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F Guthmann

Publications and source records attributed to F Guthmann.

9 recordsLinked to original sources
PubMed1999

Fatty acid translocase/CD36 mediates the uptake of palmitate by type II pneumocytes.

Type II pneumocytes, which synthesize, store, and secrete pulmonary surfactant, require exogenous fatty acids, in particular palmitic acid, for maximum surfactant synthesis. The uptake of palmitate by type II pneumocytes is thought to be protein mediated, but the protein involved has not been characterized. Here we show by RT-PCR and Northern blot analysis that rat type II pneumocytes express the mRNA for fatty acid translocase (FAT/CD36), a membrane-associated protein that is known to facilitate the uptake of fatty acids into adipocytes. The deduced amino acid sequence from rat type II pneumocytes reveals 98% identity to the FAT/CD36 sequence obtained from rat adipocytes. The uptake of palmitate by type II pneumocytes follows Michaelis-Menten kinetics (Michaelis-Menten constant = 11.9 +/- 1.8 nM; maximum velocity = 62.7 +/- 5.8 pmol. min(-1). 5 x 10(5) pneumocytes(-1)) and decreases reversibly under conditions of ATP depletion to 35% of control uptake. Incubation of cells at 0 degrees C inhibited the uptake of palmitate almost completely, whereas depletion of potassium was without effect. Preincubation of the cells with bromobimane or phloretin decreases the uptake of palmitate significantly as does preincubation with sulfo-N-succinimidyl oleate, the specific inhibitor of FAT/CD36 (C. M. Harmon, P. Luce, A. H. Beth, and N. A. Abumrad. J. Membr. Biol. 121: 261-268, 1991). From these data, we conclude that FAT/CD36 is expressed in type II pneumocytes and mediates the uptake of palmitate in a saturable and energy-dependent manner. The data suggest that the uptake process is independent of the formation of coated pits and endocytotic vesicles.

Amino Acid Sequence↗
PubMed1998

alpha- and delta-tocopherol induce expression of hepatic alpha-tocopherol-transfer-protein mRNA.

alpha-Tocopherol transfer protein (alpha-TTP) supplements nascent very-low-density lipoprotein (VLDL) preferentially with alpha-tocopherol by selecting the alpha-isomers against other stereoisomers of tocopherol. It is exclusively expressed in liver. We investigated whether the expression of the hepatic alpha-TTP can be induced by dietary tocopherols. Vitamin E-depleted rats were fed with a diet containing alpha- and delta-tocopherol (ratio 1:3). The expression of alpha-TTP mRNA was measured in liver tissue. The ratio of tocopherol stereoisomers was determined in plasma, plasma lipoproteins and tissues to measure the metabolic action of alpha-TTP. Refeeding a diet containing either alpha- or delta-tocopherol, or both, caused a steady increase of the expression of alpha-TTP mRNA. In parallel the alpha/delta-tocopherol ratio increased in plasma, VLDL, high-density lipoprotein and low-density lipoprotein as well as in liver tissue, when the diet was fed containing both isomers. The alpha-tocopherol/delta-tocopherol ratio of heart, kidney, lung, lamellar bodies of lung and in lung lavage showed no or a comparatively low increase. The data show that both tocopherol isomers were able to induce alpha-TTP mRNA in rat liver and, thus, the ability of liver to select for the alpha-isomer. On the other hand, tocopherol depletion did not change the expression of hepatic alpha-TTP mRNA in the rat.

Animals↗
PubMed1998

Expression of fatty-acid-binding proteins in cells involved in lung-specific lipid metabolism.

