Biomedical subjects

A A Spector

Publications and source records attributed to A A Spector.

At least 145 records · Page 8Linked to original sources

PubMed1984

Plasma lipid transport.

The main plasma lipid transport forms are free fatty acid, triglyceride and cholesteryl ester. Free fatty acid, derived primarily from adipocyte triglycerides, is transported as a physical complex with plasma albumin. Triglycerides and cholesteryl esters are transported in the core of plasma lipoproteins. The intestine secretes dietary fat in chylomicrons, lipoproteins that transport triglyceride to tissues for storage. Dietary cholesterol is transported to the liver by chylomicron remnants which are formed from chylomicrons. Triglyceride is released from the liver in very low density lipoproteins for utilization and storage in extrahepatic tissues. Very low density lipoproteins are converted to low density lipoproteins in the plasma; in the process, they become enriched in cholesteryl esters. High density lipoproteins take up cholesterol from tissues and other plasma lipoproteins. After the cholesterol is esterified, it is transferred ultimately to low density lipoproteins for uptake by the tissues. Phospholipids are structural components of lipoproteins and provide fatty acid for cholesteryl ester formation in the plasma, but they are not a primary transport form of lipid. Six enzymes, together with apolipoprotein cofactors and lipid transfer proteins, facilitate the plasma lipid transport process.

Albumins↗

PubMed1983

Phospholipid fatty acid modification of rat liver microsomes affects acylcoenzyme A:cholesterol acyltransferase activity.

The effect of phospholipid fatty acyl composition on the activity of acylcoenzyme A:cholesterol acyltransferase was investigated in rat liver microsomes. Specific phosphatidylcholine replacements were produced by incubating the microsomes with liposomes and bovine liver phospholipid-exchange protein. Although the fatty acid composition of the microsomes was modified appreciably, there was no change in the microsomal phospholipid or cholesterol content. As compared to microsomes enriched for 2 h with dioleoylphosphatidylcholine, those enriched with dipalmitoylphosphatidylcholine exhibited 30-45% less acyl-CoA:cholesterol acyltransferase activity. Enrichment with 1-palmitoyl-2-linoleoylphosphatidylcholine increased acyl-CoA:cholesterol acyltransferase activity by 20%. By contrast, dilinoleoylphosphatidylcholine abolished microsomal acyl-CoA:cholesterol acyltransferase activity almost completely. Addition of cofactors that stimulated microsomal lipid peroxidation inhibited acyl-CoA:cholesterol acyltransferase activity by only 10%, however, and did not increase the inhibition produced by submaximal amounts of dilinoleoylphosphatidylcholine. Certain of the phosphatidylcholine replacements produced changes in palmitoyl-CoA hydrolase, NADPH-dependent lipid peroxidase, glucose-6-phosphatase and UDPglucuronyl transferase activities, but they did not closely correlate with the alterations in acyl-CoA:cholesterol acyltransferase activity. Electron spin resonance measurements with the 5-nitroxystearate probe indicated that microsomal lipid ordering was reduced to a roughly similar extent by dioleoyl- or by dilinoleoylphosphatidylcholine enrichment. Since these enrichments produce widely different effects on acyl-CoA:cholesterol acyltransferase activity, changes in bulk membrane lipid fluidity cannot be the only factor responsible for phospholipid fatty acid compositional effect on acyl-CoA:cholesterol acyltransferase. The present results are more consistent with a modulation resulting from either changes in the lipid microenvironment of acyl-CoA:cholesterol acyltransferase or a direct interaction between specific phosphatidylcholine fatty acyl groups and acyl-CoA:cholesterol acyltransferase.

Acyl Coenzyme A↗

PubMed1983

Glycine release from Y79 retinoblastoma cells.

