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Biomedical subjects

O Stein

Publications and source records attributed to O Stein.

At least 109 records · Page 6Linked to original sources
PubMed1983

Lipoprotein lipase activity in cultured macrophage cell line J774(2) and its increase in variants deficient in adenylate cyclase and cyclic AMP-dependent protein kinase.

Three macrophage cell lines, J774(2), CT2 and J7H1 were compared with respect to synthesis and secretion of lipoprotein lipase. The enzyme activity measured was characterized as lipoprotein lipase on the basis of serum dependence and inhibition by 1 M NaCl. Enzyme activity in all three lines increased with time in culture and the highest activity was found in the medium of the CT2 line which is adenylate cyclase deficient while that in the J7H1 line, cyclic AMP-dependent protein kinase deficient, was intermediate. The half life of the enzyme activity in conditioned medium from all three lines was 30-40 min, suggesting that the different levels of activity observed do represent different levels of enzyme production by the cells. About 80% of the lipoprotein lipase activity from all three lines was present in the medium and 50-70% of cellular activity could be released into the medium by a 3-min exposure to heparin. In addition, 24 h incubation with heparin enhanced enzyme secretion in all three lines. To determine the role of cyclic AMP in the regulation of lipoprotein lipase activity use was made of dibutyryl cAMP, methyl isobutylxanthine (IBMX) and cholera toxin. These agents strikingly depressed lipoprotein lipase activity in the J774(2) line but only dibutyryl cAMP was active in the CT2 line (adenylate cyclase deficient). In the J7H1 (protein kinase deficient) line there was no response to dibutyryl cAMP or IBMX over the first 4 h of incubation. Addition of these agents did not affect total cell protein synthesis. The present findings indicate that in the intact cells changes in cyclic AMP levels are associated with a change in the activity of lipoprotein lipase.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Hepatic retention and elimination of cholesteryl linoleyl ether after injection of labeled acetylated LDL or chylomicrons.

Rat mesenteric duct chylomicrons labeled with [3H]cholesteryl linoleyl ether and human acetylated low density lipoproteins labeled with [14C]cholesteryl linoleyl ether were injected simultaneously into rats. 3 h after injection 80-90% of the injected radioactivity were recovered in the liver and the ratio of 3H/14C in the liver was the same as in the injected material. The 3H/14C ratio declined gradually over a period of 18 days due to loss of [3H]cholesteryl ether which had been injected with the chylomicrons, and retention of the same compound injected bound to acetylated LDL. The loss from the liver of the chylomicron-bound cholesteryl linoleyl ether was shown to occur through the bile, and its elimination from the body was verified by monitoring fecal excretion. The present results provide evidence that hepatic persistence of a nonhydrolyzable analog of cholesteryl ester is a function of the cell type which has ingested the lipid. Thus, the uptake of labeled chylomicrons by hepatocytes results in a slow but progressive excretion of the nonhydrolyzable lipid through the bile, while the preferential uptake of acetylated LDL by nonparenchymal cells of liver and by the spleen leads to persistence of the lipid in the organ.

Acetylation↗
PubMed1983

Transfer of cholesteryl linoleyl ether from phosphatidylcholine and phosphatidylethanolamine liposomes to cultured cells catalyzed by lipoprotein lipase.

