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Pulmonary surfactant protein C (SP-C) is one of the pulmonary surfactant proteins. It is a small, extremely hydrophobic peptide and has a highly conservative primary structure. The protein is normally palmitoylated on two adjacent cysteine residues. One arginine and one lysine residue are positively charged. SP-C enhances the adsorption of phospholipids into the air-water interface of the alveolar space. Structure and function of SP-C were investigated. Palmitoylated and non-palmitoylated human recombinant SP-C had a high α-helix content, but the orientation of the α-helix was dependent on the presence of palmitoyl chains. The physical properties of the acylated SP-C were different from the non-palmitoylated SP-C. Two forms of canine SP-C were isolated: monomeric palmitoylated SP-C, and dimeric, non-palmitoylated SP-C. The dimerization of SP-C had no impact on the physical properties of the protein. Binding of phospholipid-vesicles to the monolayer, and insertion of phospholipids into the monolayer was dependent on the positively charged amino acids.

1 The distribution of phospholipids between inside and outside of rat erythrocyte membranes was studied by incubating the cells with phospholipase A2 from Naja naja venom and sphingomyelinase from Staphylococcus aureus. 2 Choline-containing phospholipids were found to comprise the majority of the outer laver of the membrane. 3 The incorporation of radioactive fatty acids into phospholipids occurred predominantly at the inside of the membrane. 4 Exchange of phospholipids between red cell membranes and plasma lipoproteins occurred at the outside of the membrane. 5 Indications were found for a rather slow flip-flop of lecithin across the membrane.

1. 1. The rate-limiting reaction in the biosynthesis of surfactant phosphatidylcholine by type II cells isolated from adult rat lung was examined. 2. 2. Studies on the uptake of [Me-14C]choline and its incorporation into its metabolites over a 5 h period indicated that in these cells the Cholinephosphate pool was much larger than both the choline and CDPcholine pool. This is consistent with the idea that the rate-limiting reaction is that catalyzed by Cholinephosphate cytidylyltransferase. 3. 3. Evidence that Cholinephosphate cytidylyltransferase is the slowest of the three enzymes incorporating choline into phosphatidylcholine was also obtained from pulse-chase experiments. [Me-14C]Choline taken up by the cells was rapidly converted into Cholinephosphate during the pulse period. As the radioactivity disappeared from Cholinephosphate during the chase period, the label was incorporated immediately into phosphatidylcholine, without much change in the labelling of CDPcholine. This indicates that the cholinephosphotransferase is at least as fast as the Cholinephosphate cytidylyltransferase. 4. 4. Inclusion of palmitate in the chase medium accelerated the conversion of labelled Cholinephosphate into phosphatidylcholine and decreased the radioactivity associated with CDPcholine. This indicates that under these conditions the Cholinephosphate cytidylyltransferase reaction cannot keep up with increased utilization of the CDPcholine in the terminal step of the CDPcholine pathway.

In the present study pulse-label and pulse-chase experiments with isolated rat hepatocytes in suspension were designed to investigate the effects of the presence of either serine or ethanolamine in the medium on the rate of phosphatidylethanolamine synthesis via the CDPethanolamine pathway and by decarboxylation of phosphatidylserine. Addition of serine to the medium did not affect the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamine. Pulse-label experiments showed that the incorporation of [3H]serine into phosphatidylserine decreased in the presence of ethanolamine with a corresponding decrease of the incorporation of label into the ethanolamine base moiety of phosphatidylethanolamine. However, the radioactivity in the diacylglycerol part of phosphatidylethanolamine was considerably higher in the presence of ethanolamine than in its absence. Pulse-chase experiments with labelled serine demonstrated that the conversion of phosphatidylserine to phosphatidylethanolamine was not affected by varying concentrations of ethanolamine. Our observations indicate that in the presence of ethanolamine the biosynthesis of phosphatidylethanolamine via the CDPethanolamine pathway is enhanced relative to the synthesis by decarboxylation of phosphatidylserine.

Pulmonary surfactant is essential for a proper functioning of the mammalian lung. It provides mechanical stability to the alveoli, prevents alveolar edema, and plays a role in the pulmonary defense system. Because of this vital role, alterations in the surfactant system may account for some of the observed toxic effects of environmental agents and drugs on the lung. In this review we will discuss the effects of several noxious agents such as oxidant gases, smoke, dusts, and herbicides on the quality and production of surfactant.