Glycine release, induced by a high concentration of potassium chloride (K+), was investigated in cultured human Y79 retinoblastoma cells. The cells were labeled by incubation with [2-3H]glycine prior to K+ depolarization. Depolarization with 55 mM K+ caused an immediate, Ca2+-dependent release of approximately 20% of the cellular radiolabeled glycine content. Chemical analysis of the intracellular free glycine content also showed that approximately 20%, 2.4 nmol/mg protein, was released after K+ depolarization. Glycine release from labeled Y79 cells was not stimulated by incubation with 55 mM choline chloride. Based on measurements with an amino acid analyzer, it is concluded that of the free amino acids contained in the Y79 cell, only glycine is specifically released into the extracellular fluid by K+ depolarization. Although the intracellular content of serine and glutamate decreased, these amino acids were not released from the cells. Further studies with [U-14C]serine suggest that serine is converted into glycine in Y79 cells. Veratridine also caused an immediate release of [2-3H]glycine from the cells, and this was blocked by tetrodotoxin. This suggests that the Y79 cells possess voltage-dependent Na+ channels. These results indicate that K+- and veratridine-stimulated glycine release occurs in Y79 retinoblastoma cells, providing additional evidence that this continuously cultured line may be a useful model for certain human retinal and central nervous system functions.

Amino Acids↗

PubMed1983

Glycine uptake by cultured human Y79 retinoblastoma cells: effect of changes in phospholipid fatty acid unsaturation.

Glycine uptake was investigated in cultured Y79 retinoblastoma cells containing different degrees of phospholipid fatty acid unsaturation. The modifications were produced by growing the retinoblastoma cells in medium supplemented with various unsaturated fatty acids. Glycine was taken up by the retinoblastoma cells through two kinetically distinguishable process. The high-affinity system is totally dependent upon extracellular Na+ and partially dependent upon Ca2+. Of the glycine taken up by retinoblastoma cells, 85-90% remains as free intracellular glycine and less than 30% is incorporated into cellular protein. When the cells are grown in a medium containing 10% fetal bovine serum as the only source of fatty acids, the phospholipids contained 23% polyunsaturated fatty acids. Under these conditions the high-affinity system has a K'm of 34.2 +/- 3.7 micrometers and a V'max of 91.2 +/- 16.2 pmol min-1 mg protein -1. The low-affinity system has a K'm of 2.7 +/- 0.4 mM and a V'max of 4.1 +/- 0.5 nmol min-1 mg protein-1. When the polyunsaturated fatty acid content of the phospholipids was increased by supplementing the medium with linolenic or docosahexaenoic acids (n-3 polyunsaturates) or linoleic or arachidonic acids (n-6 polyunsaturates), the K'm and V'max of the high-affinity glycine uptake system were increased three- to fourfold. By contrast, supplementing the medium with oleic acid, and n-9 monounsaturate, did not significantly alter the K'm or V'max for glycine uptake. The results with this model system suggest that one of the effects of the high polyunsaturated fatty acid content normally present in neural cell membranes may be a modulation of the high-affinity transport system so that it functions more efficiently in regulating glycine uptake.

Biological Transport↗

PubMed1983

Eicosapentaenoic acid and prostacyclin production by cultured human endothelial cells.

Human umbilical vein endothelial cells incorporate eicosapentaenoic acid (EPA) when this fatty acid is present in the culture medium. From 30 to 70% of the uptake remains as EPA, and much of the remainder is elongated to docosapentaenoic acid. All of the cellular glycerophospholipids become enriched with EPA and docosapentaenoic acid, with the largest increase in EPA occurring in the choline glycerophospholipids. When this fraction is enriched with EPA, it exhibits a large decrease in arachidonic acid content. Cultures exposed to tracer amounts of [1-14C]linolenic acid in 5% fetal bovine serum convert as much as 17% of the radioactivity to EPA. The conversion is reduced, however, in the presence of either 20% fetal bovine serum or 50 microM linolenic acid. Like arachidonic acid, some newly incorporated EPA was released from the endothelial cells when the cultures were exposed to thrombin. However, as compared with arachidonic acid, only very small amounts of EPA were converted to prostaglandins. Cultures enriched with EPA exhibited a 50 to 90% reduction in capacity to release prostacyclin (PGI2) when subsequently stimulated with thrombin, calcium ionophore A23187, or arachidonic acid. The degree of inhibition was dependent on the time of exposure to EPA and the EPA concentration, and it was not prevented by adding a reversible cyclooxygenase inhibitor, ibuprofen, during EPA supplementation. EPA appears to decrease the capacity of the endothelial cells to produce PGI2 in two ways: by reducing the arachidonic acid content of the cell phospholipid precursor pools and by acting as an inhibitor of prostaglandin production. These findings suggest that regimens designed to reduce platelet aggregation and thrombosis by EPA enrichment may also reduce the capacity of the endothelium to produce PGI2.