Unilamellar liposomes prepared from purified phospholipids (phosphatidylcholine, phosphatidylethanolamine or sphingomyelin) and labeled cholesteryl linoleyl ether were used to study lipoprotein lipase-catalyzed transfer of cholesteryl ester into cells in culture. In mesenchymal rat heart cell cultures, the transfer of cholesteryl linoleyl ether and cholesteryl linoleate was similar and related to the activity of endogenously produced lipoprotein lipase. In human skin fibroblasts transfer of labeled cholesteryl linoleyl ether was proportional to the concentration of milk lipoprotein lipase added to the incubation medium. Liposomes prepared from phosphatidylcholine or phosphatidylethanolamine were much better donors of cholesteryl ether to normal and apolipoprotein E-B receptor-negative fibroblasts and to endothelial cells than those prepared from sphingomyelin. Lysophosphatidylcholine was formed during incubation with milk lipoprotein lipase but was not considered to be directly responsible for the lipoprotein lipase-catalyzed transfer of cholesteryl ether. This conclusion was drawn because in the absence of lipoprotein lipase addition of lysophosphatidylcholine to liposomes, or almost complete phospholipolysis by phospholipase A2, did not result in the transfer of cholesteryl linoleyl ether from liposomes to cells. Attachment of lipoprotein lipase to the cell surface was mandatory for the transfer of cholesteryl ether and could be prevented by heparin. High density apolipoprotein reduced also the transfer of cholesteryl linoleyl ether, even though it did not interfere with the binding of labeled milk lipoprotein lipase to cultured fibroblasts. The present results provide evidence that lipoprotein lipase, and not the products of phospholipid hydrolysis, is the ligand for the non-apolipoprotein E-B receptor-mediated transfer of cholesteryl ester to cells.

Animals↗
PubMed1982

Binding of lipoprotein lipase to the cell surface is essential for the transmembrane transport of chylomicron cholesteryl ester.

Four cell types, F1 rat heart cells, rat preadipocytes, human skin fibroblasts and bovine endothelial cells, were used to investigate whether surface binding of lipoprotein lipase was essential in the transmembrane transport of chylomicron cholesteryl ester. Exposure of F1 heart cells to colchicine resulted in decrease in endogenous surface-bound lipoprotein lipase and a concomitant fall in the uptake of chylomicron cholesteryl linoleyl ether, a nondegradable analog of cholesteryl ester. Uptake of chylomicron cholesteryl linoleyl ether was enhanced by addition of milk lipoprotein lipase and this enhancement also persisted in the presence of colchicine. The drug did not reduce surface binding to the enzyme. Milk lipoprotein lipase was bound to the cell surface of the different cell types and its fate during chase in enzyme-free medium was determined. The t 1/2 of surface-bound enzyme in endothelial cells and in F1 heart cells was about 2 h; it was 4 h in skin fibroblasts. The decrease in surface-bound lipoprotein lipase was accompanied by a parallel fall in the binding and uptake of chylomicron cholesteryl linoleyl ether by the various cell types examined. This decrease in the uptake of cholesteryl linoleyl ether occurred even though lipoprotein lipase activity in the medium was present, as evidenced by the hydrolysis of [14C]triacylglycerol. Release of surface-bound endogenous or exogenous lipoprotein lipase by heparin was accompanied by almost complete elimination of uptake of cholesteryl linoleyl ether in presence of complete hydrolysis of [14C]triacylglycerol. The present results indicate that the transmembrane transport of cholesteryl ester is catalyzed by lipoprotein lipase only when the enzyme is bound to the cell membrane.

Adipose Tissue↗
PubMed1982

Fate of milk 125I-labelled lipoprotein lipase in cells in culture. Comparison of lipoprotein lipase- and non-lipoprotein lipase-synthesizing cells.

Radioiodinated lipoprotein lipase, isolated from bovine milk (125I-labeled milk lipoprotein lipase) was shown to retain full hydrolytic activity towards its native substrate, i.e., chylomicron triacylglycerol. The 125I-labeled enzyme interacted with various cells in culture by being bound to the cellular surface, internalized and degraded. Cellular binding of the labeled enzyme occurred in the presence or absence of substrate and was related to enzyme concentration. Heparin reduced cellular binding by 50% but inhibited uptake and degradation more extensively. Cellular uptake was not affected by chloroquine or NH4Cl, but degradation of the labeled enzyme was blocked. Uptake and degradation were not inhibited by mannose 6-phosphate. The interaction between the exogenous enzyme and cells which do not synthesize lipoprotein lipase, i.e., fibroblasts and endothelial cells, resulted in a high ratio of surface binding to degradation. In heart cell cultures and preadipocyte cultures, which produce lipoprotein lipase, the ratio of enzyme catabolized to that bound was high at all time points examined. Since in the intact organism lipoprotein lipase acts at the luminal surface of vascular endothelium, it seems expedient that these cells are able to bind the enzyme, but will catabolize it only slowly. The rapid and extensive degradation of the 125I-labeled lipoprotein lipase in heart cells and preadipocytes may be related to the metabolism of the endogenously produced lipoprotein lipase.

