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Biomedical subjects

A A Spector

Publications and source records attributed to A A Spector.

At least 163 records · Page 9Linked to original sources
PubMed1981

Accumulation of (n-9)-eicosatrienoic acid in confluent 3T3-L1 and 3T3 cells.

The 3T3-L1 preadipocyte cultured cell line accumulates the n-9 class of eicosatrienoic acid (20:3 n-9) after becoming confluent when the medium contains 10% fetal bovine serum. Accumulation of 20:3 n-9 also occurred in confluent, nondifferentiating 3T3 cells but not in eight other cultured cell lines that were tested. Radioisotope experiments indicate that confluent 3T3-L1 cells synthesized 20:3 n-9 from [1-14C]oleic acid. 20:3 n-9 comprised 9.8% of the cell phospholipid fatty acids, and its accumulation was associated with a 40% reduction in the phospholipid arachidonic acid content. The highest percentage of 20:3 n-9 was present in the ethanolamine and inositol glycerophospholipid fractions. Eicosatrienoic acid also accumulated in 3T3-L1 cells that were treated with dexamethasone, methylisobutylxanthine, and insulin to achieve adipocyte morphology. Supplementation of the culture medium with 0.03 mumol/ml of either linoleic or arachidonic acid prevented 20:3 n-9 accumulation. Partial reversal of the 20:3 n-9 accumulation occurred when confluent cells were either replated at a low density to initiate logarithmic growth or supplemented with 0.03 mumol/ml of arachidonic acid. The accumulation of 20:3 n-9, a polyunsaturate that ordinarily is found only in essential fatty acid deficiency, may be an important variable in studies involving 3T3-L1 and 3T3 cells.

8,11,14-Eicosatrienoic Acid↗
PubMed1981

Differences in types of prostaglandins produced by two MOCK canine kidney cell sublines.

The pattern of prostaglandins produced from arachidonic acid by two sublines of MDCK canine kidney epithelial cells was different. In one subline designated MDCK1, the most prevalent prostaglandin product was PGE2, whereas the most prevalent product in the subline designated MDCK2 was PGF2 alpha. This difference was observed when cells previously labeled with [1-14C]arachidonic acid were stimulated with either bradykinin or the calcium ionophore A23187, or when prostaglandins were produced from labeled arachidonic acid added directly to the assay medium. In the latter case, the difference was maintained over a 38-fold range of extracellular arachidonate concentrations. These findings indicate that there is a persistent difference in the distribution of prostaglandins produced by the two commonly used sublines of MDCK cells.

Animals↗
PubMed1981

Effect of essential polyunsaturated fatty acid modifications on prostaglandin production by MDCK canine kidney cells.

Supplementation of growing MDCK canine kidney tubular epithelial cultures with linoleic acid produced a 3.6- to 4.9-fold increase in bradykinin-stimulated PGE2 release as measured by radioimmunoassay. Under these conditions the cell phospholipids contained 3.9- times more linoleic acid and 5.6-times more arachidonic acid, with the inositol, ethanolamine and choline phosphoglyceride fractions becoming enriched in arachidonic acid. By contrast, supplementation with arachidonic acid did not enhance bradykinin-stimulated PGE2 release even though the arachidonic acid content of the cell phospholipids was increased 8.8-fold. The distribution of radioactive prostaglandin products was unchanged by these fatty acid enrichments, with PGE2 accounting for 55 to 68% of the total output from [1-14C]arachidonic acid. Linoleic acid supplementation also produced a 2.5-fold increase in PGE2 formation stimulated by extracellular arachidonic acid, whereas supplementation during culture with arachidonic acid caused a 55 to 80% inhibition. This difference cannot be accounted for by changes in the ability of the cells to incorporate extracellular arachidonic acid. It is suggested that at least some of the effects of linoleate supplementation on prostaglandin production are due to the resulting enrichment of the intracellular phospholipid substate pools with arachidonic acid. In addition, it appears that prolonged exposure to arachidonic acid during culture has an overriding inhibitory effect on prostaglandin production even though the total cell lipids become highly enriched in arachidonate.

Animals↗
PubMed1981

Accumulation of N-3 polyunsaturated fatty acids cultured human Y79 retinoblastoma cells.