6-Ketoprostaglandin F1 alpha↗

PubMed1983

Role of triglycerides in endothelial cell arachidonic acid metabolism.

Arachidonic acid was incorporated into triglycerides by cultured bovine endothelial cells in a time- and concentration-dependent manner. At 75 microM or higher, more arachidonic acid was incorporated into triglycerides than into phospholipids. The triglyceride content of the cells increased as much as 5.5-fold, cytoplasmic inclusions appeared, and arachidonic acid comprised 22% of the triglyceride fatty acids. Triglyceride turnover occurred during subsequent maintenance culture; there was a 60% decrease in the radioactive arachidonic acid contained in triglycerides and a 40% decrease in triglyceride content in 6 hr. Most of the radioactivity was released into the medium as free fatty acid. The turnover of arachidonic acid, but not oleic acid in cellular triglycerides, decreased when supplemental fatty acid was added to the maintenance medium. Incorporation and turnover of radioactive arachidonic acid in triglycerides also was observed in human skin fibroblasts, 3T3-L1 cells, and MDCK cells. Other fatty acids were incorporated into triglycerides by the endothelial cells; the amounts after a 16-hr incubation with 50 microM fatty acid were 20:3 greater than 20:4 greater than 18:1 greater than 18:2 greater than 22:6 greater than 16:0 greater than 20:5. These findings indicate that triglyceride formation and turnover can play a role in the fatty acid metabolism of endothelial cells and that arachidonic acid can be stored in endothelial cell triglycerides.

Animals↗

PubMed1983

A rapid, isocratic method for phospholipid separation by high-performance liquid chromatography.

A rapid, isocratic method for separating the most prevalent phospholipids by high-performance liquid chromatography is described. Baseline resolution of phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin is achieved in less than 40 min on a silica column. Lipids are injected in 10 microliter of chloroform-diethyl ether 1:2 (v/v) and eluted with a solvent mixture of acetonitrile-methanol-sulfuric acid 100:3:0.05 (v/v/v) at a flow rate of 1 ml/min. Neutral lipids and cardiolipin elute with the solvent front. Chromatography of a radioactive cell lipid extract indicates a recovery of better than 97%. The procedure is sensitive enough to permit the analysis of the main phospholipids present in a monolayer culture containing about 100 micrograms of cell protein.

Animals↗

PubMed1982

Mechanism of defective sterol synthesis in human leukocytes.

When human lymphocytes, granulocytes and monocyte-enriched cell preparations were incubated with [2-14C]acetate, only 10-36% of the radioactivity incorporated into the nonsaponifiable lipid fraction was present as digitonin-precipitable sterols. This percentage is considerably less than that observed for rat hepatocytes (95%) or human liver slices (68%). Even though a marked increase in the incorporation of labeled acetate into both nonsaponifiable lipids and digitonin-precipitable sterols resulted from stimulation of lymphocytes with concananvalin A or granulocytes by phagocytosis, the proportion of nonsaponifiable lipid radioactivity that was digitonin-precipitable remained low. These findings suggest that rate-limiting steps beyond the 3-hydroxy-3-methylglutaryl coenzyme A reductase reaction exist in the sterol synthetic pathway of leukocytes. Pulse-chase experiments demonstrated a precursor product relationship in leukocytes between lanosterol and cholesterol, but some squalene appears to be in a pool that is not further metabolized. A subcellular fraction prepared from mixed leukocytes was incapable of converting appreciable amounts of [3H]squalene to lanosterol or cholesterol, suggesting an enzyme deficiency in this segment of the sterol synthetic pathway. With isolated liver microsomes, 50% of the nonsaponifiable lipid radioactivity synthesized from [3H]squalene was recovered in cholesterol when the system contained added liver cytosol. By contrast, if the liver cytosol was replaced by leukocyte cytosol, 14-fold less radioactivity was incorporated into nonsaponifiable lipids, and only 17% of the radioactivity was recovered in cholesterol. When sterol-carrier protein 1, partially purified from rat liver, was added to leukocyte cytosol, [3H]squalene incorporation into lanosterol increased more than 3-fold. With the addition of both sterol-carrier protein 1 and sterol-carrier protein 2, cholesterol synthesis increased 2-fold. These results suggest that the low cholesterol synthetic activity in human leukocytes is due to a defect in one or more of the microsomal enzymes that operate between squalene and cholesterol, as well as to a deficiency of sterol-carrier proteins in leukocyte cytosol.

