Effect of 25-hydroxycholesterol and bile acids on the regulation of cholesterol metabolism in Hep G2 cells.

The effect of 25-hydroxycholesterol (25-OH-cholesterol) and chenodeoxycholic (CDC) acid on apoprotein secretion, low-density lipoprotein receptor activity, and [3H]triacylglycerol secretion in Hep G2 cells was studied. Both 25-OH-cholesterol and CDC acid increased the secretion of apolipoprotein (apo) E by Hep G2 cells. The secretion of apo A-I was slightly lowered (less than 10% disease). The maximal increase in apo E secretion was observed in culture medium containing 2 micrograms of 25-OH-cholesterol/ml or 10 micrograms of CDC acid/ml plus 10% fetal calf serum. Cholesterol, 7-OH-cholesterol and other bile acids were ineffective in inducing increases in apo E secretion. Another cholesterol synthesis inhibitor, mevinolin, was also ineffective in generating an increase in apoprotein secretion. The data indicated a specific interaction between 25-OH-cholesterol or CDC acid and apo E secretion in Hep G2 cells. Cholesterol synthesis, as measured by the incorporation of [14C]acetic acid into sterols, was repressed in Hep G2 cells in the presence of 25-OH-cholesterol (17% of control value). CDC acid, on the other hand, increased [14C]acetic acid incorporation (156% of control value). The number of LDL receptors in Hep G2 cells was decreased after incubation with 25-OH-cholesterol (62% of control value), but increased significantly after incubation with CDC acid (149% of control value). The secretion of [3H]triacylglycerol by Hep G2 cells incubated with 25-OH-cholesterol was greatly increased (248% of control value). On the contrary, CDC acid did not cause any increase in [3H]triacylglycerol secretion. The above results suggest that 25-OH-cholesterol and CDC acid have different effects on lipid metabolism in Hep G2 cells. The mRNA levels of apo E increased in cells preincubated with 25-OH-cholesterol and CDC acid, which suggested that the increase in apo E secretion is at least partly due to an increase in synthesis.     

Metabolism of oxygenated derivatives of arachidonic acid by Caco-2 cells.

Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated separately with 3H8-labeled preparations of three different lipid mediators of inflammation: 5-hydroxyeicosatetraenoic acid, 12-hydroxyeicosatetraenoic acid, and leukotriene B4. Both [3H8]5-hydroxyeicosatetraenoic and [3H8]12-hydroxyeicosatetraenoic acids were taken up and metabolized by Caco-2 cells, but [3H]leukotriene B4 remained unmetabolized in the incubation medium. [3H]5-hydroxyeicosatetraenoic acid was esterified into cellular phospholipids (15%) and triglycerides (4%) but did not undergo beta-oxidation. When [3H]12-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, 14% underwent two cycles of beta-oxidation to form [3H]8-hydroxyhexadecatrienoic acid, and 3% underwent three cycles of beta-oxidation to form [3H]6-hydroxytetradecadienoic acid, both of which were released into the media. [3H]12-Hydroxyeicosatetraenoic acid was also esterified into cellular phospholipids (13%), but none was esterified into cellular triglycerides.     

The effect of interleukin 1 beta on the biosynthesis of cholesterol, phosphatidylcholine, and sphingomyelin in fibroblasts, and on their efflux from cells to lipid-free apolipoprotein A-I.

In this study we have investigated the effect of interleukin 1beta (IL-1beta) on the metabolism of cholesterol and choline-phospholipids in cultured fibroblasts, and also measured efflux of these lipids to lipid-free apo A-I as a function of IL-1beta treatment. Long-term exposure (up to 48 h) of cells to IL-1beta (1 ng.mL-1) markedly increased the rate of cholesterol esterification, as determined by the incorporation of [3H]oleic acid into cholesteryl esters. This treatment also led to a substantially increased mass of cholesteryl esters in the cells. The accumulation of cholesteryl esters in IL-1beta-treated cells could be blocked using compound 58-035 to inhibit the activity of acyl-CoA cholesterol acyl transferase. The activation of cholesterol esterification by IL-1beta was evident within a few hours after initiation of the IL-1beta treatment. Cholesterol biosynthesis was inhibited by 25% by IL-1beta (after 48 h exposure), and this eventually led to a 20% decrease in cell cholesterol mass. Treatment of cells with IL-1beta for 48 h also reduced the synthesis of sphingomyelin and caused a 30% decrease in cell sphingomyelin mass (after 48 h at 1 ng.mL-1 of IL-1beta). IL-1beta did not stimulate an acute (within a few minutes up to an hour) degradation of cell [3H]sphingomyelin. This suggests that IL-1beta did not activate an endogenous sphingomyelinase in these cells, but only affected rates of synthesis. The rate of phosphatidylcholine synthesis was barely affected, but mass was moderately reduced by a 48-h treatment of cells with IL-1beta. Finally, the efflux of cell [3H]cholesterol, [3H]sphingomyelin, and [3H]phosphatidylcholine to lipid-free apolipoprotein A-I was markedly increased from cells treated with IL-1beta for 24 and 48 h. We conclude that long-term exposure of cells to IL-1beta had marked effects on the cellular homeostasis of cholesterol and choline-containing phospholipids.     

