Phosphatidylinositol phosphate kinases.

Phosphatidylinositol 4,5-bisphosphate is a multi-functional lipid, whose proposed functions now number more than twenty, covering many aspects of cell biology in several different subcellular compartments. The enzymes primarily responsible for synthesizing this lipid, the Type I phosphatidylinositol 4-phosphate 5-kinases, are therefore a tightly regulated and diverse family. Here we review our current knowledge about these enzymes and how they may be regulated.

Metabolic fate of extracellular NAD in human skin fibroblasts.

Extracellular NAD is degraded to pyridine and purine metabolites by different types of surface-located enzymes which are expressed differently on the plasmamembrane of various human cells and tissues. In a previous report, we demonstrated that NAD-glycohydrolase, nucleotide pyrophosphatase and 5'-nucleotidase are located on the outer surface of human skin fibroblasts. Nucleotide pyrophosphatase cleaves NAD to nicotinamide mononucleotide and AMP, and 5'-nucleotidase hydrolyses AMP to adenosine. Cells incubated with NAD, produce nicotinamide, nicotinamide mononucleotide, hypoxanthine and adenine. The absence of ADPribose and adenosine in the extracellular compartment could be due to further catabolism and/or uptake of these products. To clarify the fate of the purine moiety of exogenous NAD, we investigated uptake of the products of NAD hydrolysis using U-[(14)C]-adenine-NAD. ATP was found to be the main labeled intracellular product of exogenous NAD catabolism; ADP, AMP, inosine and adenosine were also detected but in small quantities. Addition of ADPribose or adenosine to the incubation medium decreased uptake of radioactive purine, which, on the contrary, was unaffected by addition of inosine. ADPribose strongly inhibited the activity of ecto-NAD-hydrolyzing enzymes, whereas adenosine did not. Radioactive uptake by purine drastically dropped in fibroblasts incubated with (14)C-NAD and dipyridamole, an inhibitor of adenosine transport. Partial inhibition of [(14)C]-NAD uptake observed in fibroblasts depleted of ATP showed that the transport system requires ATP to some extent. All these findings suggest that adenosine is the purine form taken up by cells, and this hypothesis was confirmed incubating cultured fibroblasts with (14)C-adenosine and analyzing nucleoside uptake and intracellular metabolism under different experimental conditions. Fibroblasts incubated with [(14)C]-adenosine yield the same radioactive products as with [(14)C]-NAD; the absence of inhibition of [(14)C]-adenosine uptake by ADPribose in the presence of alpha-beta methyleneADP, an inhibitor of 5' nucleotidase, demonstrates that ADPribose coming from NAD via NAD-glycohydrolase is finally catabolised to adenosine. These results confirm that adenosine is the NAD hydrolysis product incorporated by cells and further metabolized to ATP, and that adenosine transport is partially ATP dependent.

Transport of sphingomyelin to the cell surface is inhibited by brefeldin A and in mitosis, where C6-NBD-sphingomyelin is translocated across the plasma membrane by a multidrug transporter activity.

Sphingomyelin is a major lipid of the mammalian cell surface. The view that sphingomyelin, after synthesis in the Golgi lumen, reaches the outer leaflet of the plasma membrane on the inside of carrier vesicles has been challenged by inconsistencies in the results of transport studies. To investigate whether an alternative pathway to the cell surface exists for sphingomyelin, brefeldin A and mitotic cells were used to block vesicular traffic between the Golgi complex and the plasma membrane. Exogenous sphingomyelinase was applied in the cold to assay for the presence of sphingomyelin on the surface of CHO cells. Newly synthesized radiolabeled sphingomyelin was found to equilibrate with cell surface sphingomyelin within 1.5 hours at 37 degrees C. Brefeldin A and mitosis inhibited this transport but, surprisingly, not the surface appearance of the short-chain sphingomyelin analog N-6[7-nitro-2,1,3-benzoxadiazol-4-yl]aminohexanoyl(C6-NBD)-sphingo myelin as assayed by depletion of this lipid in the medium by the scavenger albumin. Transport of C6-NBD-sphingomyelin in the presence of brefeldin A was blocked by cyclosporin A and PSC 833, inhibitors of the multidrug resistance P-glycoprotein. The same was observed in HepG2 and HeLa cells, and for short-chain glucosylceramide, which demonstrates the general nature of the transporter-dependent sphingolipid translocation across the plasma membrane.

Synthesis of sphingomyelin by oligodendrocytes--how and where?

Sphingomyelin (SM) biosynthesis in cultured oligodendrocytes (OC) was evaluated: (i) with [14C] tracers (choline, ethanolamine, serine) to pinpoint the major metabolic routes; (ii) with fluorescent and truncated, radiolabeled ceramide analogs to determine the relative activities of SM-synthase in intra-and extra-Golgi compartments of OC. In contrast to a general contention in the literature that SM synthase is absent from the brain, our data show that (choline-->CDP-choline-->phosphatidylcholine (PC)-->SM) is the major anabolic route with only a minor contribution to PC via methylation of phosphatidylethanolamine (PE). SM synthase activity was found to be equally divided between intra- and extra-Golgi compartments of OC. Moreover, significant SM-synthase activity was recovered in purified myelin preparations. Our results shed new light on the possible involvement of sphingolipid-derived mediators in myelination.

