Inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis in rat-2 fibroblasts by cell-permeable ceramides.

Phospholipids and sphingolipids are important precursors of lipid-derived second messengers such as diacylglycerol and ceramide, which participate in several signal transduction pathways and in that way mediate the effects of various agonists. The cross-talk between glycerophospholipid and sphingolipid metabolism was investigated by examining the effects of cell-permeable ceramides on phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) synthesis in Rat-2 fibroblasts. Addition of short-chain C6-ceramide to the cells resulted in a dose- and time-dependent inhibition of the CDP-pathways for PtdCho and PtdEtn synthesis. Treatment of cells for 4 h with 50 microM C6-ceramide caused an 83% and a 56% decrease in incorporation of radiolabelled choline and ethanolamine into PtdCho and PtdEtn, respectively. Exposure of the cells for longer time-periods (>/= 16 h) to 50 microM C6-ceramide resulted in apoptosis. The structural analogue dihydro-C6-ceramide did not affect PtdCho and PtdEtn synthesis. In pulse-chase experiments, radioactive choline and ethanolamine accumulated in CDP-choline and CDP-ethanolamine under the influence of C6-ceramide, suggesting that synthesis of both PtdCho and PtdEtn were inhibited at the final step in the CDP-pathways. Indeed, cholinephosphotransferase and ethanolaminephosphotransferase activities in membrane fractions from C6-ceramide-treated cells were reduced by 64% and 43%, respectively, when compared with control cells. No changes in diacylglycerol mass levels or synthesis of diacylglycerol from radiolabelled palmitate were observed. It was concluded that C6-ceramide affected glycerophospholipid synthesis predominantly by inhibition of the step in the CDP-pathways catalysed by cholinephosphotransferase and ethanolaminephosphotransferase.     

Interaction of lipid vesicles with monomolecular layers containing lung surfactant proteins SP-B or SP-C

Pulmonary surfactant contains two families of hydrophobic proteins, SP-B and SP-C. Both proteins are thought to promote the formation of the phospholipid monolayer at the air-fluid interface of the lung. The Wilhelmy plate method was used to study the involvement of SP-B and SP-C in the formation of phospholipid monolayers. The proteins were either present in the phospholipid vesicles which were injected into the subphase or included in a preformed phospholipid monolayer. In agreement with earlier investigators, we found that SP-B and SP-C, present in phospholipid vesicles, were able to induce the formation of a monolayer, as became apparent by an increase in surface pressure. However, when the proteins were present in a preformed phospholipid monolayer (20 mN/m) at similar lipid to protein ratios, the rate of surface pressure increase after injection of pure phospholipid vesicles into the subphase at similar vesicle concentrations was 10 times higher. The process of phospholipid insertion from phospholipid vesicles into the protein-containing monolayers was dependent on (1) the presence of (divalent) cations, (2) the phospholipid concentration in the subphase, (3) the size of the phospholipid vesicles, (4) the protein concentration in the preformed monolayer, and (5) the initial surface pressure at which the monolayers were formed. Both in vesicles and in preformed monolayers, SP-C was less active than SP-B in promoting the formation of a phospholipid monolayer. The use of preformed monolayers containing controlled protein concentrations may allow more detailed studies on the mechanism by which the proteins enhance phospholipid monolayer formation from vesicles.     

An IGF binding protein is an inhibitor of FGF stimulation.

We purified to homogeneity a growth inhibiting diffusible factor (IDF45) secreted by dense cultures of mouse 3T3 cells and which was able to inhibit 100% of DNA synthesis stimulated by serum in chick embryo fibroblasts (CEF) (Blat et al., 1989a). We then demonstrated that this factor was an IGF-binding protein (Blat et al., 1989b). Indeed, its N-terminal amino acid sequence was homologous to that of rat IGFBP-3. Our present results show that basic fibroblast growth factor (bFGF) induced, respectively, a fivefold and threefold increase in DNA synthesis in mouse embryo fibroblasts (MEF) and CEF. IDF-45 inhibited the stimulation induced by bFGF by about 65%, while stimulation induced by insulin, PDGF, or EGF was only weakly or not at all inhibited by IDF45. When bFGF stimulation was determined in the presence of a high concentration of insulin in conditions which minimize the effect of endogenous IGF-I or -II, this stimulation was decreased by about 50% in the presence of IDF45. This result suggests that addition of bFGF stimulates IGF secretion, thereby resulting in partial loss of inhibition, by IDF45, of bFGF stimulation.     

