Biomedical subjects

A A Spector

Publications and source records attributed to A A Spector.

At least 127 records · Page 7Linked to original sources

PubMed1986

12-Hydroxyeicosatetraenoic acid reduces prostacyclin production by endothelial cells.

12-Hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase product released by activated platelets and macrophages, reduced prostacyclin (PGI2) formation in bovine aortic endothelial cultures by as much as 70%. Maximal inhibition required 1 to 2 h to occur and after 2 hr, a concentration of 1 microM 12-HETE produced 80% of the maximum inhibitory effect. 5-HETE and 15-HETE also inhibited PGI2 formation. The inhibition was not specific for PGI2; 12-HETE reduced the formation of all of the radioactive eicosanoids synthesized from [1-14C]arachidonic acid by human umbilical vein endothelial cultures. Inhibition occurred in the human cultures when PGI2 formation was elicited with arachidonic acid, ionophore A23187 or thrombin. These findings suggest that prolonged exposure to HETEs may compromise the antithrombotic and vasodilator properties of the endothelium by reducing its capacity to produce eicosanoids, including PGI2.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1986

Effect of ethanolamine on choline uptake and incorporation into phosphatidylcholine in human Y79 retinoblastoma cells.

The effect of physiological concentrations of ethanolamine on choline uptake and incorporation into phosphatidylcholine was investigated in human Y79 retinoblastoma cells, a multipotential, undifferentiated retinal cell line that has retained many neural characteristics. These cells have a high-affinity uptake system for choline, and the majority of the choline taken up was incorporated into phosphatidylcholine via the CDP-choline pathway. The presence of extracellular ethanolamine significantly decreased high-affinity choline uptake and, subsequently, the amount of choline incorporated into phosphatidylcholine. When 100 mumol/L ethanolamine was added, there was a decrease of about 8% in the phosphatidylcholine content. Ethanolamine had no effect on choline incorporation into phosphatidylcholine, however, once choline was taken up by the cell. The K'M and V'max for high-affinity choline uptake was increased from 0.93 to 9.74 microM and 19.60 to 79.25 pmol/min per mg protein, respectively, by the presence of 25 mumol/L ethanolamine. In contrast, 25 mumol/L choline had no effect on the kinetic parameters of high-affinity ethanolamine uptake. Therefore, the reduction in high-affinity choline transport by ethanolamine apparently is not simply due to competitive inhibition. 2,2-Dimethylethanolamine and 2-methylethanolamine both reduced choline uptake to a greater extent than ethanolamine. However, because these compounds exist at much lower concentrations than ethanolamine, they probably have little physiological influence. These results suggest that changes in ethanolamine concentration within the physiologic range can regulate the synthesis and content of phosphatidylcholine in a neural cell by influencing the uptake of choline.

Biological Transport↗

PubMed1986

Free fatty acid release from endothelial cells.

Cultured bovine aortic endothelial cells that have been previously enriched with fatty acid are able to release free fatty acid (FFA) into the extracellular fluid. No stimulus other than the presence of albumin in the medium is needed to elicit the FFA release. Intracellular triglycerides appear to be the source of most of the FFA that is released. The released FFA is composed of a mixture of fatty acids, with the fatty acid used to enrich the cells contributing about half of the total. Under certain conditions sufficient fatty acid can be released to increase the FFA concentration of the extracellular fluid. Cells enriched initially with arachidonic acid released 1.7- to 2.9-times more FFA as compared to cells enriched with corresponding amounts of oleic acid. Neither prostaglandins nor lipoxygenase products contributed appreciably to the amount of FFA released from cells enriched with arachidonic acid. Porcine pulmonary artery endothelial cells also can release net amounts of FFA. These findings indicate that endothelial cells have the capacity to release fatty acid in the form of FFA. This process could possibly play a role in the transfer of fatty acids, particularly arachidonic acid, across the endothelium.

Animals↗

PubMed1985

Incorporation of the purified human placental insulin receptor into phospholipid vesicles.