The metabolism of the n-3 class of polyunsaturated fatty acids, which occur in relatively high quantities in neural tissues, was studied in human Y79 retinoblastoma cells. These cells contained low levels of n-3 polyunsaturates when grown in culture media supplemented with fetal bovine serum. The cells readily incorporated performed docosahexaenoic acid (22.6 n-3) into phospholipids, but human skin fibroblasts did this to a similar extent. When 10 to 30 mumol/ml linolenic acid (18:3 n-3) was added, the cell also accumulated 22:6 in phospholipids. The capacity to convert appreciable amounts of 18:3 to 22:6 appears to be a unique property of the retinoblastoma cells as compared with other continuously cultured cell lines. More 18:3 than linoleic acid (18:2 n-6) was incorporated into phospholipids by the retinoblastoma cultures, and 18:3 was channeled to a larger extent into the ethanolamine glycerophospholipid fraction. These findings indicate that retinoblastoma cells handle n-3 polyunsaturated fatty acids in a manner very similar to neural tissue in vivo. Based on the results obtained with this model system, it appears that three processes may contribute to the accumulation of 22:6 in retina and neural tissue: increased ability to incorporate 18:3, the capacity to convert 18:3 to 22:6, and channeling of 18:3 and its metabolites into ethanolamine glycerophospholipids.

Cell Division↗
PubMed1981

Utilization of arachidonic and linoleic acids by cultured human endothelial cells.

When cultured human umbilical vein endothelial cells are supplemented with linoleic acid, the arachidonic acid content of the cellular phospholipids is reduced approximately 35%. Most of the fatty acid compositional change occurs during the first 24 h. One factor responsible for this effect is the inability of the endothelial cells to convert appreciable amounts of linoleic to arachidonic acid, due to a fatty acid delta 6-desaturase deficiency. By contrast, these endothelial cultures contain delta 5- and delta 9-desaturase activity and are able to elongate long-chain polyunsaturated fatty acids. The other factor that contributes to the decrease in arachidonic acid is that high concentrations of linoleic acid reduce the incorporation of arachidonate into cellular phospholipids. Stearic acid, a long-chain saturate, does not produce any reduction, whereas eicosatrienoic acid is an even more effective inhibitor than linoleic acid. In spite of the fact that high concentrations of these polyunsaturates produced inhibition, the endothelial cells were found to efficiently incorporate exogenous arachidonic acid into cellular phospholipids and triglycerides. This may serve to compensate for the inability of these cells to synthesize arachidonic acid from linoleic acid. These findings suggest that the endothelium obtains arachidonic acid from an extracellular source, that this cannot be provided in the form of linoleic acid and, in fact, that high concentrations of linoleic acid actually may interfere with the ability of the endothelium to maintain an adequate supply of intracellular arachidonic acid.

Arachidonic Acid↗
PubMed1981

Effect of fatty acid saturation on NADPH-dependent lipid peroxidation in rat liver microsomes.

NADPH-dependent lipid peroxidation in liver microsomes, as measured by malondialdehyde formation, decreased by 90% when rats were fed a diet containing 16% coconut oil. This reduction occurred within 1 to 3 days after the rats were placed on this highly saturated diet. The decrease in peroxidation activity was associated with a reduction in the polyunsaturated fatty acid content of the microsomal phospholipids, particularly arachidonic acid. When the rats were transferred to a highly polyunsaturated diet containing 16% sunflower seed oil, microsomal lipid peroxidation and arachidonic acid content were restored to normal values within 10 days. Arachidonic acid contained in the microsomal choline and ethanolamine phosphoglycerides was the main substrate for peroxidation. Addition of diarachidonyl phosphatidylcholine, but not free arachidonic acid, to the assay system restored peroxidation activity in tahe micraosomes prepared from ahe livers of the rats fed saturated fat. Likewise, prior incubation of these microsomes with a mixture of phospholipid exchange protein and liposomes containing diarachidonyl phosphatidylcholine restored peroxidation activity. These results indicate that diets rich in saturated fat reduce microsomal lipid peroxidation by decreasing the availability of polyunsaturated fatty acids in substrate phospholipids.

Adenosine Diphosphate↗
PubMed1981

Hepatic acylcoenzyme A: cholesterol acyltransferase activity during diet-induced hypercholesterolemia in cynomolgus monkeys.