Sphingomyelin is synthesized at the plasma membrane of oligodendrocytes and by purified myelin membranes: a study with fluorescent- and radio-labelled ceramide analogues.

In most cell types sphingomyelin is synthesized predominantly in the cis-medial compartments of the Golgi stacks whereas the contribution of the plasma membrane is much lower. The aim of this study was to assess the contribution of both compartments to the synthesis of sphingomyelin in myelinating cells. Therefore, oligodendrocytes from rat spinal cord were incubated in culture with fluorescently- or radiolabelled ceramides, and the effects of a block in the vesicular flow (monensin, brefeldin A, low temperature) on surface synthesis of sphingomyelin were evaluated. The results indicate that approximately 50% of the sphingomyelin synthase is present at the plasma and myelin membranes of oligodendrocytes.

Uptake and metabolism of fluorescent ceramide analogs by rat oligodendrocytes in culture.

We studied the metabolism of sphingolipids by oligodendrocytes derived from rat spinal cord by providing lipid vesicles with either N-lissamine-rhodaminyl-ceramide (LRh-Cer) or N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-ceramide (NBD-Cer) to the cells cultured in a chemically-defined medium. With both probes the major fluorescent product turned out to be sphingomyelin (SM). Most of LRh-SM was not cell-associated but recovered from the culture medium, probably due to back-exchange to the lipid vesicles. The accumulation of LRh-SM, both in the cells and in the medium, was inhibited in the presence of monensin or brefeldin A, whereas the production of NBD-SM was much less affected by these Golgi perturbing drugs. With LRh-Cer as substrate, LRh-labelled fatty acid (FA), galactosyl- and sulfogalactosyl-ceramides (GalCer and SGalCer) were also formed. NBD-Cer, however, was metabolized to glucosylceramide (GlcCer) and GalCer but not to SGalCer or NBD-FA. These data demonstrate that chemical modifications of ceramide alter its metabolism in oligodendrocytes and that the metabolites of LRh-Cer reflect the glycolipid composition of myelin more closely than those of NBD-Cer.

A novel fluorescent pH indicator for the acidic range.

Reaction of a commercial preparation of lissamine-rhodamine B sulfonyl chloride with primary amines gives rise to two sulfonamide derivatives, of which one behaves as a pH indicator with maximal fluorescence emission in the acidic range and a pKa of about 4.6. The structure of this derivative and the mechanism of fluorescence dependence on pH, elucidated by means of NMR, is presented.

Cultured oligodendrocytes metabolize a fluorescent analogue of sulphatide; inhibition by monensin.

It has been suggested that oligodendrocytes can actively phagocytose myelin debris during active myelination or after injury and experimental demyelination. Therefore, we have used a fluorescent analogue (N-lissamine rhodaminyl-(12-aminododecanoyl) cerebroside 3-sulphate) to study the metabolic fate of sulphatide, a galactosphingolipid that is highly enriched in myelin membranes. The fluorescent sulphatide was incorporated in small unilamellar vesicles and administered to cultured oligodendrocytes. The association of the lipid probe to the cells in culture was saturable in time and with the concentration of the probe. The processes of association, internalization and subcellular distribution were followed by confocal scanning laser microscopy and appeared to be very rapid. Within 20 min a marked perinuclear staining was seen. After prolonged incubation the fluorescence distributed gradually over the cytoplasm and into cellular branches along structures suggestive of cytoskeletal elements. Lipid analysis demonstrated that ceramide was the major metabolite present in the cells but galactosylceramide, sphingomyelin and free fatty acid were also detected. In the culture medium only free fatty acid and sphingomyelin were found. Monensin did not affect the cellular association and internalization of the fluorescent sulphatide but markedly reduced its conversion to metabolic products. These results indicate that exogenous sulphatide is targeted to the Golgi apparatus prior to its lysosomal degradation.

Rapid preparation of a distinct lysosomal population from myelinating mouse brain using Percoll gradients.

To study the vesicular lysosome-associated transport and the metabolism of some brain macromolecules (in particular, sialoglycoconjugates), we developed a rapid procedure to obtain a distinct lysosomal population starting from myelinating mouse brain. This procedure is based on an initial differential centrifugation step producing a 1,000-17,500-g fraction (P2), followed by isopycnic centrifugation of fraction P2 on a self-generated colloidal silica gel (Percoll) gradient. The heaviest subfraction thus obtained is very rich in acid hydrolase activities like beta-galactosidase, arylsulfatase A, and acid phosphatase. The enrichment of these enzymes is approximately 100-fold as compared with the starting homogenate, whereas the markers of other subcellular organelles, such as mitochondria, plasma membranes, or the Golgi apparatus, are virtually absent. The lysosomal preparation contains approximately 12-14% of the total acid hydrolase activities, with a protein yield of approximately 0.12%. Electron microscopy shows that the lysosomal fraction is composed of an approximately 90% pure population of lysosomes. Therefore, the procedure described here is suitable for obtaining a highly purified lysosome preparation from myelinating mouse brain.