Acetates↗

PubMed1982

Prostaglandin production by 3T3-L1 cells in culture.

Rapidly growing cultures of 3T3-L1 preadipocytes produce large quantities of prostaglandins when they are either stimulated with the calcium ionophore A23187 or incubated with arachidonic acid. The main prostaglandin produced under all conditions was prostaglandin E2. Prostaglandin production in response to ionophore stimulation or incubation with arachidonic acid decreased markedly, however, as the cultures approached confluence, were maintained in the confluent state, or were stimulated to differentiate. Enrichment of confluent, differentiated cultures with arachidonic acid did not enhance prostaglandin production. Recovery of prostaglandin production occurred when logarithmic growth was reinstituted by reseeding confluent cultures at low cell densities, but sparse cultures maintained in a low-growth phase did not recover the ability to produce large amounts of prostaglandin E2. Therefore, the decline in prostaglandin synthetic capacity appears to be associated with the decrease in growth rate as the cells approach confluence. Media conditioned by confluent cells reduced prostaglandin E2 production when added to rapidly growing cells, suggesting that an inhibitor of prostaglandin synthesis may be formed by the confluent cultures. Nondifferentiating 3T3 fibroblasts, which similarly release mainly prostaglandin E2, also exhibited a decrease in prostaglandin production as the cultures became confluent. The amounts of prostaglandins produced by 3T3 cells in the confluent state were much greater, however, than those produced by confluent or differentiated 3T3-L1 cultures. These findings suggest that the low capacity to produce prostaglandins may be involved in either the induction or maintenance of differentiation in 3T3-L1 cells.

Adipose Tissue↗

PubMed1982

Effect of alterations in membrane lipid unsaturation on the properties of the insulin receptor of Ehrlich ascites cells.

We have altered the phospholipid composition of the plasma membranes of Ehrlich ascites cells, grown in mice and studied the effects on the properties of the insulin receptor of this cell. The insulin receptor of the Ehrlich cell demonstrated all of the binding characteristics of mammalian insulin receptors: specificity for insulin and insulin analogs, saturability, inverse relationship of steady-state binding levels to temperature, and negative cooperativity. Cellular phospholipids enriched in monounsaturated fatty acyl groups were produced by growth in animals that were maintained on a diet rich in coconut oil; cellular phospholipids enriched in polyunsaturated fatty acyl groups were produced in animals fed sunflower oil. Insulin receptors were present in the normal cells at 180,000 sites/cell but this fell to 125000 (P less than 0.001) in cells enriched in monounsaturated fatty acids and rose to 386,000 (P less than 0.001) in cells enriched in polyunsaturated fatty acids. The normal cells had affinity constants (Ke and Kf) of 0.03 and 0.01 nM-1. The cells enriched in monounsaturated fatty acids had an increase in these affinity constants to 0.06 and 0.03 nM-1 whereas values of 0.01 and 0.005 mM-1 were obtained in the cells enriched in polyunsaturated fatty acids (all comparison P less than 0.001). Thus, increased unsaturation of plasma membrane phospholipids, produced by dietary manipulations, was associated with an increase in insulin receptor number but a decrease in binding affinity. In contrast, increased saturation of the phospholipids of the plasma membrane was associated with a decrease in receptor number and an increase in affinity. The results can be explained by a model in which the insulin receptor is assumed to be multimeric.

Animals↗

PubMed1982

Lipid effects on the binding properties of a reconstituted insulin receptor.