Ca2(+)-dependent synthesis of prostaglandin I2 and mobilization of arachidonic acid from phospholipids in cultured endothelial cells permeabilized with saponin

The feasibility of using saponin as a permeabilization agent to study the effect of free Ca2+ concentration ([Ca2+]f) on prostaglandin I2 (PGI2) synthesis and mobilization of arachidonic acid from membrane phospholipids was investigated in cultured bovine pulmonary artery endothelial cells (BPAEC). Treatment of BPAEC with 20 micrograms/ml saponin caused selective permeabilization of the plasma membrane as determined by measurements of the release of lactate dehydrogenase and beta-hexosaminidase. In cells prelabeled with [3H]arachidonic acid for 22 h, permeabilization with 20 micrograms/ml saponin induced PGI2 synthesis and release of [3H]arachidonic acid from membrane phospholipids. These effects were dependent upon [Ca2+]f in the range 72 nM to 5 microM. Release of [3H]arachidonic acid from phospholipid classes was determined in suspensions of BPAEC prelabeled with [3H]arachidonic acid and permeabilized with 20 micrograms/ml saponin. At [Ca2+]f optimal for PGI2 synthesis, 16.2% of the total incorporated [3H]arachidonic acid was released from phosphatidylinositol (3.4%), phosphatidylethanolamine (3.5%) and phosphatidylcholine (9.3%). The time course and dependence upon [Ca2+]f of [3H]arachidonic acid release from phospholipids correlated with PGI2 synthesis. The amount of PGI2 synthesized in permeabilized BPAEC was similar to that in cell cultures treated with the calcium ionophore A23187. In comparison, however, PGI2 synthesis induced by A23187 was associated with less release of [3H]arachidonic acid from membrane phospholipids, e.g., 2.3% versus 16.2%. The greater loss of [3H]arachidonic acid from phospholipids in saponin-permeabilized BPAEC was most likely due to the loss of cell integrity and/or nonspecific effects of the detergent on phospholipases. Despite these limitations, the Ca2+ dependence observed for PGI2 synthesis and [3H]arachidonic acid mobilization suggest that saponin-permeabilization may provide a useful system for studies of the intracellular events triggered by the rise in intracellular Ca2+ which culminate in PGI2 synthesis.     

Phosphatidylinositol-anchored glycoproteins of PC12 pheochromocytoma cells and brain

PC12 pheochromocytoma cells and cultures of early postnatal rat cerebellum were labeled with [3H]glucosamine, [3H]fucose, [3H]leucine, [3H]ethanolamine, or sodium [35S]sulfate and treated with a phosphatidylinositol-specific phospholipase C. Enzyme treatment of [3H]glucosamine- or [3H]fucose-labeled PC12 cells led to a 15-fold increase in released glycoproteins. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, most of the released material migrated as a broad band with an apparent molecular size of 32,000 daltons (Da), which was specifically immunoprecipitated by a monoclonal antibody to the Thy-1 glycoprotein. A second glycoprotein, with an apparent molecular size of 158,000 Da, was also released. After treatment with endo-beta-galactosidase, 40-45% of the [3H]glucosamine or [3H]fucose radioactivity in the phospholipase-released glycoproteins was converted to products of disaccharide size, and the molecular size of the 158-kDa glycoprotein decreased to 145 kDa, demonstrating that it contains fucosylated poly-(N-acetyllactosaminyl) oligosaccharides. The phospholipase also released labeled Thy-1 and the 158-kDa glycoprotein from PC12 cells cultured in the presence of [3H]ethanolamine, which specifically labels this component of the phosphatidylinositol membrane-anchoring sequence, while in the lipid-free protein residue of cells not treated with phospholipase, Thy-1 and a doublet at 46/48 kDa were the only labeled proteins. At least eight early postnatal rat brain glycoproteins also appear to be anchored to the membrane by phosphatidylinositol. Sulfated glycoproteins of 155, 132/134, 61, and 21 kDa are the predominant species released by phospholipase, which does not affect a major 44-kDa protein seen in [3H]ethanolamine-labeled brain cultures. The 44-48- and 155/158-kDa proteins may be common to both PC12 cells and brain.     