Acylcoenzyme A: cholesterol acyltransferase (ACAT) activity was studied in hepatic microsomes of cynomolgus monkeys fed either commercial chow or an atherogenic diet of high cholesterol and saturated fat content. ACAT activity (pmol/min per mg protein) was 35 in liver microsomes from control monkeys, and 142 and 161 at 10 and 100 days, respectively, after starting the high cholesterol diet. The cholesterol-fed monkeys had about 1.5-fold increase in cholesterol content of hepatic microsome was compared to control monkeys (94 nmol/mg protein in controls versus 142 nmol/mg protein in the cholesterol fed group). There was no difference between the two groups in microsomal fatty acids in saturated, monoenoic, or polyenoic acid classes. However, the cholesterol-fed monkeys had relatively lower amounts of linoleic acid and higher amounts of arachidonic acid in the microsomes. To determine whether the increased microsomal cholesterol content might be responsible for the increase in ACAT activity, liver microsomes from control monkeys were incubated for 15-120 min with liposomes composed of cholesterol and dipalmitoyl phosphatidylcholine, 2:1 (mol/mol). The microsomal cholesterol content increased from 90 to 128 nmol/mg protein as the incubation progressed. There was a corresponding increase in ACAT activity from 80 to 240 pml/min per mg protein. This observation is consistent with the view that the high hepatic ACAT activity in the cholesterol-fed monkeys is due to the larger amount of cholesterol contained in the microsomes. The increase in hepatic ACAT activity occurs soon after cholesterol feeding is started; this response may be involved in the production of cholesteryl ester-rich lipoprotein by the liver, and thereby may be related to the atherogenic process in these primates.

Acyltransferases↗
PubMed1980

Retention of human skin fibroblast fatty acid modifications during maintenance culture.

The fatty acid composition of cultured human skin fibroblasts was modified by adding either oleic or linoleic acid to the growth medium. After the cultures became confluent, they were washed and transferred to different maintenance media in order to determine the stability of the various fatty acyl modifications. Some changes in fatty acid composition occurred under all conditions. When the maintenance medium was supplemented with fatty acid, the cellular neutral lipid and phospholipid fatty acyl composition were altered markedly within 16 to 24 hr. If no supplemental fatty acid was available during the maintenance period, however, the modified fatty acyl compositions were sufficiently retained so that appreciable differences between the cells enriched with oleate and linoleate persisted for at least 48 to 72 hr. This considerable degree of stability occurred when either 10% delipidized fetal bovine serum or 10% fetal bovine serum containing its inherent lipids were present in the maintenance medium. Although the triglyceride content of the fatty acid-modified cells was quite labile, neither the cholesterol nor phospholipid content changed appreciably during culture in any of the maintenance media. Since the fatty acid compositional differences persisted during several days of maintenance under certain conditions, these modified cultures appear to be a useful experimental system for assessing the effect of lipid structure on fairly long-term cellular functions.

Cells, Cultured↗
PubMed1980

Effect of fatty acid modification on prostacyclin production by cultured human endothelial cells.

We have investigated whether changes in cellular fatty acid saturation can influence prostacyclin (PGI2) production by cultured human umbilical vein endothelial cells. As compared to control cells, those enriched with linoleic acid released 60--75% less PGI2 in response to thrombin or the calcium ionophore A23187. A similar but considerably smaller effect was observed when the cells were enriched with oleic or linolenic acid, but no reduction occurred with palmitic or linoelaidic acids. Some reduction in PGI2 release was noted as early as 1 h after exposure to linoleic acid. When the culture medium was supplemented with linoleic acid, the cell phospholipids contained four to five times more linoleate and 25--40% less arachidonate. These changes were most marked in the choline and serine plus inositol phosphoglyceride fractions. When the fatty acid composition of the cells enriched with linoleic acid was allowed to revert, there was a progressive increase in the capacity of the cells to release PGI2 in response to thrombin. The increase correlated with a reduction in linoleate content of the cell lipids, but there was no change in arachidonate content. This suggests that linoleic acid may act as an inhibitor of PGI2 production. The cultured endothelial cells were also able to produce PGI2 directly from added arachidonic acid. As the arachidonic acid concentration of the medium was raised, PGI2 formation by the linoleate-enriched cells increased relative to control cells, suggesting that the inhibition produced by linoleic acid may be competitive.

Arachidonic Acids↗
PubMed1980

Effect of dietary fat saturation on acylcoenzyme A:cholesterol acyltransferase activity of rat liver microsomes.