Characterization of the soluble human granulocyte-macrophage colony-stimulating factor receptor complex.

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GM-R) is expressed on both hematopoietic and non-hematopoietic tissues. Although the receptor has been identified by cross-linking studies as an 84,000-dalton protein, very little is known about its biochemistry. In this report, we describe a soluble binding assay for the human GM-R which allowed us to characterize the receptor complex from various sources, including plasma membranes of placenta, neutrophils, and human myeloid leukemia cell lines. Preparation of membranes as well as solubilization by Triton X-100 and N-octylglucoside resulted in a 5-10-fold lower affinity of the receptor for GM-CSF. The Kd decreased from 20 to 80 pM in intact cells to 200-500 pM in both intact and solubilized membranes. Binding in solution was rapid, specific for GM-CSF, and best fit a "one-site" model with an approximate Kd of 500 pM. The dissociation rate constant for the soluble GM-R was very similar to that of intact cells (k2 = 0.013 min-1 versus 0.017 min-1, respectively). As expected, solubilized membranes obtained from those cells expressing the highest number of GM-R (neutrophils and dimethyl sulfoxide-induced HL-60 cells; approximately 500-800 sites/cell) possessed the highest concentration of soluble GM-R (approximately 2-3 x 10(8) GM-R/micrograms). Cross-linking of 125I-GM-CSF to soluble GM-R resulted in the appearance of two specifically labeled complexes. A major 110-kDa receptor-ligand complex is found when cross-linking is performed with intact cells; both 110- and 200-kDa species are seen when cross-linking is performed with either intact membranes or soluble GM-R. These studies define methods by which intact GM-R can be solubilized and measured in solution, permitting a more complete biochemical characterization of the intact GM-R complex.     

An improved procedure for the isolation of lamellar bodies from human lung. Lamellar bodies free of lysosomes contain a spectrum of lysosomal-type hydrolases

We have recently shown that lamellar body fractions purified from human lung contain a distinct acid alpha-glucosidase distinguishable from lysosomal acid alpha-glucosidase in that it does not cross-react with antibodies raised against the lysosomal enzyme and does not bind to concanavalin A (De Vries, A.C.J., Schram, A.W., Tager, J.M., Batenburg, J.J. and Van Golde, L.M.G. (1985) Biochim. Biophys. Acta 837, 230-238). In order to study the relationship between the non-concanavalin A-binding alpha-glucosidase and lamellar bodies more closely a method was developed for the further purification of the organelles. A purified lamellar body preparation isolated from human lung homogenate by discontinuous sucrose density centrifugation was subjected to gel filtration with Sepharose 4B followed by Percoll density gradient centrifugation, which yielded a lamellar body preparation with a phospholipid phosphorus/protein ratio of 12.57 +/- 0.38 (mumol/mg) (n = 3) as compared to a ratio of 3.34 +/- 0.16 (mumol/mg) (n = 3) in the sucrose density gradient preparation. Concomitantly there was a 3.3 +/- 0.1 (n = 3)-fold enrichment in the content of total acid alpha-glucosidase and a 3.2 +/- 0.1 (n = 3) -fold enrichment of non-concanavalin A-binding acid alpha-glucosidase. The new purification method removes adhering proteins without changing the phospholipid composition. During the successive purification steps the concanavalin A-sensitive and -insensitive alpha-glucosidases remained fully lamellar body fraction associated. Differences between a lysosome-enriched fraction and a lamellar body preparation at varying stages of purification with respect to the ratio between soluble acid hydrolases and the membrane-associated lysosomal enzyme glucocerebrosidase indicate that the purified lamellar bodies were not contaminated with lysosomes. The absence of lysosomes in the purified lamellar body fraction was confirmed by experiments with the weak base glycyl-L-phenylalanine-beta-naphthylamide, which is an artificial substrate for the lysosomal enzyme cathepsin C and brings about lysis of lysosomes. Morphological examination by electron microscopy endorses the absence of contaminating vesicles and organelles and showed a structural integrity of the lamellar bodies in the final preparation. The improved isolation procedure strongly suggests that the concanavalin A-insensitive acid alpha-glucosidase is endogenous to lamellar bodies and supports our earlier idea that it can be used as a lamellar body-specific marker enzyme. In addition, the experiments show that lamellar bodies free of lysosomes contain a spectrum of lysosomal-type enzymes.     