Soluble CD14 mediates efflux of phospholipids from cells

Soluble CD14 (sCD14), a 55-kDa glycoprotein found in plasma, has been shown to act as a shuttle for bacterial LPS and phospholipids, transporting LPS and phospholipid monomers from LPS aggregates or liposomes to high density lipoprotein particles. sCD14 has also been shown to mediate the transport of LPS and phosphatidylinositol into cells. Here we show that sCD14 mediates not only the influx but also the efflux of cellular phospholipids. Addition of sCD14 enhanced efflux of cellular phospholipids labeled with [(3)H]palmitic acid, [(3)H]oleic acid, or [(3)H]choline chloride from differentiated THP-1 monocytic cells. Efflux was dependent on the concentration of sCD14 added and was essentially complete in 30 min. The role of membrane-bound CD14 (mCD14) in lipid efflux was assessed using matched pairs of cell lines that express or fail to express this protein. While efflux was very dependent on mCD14 in U373 cells, it was not dependent on mCD14 in Chinese hamster ovary cells, suggesting a role for additional cellular proteins in determining the pathway of phospholipid efflux. A deletion mutant of sCD14 lacking the LPS binding site had less ability to efflux phospholipids than intact sCD14, suggesting that this site is needed for CD14 to serve in phospholipid transport. [(3)H]Palmitate-labeled lipids released by sCD14 were precipitated with anti-CD14 then analyzed by HPLC. Phosphatidylcholine was the dominant phospholipid exported and bound to sCD14. These results demonstrate that sCD14 mediates efflux of phospholipids from cells and suggest that sCD14 contributes to phospholipid transport in blood.     

Synthesis and secretion of lipoproteins by human hepatocytes in culture

Summary Confluent monolayers of normal human hepatocytes obtained by collagenase perfusion of liver pragments were incubated in a serum-free medium. Intracellular apolipoproteins apo AI, apo C, apo B, and apo E were detected between Day 1 and Day 6 of the culture by immunoenzymatic staining using polyclonal antibodies directed against these apoproteins and monoclonal antibodies directed against both forms of apo B (B100 and B48). Translation of mRNA isolated from these hepatocytes in an acellular system revealed that apo AI and apo E were synthesized as the precusor forms of mature plasma apo AI and apo E. Three lipoprotein fractions corresponding to the density of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) were isolated from the medium at Day 5 of culture and examined by electron microscopy after negative staining. VLDL and LDL particles are similar in size and shape to plasma lipoproteins; spherical HDL are larger than normal plasma particles isolated at the same density. Their protein represented 44, 19.5, and 36.5% respectively, of the total lipoprotein protein. The secretion rate of VLDL protein corresponded to that measured in primary cultures of rat hepatocytes. After incorporation of [³H]glycerol, more than 92% of the [³H]triglyceride secreted into the medium was recovered in the VLDL fraction. These results demonstrate that primary cultures of normal human hepatocytes are able to synthesize and secrete lipoproteins and thus could be a useful model to study lipoprotein metabolism in human liver.     

Effect of apolipoproteins E and C-III on the interaction of chylomicrons with parenchymal and non-parenchymal cells from rat liver.