Purified human placental insulin receptors were incorporated into small unilamellar phospholipid vesicles by the addition of n-octyl beta-glucopyranoside solubilized phospholipids, followed by removal of the detergent on a Sephadex G-50 gel filtration column and extensive dialysis. The vesicles have an average diameter of 142 +/- 24 nm by Sephacryl S-1000 gel filtration chromatography and 119 +/- 20 nm by transmission electron microscopy. These vesicles are impermeant to small molecules as indicated by their ability to retain [gamma-32P]ATP, which could be released by the addition of 0.05% Triton X-100. Detergent permeabilization or freeze-thawing of the insulin receptor containing vesicles in the presence of 125I-insulin indicated that approximately 75% of the insulin binding sites were oriented right side out (extravesicularly). Sucrose gradient centrifugation of insulin receptors incorporated at various protein to phospholipid mole ratios demonstrated that the insulin receptors were inserted into the phospholipid bilayer structure in a concentration-dependent manner. Addition of [gamma-32P]ATP to the insulin receptor containing vesicles was relatively ineffective in promoting the autophosphorylation of the beta subunit in the absence or presence of insulin. Permeabilization of the vesicles with low detergent concentrations, however, stimulated the beta-subunit autophosphorylation approximately 2-fold in the absence and 10-fold in the presence of insulin. Insulin-stimulated beta-subunit autophosphorylation was also observed under conditions such that 94% of those vesicles containing insulin receptors had a single receptor per vesicle, suggesting that the initial beta-subunit autophosphorylating activity is intramolecular. Phospho amino acid analysis of the vesicle-incorporated insulin receptors demonstrated that the basal and insulin-stimulated beta-subunit autophosphorylation occurs exclusively on tyrosine residues. It is concluded that when purified insulin receptors are incorporated into a phospholipid bilayer, they insert into the vesicles primarily in the same orientation as occurs in the plasma membrane of intact cells and retain insulin binding as well as insulin-stimulated beta-subunit autophosphorylating activities.

Electrophoresis, Polyacrylamide Gel↗

PubMed1985

Increased production of lipoxygenase products by cholesterol-rich mouse macrophages.

The metabolism of arachidonic acid by cholesterol-enriched resident mouse peritoneal macrophages was investigated. The amounts of monohydroxyeicosatetraenoic acid (mono-HETE) produced by the cholesterol-rich macrophages were 2.5-fold greater when compared to control macrophages. The major lipoxygenase product, identified by high-performance liquid chromatography in both macrophages was 12-HETE. Since macrophages are important participants in the formation of atheromatous lesions, the increased metabolism of arachidonic acid to HETE products by cholesterol-rich macrophages could contribute to the initiation and progression of the atherosclerotic process.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1985

The utilization of ethanolamine and serine for ethanolamine phosphoglyceride synthesis by human Y79 retinoblastoma cells.

Phospholipid synthesis was investigated in human Y79 retinoblastoma cells, a cultured cell line of retinal origin that retains many neural characteristics. Ethanolamine is taken up by Y79 cells through a high-affinity transport system and is utilized to synthesize ethanolamine and choline phosphoglycerides. High-affinity ethanolamine uptake has a K'm of 40.6 microM and a V'max of 1.06 nmol/min/mg protein, and the process is Na+ dependent. Choline is the only compound tested that reduced ethanolamine uptake, and very high choline concentrations were required to produce this effect. The cells incorporate ethanolamine into phosphatidylethanolamine and ethanolamine plasmalogen at equivalent rates, and the rates of catabolism of these phospholipids are similar. Only a small quantity of ethanolamine is incorporated into phosphatidylcholine, but the amount is not reduced by the addition of choline. Serine is incorporated into phosphatidylserine, which then is converted to phosphatidylethanolamine. Ethanolamine reduces but does not abolish this conversion. Unlike ethanolamine, only a small amount of serine is incorporated into ethanolamine plasmalogen. It is possible that the ethanolamine high-affinity uptake system is necessary to provide a neural cell with enough free ethanolamine for ethanolamine plasmalogen synthesis.

