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A A Spector

Publications and source records attributed to A A Spector.

At least 109 records · Page 6Linked to original sources
PubMed1988

Eicosanoid metabolism in cerebromicrovascular endothelium.

Cultured murine cerebral microvessel endothelia produce predominantly prostacyclin and prostaglandin (PG) E2 when exposed to trace amounts of arachidonic acid. At higher concentrations of arachidonate or with ionophore A23187, they produce more PGE2 than prostacyclin and additionally make PGF2 alpha and small amounts of eicosanoids comigrating with hydroxylated derivatives of arachidonate. Endothelia grown on micropore filters release prostaglandins from both apical and basal surfaces; however, the ratio of basal to apical release is as high as 4:1. This polarity suggests that cerebral endothelial prostaglandins can interact with neighboring cells of the vessel wall and brain parenchyma, where they may play important roles in the control of cerebrovascular tone and neuroglial function. These eicosanoids also are produced by mouse brain microvessels, but the major microvessel product is 12-hydroxyeicosatetraenoic acid. This suggests that 12-hydroxyeicosatetraenoic acid may be abundant in brain injuries after arachidonate release. Cultured cerebral endothelia rapidly convert 12-hydroxyeicosatetraenoic to more polar metabolites and thus may prevent the accumulation of this potentially deleterious hydroxyacid.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1988

Transfer of functional insulin receptors to receptor-deficient target cells.

Purified human insulin receptors incorporated into phospholipid vesicles have previously been shown to retain insulin binding as well as insulin-stimulated beta-subunit autophosphorylating activity. These vesicles were used as a vehicle to transfer receptors to the insulin receptor-deficient Madin-Darby canine kidney (MDCK) cell line. Fusion of control [14C] dioleoylphosphatidylcholine-labeled phospholipid vesicles with MDCK cells was found to be dependent on both the amount of time and the concentration of polyethylene glycol used for fusion. Optimal insulin receptor transfer, as determined by recovery of [125I]insulin binding, occurred when MDCK cells were incubated for 45 min at 37 C in the presence of 15% polyethylene glycol plus receptor-containing vesicles. Scatchard analyses for insulin receptor binding before and after vesicle fusion demonstrated no postfusion alteration in insulin receptor affinity and a 10-fold increase in the number of insulin receptors present in the MDCK cells. Fusion transfer of insulin receptors to MDCK cells rendered the cells sensitive to insulin (10-100 nM) for stimulation of glycogen synthesis. Chloroquine (0.1 mM) was found to block endosomal processing of receptor-bound [125I] insulin within 1 h. These findings indicate that insulin receptors function as dissociable units which can be inserted into target plasma membranes with resultant recoupling to cellular systems.

Animals↗
PubMed1987

Depletion of arachidonic acid from GH3 cells. Effects on inositol phospholipid turnover and cellular activation.

We have adapted rat pituitary GH3 cells to grow in delipidated culture medium. In response, esterfied linoleic acid and arachidonic acid become essentially undetectable, whereas eicosa-5,8,11-trienoic acid accumulates and oleic acid increases markedly. These changes occur in all phospholipid classes, but are particularly pronounced in inositol phospholipids, where the usual stearate/arachidonate profile is replaced with oleate/eicosatrienoate (n - 9) and stearate/eicosatrienoate (n - 9). Incubation of arachidonate-depleted cells with 10 microM-arachidonic acid for only 24 h results in extensive remodelling of phospholipid fatty acids, such that close-to-normal compositions and arachidonic acid content are achieved for the inositol phospholipids. In comparison studies with arachidonic acid-depleted or -repleted cells, it was found that the arachidonate content does not affect thyrotropin-releasing-hormone (TRH)-stimulated responses measured at long time points, including [32P]Pi labelling of phosphatidylinositol and phosphatidic acid, stimulation of protein phosphorylation, and basal or TRH-stimulated prolactin release. However, transient events such as stimulated breakdown of inositol phospholipids and an initial rise in diacylglycerol are enhanced by the presence of arachidonate. These results show that arachidonic acid itself is not required for operation of the phosphatidylinositol cycle and is not an obligatory intermediate in TRH-mediated GH3 cell activation. It is possible that any structural or functional role of arachidonic acid in these processes is largely met by replacement with eicosatrienoate (n - 9). However, since arachidonate in inositol phospholipids facilitates their hydrolysis upon stimulation by TRH, arachidonic acid apparently may have a specific role in the recognition of these lipids by phospholipase C.

Animals↗
PubMed1987

Biological and therapeutic potential of membrane lipid modification in tumors.

The membrane fatty acid composition of cancer cells can be modified either in culture or during growth in animals without disrupting basic membrane or cellular integrity. Only fatty acids are affected; no changes occur in membrane cholesterol, phospholipid, or protein content. There are changes in membrane physical properties and certain cellular functions, including carrier-mediated transport, receptor binding, ion channels, and eicosanoid production. Fatty acid modification also can enhance the sensitivity of the cells to hyperthermia and Adriamycin. This technique provides a new approach to understanding the membrane properties of neoplastic cells. Membrane fatty acid modification also may be of potential value as a therapeutic approach designed to augment the cytotoxicity of other antineoplastic therapies.

Animals↗
PubMed1987

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.

Animals↗
PubMed1987

Lipid transfer between endothelial and smooth muscle cells in coculture.