Pulmonary surfactant prevents alveolar collapse and transudation at low lung volumes by diminishing the surface tension at the air-liquid interface (Goerke, 1974). Pulmonary surfactant consists of approx. 90% of lipids and it is generally accepted that dipalmitoylphosphatidylcholine, which comprises almost half of total surfactant, is the major surface active component of this material. Noteworthy is the relatively high content of phosphatidylglycerol, a phospholipid which is found in other mammalian tissues in minute amounts only. It is known from numerous studies with a variety of mammalian species that the production of surfactant in the developing lung is initiated during the terminal part of gestation (for a review see Possmayer, 1984). This results in a surge of surfactant accumulation in the airways in this period, which coincides with the ability of the neonate to establish regular airbreathing. Surfactant deficiency is indeed the prime factor responsible for the occurrence of respiratory distress syndrome in premature newborns (Farrell & Avery, 1975). The formation of surfactant takes place in the alveolar type-I1 cell, one of the 40 different cell types in lung tissue. Studies on the biosynthesis of surfactant have long been hampered by the enormous cellular diversity of lung tissue. The development of techniques to isolate type-I1 cells from adult lung (Mason, 1982) and from fetal lung at different stages of development (Douglas & Teel, 1976) has given momentum to studies on the pathways operating in surfactant-lipid synthesis in the adult and fetal lung and on the regulation of these processes.

1. The distribution of radioactivity among lipid classes of myelin and other subcellular brain fractions of young rats (18-21 days) was determined after in vivo injection of (3-(14)C-labelled ketone bodies, [U-(14)C] glucose or [2-(14)C] glucose. 2. The incorporation ratios (sterol/fatty acids) were 0.67, 1.48, 0.25, 0.62 and 0.54 for whole brain, myelin, mitochondria, microsomes and synaptosomes, respectively, with (3-(14)C)-labelled ketone bodies as substrate and 0.37, 0.89, 0.19, 0.34 and 0.29 with [U-(14)C] glucose as substrate. These data show that, both in whole brain and in subcellular brain fractions, acetyl groups derived from ketone bodies are used for sterol synthesis to a large extent than acetyl groups originating from glucose. 3. The specific radioactivity of cholesterol is much higher in myelin than in whole brain or in the other brain fractions, particularly after administration of labelled ketone bodies as substrate. 4. The incorporation patterns of acetoacetate and D-3-hydroxybutyrate were very similar, indicating that both ketone bodies contribute acetyl groups for lipid synthesis via the same metabolic route. 5. Our data suggest that a direct metabolic path from ketone bodies towards cholesterol exists - possibly via acetoacetyl-CoA formation in the cytosol of brain cells - and that this process is most active in oligodendrocytes.

In lamellar bodies isolated from adult human lung at least two acid alpha-glucosidases are present: one similar to the lung lysosomal alpha-glucosidase, and another lamellar body-specific isoenzyme. In the present study we measured the activity of this lamellar body-specific alpha-glucosidase and of lysosomal alpha-glucosidase in a patient with an inherited deficiency of lysosomal alpha-glucosidase. The activity of the lamellar body-specific alpha-glucosidase was not affected in the patient, whereas the lysosomal alpha-glucosidase activity was strongly depressed. The results strongly suggest that the lysosomal alpha-glucosidase and the lamellar body-specific alpha-glucosidase are different gene products.

The glycolytic enzyme 6-phosphofructokinase (EC 2.7.1.11) was studied in adult and fetal type II pneumocytes which had been isolated from rat lung at different days of development. In addition, the activities of the enzymes hexokinase (EC 2.7.1.1), enolase (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40) were assayed. The specific activities of the latter enzymes decrease during perinatal development and reach about adult values shortly after birth. In contrast, 6-phosphofructokinase activity increases slightly until 2 days before birth, and drops sharply afterwards. The 6-phosphofructokinase subunit composition was determined in fetal and adult type II cells. The ratio of the three subunits of 6-phosphofructokinase in type II cells isolated on fetal days 19 and 21 (term is at day 22) and in adult type II cells was identical: the three subunits were present in a ratio of 68: 14: 18 for types L, M and C, respectively. In addition, we investigated some regulatory properties of 6-phosphofructokinase from alveolar type II cells. 6-Phosphofructokinase from alveolar type II cells is strongly inhibited by increasing MgATP concentrations. This inhibition is reflected by an increase in the S0.5 for fructose 6-phosphate. Fructose 2,6-bisphosphate stimulates alveolar type II 6-phosphofructokinase. Half-maximal stimulation occurs at 1.6 and 2.0 microM fructose 2,6-bisphosphate for fetal and adult type II cells, respectively. The level of the most potent positive effector of 6-phosphofructokinase, fructose 2,6-bisphosphate, was also determined. The level of the hexose bisphosphate decreases during prenatal development; however, the level in the adult type II cells is considerably lower. The concentration of fructose 2,6-bisphosphate appears to be sufficient to fully activate 6-phosphofructokinase both in fetal and adult type II cells.