The turkey erythrocyte membrane insulin receptor was solubilized and reconstituted into vesicles composed of either soy or dimyristoyl phosphatidylcholine. Reconstitution with soy phosphatidylcholine provided a lipid environment containing 43% unsaturated fatty acids, as compared with 82% saturated fatty acids in the dimyristoyl phosphatidylcholine preparation. After reconstitution, both species of vesicles were isolated from a 2 to 30% continuous sucrose gradient at a density of 1.071 g/ml. Scatchard analysis of binding data obtained at 15 degrees C revealed that the reconstituted receptor had a greater affinity for [125I]iodoinsulin in the saturated lipid environment (Ke = 0.167 nM-1; K1 = 2.18 nm-1) than in the unsaturated lipid environment (Ke = 0.0162 nM-1; K1 = 0.479 nm-1). Low affinity binding also was increased in the saturated vesicles. These increases were paralleled by a reduction in the number of available insulin binding sites in the saturated lipid environment. There was no difference, however, in the relative affinity of the reconstituted receptor preparations for insulin or proinsulin. Electron microscopy and gel filtration indicated that the binding differences are not due to differences in vesicle size. They also are not due to differences in the orientation of the receptor within the lipid bilayer, for its sensitivity to trypsin digestion was similar in both types of vesicles. Solubilization studies with 1% beta-octylglucoside indicated, however, that the dimyristoyl phosphatidylcholine vesicles incorporated a slightly lesser amount of insulin receptor. Similar results were also observed at 37 degrees C. These results suggest that the membrane lipid environment, especially the degree of unsaturation of the phospholipid fatty acyl chains, can influence the binding properties of the insulin receptor.

Animals↗

PubMed1982

Enrichment of human platelet phospholipids with linoleic acid diminishes thromboxane release.

We have investigated whether exposure of human platelets to elevated concentrations of linoleic acid, the principal dietary polyunsaturate, would influence platelet thromboxane A2 release. Platelets were incubated with albumin-bound linoleic acid at 30 degrees C for 24 h, with prostaglandin E1 added to prevent aggregation. The linoleic acid supplemented platelets released, on average, 50% less thromboxane A2 in response to stimulation with thrombin than corresponding control platelets. Other fatty acids were without appreciable effect. The inhibition of thrombin-stimulated thromboxane A2 release was dependent on the time and temperature of incubation, as well as on the concentration of added linoleic acid. Supplementation increased the amount of linoleic acid in the platelet phospholipids, but the arachidonic acid content of the phospholipids was reduced. [1-14C]Linoleic acid was not converted to arachidonic acid by the platelets. Linoleic acid was released exclusively from the inositol phosphoglycerides when the enriched platelets were stimulated with thrombin. The linoleate-enriched platelets converted less [1-14C]arachidonic acid to all prostaglandin products, suggesting that the platelet cyclooxygenase was partially inhibited.

Arachidonic Acid↗

PubMed1982

Choline uptake in cultured human Y79 retinoblastoma cells: effect of polyunsaturated fatty acid compositional modifications.

Choline uptake in Y79 human retinoblastoma cells occurs through two kinetically distinguishable processes. The high-affinity system shows little sodium or energy dependence, and it does not appear to be linked to acetyl CoA; choline O-acetyltransferase. When the cells are grown in a culture medium containing 10% fetal bovine serum, the high-affinity system has a K'm= 2.16 +/- 0.13 microM and V'max = 27.0 +/- 2.9 pmol min-1 mg-1, whereas the low-affinity system has a k'm = 20.4 +/- 1.3 microM and V'max = 402 +/- 49 pmol min-1 mg-1. Under these conditions, the polyunsaturated fatty acid content of the cell membranes is relatively low. When the polyunsaturated fatty acid content of the microsomal membrane fraction was increased by supplementing the culture medium with linolenic or docosahexaenoic acids (n-3 polyunsaturated fatty acids) or arachidonic acid (n-6 polyunsaturated fatty acid), the K'm of the high-affinity choline transport system was reduced by 40--60%. The V'max also was reduced by 20--40%. Supplementation with oleic acid, the most prevalent monounsaturated fatty acid, did not affect either kinetic parameter. The results suggest that one functional effect of the high unsaturated fatty acid content of neural cell membranes is to facilitate the capacity of the high-affinity choline uptake system to transport low concentrations of choline. This effect appears to be specific for polyunsaturated fatty acids but not for a single type, for it is produced by members of both the n-3 and n-6 classes of polyunsaturated fatty acids.