Members of the fatty-acid-binding protein (FABP) family are thought to play an important role in fatty acid transport within the cytosol and thus to be involved in lipid metabolism. As previous data on the occurrence of distinct FABP types in total lung are contradictory, we determined the expression of FABP types in three isolated cell types of rat lung, which are characterised by active lipid metabolism. Alveolar type-II cells synthesise, store and secrete pulmonary surfactant, a phospholipid-rich surface-tension-lowering agent, whereas lung fibroblasts, localised adjacent to the alveolar type-II cells, are assumed to provide neutral lipid substrate to alveolar type-II cells around birth, and alveolar macrophages are known to degrade complex lipids. Initial screening by reverse transcriptase PCR revealed the occurrence of heart (H-), epidermal (E-) and liver FABP in rat lung, the latter being not detectable in the three cell types studied. Cells were analysed by northern and western blotting, then quantitatively by sandwich ELISA, for which recombinant rat E-FABP was prepared. E-FABP mRNA was found in all three cell types, and E-FABP was detected in the following amounts: 240.9 +/- 19.0 ng/mg cytosolic protein in alveolar type-II cells; 172.3 +/- 0.7 ng/mg protein for lung fibroblasts; and 36.9 +/- 3.5 ng/mg protein for alveolar macrophages. This indicates a basic function of E-FABP in cellular lipid metabolism. In contrast, H-FABP probably is involved in the metabolism of neutral lipids because H-FABP mRNA was found only in lung fibroblasts with a corresponding protein level of 315.5 +/- 6.9 ng/mg. Small amounts of H-FABP protein were present in alveolar type-II cells and alveolar macrophages.

Animals↗
PubMed1997

Interaction of lipoproteins with type II pneumocytes in vitro: morphological studies, uptake kinetics and secretion rate of cholesterol.

Apart from dipalmitoyl phosphatidylcholine, cholesterol is the most abundant surfactant lipid. About 90 to 99% of cholesterol of the alveolar surfactant is derived from serum lipoproteins. The aim of this study was to identify the lipoprotein which preferentially supplements type II pneumocytes with cholesterol destined for surfactant production. Ultrastructural investigations revealed that type II pneumocytes bind and take up HDL, LDL and VLDL. Binding and uptake of VLDL occurred even in the presence of excess LDL indicating that, besides LDL receptors, type II pneumocytes express additional binding sites for VLDL. Type II pneumocytes in primary culture are able to take up cholesterol added in the form of HDL, LDL and VLDL. Cholesterol uptake was lowest from HDL and highest from VLDL. The maximal velocity of cholesterol uptake from VLDL was more than three times that of cholesterol uptake from LDL. The half-maximal saturation of cholesterol uptake from VLDL was nearly half that of LDL. From these kinetic data and the distribution of free cholesterol among the serum lipoproteins, we calculated that the cholesterol uptake from VLDL is more than three times that of cholesterol uptake from LDL. In double-labeling experiments type II pneumocytes secreted palmitic acid-labeled phospholipids together with labeled free cholesterol taken up from lipoproteins. The secretion rates of both phospholipids and free cholesterol were stimulated to nearly the same extent by isoproterenol. From our results we conclude that type II pneumocytes interact specifically with HDL, LDL and VLDL. Cholesterol taken up in the form of the individual lipoproteins shows no difference in its availability for the formation of cholesterol ester and surfactant by type II pneumocytes in vitro. Based on the kinetic studies, it appears that VLDL is the major gateway through which cholesterol is provided to satisfy the cholesterol requirements of type II pneumocytes for the synthesis of surfactant.

Animals↗
PubMed1995

Alveolar surfactant subfractions differ in their lipid composition.