Adipose Tissue↗
PubMed1982

Long-lived labeling of phagocytic cells with analogs of atheroma lipids.

[3H]Trioleyl glyceryl ether and [3H]cholesteryl linoleyl ether were bound to Intralipid and injected intraperitoneally into mice. About 20% of the injected label was recovered from peritoneal macrophages up to 2 weeks after injection, and there was a gradual appearance of the label in the liver. Following injection of acetylated low density lipoprotein, labeled with [3H]trioleyl glyceryl ether, into rats, about 90% of the label appeared in the liver shortly after injection and all label was retained up to 73 days. The findings indicate that [3H]cholesteryl linoleyl ether and [3H]trioleyl glycerol can serve as non-degradable analogs of atheroma lipids, which are readily taken up by macrophages when presented in the form of appropriate substrates. These preliminary results serve as basis for experiments designed to study the role of macrophages in the transport of atheroma lipids.

Animals↗
PubMed1981

Interaction between macrophages and aortic smooth muscle cells. Enhancement of cholesterol esterification in smooth muscle cells by media of macrophages incubated with acetylated LDL.

Mouse peritoneal macrophages were cultured for 24 h in Dulbecco-Vogt medium containing 10% calf serum. This medium was replaced with Dulbecco-Vogt medium containing 1% bovine serum albumin to which all subsequent additions were made. Medium changes, accompanied by appropriate additions, were made every 48 or 72 h and the media were used for incubation of aortic smooth muscle cells, prelabeled with [3H]cholesterol. The amount of labeled cholesteryl ester in the smooth muscle cells incubated for 48 h with macrophage media which had been collected 48-144 h after addition of acetylated LDL was increased 3-4 times above that present prior to postincubation. A marked increment in cholesteryl ester mass occurred also after incubation of smooth muscle cells with macrophage media conditioned with acetylated LDL and this effect was shared by maleylated LDL, but not by other negatively charged compounds. The increase in labeled cholesteryl ester in smooth muscle cells was more pronounced with media collected at later time intervals of incubation with macrophages and was evident 8 hr after postincubation. Only the d less than 1.063 fraction of the medium enhanced cholesterol esterification in smooth muscle cells. The acetylated LDL reisolated from macrophage media at d less than 1.063 did not compete with native LDL for degradation by smooth muscle cells. No increase in degradation of 125I-labeled acetylated LDL preincubated with macrophages was observed above that of non-preincubated acetylated LDL. The macrophage medium conditioned with acetylated LDL depressed [14C]acetate incorporation into sterols in smooth muscle cells and this effect was abolished by extraction of the medium with diethyl ether. The ratio of free to total cholesterol in the macrophage media collected after incubation with acetylated LDL increased from 28-70%, and a decrease occurred after incubation with smooth muscle cells. The enhancement of cholesterol esterification could be abolished by addition of high density apolipoprotein/sphingomyelin mixture during incubation with macrophages, even though excretion of free cholesterol into the medium increased 3-fold. It is proposed that when smooth muscle cells are presented with a lipoprotein in which an increase in the free to esterified cholesterol ratio occurred, and which is not recognized by a specific receptor, the enhancement of cellular cholesterol esterification is due mostly to a surface transfer of lipoprotein-free cholesterol. The present results offer another view of the possible interactions between macrophages and smooth muscle cells. A modified lipoprotein, not recognized by smooth muscle cells, is ingested by macrophages, which leads to accumulation of esterified cholesterol. Part of the esterified cholesterol undergoes hydrolysis and is excreted back into the medium, leading to enrichment of the lipoproteins in the medium with free cholesterol. This enrichment with free cholesterol promotes cholesterol esterification in smooth muscle cells.

Acylation↗
PubMed1981

The fate of cholesteryl linoleyl ether and cholesteryl linoleate in the intact rat after injection of biologically labeled human low density lipoprotein.