Cell Line↗

PubMed1985

Morphological alterations in cultured endothelial cells induced by arachidonic acid.

The addition of arachidonic acid (20:4), but not other fatty acids, including the structurally similar eicosapentaenoic acid (20:5), induced specific morphological changes in cultured endothelial cells derived from bovine aorta and pulmonary artery. Cells exhibited a time- and dose-dependent change from their normal, epithelioid morphology to become elongated, polygonal, and spindle-shaped. Cells isolated from aorta appeared more sensitive to these changes than those from pulmonary artery. The effect was observed as early as 12 h after exposure to 20:4, required 48 h for maximal expression, and could be reversed in 2-5 h after change to normal media. The morphological alteration was not observed in cells treated with leukotrienes or PGE2. When cells were pretreated with ibuprofen, aspirin, or indomethacin to block prostaglandin synthesis and then exposed to 20:4, the dose-response effect was shifted to the left. This increased sensitivity to 20:4 suggests either a direct effect of 20:4 on cell morphology or an indirect effect due to metabolites of 20:4 which are not dependent on the cyclooxygenase pathway.

Animals↗

PubMed1985

Characterization of an insulin receptor in human Y79 retinoblastoma cells.

Cultured human Y79 retinoblastoma cells bind [125I]iodoinsulin in a manner similar to that of other CNS and peripheral tissues. The only difference noted between the insulin binding properties of the Y79 cells and other CNS preparations is that insulin binding to Y79 cells is down-regulated by prolonged exposure of the cells to insulin. By contrast, studies with the various brain preparations indicate that the brain insulin receptor is not down-regulated by circulating levels of insulin. Insulin binding to Y79 cells exhibits negative cooperativity, has a pH optimum of 7.8, is responsive to cations, and gives a curvilinear Scatchard plot. Y79 cell insulin binding capacity is 26 fmol/100 micrograms of cell protein, corresponding to about 125,000 binding sites per cell. These findings are the first to report insulin binding in a human cell line of retinal origin. The characterization of the insulin binding in this cell line may facilitate an understanding of the relationship between insulin and its specific functions in the human retina.

Binding, Competitive↗

PubMed1985

Exposure to fatty acid increases human low density lipoprotein transfer across cultured endothelial monolayers.

Human low density lipoproteins radiolabeled with 125I transfer across confluent monolayers of cultured porcine pulmonary artery endothelial cells. The amount transferred was dependent on the low density lipoprotein concentration and was not saturable at concentrations up to 300 micrograms protein per 0.5 ml medium. Gel filtration showed that more than 90% of the 125I which crossed the endothelial monolayer remained associated with low density lipoproteins, indicating that appreciable amounts of lipoprotein were not degraded during the transfer process. When the endothelial monolayer was exposed for 24 hours to culture media supplemented with 100-300 microM fatty acid complexed with 100 microM albumin, the amount of low density lipoprotein subsequently transferred increased by 65% to 150%. The extent of the increase was dependent on the type of fatty acid added and its concentration. At 200 microM, albumin-bound oleic and linoleic acids increased low density lipoprotein transfer, whereas palmitic, linolenic, arachidonic, and eicosapentaenoic acids did not. These results are consistent with the hypothesis that exposure of the endothelium to elevated concentrations of fatty acid may allow excessive amounts of cholesterol-rich lipoproteins to enter the arterial intima.

Animals↗

PubMed1985

Membrane lipid composition and cellular function.