1. 1. Alveolar type II cells isolated from adult rat lung incorporated radioactively labelled palmitate predominantly into the 2-position of disaturated phosphatidylcholine. In disaturated diacylglycerol, however, the radioactivity was almost equally distributed between the 1- and 2-positions. 2. 2. Exposure of isolated type II cells to the phospholipase A2 inhibitors 4-bromophenacylbromide or quinacrine dihydrochloride led to a decreased synthesis of total phosphatidylcholines from various labelled precursors. Interestingly, it also led to an increased degree of unsaturation of the phosphatidylcholines synthesized by the cells. 3. 3. Incubation of type II cell sonicates with radioactively labelled CDPcholine resulted in the formation of labelled phosphatidylcholine; 56% of this phosphatidylcholine appeared to be disaturated. In similar experiments with homogenates from whole lung, 20% of the synthesized phosphatidylcholine was disaturated. 4. 4. These results suggest that both direct synthesis de novo and remodeling of 1-saturated-2-unsaturated phosphatidylcholines contribute to the biosynthesis of disaturated phosphatidylcholine in isolated alveolar type II cells.

Rat pups, 3 weeks old, were injected i.p. with combinations of ³H₂0 and either (3-¹⁴C]acetoacetate or [¹⁴C]glucose. ³H/¹⁴C incorporation ratios were measured in lipid fractions of homogenates and myelin prepared from whole brain and spinal cord. Spinal cord synthesized at least twice as much fatty acids and 3-fold more sterols than whole brain. Both tissues used acetoacetate preferentially for sterol synthesis, whereas label from [¹⁴C]glucose was distributed between fatty acids and sterols in the same way as ³H from ³H₂O. The relative contributions of acetoacetate to sterol synthesis in whole tissue and in the purified myelin fraction were about the same, both for the cerebrum and for the spinal cord.

We developed a new method for the determination of choline- and ethanolamine-containing precursors of phosphatidylcholine and phosphatidylethanolamine after their separation by HPLC and we have studied the effects of different dietary conditions on the pool sizes of these metabolites in rat liver. Fasting for 48 h induced only a small decrease in the amounts of phosphatidylethanolamine and its water-soluble precursors. Upon refeeding with a high-sucrose, fat-free diet for 72 h, the levels of ethanolamine-containing compounds were only slowly restored. The effects of various dietary conditions on the amounts of phosphatidylcholine and its water-soluble precursors were much more pronounced. Fasting induced a sharp decrease, especially of the amount of cholinephosphate. However, the levels of phosphatidylcholine and the choline-containing precursors were rapidly restored upon refeeding for 24 h. Continued refeeding for an additional 48 h enhanced the cholinephosphate pool size to a level more than double that found in normally fed rats. The latter effect was accompanied by an inhibition of the enzyme CTP: choline-phosphate cytidylyltransf erase. The results are discussed in view of a possible regulatory mechanism that may balance the amounts of phosphatidylcholine and phosphatidylethanolamine.

Isolated rat hepatocytes in suspension secrete very low density lipoproteins (VLDL) at a rate comparable with that of the perfused liver. The apoproteins of these lipoproteins are mainly of the B and E type. The amount of apoprotein C in VLDL secreted by hepatocytes is much less than that present in VLDL obtained from rat serum. Incubation of hepatocytes in the presence of fatty acids stimulates the intracellular synthesis of triacylglycerols and their secretion in VLDL. This stimulation is a linear function of the palmitic acid concentration up to 1.6 m m , the highest concentration tested. Colchicine (50 μ m ) reduced VLDL secretion by 90%. The stimulation of triacylglycerol synthesis and VLDL secretion upon incubation of hepatocytes with fatty acids is paralleled by an enhanced activity of microsomal diacylglycerol acyltransferase (DGAT, EC 2.3.1.20), the only enzyme exclusively involved in the synthesis of triacylglycerols. A mixture of oleic (0.2 m m ) and palmitic (0.2 m m ) acid added to the cell medium stimulates the activity of DGAT by 354%. This increase in enzyme activity persisted during cell homogenization and subsequent preparation of microsomes to assay the enzyme. It is concluded that freshly isolated hepatocytes in suspension represent a good system to study triacylglycerol synthesis and VLDL secretion, and that the stimulatory effects of fatty acids on these processes are, at least partially, mediated by enhanced activities of DGAT.

1. 1. The pool sizes of the choline intermediates and cofactors involved in the CDPcholine pathway were studied in alveolar type II cells from adult rat lung. 2. 2. The choline phosphate pool was much larger than both the choline and CDPcholine pools. 3. 3. Kinetic analysis of the pool sizes revealed that cholinephosphate cytidylyltransferase catalyzes a non-equilibrium reaction. 4. 4. These results are consistent with the idea that cholinephosphate cytidylyltransferase regulates the choline flux into phosphatidylcholine in adult rat lung type II cells.