[3H]Triacylglycerol-labelled chylomicrons were isolated from intestinal lymph, obtained from rats made hypolipidaemic by treatment with pharmacological amounts of 17 alpha-ethynyloestradiol. Oestrogen treatment results in a large reduction in the content of apolipoproteins (apo) E and C of lymph chylomicrons. Upon incubation in vitro with freshly isolated parenchymal and non-parenchymal cells the apo E-, apo C-poor chylomicrons became readily cell-associated. With increasing chylomicron concentrations this cell-association was saturable and half-maximal cell-association was achieved at about 0.55 mg of triacylglycerol/ml. The cell-association was time- and temperature-dependent. A more than 90% inhibition of the cell-association of the [3H]triacylglycerol moiety was observed with both parenchymal and non-parenchymal cells when pure apo C-III (12.6 micrograms/mg of triacylglycerol) was incorporated into the chylomicrons. These data indicate that apo E-, apo C-poor chylomicrons are bound to both parenchymal and non-parenchymal liver cells at a high-affinity site of limited capacity and that binding to this site is strongly inhibited by apo C-III. With apo C-III-enriched chylomicrons simultaneous determination of the cell-association of the 125I-apo C-III and the [3H]triacylglycerol moiety indicated that more 125I-apo C-III becomes associated to the cells than expected on the basis of [3H]triacylglycerol radioactivity measurements. It is suggested that upon cell-association of apo C-III its binding to the chylomicron particles is lost. Consequently the occupation of the cellular recognition site by apo C-III prevents further chylomicron binding and thus leads to a decrease of the cell-association level of the [3H]triacylglycerol moiety. Apo E enrichment of the chylomicrons led to an increased cell-association rate with parenchymal cells and to a marked increase of the cell-association level with non-parenchymal cells. The cell-association of the apo E radioactivity followed closely the [3H]triacylglycerol radioactivity, indicating that the particle-apo E complex is bound as a unity. The apo E effects were opposed by apo C-III. With apo E-, apo C-III-enriched chylomicrons more 125I-apo E became associated with the cells than could be expected on the basis of the [3H]triacylglycerol measurements. It is concluded that apo C-III can weaken the interaction of apo E with the chylomicrons leading to the cell-association of free apo E. It appears that subtle changes in the apo E and/or apo C-III content of chylomicrons can influence the interaction with both parenchymal and non-parenchymal liver cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of apolipoprotein A-IV in reverse cholesterol transport studied with cultured cells and liposomes derived from an ether analog of phosphatidylcholine

Cholesterol efflux was studied in a model system in culture using apolipoproteins and phospholipids added in the form of liposomes at concentrations expected to be present in the extracellular fluid. Fibroblasts were seeded in medium containing [3H]cholesterol-labeled serum, grown till confluent, and the [3H]cholesterol efflux was studied in serum-free medium. Addition of delipidated HDL apolipoprotein resulted in a very low release of [3H]cholesterol, which did not increase with time of exposure or concentration of apolipoproteins. Addition of increasing amounts of HDL apolipoprotein to liposomes prepared from either dioleoylphosphatidylcholine (PC) or its nonhydrolysable ether analog, dioleylphosphatidylcholine (DOEPC) resulted in a 3-5-fold increase of [3H]cholesterol efflux, over that achieved with liposomes alone. This model system permitted the test of the putative role of apolipoprotein A-IV in cholesterol removal from cells. The ability of apolipoprotein A-IV to enhance [3H]cholesterol efflux from cells by DOEPC liposomes was compared to that of apolipoproteins A-I, E and C, which were added at equimolar concentrations. At nM concentrations, apolipoproteins A-IV, A-I and E were equally able to enhance cholesterol efflux, while C apolipoproteins were less effective at these low concentrations. Mixtures prepared from apolipoprotein A-IV, A-I and E and PC or DOEPC liposomes were equally effective in cholesterol removal, while phosphatidylethanolamine liposome apolipoprotein mixtures had a much lower capacity. The present study provides the first evidence that apolipoprotein A-IV can play a role in reverse cholesterol transport as was suggested on the basis of high concentrations of this apolipoprotein in nonlipoprotein form in plasma and extracellular fluid. The efficacy of DOEPC liposomes to serve as cholesterol acceptors might be of potential value for enhancement of reverse cholesterol transport in vivo.     

Efflux of lipid from fibroblasts to apolipoproteins: dependence on elevated levels of cellular unesterified cholesterol.