Biological Transport↗

PubMed1982

Effect of liposome composition on the activity of detergent-solubilized acylcoenzyme A: cholesterol acyltransferase.

Acylcoenzyme A:cholesterol acyltransferase (ACAT) was solubilized from Ehrlich ascites cell microsomes with Triton X-100. After removal of the detergent, ACAT activity per mg protein was reduced by 50 to 65% as compared with untreated microsomes. When this microsomal extract was combined with liposomes composed of cholesterol and egg phosphatidylcholine, the ACAT activity increased 5.4- to 6.7-fold. Under these conditions sucrose density gradient centrifugation indicated that more than 50% of the added lipid was incorporated into vesicles having the same density as the ACAT activity, suggesting the formation of a complex. ACAT activity increased 2.9-fold when the phosphatidylcholine content of the liposomes was raised from 0.5 to 5.0 mumol/mg microsomal protein. By contrast, the ACAT activity increased only 42% when the cholesterol content of the liposomes was raised from 0.17 to 0.57 mumol/mg microsomal protein. Addition of phosphatidylethanolamine to the liposomes produced little change in ACAT activity, whereas the activity was reduced by 25 and 50%, respectively, when sphingomyelin or phosphatidylserine was added. ACAT activity was five times higher when the liposomes were prepared from dioleoylphosphatidylcholine than from saturated phosphatidylcholines, including hydrogenated egg yolk, dimyristoyl or dipalmitoyl phosphatidylcholine. Likewise, the ACAT activity with liposomes made from soybean or egg yolk phosphatidylcholine was almost 3.5-fold greater than with those prepared from the saturated phosphatidylcholines. These results are consistent with the view that the activity of ACAT can be modified by changes in the composition of the membrane lipids with which the enzyme is associated.

Acyltransferases↗

PubMed1982

Electron spin resonance studies on intact cells and isolated lipid droplets from fatty acid-modified L1210 murine leukemia.

It has been suggested that the formation of cytoplasmic lipid droplets may produce an artifact and be responsible for the differences in membrane physical properties detected in lipid-modified cells using fluorescence polarization or spin label probes. To investigate this, the electron spin resonance spectra of lipid droplets isolated from the cytoplasm of L1210 leukemia cells were compared with spectra obtained from the intact cell. Mice bearing the L1210 leukemia were fed diets containing either 16% sunflower oil or 16% coconut oil in order to modify the fatty acid composition of the tumor. A microsome-rich fraction prepared from L1210 cells grown in animals fed the sunflower oil-rich diet contained more polyenoic fatty acids (52 versus 29%), while microsomes from L1210 cells grown in animals fed the coconut oil-rich diets contained more monoenoic fatty acids (37 versus 12%). The order parameter calculated for lipid droplets labeled with the 5-nitroxystearic acid spin probe was only about one-half that of intact cells, whereas it was similar to that obtained for pure triolein droplets suspended in buffer. Order parameters of the inner hyperfine splittings calculated from the spectra of cells grown in the sunflower oil-fed animals [0.543 +/- 0.001 (S.E.)] were lower than those from the cells grown in animals fed the coconut oil diets (0.555 +/- 0.002) (p less than 0.005). In contrast, the order parameters of the lipid droplets isolated from the cells grown in animals fed sunflower oil (0.303 +/- 0.029) or coconut oil (0.295 +/- 0.021) were not significantly different, indicating that motion of a spin label probe in the highly fluid cytoplasmic lipid droplets is not affected by these types of modifications in cellular fatty acid composition. Therefore, the electron spin resonance changes that are observed in the intact cells cannot be due to localization of the probe in cytoplasmic lipid droplets. These results support the conclusion that the electron spin resonance changes observed with the 5-nitroxystearic acid spin probe are due to changes in membrane fluidity produced by the modification in cellular lipid composition.

Animals↗

PubMed1982

Effect of fatty acid modification on prostaglandin production by cultured 3T3 cells.