Membrane fatty acid composition, phospholipid composition, and cholesterol content can be modified in many different kinds of intact mammalian cells. The modifications are extensive enough to alter membrane fluidity and affect a number of cellular functions, including carrier-mediated transport, the properties of certain membrane-bound enzymes, binding to the insulin and opiate receptors, phagocytosis, endocytosis, depolarization-dependent exocytosis, immunologic and chemotherapeutic cytotoxicity, prostaglandin production, and cell growth. The effects of lipid modification on cellular function are very complex. They often vary from one type of cell to another, and they do not exert a uniform effect on all processes in a single cell line. Therefore, it is not yet possible to make any generalizations or to predict how a given system will respond to a particular type of lipid modification. Many of the functional responses probably are caused directly by the membrane lipid structural changes, which affect either bulk lipid fluidity or specific lipid domains. The conformation or quaternary structures of certain transporters, receptors, and enzymes probably are sensitive to changes in the structure of their lipid microenvironment, leading to changes in activity. Prostaglandin production is modulated by the availability of substrate fatty acids stored in the membrane phospholipids, but the underlying chemical mechanism still involves a change in membrane lipid structure. While this is the most likely mechanism, the possibility that the membrane lipid compositional change is an independent event that occurs concurrently but is not causally related to the functional perturbations also must be considered.

Animals↗

PubMed1985

Effect of human plasma lipoproteins on prostacyclin production by cultured endothelial cells.

Prostacyclin (PGI2) production by bovine aortic or human umbilical vein endothelial cells increased when either human high density lipoproteins3 (HDL3) or low density lipoproteins (LDL) were added to a serum-free culture medium. At low concentrations and short incubation times, HDL3 produced more PGI2 than LDL, but LDL was just as effective as HDL3 in 18-hr incubations with high concentrations of lipoproteins. Neither lipoprotein was toxic to the cultures as assessed by [3H]leucine incorporation into cell protein. The stimulatory effect of HDL3 and LDL on PGI2 production decreased as growing cultures became confluent. Incubation with lipoproteins neither enhanced arachidonic acid release nor increased PGI2 formation when the cells were stimulated subsequently with ionophore A23187, indicating that the lipoproteins do not affect the intracellular processes involved in PGI2 production. The addition of albumin reduced the amount of PGI2 formation elicited by HDL3 or LDL. As compared with albumin-bound arachidonic acid, from 6- to 13-fold less PGI2 was produced during incubation with the lipoproteins. Furthermore, the amount of PGI2 formation elicited by the lipoproteins in 18 hr was 4-fold less than that produced during incubation with a fatty acid mixture containing only 5% arachidonic acid, and 3-fold less than when the cells were stimulated with the ionophore A23187 for 20 min. Taken together, our results indicate that human HDL and LDL contribute to endothelial PGI2 production only in a modest way and suggest that this process is not specific for either of these two plasma lipoproteins. In view of the greater participation of albumin-bound arachidonic acid in PGI2 production, plasma lipoproteins may not play as important a role in endothelial prostaglandin formation as has been suggested.

Animals↗

PubMed1985

A comparison of lipid metabolism in two human retinoblastoma cell lines.

A number of differences have been observed in the lipid metabolism of cultured Y79 and WERI Rb-1 retinoblastoma cells. The Y79 cells have a higher phospholipid content and a higher percentage of ethanolamine phosphoglycerides, whereas the WERI Rb-1 cells have more triglycerides. This is consistent with the finding that most of the polyunsaturated fatty acid taken up by the Y79 cells is incorporated into phospholipids, while a sizable amount of the uptake in WERI Rb-1 cells is incorporated into triglyceride. Polyunsaturated fatty acids of the n-6 class are taken up at about the same rate by both cells, but the n-3 polyunsaturates are taken up more rapidly by the Y79 cells. In addition, the Y79 cells convert a larger percentage of linoleic acid (18:2, n-6) to arachidonic acid (20:4, n-6) and of linolenic acid (18:3, n-3) to docosahexaenoic acid (22:6, n-3), and they accumulate these products when the precursors are added to the culture medium. When grown in 10% fetal bovine serum without supplemental fatty acid, however, the WERI Rb-1 contain more arachidonic and docosahexaenoic acids. These differences in lipid content and metabolism suggest that although the Y79 and WERI Rb-1 cell lines are derived from the same type of retinal tumor, they either originated from functionally different retinal neurons or have diverged biochemically during propagation in culture.

Cell Line↗

PubMed1985

Taurine transport and metabolism in human retinoblastoma cells.