Earlier work from this laboratory showed that enrichment of cells with free cholesterol enhanced the efflux of phospholipid to lipoprotein acceptors, suggesting that cellular phospholipid may contribute to high density lipoprotein (HDL) structure and the removal of sterol from cells. To test this hypothesis, we examined the efflux of [3H]cholesterol (FC) and [32P]phospholipid (PL) from control and cholesterol-enriched fibroblasts to delipidated apolipoproteins. The percentages of [3H]cholesterol and [32P]phospholipid released from control cells to human apolipoprotein A-I were 2.2 +/- 0.5%/24 h and 0.8 +/- 0.1%/24 h, respectively. When the cellular cholesterol content was doubled, efflux of both lipids increased substantially ([3H]FC efflux = 14.6 +/- 3.6%/24 h and [32P]PL efflux = 4.1 +/- 0.3%/24 h). Phosphatidylcholine accounted for 70% of the radiolabeled phospholipid released from cholesterol-enriched cells. The cholesterol to phospholipid molar ratio of the lipid released from cholesterol-enriched cells was approximately 1. This ratio remained constant throughout an incubation time of 3 to 48 h, suggesting that there was a coordinate release of both lipids. The concentrations of apoA-I, A-II, A-IV, E, and Cs that promoted half-maximal efflux of phospholipid from cholesterol-enriched fibroblasts were 53, 30, 68, 137, and 594 nM, respectively. With apoA-I and A-IV, these values for half-maximal efflux of phospholipid were identical to the concentrations that resulted in half-maximal efflux of cholesterol. Agarose gel electrophoresis of medium containing apoA-I that had been incubated with cholesterol-enriched fibroblasts revealed a particle with alpha to pre-beta mobility. We conclude that the cholesterol content of cellular membranes is an important determinant in the ability of apolipoproteins to promote lipid removal from cells. We speculate that apolipoproteins access cholesterol-phosphatidylcholine domains within the plasma membrane of cholesterol-enriched cells, whereupon HDL is generated in the extracellular compartment. The release of cellular lipid to apolipoproteins may serve as a protective mechanism against the potentially damaging effects of excess membrane cholesterol.     

Specific incorporation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid into phosphatidylcholine in human endothelial cells

The incorporation of hydroxyeicosatetraenoic acids (HETEs) into cellular lipids was studied in cultures of human umbilical vein endothelial cells. 5-[3H]HETE was incorporated into the phospholipids (8%) and neutral lipids (15.5%). The uptake was at half maximum after 15 min and reached a plateau after 1 h. The incorporation occurred mainly into phosphatidylcholine (6.3%) with minimal uptake into phosphatidylserine and phosphatidylinositol (0.6%) or phosphatidylethanolamine (1.2%). There was no uptake of 12-[3H]HETE, 15-[3H]HETE or [3H]leukotriene B4 into phospholipids. Treatment of the phosphatidylcholine fraction with phospholipase A2 released 64% of the 5-[3H]HETE with 26% remaining in the lysophosphatidylcholine fraction. This indicates that the majority of the 5-HETE was in the sn-2 position. Unlabeled 5-HETE and arachidonic acid inhibited the uptake of 5-[3H]HETE into phosphatidylcholine with an ID50 of 2.5 and 1.25 microM, respectively. Stearic acid and 15-HETE were not effective inhibitors. Histamine, which activates phospholipases, increased the uptake of 5-[3H]HETE into phosphatidylcholine by 3-fold. Both 5-[3H]HETE and 12-[3H]HETE were incorporated into the neutral lipids of the cells. Analysis of the neutral lipid fraction revealed that 5-[3H]HETE was incorporated into mono-, di- and triacylglycerols but not cholesterol esters. Incorporation of 5-HETE into cellular lipids reduced histamine- and arachidonic acid-stimulated synthesis of 6-ketoprostaglandin F1 alpha and prostaglandin E2 in a concentration-related manner. Angiotensin I converting enzyme activity was not changed. Thus, 5-HETE is incorporated specifically into phosphatidylcholine and glycerol esters of human endothelial cells and this incorporation inhibits prostaglandin synthesis in these cells.     

Glycoproteins released into the culture medium of differentiating murine neuroblastoma cells.

C-1300 murine neuroblastoma cells release glycoproteins into the culture medium. The process was studied by prelabeling spinner cultures for 12 to 60 hours with [3H]glucosamine. Then, the medium was removed and replaced with fresh medium lacking radioactive isotope. Soluble material released into the medium during the subsequent 2-hour incubation was collected by trichloroacetic acid precipitation. The released proteins were then separated by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulfate. The electrophoretograms of glycoproteins obtained from cultures labeled for different lengths of time were very similar; three major radioactive regions centered about molecular weights 87,000, 66,000, and 55,000 were present. When spinner cells were transferred to monolayer culture in the presence of N6,O2' dibutyryl adenosine 3':5'-monophosphate (Bt2cAMP), differentiation (extension of neurites twice the diameter of the perikaryon) was observed. Monolayer cultures grown in the presence of Bt2cAMP and [3H]glucosamine for 12 hours released glycoproteins which gave a gel electrophoresis pattern similar to that obtained using spinner cultures. However, after 60 hours in the presence of Bt2cAMP and [3H]glucosamine, the released radioactive material consisted almost exclusively of glycoproteins of the 66,000 molecular weight class. Similar results were obtained if [3H]fucose was substituted for [3H]glucosamine, or if bromodeoxyuridine (which also induced differentiation) was substituted for Bt2cAMP. Similar experiments using radioactive amino acids were conducted with both spinner and monolayer cultures. Much of the released radioactive material was contained in the same three molecular weight classes as the glycoproteins released by spinner cells prelabeled with [3H]glucosamine, and this pattern did not vary with length of labeling period or type of culture. These results may imply that the glycosylation of released proteins is influenced by agents which can induce differentiation. The origin of this released material is discussed. [3H]Glucosamine-labeled glycoproteins of the molecular weight class centered about 55,000 (discussed above) were isolated by preparative gel electrophoresis. They co-migrated with authentic mouse brain microtubular protein as two closely spaced bands on a number of different electrophoretic systems. This protein fraction was also characterized as complexing with a monospecific antitubulin antibody.     

