydrophobic body from unfavorable interactions with water, accommodation of ceramide under the larger phosphocholine headgroup of SM could contribute to their favorable interactions. To elucidate the role of SM headgroup for SM/ceramide interactions, we explored the effects of reducing the size of the phosphocholine headgroup (removing one, two, or three methyls on the choline moiety, or the choline moiety itself). Using differential scanning calorimetry and fluorescence spectroscopy, we found that the size of the SM headgroup had no marked effect on the thermal stability of ordered domains formed by SM analog/palmitoyl ceramide (PCer) interactions. In more complex bilayers composed of a fluid glycerophospholipid, SM analog, and PCer, the thermal stability and molecular order of the laterally segregated gel domains were roughly identical despite variation in SM headgroup size. We suggest that that the association between PCer and SM analogs was stabilized by ceramide’s aversion for disordered phospholipids, by interfacial hydrogen bonding between PCer and the SM analogs, and by attractive van der Waals’ forces between saturated chains of PCer and SM analogs.     

Stabilization of sphingomyelin interactions by interfacial hydroxyls — A study of phytosphingomyelin properties

D-ribo-phytosphingosines are biologically significant long-chain bases present in various sphingolipids from yeasts, fungi, plants and mammals. In this study we prepared phytopalmitoylsphingomyelin (phytoPSM) analogs based on the D-ribo-phytosphingosine base. The N-linked acyl chains were either 16:0, 2OH(R)16:0 (natural isomer), or 2OH(S)16:0. The gel-phase of phytoPSM was more stable than that of PSM (T_m 48.6 °C and 41.0 °C, respectively). The gel-liquid crystalline phase transition enthalpies were 9.1 ± 0.4 and 6.1 ± 0.3 kcal/mol for phytoPSM and PSM, respectively. An N-linked 2OH(R)16:0 in phytoPSM destabilized the gel phase relative to phytoPSM (by ~+ 6 °C, based on DPH anisotropy measurements), whereas 2OH(S)16:0 in phytoPSM stabilized it (by ~? 6 °C). All phytoPSM analogs formed sterol-enriched ordered domains in a fluid ternary bilayer, and those containing phytoPSM or 2OH(S)phytoPSM were more thermostable than the domains containing 2OH(R)phytoPSM or PSM. The affinity of cholestatrienol for POPC bilayers containing 20 mol% phytoPSM was higher than for comparable bilayers with an equal amount of PSM. The 2-hydroxylated acyl chains in phytoPSM did not markedly alter sterol affinity. We conclude that phytoPSM is a more ordered sphingolipid than PSM, and is fully capable of interacting with cholesterol.     

Complexation of C6-Ceramide with Cholesteryl Phosphocholine – A Potent Solvent-Free Ceramide Delivery Formulation for Cells in Culture

Ceramides are potent bioactive molecules in cells. However, they are very hydrophobic molecules, and difficult to deliver efficiently to cells. We have made fluid bilayers from a short-chain D-erythro-ceramide (C6-Cer) and cholesteryl phosphocholine (CholPC), and have used this as a formulation to deliver ceramide to cells. C6-Cer complexed with CholPC led to much larger biological effects in cultured cells (rat thyroid FRTL-5 and human HeLa cells in culture) compared to C6-Cer dissolved in dimethyl sulfoxide (DMSO). Inhibition of cell proliferation and induction of apoptosis was significantly more efficient by C6-Cer/CholPC compared to C6-Cer dissolved in DMSO. C6-Cer/CholPC also permeated cell membranes and caused mitochondrial Ca²⁺ influx more efficiently than C6-Cer in DMSO. Even though CholPC was taken up by cells to some extent (from C6-Cer/CholPC bilayers), and was partially hydrolyzed to free cholesterol (about 9%), none of the antiproliferative effects were due to CholPC or excess cholesterol. The ceramide effect was not limited to D-erythro-C6-Cer, since L-erythro-C6-Cer and D-erythro-C6-dihydroCer also inhibited cell priolifereation and affected Ca²⁺ homeostasis. We conclude that C6-Cer complexed to CholPC increased the bioavailability of the short-chain ceramide for cells, and potentiated its effects in comparison to solvent-dissolved C6-Cer. This new ceramide formulation appears to be superior to previous solvent delivery approaches, and may even be useful with longer-chain ceramides.