These findings suggest that the Y79 retinoblastoma cell may be a useful experimental system in which to study certain aspects of taurine metabolism. The cells contain high concentrations of taurine when they are grown under normal conditions. This can occur in two ways. One is through synthesis of taurine from serine; the other is by taurine uptake facilitated by a high-affinity transport system. These results regarding synthesis and uptake are consistent with what has been reported for retinal preparations from other species (Lombardini 1980; Sabceda 1980; Schmidt 1980; Pourcho 1981; Adler 1983). The retinoblastoma cells did not release taurine, however, when they were depolarized. Cultured chick embryo retinal neurons also do not release taurine following depolarization (Adler 1983). Therefore, neither of these culture systems provides any evidence for a direct role of taurine release in retinal neurotransmission. The retinoblastoma cells take up relatively low concentrations of taurine more efficiently through the high-affinity transport system when they are enriched in docosahexaenoic acid (22:6). This suggested the possibility that the high 22:6 content of the retina may be related specifically to taurine utilization. Additional studies revealed, however, that the transport effect was not specific for either 22:6 or taurine. Enrichment with arachidonic acid, an n-6 polyunsaturate that cannot be converted to 22:6, produced a similar enhancement of taurine uptake (Yorek et al. 1984).(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport, Active↗

PubMed1984

Comparative utilization of n-3 polyunsaturated fatty acids by cultured human Y-79 retinoblastoma cells.

The Y-79 retinoblastoma cell, a cultured human line derived from the retina, was utilized as a model for investigating the metabolism of n-3 polyunsaturated fatty acids in neural tissue. When cultures were incubated with 5 microM linolenic (18:3), eicosapentaenoic (20:5) or docosahexaenoic (22:6) acids, a low concentration probably representative of physiologic levels, the amount incorporated was 20:5 congruent to 18.3 greater than 22:6. Regardless of which fatty acid was provided, 65-75% of the total uptake accumulated in phosphatidylethanolamine and ethanolamine plasmalogen, suggesting that these phospholipids play an important role in n-3 polyunsaturated fatty acid metabolism. A small amount of 22:6 was converted to 20:5, which was recovered in phosphatidylinositol and phosphatidylserine. Therefore, one metabolic function of 22:6 may be to serve as an intracellular storage pool for the formation of 20:5 through retroconversion. When any of the n-3 polyunsaturates was available, the main fatty acid that accumulated in the cell phospholipids was 22:6. The extent to which 22:6 accumulated, however, depended on the particular n-3 polyunsaturated fatty acid that was available. This suggests that the 22:6 content of a neural cell, and any cellular function dependent on 22:6 content, may be regulated by changes in the type of n-3 polyunsaturate available to the nervous system.

Carbon Radioisotopes↗

PubMed1984

Effects of membrane lipid unsaturation on the interactions of insulin and multiplication stimulating activity with endothelial cells.

Modification of plasma membrane fatty acyl composition has resulted in major changes in insulin binding and insulin action in several cell types. In the present study, endothelial cells, which in vivo are directly bathed by the changing fatty acid and insulin environment of the bloodstream, were grown in media enriched in specific saturated, monounsaturated and polyunsaturated fatty acids. These media conditions resulted in major and specific alteration in fatty acyl unsaturation of both neutral lipids and phospholipids of the endothelial cells. Despite the extensive fatty acyl changes, the lipid-modified cells demonstrated no change in the binding of insulin or the insulin-like growth factor, multiplication stimulating activity, and little alteration in insulin-induced down-regulation of the insulin receptor, or in cell processing of insulin. We suggest that the insulin receptor of the endothelial cell responds in a different manner than other cell types to similar alterations of membrane fatty acyl composition.

Animals↗

PubMed1984

Electron-spin resonance studies of lipid-modified microsomes from Friend erythroleukemia cells.