Metabolism of platelet activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and 1-alkyl-2-acetyl-sn-glycerol by human endothelial cells

The metabolism of platelet activating factor (1-[1,2-3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine) and 1-[1,2-3H]alkyl-2-acetyl-sn-glycerol was studied in cultures of human umbilical vein endothelial cells. Human endothelial cells deacetylated 1-[1,2-3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine to the corresponding lyso compound (1-[1,2-3H]alkyl-2-lyso-sn-glycerol-3-phosphocholine) and a portion was converted to 1-[1,2-3H]alkyl-2-acyl(long-chain)-sn-glycero-3-phosphocholine. Lyso platelet activating factor (lyso-PAF) (1-[1,2-3H]alkyl-2-lyso-sn-glycero-3-phosphocholine) was detected in the media very early during the incubation and the amount remained higher than the level of the lyso product observed in the cells. Cellular levels of 1-[1,2-3H]alkyl-2-lyso-sn-glycero-3-phosphocholine were significantly higher than the acylated product (1-[1,2-3H]alkyl-2-acyl(long-chain)-sn-glycero-3-phosphocholine) at all times during the 60-min incubation period, which suggests that the ratio of acetylhydrolase to acyltransferase activities is greater in endothelial cells than in most other cells. When endothelial cells were incubated with 1-[1,2-3H]alkyl-2-acetyl-sn-glycerol, a known precursor of PAF, 1-[1,2-3H]alkyl-sn-glycerol was the major metabolite formed (greater than 95% of the 3H-labeled metabolites during 20- and 40-min incubations). At least a portion of the acetate was removed from 1-[1,2-3H]alkyl-2-acetyl-sn-glycerol by a hydrolytic factor released from the endothelial cells into the medium during the incubations. Only negligible amounts of the total cellular radioactivity (0.2%) was incorporated into platelet activating factor (1-[1,2-3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine); therefore, it is unlikely that the previously observed hypotensive activity of 1-alkyl-2-acetyl-sn-glycerols can be explained on the basis of the conversion to platelet activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by endothelial cells. Results of this investigation indicate that endothelial cells play an important role in PAF catabolism. Undoubtedly, the endothelium is important in the regulation of PAF levels in the vascular system.     

Synthesis and transport of different sphingomyelin species in rat Sertoli cells

Cellular phospholipids of Sertoli cells from immature rats were labeled with [¹⁴C]-choline. Two sphingomyelin bands (SM1 and SM2) were identified by TLC. The incorporation of [¹⁴C]-choline over a 45 h period of incubation demonstrated that there are differences in labeling kinetics between SM1 and SM2. The subcellular location of SM1 and SM2 was investigated by accessibility to bacterial sphingomyelinase. The results showed the existence of two SM pools in Sertoli cells, but an equal cellular distribution of SM1 and SM2. SM2 is characterized by a relatively high content of unsaturated fatty acids. The inhibition of vesicular flow by monensin determines a decrease of about 60–70% in incorporation into SM1 and SM2, suggesting the existence of at least two sites of sphingomyelin synthesis. Pulse-chase and time-course experiments indicated a phosphatidylcholine SM precursor product relationship and differences in kinetic properties between SM1 and SM2. Resynthesis experiments showed that monensin had only a partial inhibitory effect on SM1 resynthesis, and a second sphingomyelinase treatment demonstrated that the resynthesized fraction reached the outer leaflet of the plasma membrane. The 60–70% inhibition of SM synthesis by monensin showed that the trans-Golgi cisternae and the trans-Golgi network are the most likely sites of bulk SM synthesis, and that about 15% of SM was synthesized in the cis/medial Golgi apparatus. Additionally the results indicated that plasma membrane SM synthase activity could be the site of about 15% of SM synthesis in Sertoli cells.     