Alveolar surfactant consists of subfractions which are generated during normal lung function. Although subfractions obtained by differential centrifugation of lung lavage differ in structure, function and protein content, the phospholipid-pattern shows only minor differences. To correlate possible differences in composition between subfractions to their functional properties we did a more detailed analysis of lipid pattern. Subfractions of lung lavages from Wistar rats were obtained by differential centrifugation, lipid classes were separated by thin layer chromatography (TLC). Fatty acids and plasmalogens were determined as methylester and dimethylacetals by gas chromatography, respectively. Cholesterol and vitamin E were determined enzymatically and by HPLC, respectively. The patterns of fatty acids of total lipids and of the molecular species of phosphatidylcholine and phosphatidylethanolamine were very similar among the subfractions. The distribution of individual lavage lipids varied considerably. Three types of subfractions can be distinguished: The two dense subfractions (1000 g and 60,000 g) contain 70-88% of total phospholipids, dipalmitoylphosphatidylcholine, polyunsaturated phospholipids and polyunsaturated fatty acids present in lung lavage. The less dense subfraction (100,000 g) contains 44-60% of total cholesterol, choline plasmalogen, ethanolamine plasmalogen and vitamin E. The 100,000 g supernatant contains 40-50% of total tri-, diacylglycerols and free fatty acids. Our results support the concept that the 1000 g subfraction contains freshly secreted surfactant. The 60,000 g subfraction likely contains the monolayer and freshly secreted surfactant. The 100,000 g pellet probably contains material "squeezed out" from the monolayer at expiration. Most likely, the supernatant contains material destined for removal from the airspace.

Animals↗
PubMed1994

Synthesis and secretion of plasmalogens by type-II pneumocytes.

Alveolar surfactant (exposed to air and therefore a prime target of air oxidants) is supplied with antioxidants during its intracellular formation on type-II pneumocytes [Rüstow, Haupt, Stevens and Kunze (1993) Am. J. Physiol. 265, L133-L139]. Plasmalogens can protect animal cells against lipid peroxidation caused by u.v. radiation. It has been suggested that plasmalogens play a direct role in protecting animal cell membranes against oxidative stress [Zoeller, Morand and Raetz (1988) J. Biol. Chem. 263, 11590-11596]. We investigated biosynthesis and secretion of plasmalogens and phospholipids by type-II cells of adult rat lungs. The plasmalogens of type-II cells consist of 93% ethanolamine plasmalogens (EthPlas) and 7% choline plasmalogens (ChoPlas). Plasmalogens isolated from alveolar surfactant, however, consist of 36.5% ChoPlas and 63.5% EthPlas. The different incorporation rates of [14C]hexadecanol into both types of plasmalogen by type-II pneumocytes are reflected in the relative proportions of their total cellular plasmalogen content. Type-II cells cultured in the presence of labelled hexadecanol or labelled hexadecylglycerol and of labelled palmitate secrete labelled ChoPlas and labelled phospholipids, both spontaneously and in response to isoprenaline. The spontaneous and stimulated secretion rates of labelled ChoPlas are 3-6 times higher than those of labelled EthPlas. This higher relative secretion rate of ChoPlas corresponds to its higher proportion in the total plasmalogen content of alveolar surfactant compared with type-II cells. Added extracellular surfactant-specific protein A inhibits the secretion of plasmalogens as well as that of phospholipids by type-II cells. The molecular species of EthPlas and ChoPlas isolated from type-II cells or lung lavage do not differ significantly and consist mainly of molecular species containing poly-unsaturated fatty acids. We conclude that ChoPlas are secreted partly as integral constituents of the alveolar surfactant. Type-II cells select between both types of plasmalogens for secretion as a constituent of surfactant. The intramolecular sorting signal presumably is the choline moiety.

Animals↗
PubMed1992

Characterization of soluble platelet guanylyl cyclase with peptide antibodies.

Soluble guanylyl cyclase partially purified from bovine and human platelets was characterized with antibodies raised against synthetic peptides corresponding to different sequences of the alpha 1- and beta 1-subunits of the bovine lung enzyme. On immunoblots, the platelet guanylyl cyclase was recognized by the four antisera used, with the exception of an antiserum against the C-terminus of the beta 1-subunit which did not react with the human platelet but with the bovine platelet beta 1-subunit. Furthermore the human platelet beta 1-subunit exhibited a slightly lower molecular mass than the bovine protein. The C-terminal antibodies precipitated native platelet and lung guanylyl cyclase activity. In contrast an antibody against a peptide out of the putative catalytic domain, which is highly conserved between all guanylyl cyclases sequenced so far, did not precipitate native guanylyl cyclase, although it recognized both subunits on immunoblots, suggesting that the respective amino acid sequence is located in an inner site of the protein.

Amino Acid Sequence↗