The saturation of the fat contained in the diet has been observed to affect the acylcoenzyme A:cholesterol acyltransferase (ACAT) activity of rat liver microsomes. ACAT activity in microsomes (Mp) prepared from livers of rats fed a polyunsaturated fat-enriched diet containing 14% sunflower seed oil was 70-90% higher than in microsomes (Ms) prepared from livers of rats fed a saturated fat-enriched diet containing 14% coconut oil. This difference was observed within 20 days after the diets were begun, the earliest time tested, and persisted throughout the 70-day experimental period. The difference was noted at all [1-14C]palmitoyl CoA concentrations tested, 2.5-33 micronM, and at temperatures between 18 and 40 degrees C. Arrhenius plots revealed a single transition in enzyme activity, occurring at 29 degrees C in both microsomal preparations. Likewise, the activation energy above this transition was the same in Mp and Ms, 12.5 KCal/mol. Addition of albumin to the incubation medium increased the ACAT activity of both microsome preparations, but the difference between Mp and Ms persisted. Mp was enriched in polyenoic fatty acids, primarily 18:2 and 20:4, while Ms was enriched in monoenoic acids. Although the 20:4 increase in Mp occurred in all phosphoglycerides, it was especially pronounced in the serine and inositol phosphoglyceride fraction. There were no differences in the phospholipid or cholesterol content, phospholipid head group composition, or protein composition of the two microsomal preparations. The possibility is discussed that the changes in ACAT activity result from the differences in fatty acid composition of the microsomes. Other microsomal enzymes exhibited varying responses to these dietary fatty acid modifications. Palmitoyl CoA hydrolase and NADPH cytochrome c reductase activities were unchanged. UDP glucuronyl transferase activity was 50% higher in Mp, but glucose-6-phosphatase and NADH cytochrome b5 reductase activities were 25% higher in Ms. Therefore, dietary fat modifications do not produce a uniform effect on the activity of microsomal enzymes.

Acyltransferases↗
PubMed1980

Effect of fatty acid modifications of cultured hepatoma cells on susceptibility to complement-mediated cytolysis.

The fatty acid composition of Morris Hepatoma 7777 cells was modified by exposure to culture media that were supplemented with 0.1 to 0.36 mM oleic or linoleic acid for 5 days. Changes occurred in the fatty acid composition of both the cellular phospholipid and the neutral lipid fractions. Exposure to linoleic acid caused a large increase in the polyunsaturated fatty acid content of the cell lipids, whereas enrichment in monoenoic fatty acids occurred when the cells were exposed to high levels of oleic acid. Cellular phospholipid content decreased, cholesterol content did not change, and triglyceride content increased as a result of fatty acid supplementation. The fatty acid-modified cells showed increased susceptibility to complement-mediated cytolysis as compared with control cells grown in unsupplemented culture media. The extent of the increase in susceptibility to cytolysis depended on the degree of lipid modification and also on the cell number and antibody titer used in the assay. Cells enriched with linoleic acid were the most susceptible, but oleic acid enrichment also produced increased susceptibility to immune cytolysis. The kinetic study showed that the initial rate of cytolysis was higher in fatty acid-modified cells than in the control cells. There was no difference in the osmotic fragility of the control and fatty acid-modified cells. These results indicate that changes in the lipid composition of a target cell can influence its susceptibility to complement-dependent cytolysis.

Animals↗
PubMed1979

Evidence for rate-limiting steps in sterol synthesis beyond 3-hydroxy-3-methylglutaryl-coenzyme A reductase in human leukocytes.

When human blood leukocytes are incubated with [2-14C]acetate only about 32% of the nonsaponifiable lipid radioactivity is recovered in digitonin-precipitable material. Using thin-layer chromatography and gas-liquid radiochromatography, we have determined that most of the label from [2-14C]acetate in the nonsaponifiable fractions is in lanosterol, squalene and an unidentified sterol. Only 11% of the acetate radioactivity is contained in cholesterol. This distribution does not change when cholesterol synthesis is depressed by the addition of lipoproteins to the medium. These findings are in marked contrast to studies with liver, where most of the nonsaponifiable radioactivity derived from acetate is recovered in digitonin-precipitable sterols. Furthermore, they suggest that rate-limiting steps beyond the 3-hydroxy-3-methylglutaryl coenzyme A reductase reaction exist in the sterol synthesis pathway of human leukocytes.

Acetates↗