Inhibition of choline efflux results in enhanced acetylcholine synthesis and release in the guinea-pig corticocerebral synaptosomes.

Synthesis and release of [3H]acetylcholine ([3H]ACh) were measured in synaptosomes from the guinea pig cerebral cortex after preloading with [3H]choline ([3H]Ch). We demonstrate here that inhibition of choline (Ch) efflux results in an increase in acetylcholine (ACh) synthesis and release. Our findings are as follows: (1) inhibition of [3H]Ch efflux by hemicholinium-3 (HC-3) (100 microM), increased the levels of both the released (116% of control) and the residing (115% of control) [3H]ACh. (2) The muscarinic agonist, McN-A-343 (100 microM), which was previously shown to inhibit Ch efflux, also increased the released (121% of control) and the residing (109% of control) [3H]ACh. (3) Omission of Na+ ions (which are required for Ch transport) from the incubation medium had similar effects to those observed with McN-A-343 and HC-3. These results suggest inverse relationships between Ch efflux on one hand, and ACh synthesis and release on the other hand. (4) Depolarization with 50 mM K+, or with the K+ channel blocker, 4-aminopyridine (100 microM), also increased the total level of [3H]ACh (113 and 107% of nondepolarized synaptosomes, respectively). However, whereas conditions that inhibit Ch transport such as HC-3, McN-A-343 and "no sodium" increased both the residing and the released [3H]ACh depolarization with high K+ or 4-aminopyridine reduced the residing (79 and 87% of control, respectively) and increased only the released [3H]ACh (182 and 148% of control, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific pools of phospholipids are used for lipoprotein secretion by cultured rat hepatocytes

Since phospholipids are major components of all serum lipoproteins, the role of phospholipid biosynthesis in lipoprotein secretion from cultured rat hepatocytes has been investigated. In liver, phosphatidylcholine is made both by the CDP-choline pathway and by the methylation of phosphatidylethanolamine, which in turn is derived from both serine (via phosphatidylserine) and ethanolamine (via CDP-ethanolamine). Monolayer cultures of rat hepatocytes were incubated in the presence of [methyl-3H]choline, [1-3H] ethanolamine, or [3-3H]serine. The specific radioactivity of the phospholipids derived from each of these precursors was measured in the cells and in the secreted lipoproteins of the cultured medium. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine derived from [1-3H]ethanolamine were markedly lower (approximately one-half and less than one-tenth, respectively) in the secreted phospholipids than in the cellular phospholipids. Thus, ethanolamine was not an effective precursor of the phospholipids in lipoproteins. On the contrary, the specific radioactivity of phosphatidylcholine made from [methyl-3H]choline was approximately equal in cells and lipoproteins. In addition, over the first 4 h of incubation with [3-3H]serine, the specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were significantly higher in the lipoproteins than in the cells. These data indicate that there is not a random and homogeneous labeling of the phospholipid pools from the radioactive precursors. Instead, specific pools of phospholipids are selected, on the basis of their routes of biosynthesis, for secretion into lipoproteins.     

Phorbol ester inhibits 13-cis-retinoic acid-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate in cultured murine keratinocytes: a possible negative feedback via protein kinase C-activation.

The incubation of double-labelled [( 14C]-glycerol and [3H]-myoinositol) keratinocytes with 13-cis retinoic acid induced the transient and simultaneous release of [3H]-inositol trisphosphate ([3H]-InsP3) and [14C]-diacylglycerol ([14C]-DAG) indicating that a possible mode of action of this retinoid on murine keratinocytes may be at least in part the early transient release of the two putative messengers (InsP3 and DAG) from phosphatidylinositol-4,5 bisphosphate (PtdIns4, 5P2). In contrast, the preincubation of the keratinocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA) prior to incubation with 13-cis-RA suppressed the 13-cis-RA-induced release of [3H]-InsP3 and [14C]-DAG. The specificity of the TPA effect was established by the lack of effect of the biologically inactive 4 alpha-phorbol 12, 13-didecanoate. Furthermore, the incubation of the TPA-primed keratinocytes with 13-cis-RA caused a delayed and sustained accumulation of [14C]-DAG. An exploration of the source of this late release of [14C]-DAG revealed that this [14C]-DAG was released from non-inositol containing phospholipids, particularly, phosphatidylcholine. This latter DAG released in the TPA-primed cells correlated with the translocation of the cytoplasmic protein kinase C (PKC) activity to the membrane associated PKC activity. Taken together, these results suggest that alteration of PKC activity, presumably induced by DAG released from non-inositol phospholipids, may play a major role in the TPA-induced negative feedback inhibition of 13-cis RA-induced hydrolysis of keratinocyte PtdIns4, 5P2.     