We have investigated the extent to which modifications in the essential fatty acid content of mammalian cells can affect prostaglandin production. Swiss mouse 3T3 cells stimulated with the calcium ionophore A23187 produced 1.7 to 7 times more prostaglandin E(2) (PGE(2)) when the cultures were supplemented with linoleic acid. Increases in PGE(2) production as a result of linoleic acid supplementation occurred under all culture conditions except during the first 24 hr after attachment, when prostaglandin production was very high. Arachidonic acid supplementation produced a similar enhancement in the capacity of the cells to produce PGE(2), but no appreciable increase occurred when the cultures were supplemented with oleic acid. The phospholipids of the cells exposed to the linoleate-enriched medium contained 4 times more arachidonic acid and twice as much linoleic acid as compared with the corresponding controls. The choline phosphoglycerides were most highly enriched in arachidonic acid, but 2- to 3-fold increases also occurred in the inositol and ethanolamine phosphoglycerides. When cultures initially enriched with linoleic acid were transferred to an unsupplemented medium, the fatty acid composition as well as the capacity of the cells to produce PGE(2) reverted almost to control values. The amount of exogenous arachidonic acid converted to PGE(2) as measured by radioimmunoassay also was greater when the cells were enriched with linoleic acid. Studies with radioactive arachidonic acid indicated that the distribution of prostaglandin metabolites was not affected appreciably by linoleic acid enrichment. These findings suggest that at least two factors contribute to the increased capacity of the cultures supplemented with linoleate to produce PGE(2). One is enrichment of the phospholipid substrate pools with arachidonic acid. The other is an increased ability of the cells to synthesize PGE(2) from unesterified arachidonic acid, perhaps because the prostaglandin-forming enzymes are more active.-Denning, G. M., P. H. Figard, and A. A. Spector. Effect of fatty acid modification on prostaglandin production by cultured 3T3 cells.

Animals↗

PubMed1982

Effect of fatty acid modification of cultured hepatoma cells on susceptibility to natural killer cells.

Rat hepatoma (Morris 7777) cells modified with either oleic or linoleic acid exhibited greater susceptibility to normal spleen cell-mediated lysis in a 16-hr 51Cr release assay. At effector:target cell ratios of 300:1, the specific lysis of fatty acid-enriched target cells (cultured for 2 days in fatty acid-supplemented medium) by the normal rat spleen cells was 60% higher than the untreated target cells (P less than 0.01). Prolonging the culture in fatty acid-supplemented medium up to 6 days produced similar effects. Analysis of the fatty acid composition of cellular lipids revealed that an elevation of oleic or linoleic acid was the only significant alteration in the hepatoma cells grown in the oleic or linoleic acid-supplemented medium, respectively. The percentage of the acids was increased in the total cellular phospholipids, the choline, ethanolamine, serine, and inositol phosphoglyceride fractions, and the neutral lipids. In conclusion, we suggest that the elevation of oleic acid and linoleic acid contents in the membranes of the fatty acid-modified hepatoma cells may contribute to the increased susceptibility of these cells to natural killer cell-mediated cytotoxicity.

Animals↗

PubMed1981

Effects of octyl beta-glucoside on insulin binding to solubilized membrane receptors.

Octyl beta-glucoside (1%), a dialyzable detergent, was used to solubilize the insulin receptor of the turkey erythrocyte membrane. Insulin binding capacity was stable for at least 1 week when the receptor was kept in 1% octyl beta-glucoside at 4 degrees C. The binding properties of the solubilized receptor were examined at detergent concentrations above (1%) and below (0.6%) the critical micelle concentration. A reduction in insulin binding occurred when the detergent concentration was raised above the critical micelle concentration, due to an apparent decrease in the number of binding sites. The specificity of the receptor for insulin analogues was preserved, and the relative affinity of the solubilized receptor, desoctapeptide insulin greater than proinsulin greater than porcine insulin, was similar in 0.6% and 1% detergent. Addition of divalent cations increased insulin binding to a similar extent at both detergent concentrations, but there was a slightly greater stimulation of binding in 0.6% detergent as compared to 1% detergent. The pH optimum for binding was not affected by changes in the detergent concentration. These results indicate that the insulin receptor can be successfully solubilized by octyl beta-glucoside and that the binding activity is quite stable. Therefore, octyl beta-glucoside may be a useful detergent for purification of this receptor. In addition, the data indicate that the binding properties of the insulin receptor can be affected by changes in the physical state of the octyl beta-glucoside.

Animals↗