The fatty acid composition of cultured Friend erythroleukemia cells was modified by supplementation of the medium with oleic or linoleic acid. There was a 30% reduction in saturated and a 35% reduction in polyunsaturated fatty acids in microsomal phospholipids when the cells were grown in media supplemented with oleic acid, and a 3-fold increase in polyunsaturated fatty acids when the cells were grown in linoleic acid-supplemented media. Electron-spin resonance studies with the 5- nitroxystearate probe demonstrated that there was no appreciable change in microsomal lipid mobility as measured by the order parameters. In contrast, changes in lipid mobility were detected with the spin-label probe when microsomes were first isolated from Friend erythroleukemia cells and subsequently modified by incubation with liposomes composed of either dioleoyl- or dilinoleoylphosphatidylcholine plus bovine liver phospholipid-exchange protein. The fatty acid compositional changes produced in these microsomes were similar to those obtained when the intact cells were grown in media containing supplemental fatty acids. These findings indicate that the lipid mobility of Friend cell microsomes can be altered by phospholipid replacements in vitro, but that this does not occur when similar microsomal fatty acid modifications are produced during culture of the intact cell.

Animals↗

PubMed1984

Changes in serum influence the fatty acid composition of established cell lines.

The fatty acid composition of different kinds of commercially available serum used to supplement cell culture media differs widely. As compared with fetal bovine serum, horse and bovine calf serum have a very high content of linoleic acid (18:2) and are low in arachidonic acid (20:4). (Fatty acids are abbreviated as number of carbon atoms:number of double bonds). Swine serum contains substantial amounts of both 18:2 and 20:4. Only fetal bovine serum contains more than 1% docosahexaenoic acid (22:6). Considerable differences in fatty acid composition occur when cells are grown in media containing any of these different serum supplements. The 18:2 and 20:4 content of 3T3 mouse fibroblast phospholipids is highest when the medium contains horse serum, intermediate with bovine calf serum, and lowest with swine or fetal bovine serum. Likewise, the highest phospholipid 18:2 content in Madin-Darby canine kidney cells (MDCK) occurs when the medium contains horse serum. With MDCK cells, however, growth in swine serum produces the highest 20:4 content. The 3T3 cell phospholipids accumulate more than 1% 22:6 only when the medium contains fetal bovine serum, whereas in no case do the MDCK cell phospholipids accumulate appreciable amounts of 22:6. The fact that the cellular fatty acid composition is likely to change should be taken into account when changes are contemplated in the serum used to grow established cell lines.

Animals↗

PubMed1984

Effect of membrane polyunsaturation on carrier-mediated transport in cultured retinoblastoma cells: alterations in taurine uptake.

Neural cell membranes naturally contain a large amount of polyunsaturated fatty acid, but the functional significance of this is unknown. An increase in membrane polyunsaturation has been shown previously to affect the high-affinity transport systems for choline and glycine in cultured human Y79 retinoblastoma cells. To test the generality of membrane polyunsaturation effects on transport, we investigated the uptake of other putative neurotransmitters and amino acids by these cells. Taurine, glutamate, and leucine were taken up by both high- and low-affinity transport systems, whereas serine, gamma-aminobutyrate, and alpha-aminoisobutyrate were taken up only by low-affinity systems. The high-affinity taurine and glutamate and low-affinity serine uptake systems were Na+ dependent. Arachidonic acid (20:4) supplementation of Y79 cells produced enrichment of all the major microsomal phosphoglycerides with 20:4, while docosahexaenoic acid (22:6) supplementation produced large increases in the 22:6 content of all fractions except the inositol phosphoglycerides. Enrichment with these polyunsaturated fatty acids facilitated taurine uptake by lowering the K'm of its high-affinity transport system. By contrast, enrichment with oleic acid did not affect taurine uptake. Glutamate, leucine, serine, gamma-aminobutyrate, and alpha-aminoisobutyrate uptake were not affected when the cells were enriched with any of these fatty acids. These findings demonstrate that only certain transport systems are sensitive to the polyunsaturated fatty acid content of the retinoblastoma cell membrane. The various transport systems either respond differently to changes in membrane lipid unsaturation, or they are located in lipid domains that are modified to different extents by changes in unsaturation.

Amino Acids↗