Docosahexaenoic acid metabolism and effect on prostacyclin production in endothelial cells

Bovine aortic endothelial cultures readily take up docosahexaenoic acid (DHA). Most of the DHA was incorporated into phospholipids, primarily in ethanolamine and choline phosphoglycerides, and plasmalogens accounted for 34% of the DHA contained in the ethanolamine fraction after a 24-h incubation. The retention of DHA in endothelial phospholipids was not greater than other polyunsaturated fatty acids and unlike arachidonic and eicosapentaenoic acids, DHA did not continue to accumulate in the ethanolamine phosphoglycerides after the initial incorporation. About 15% of the [14C(U)]DHA uptake was retroconverted to docosapentaenoic and eicosapentaenoic acids in 24 h. Some of the newly incorporated [14C(U)]DHA was released when the cells were incubated subsequently in a medium containing serum and albumin. The released radioactivity was in the form of free fatty acid and phospholipids and after 24 h, 11% was retroconverted to docosapentaenoic and eicosapentaenoic acids. Total DHA uptake was decreased only 10% by the presence of a 100 microM mixture of physiologic fatty acids, but as little as 10 microM docosatetraenoic acid reduced DHA incorporation into phospholipids by 25%. DHA was not converted to prostaglandins or lipoxygenase products by the endothelial cultures. When DHA was available, however, less arachidonic acid was incorporated into endothelial phospholipids, and less was converted to prostacyclin (PGI2). Enrichment of the endothelial cells with DHA also reduced their capacity to subsequently produce PGI2. These findings indicate that endothelial cells can play a role in DHA metabolism and like eicosapentaenoic acid, DHA can inhibit endothelial PGI2 production when it is available in elevated amounts.     

Lipoprotein-mediated efflux of radiolabeled cholesterol from cells does not indicate net removal of cellular cholesterol mass

The efflux of cholesterol from human skin fibroblasts was determined using radioisotope techniques and mass measurements. When the cells were labeled with [14C]- or [3H]-cholesterol and then incubated with very low density, low density, or high density lipoproteins or with serum, 20 to 30% of the label was released into the medium in 20 h. However, when the cellular cholesterol content was determined after incubation with various lipoproteins under identical conditions, only the heavier subfraction of high density lipoproteins (HDL3) caused a significant decrease in cellular cholesterol. This net removal of cholesterol can be observed in the cells without overloading them with cholesterol, by incubation with low density lipoproteins. Time studies indicated that at least 24 h of incubation is required to detect significant removal of cellular cholesterol. These experiments show that methods using the release of labeled cholesterol from cultured cells to determine net cholesterol removal mediated by high density lipoprotein, although currently used by many investigators, can lead to erroneous conclusions when employed without the measurement of cholesterol mass.     

Synthesis and secretion of a novel binding protein for retinol by a cell line derived from Sertoli cells

An established cell line (TM-4) derived from murine Sertoli cells, the major supportive cell type of the testes, secretes a protein that binds retinol when grown in serum-free chemically defined medium. The protein that binds retinol is trypsin-sensitive and has an apparent Kd for retinol of 54 nM. Cholesterol, retinyl acetate, or UV-irradiated retinol at levels 100-fold in excess of retinol are poor competitors of [3H]retinol binding. Retinoic acid at a 100-fold molar excess inhibited [3H]retinol binding by 71%. In contrast, excess unlabeled retinol completely inhibits [3H]retinol binding. More than 80% of the total retinol-binding activity in confluent cultures is found in the culture medium. Prior to incubation with retinol, the protein that binds retinol has an apparent Mr of less than 150,000 by column chromatography; however, after incubation with retinol the protein that binds retinol exhibits an apparent Mr of 2 X 10(6) or greater and a sedimentation coefficient greater than 4 S. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that the major iodinatable component of the aggregated protein that binds retinol has an apparent Mr of 70,000. The secreted protein that binds retinol is not immunologically cross-reactive with either serum or cellular retinol-binding protein or transferrin. These findings suggest that Sertoli cells may secrete a protein that binds retinol. Such a protein could be involved in the transport of retinol either to the lumen of the seminiferous tubules or to the developing germ cells themselves.