In vitro labeling of low density lipoproteins (LDL) with [7 alpha(n)-3H]cholesteryl linoleyl ether, and with [4-14C]cholesteryl linoleate was achieved by a modification of the method developed for labeling of very low density lipoproteins. [3H]Cholesteryl linoleyl ether and [14C]cholesteryl linoleate were cosonicated with partially delipidated high density lipoprotein (HDL) and the HDL was purified by centrifugation at d = 1.063. LDL was labeled by incubation of the labeled HDL in the presence of the d greater than 1.25 fraction of human plasma and reisolated at d = 1.063. The 3H/14C ratio in the labeled LDL was the same as in the HDL. The labeled LDL had the same lipid composition and ultrastructural appearance as the non-incubated LDL. After injection into rats, both labels disappeared at similar rates and the t1/2 between 1-24 h was 7.0 h. Up to 8 h after injection of labeled LDL, 94-97% of 3H and 14C radioactivity in the plasma was precipitable by heparin-manganese. 24 h after injection, 28% of the [3H]cholesteryl linoleyl ether was recovered in the liver, 6% in small intestine and 34% in the carcass, and the rest was distributed among all other organs; total recovery of 3H label was 89 +/- 3.0%. The present findings indicate that as in the rat there is no transfer of esterified cholesterol among plasma lipoproteins, LDL is catabolized by both the liver and extrahepatic tissues.

Animals↗
PubMed1981

Metabolism of cytoplasmic triacylglycerol in cultured aortic smooth muscle cells.

A turnover of cytoplasmic triacylglycerol was studied in cultured rat, rabbit, and bovine aortic smooth muscle cells. Cytoplasmic triacylglycerol was labeled with [3H]glycerol in the presence of oleic acid in the medium and its loss from the cell was studied in the presence of carrier glycerol. Multiple additions of Isuprel or dibutyryl cyclic AMP during the chase period did not enhance the loss of labeled triacylglycerol. The rate of hydrolysis of cellular triacylglycerol was unchanged in the absence or in the presence of 100 microM chloroquine. Modulation of cellular cholesterol content by addition of low density lipoprotein or high density apolipoprotein--sphingomyelin liposomes did not affect the residence time of the cellular triacylglycerol. We conclude that cytoplasmic triacylglycerol in cultured aortic smooth muscle cells is metabolized by an extralysosomal enzyme which is neither catecholamine responsive nor affected by modulation of cellular cholesterol.

Animals↗
PubMed1980

Modulation of cytoplasmic cholesteryl ester of smooth muscle cells in culture derived from rat, rabbit and bovine aorta.

Esterification of cholesterol in smooth muscle cells, isolated from rat, rabbit and bovine aorta, was achieved by incubation with cholesterol enriched medium containing [7(n)-3H]cholesterol. The newly formed cholesteryl ester was readily hydrolyzed when the cells were post-incubated with medium containing lipoprotein deficient serum. The rate of loss of labeled cholesteryl ester was not inhibited by the presence of 100 microM chloroquine. Addition of LDL to the post-incubation medium retarded the decrease in labeled cellular cholesteryl ester in rat smooth muscle cells and this effect of LDL was abolished by chloroquine. In bovine and rabbit smooth muscle cells, enriched in cholesteryl ester, addition of LDL to post-incubation medium resulted in an increase in labeled cholesteryl ester and in cholesteryl ester mass. Retardation in the loss of labeled cellular cholesteryl ester occurred also on addition of oleic acid to the post-incubation medium. In the presence of HDL and especially of high density apolipoprotein-sphingomyelin liposomes there was an efflux of cellular free cholesterol and a reduction in cholesteryl ester. These findings indicate that the catabolism of cytoplasmic cholesteryl ester in aortic smooth muscle cells is catalyzed by extralysosomal enzymes. The cytoplasmic cholesteryl ester hydrolase is apparently not activated by cyclic AMP. The intracellular availability of unesterified cholesterol, which can be modulated by plasma lipoproteins, may determine the residence time of cellular cholesteryl ester. Thus under pathological conditions an increase in extracellular LDL accompanied by a reduction in HDL would prolong the residence time of cholesteryl esters and thus promote their intracellular accretion.

Animals↗