Activity and isoenzyme patterns of glycolytic enzymes during perinatal development of rat lung

The enzymes hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11), enolase (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40) were studied in rat lung during development starting at day 16 of gestation (day-6) until 5 days after birth. During gestation, the activities of hexokinase type II, enolase and pyruvate kinase decreased and reached adult values at birth or shortly thereafter. Hexokinase type I remained relatively constant and the decrease of soluble type II hexokinase was compensated for by an increment of particle-bound hexokinase starting at day 20 of gestation until birth. In contrast, phosphofructokinase activity increased until day 20 of gestation followed by a rapid fall in activity until 2 days after birth. Except for hexokinase no isoenzyme shifts were observed in the period of observation. The results are discussed with respect to the proposed relationship between glycogen breakdown and surfactant synthesis during the perinatal period and suggest a regulatory role for phosphofructokinase in this process.     

The predicted ATP-binding domains in the hexose transporter GLUT1 critically affect transporter activity.

The glucose transporter GLUT1 has three short amino acid sequences (domains I-III) with homology to typical ATP-binding domains. GLUT1 is a facilitative transporter, however, and transports its substrates down a concentration gradient without a specific requirement for energy or hydrolysis of ATP. Therefore, we assessed the functional role of the predicted ATP-binding domains in GLUT1 by site-directed mutagenesis and expression in Xenopus oocytes. For each mutant, we determined the level of protein expression and the kinetics of transport under zero-trans influx, zero-trans efflux, and equilibrium exchange conditions. Although all five mutants were expressed at levels similar to that of the wild-type GLUT1, each single amino acid change in domains I or III profoundly affected GLUT1 function. The mutants Gly116-->Ala in domain I and Gly332-->Ala in domain III exhibited only 10-20% of the transport activity of the wild-type GLUT1. The mutants Gly111-->Ala in domain I and Leu336-->Ala in domain III showed altered kinetic properties; neither the apparent Km nor the Vmax for 3-methylglucose transport were increased under equilibrium exchange conditions, and they did not show the expected level of countertransport acceleration. The mutant Lys117-->Arg in domain I showed a marked increase in the apparent Km for 3-methylglucose transport under zero-trans efflux and equilibrium exchange conditions while maintaining countertransport acceleration. These results indicate that the predicted ATP-binding domains I and III in GLUT1 are important components of the region in GLUT1 involved in transport of the substrate and that their integrity is critical for maintaining the activity and kinetic properties of the transporter.     

Effects of triiodothyronine on the synthesis of sulfolipids by oligodendrocyte-enriched glial cultures

Glial cultures were obtained from the brains of 1-week-old rats and were grown in a chemically defined, serum-free medium. We investigated the development of oligodendrocytes in these cultures and the synthesis of sulfolipids in the presence and absence of triiodothyronine (T3) in the medium: (1) In the presence of T3, the incorporation of [35S]sulfate into sulfolipids exhibited a developmental profile which is comparable to that found in the developing brain in vivo. A sharp peak of sulfolipid synthesis was observed at day 5 in vitro, which is equivalent to day 12 after birth. As observed in vivo, the percentage of label incorporated into sulfogalactosyldiradylglycerols decreased with time in culture. (2) Addition of T3 to the medium stimulated sulfolipid synthesis by oligodendrocytes in a dose-related manner (optimal T3 concentration, 30 nM). The hormone also enhanced the rates of cholesterogenesis and lipogenesis but to a lesser extent than sulfolipid synthesis. (3) The temporary omission of T3 from the medium resulted in lower rates of sulfolipid synthesis that could not be restored by readdition of T3. This inhibitory effect was most pronounced if the hormone was omitted from the medium on days 2 and 3 in culture. (4) Omission of T3 also resulted in the development of fewer oligodendrocytes in the cultures. Our results show that T3 is essential for the development of oligodendrocytes in our neurone-free culture system. They also indicate that the stimulation of myelination by thyroid hormones can, at least partially, be explained as a direct effect of T3 on oligodendrocytes, independent of an effect of T3 on neuronal growth.