1. Alveolar type II cells from adult rat lung have the enzymic capability to acylate 1-palmitoyl-lysophosphatidyl-glycerol, using palmitoyl-CoA to form dipalmitoylphosphatidylglycerol. 2. On a protein basis the acylation of 1-palmitoyl-lysophosphatidylglycerol is at least 2-fold more active in sonicated type II cells than in whole lung homogenates. 3. Both type II cells and whole lung homogenates show higher activity towards palmitoyl-CoA than towards oleoyl-CoA for acylation of 1-palmitoyl-lysophosphatidylglycerol. 4. Both in type II cells and in whole lung homogenates the rates of acylation of 1-palmitoyl-lysophosphatidylglycerol and 1-palmitoyl-lysophosphatidylcholine with palmitate are of the same order of magnitude, while the rate of acylation of 1-palmitoyl-lysophosphatidylethanolamine is much lower.

1. 6-Phosphofructokinase of both fetal and adult rat lung consists of L, M and C subunits in a ratio of 65:25:10. 2. 6-Phosphofructokinase was purified to homogeneity from adult rat lung and subjected to phosphorylation in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. 3. This resulted in phosphorylation of the L and M subunit of 6-phosphofructokinase. 4. The C subunit was not phosphorylated. 5. However, if the phosphorylation of 6-phosphofructokinase was studied in the cytosol fraction of either fetal or adult lung using endogenous protein kinase(s), only the L subunit was phosphorylated. 6. This phosphorylation was dependent on cyclic AMP. 7. No influence of calcium, calmodulin or phosphatidylserine/diolein on the phosphorylation was observed. 8. It is concluded that although both L and M subunits of rat lung 6-phosphofructokinase are potential substrates for cyclic AMP-dependent protein kinase, their phosphorylation in situ is differentially regulated.

Mammalian sperm cells are activated prior to fertilization by high bicarbonate levels, which facilitate lipoprotein-mediated cholesterol efflux. The role of bicarbonate and cholesterol acceptors on the cholesterol organization in the sperm plasma membrane was tested. Bicarbonate induced an albumin-independent change in lipid architecture that was detectable by an increase in merocyanine staining (due to protein kinase A-mediated phospholipid scrambling). The response was limited to a subpopulation of viable sperm cells that were sorted from the non-responding subpopulation by flow cytometry. The responding cells had reduced cholesterol levels (30% reduction) compared with non-responding cells. The subpopulation differences were caused by variable efficiencies in epididymal maturation as judged by cell morphology. Membrane cholesterol organization was observed with filipin, which labeled the entire sperm surface of non-stimulated and non-responding cells, but labeled only the apical surface area of bicarbonate-responding cells. Addition of albumin caused cholesterol efflux, but only in bicarbonate-responding cells that exhibited virtually no filipin labeling in the sperm head area. Albumin had no effect on other lipid components, and no affinity for cholesterol in the absence of bicarbonate. Therefore, bicarbonate induces first a lateral redistribution in the low cholesterol containing spermatozoa, which in turn facilitates cholesterol extraction by albumin. A model is proposed in which phospholipid scrambling induces the formation of an apical membrane raft in the sperm head surface that enables albumin mediated efflux of cholesterol.

Cultures of glial cells were prepared from the brains of one-week-old rat pups. After one day in culture, serum was omitted from the medium and replaced by a combination of growth-stimulating hormones and other factors that enhanced the percentage of oligodendrocytes in the cultures. We investigated the effects of hydrocortisone on the development of oligodendrocytes, on the activities of oligodendrocyte-specific enzymes and on glucose- and lipid-metabolism of the glial cells. (1) Hydrocortisone greatly enhanced the survival of glial cells in culture. (2) The development of galactocerebroside-positive cells and the specific activity of 2′,3′-cyclic-nucleotide 3′-phosphodiesterase were stimulated by 50 nM hydrocortisone, whereas these effects were partly reversed at higher concentrations of the hormone. (3) The specific activity of glycerol-3-phosphate dehydrogenase was markedly stimulated by hydrocortisone; 1 μM or higher concentrations of hydrocortisone were required for an optimal effect. (4) The consumption of glucose and the production of lactate were lowered by hydrocortisone whereas the oxidation of [6-14C]glucose to 14CO2 was not affected. (5) Incorporation of [35S]sulfate into sulfolipids was greatly enhanced by hydrocortisone and [14C]incorporation from [1-14C]acetate into cholesterol and fatty acids was also stimulated but to a smaller extent. These results show that hydrocortisone (i) exerts a general trophic function on glial cells in our culture system; (ii) enhances the ratio of oligodendrocytes over astrocytes, possibly by directing bipotential progenitor cells to develop into oligodendrocytes; (iii) specifically induces glycerol-3-phosphate dehydrogenase in oligodendrocytes.