A coculture system was employed to study the interactions between endothelium and vascular smooth muscle cells in arachidonic acid metabolism. Bovine aortic endothelial cells grown on micropore filters impregnated with gelatin and coated with fibronectin are mounted on polystyrene chambers and suspended over confluent smooth muscle cultures. The endothelial basal laminae are oriented toward the underlying smooth muscle, and the two layers are separated by only 1 mm. Each cell layer was assayed individually: apical and basolateral fluid also was collected separately for assay. Fatty acids, including arachidonic acid, are readily transferred between the endothelial and smooth muscle cells in this system. Distribution of the incorporated fatty acids among the lipids of each cell is the same as when the fatty acid is added directly to the culture medium. Arachidonic acid released from endothelial cells is available as a substrate for prostaglandin production by smooth muscle. In addition, fatty acids released from the smooth muscle cells can pass through the endothelium and accumulate in the fluid bathing the endothelial apical surface. These fatty acid interchanges may be involved in cell-cell signaling within the vascular wall, the clearance of lipids from the vascular wall, or the redistribution of arachidonic acid and other polyunsaturated fatty acids between adjacent cell types. Furthermore, the findings suggest that prostaglandin production by smooth muscle cells can occur in response to stimuli that cause arachidonic acid release from endothelial cells.

Animals↗
PubMed1987

Membrane fatty acid modification in tumor cells: a potential therapeutic adjunct.

The fatty acid compositions of several tumors have been modified sufficiently to alter some of their properties and functions. These modifications were produced in culture by adding specific fatty acids to the growth medium or by feeding fat-supplemented diets to tumor-bearing mice. The phospholipid fatty acid composition of the plasma membrane was modified, but there were no changes in membrane phospholipid or cholesterol content or in phospholipid head group composition. Each of the most abundant membrane phosphoglyceride fractions exhibited some degree of fatty acid modification. Electron spin resonance measurements with nitroxystearate spin probes indicated that the fatty acid modifications were sufficient to alter the physical properties of the plasma membrane. The K'm for methotrexate uptake was reduced when the L1210 leukemia cells were enriched in linoleic acid. Even when the kinetics of uptake at 37 C were not altered, such as for melphalan and phenylalanine uptake, the temperature transition of transport was modified, indicating that these transport systems also are responsive to the membrane fatty acid modifications. Enrichment with highly polyunsaturated fatty acid did not affect either the growth rate or radiosensitivity of the L1210 leukemia. However, the sensitivity of the L1210 cells to the cytotoxic effects of Adriamycin and hyperthermia was increased. These findings suggest the possibility that fatty acid modification of tumors may be a useful adjunct to certain currently available therapeutic modalities.

Animals↗
PubMed1987

Formation of 8-hydroxyhexadecatrienoic acid by vascular smooth muscle cells.

Smooth muscle cells derived from the human umbilical vein produce four radioactive metabolites when they are incubated in culture with [3H]-12-hydroxyeicosatetraenoic acid. This conversion does not require the addition of an agonist for eicosanoid formation. The main product, which accounts for 60% of the radioactivity converted to these metabolites, has been identified as 8-hydroxyhexadecatrienoic acid.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1987

Phospholipid activation of the insulin receptor kinase: regulation by phosphatidylinositol.

A soybean phospholipid mixture produced a concentration-dependent enhancement of beta subunit autophosphorylation of the detergent-soluble, purified human placental insulin receptor. Although phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine also increased insulin receptor autophosphorylation, only phosphatidylinositol (PtdIns) stimulated to a similar extent as the phospholipid mixture. The effect of PtdIns was biphasic, stimulating at low concentrations (75 microM), but having no stimulatory effect at high concentrations (1.0 mM). Phospholipids also stimulated the exogenous protein kinase activity of the insulin receptor toward histone H2B. Phosphorylation of PtdIns occurred with these purified insulin receptor preparations, but this activity was insulin-independent, and the turnover number for PtdIns phosphorylation in the presence of soybean phospholipid was 1/220th as small as the turnover number for the autophosphorylating activity. These results suggest that although PtdIns can modulate the activity of the insulin receptor kinase, PtdIns phosphorylation itself is not directly involved in this regulation.

Autoradiography↗
PubMed1987

Synthesis and high affinity uptake of serotonin and dopamine by human Y79 retinoblastoma cells.

Human Y79 retinoblastoma cells are capable of synthesizing the putative retinal neurotransmitters dopamine and serotonin. Separation of the catecholamines and indolamines by high performance liquid chromatography combined with electrochemical detection showed that the cells readily convert tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and, to a lesser extent, dopamine. When DOPA was added, a large quantity of dopamine was produced, as well as norepinephrine, epinephrine, and 3,4-dihydroxyphenylacetic acid. Exogenous tryptophan added to the cells was partially converted to 5-hydroxytryptophan and serotonin. A larger quantity of serotonin was produced when 5-hydroxytryptophan was added. Y79 cells have a high- and low-affinity uptake system for dopamine and serotonin. The K'm and V'max for the high-affinity uptake of dopamine and serotonin are 2.34 +/- 0.64 and 3.63 +/- 1.15 microM and 4.77 +/- 1.12 and 3.20 +/- 1.20 pmol min-1 mg protein-1, respectively. These kinetic parameters are similar to those reported for other retinal preparations where dopamine and serotonin have been suggested to function as neurotransmitters. Tyrosine and tryptophan, the physiologic precursors of dopamine and serotonin, respectively, and phenylalanine are also taken up by high- and low-affinity transport systems. The kinetic parameters for their high-affinity uptake systems are all very similar, suggesting that they may be taken up by the same transporter. These studies show that a tumor cell line derived from the human retina synthesizes dopamine and serotonin and has high-affinity uptake systems for these compounds and their precursors.(ABSTRACT TRUNCATED AT 250 WORDS)

3,4-Dihydroxyphenylacetic Acid↗
PubMed1987

Effects of 12-HETE on renal tubular epithelial cells.

The interaction of 12-HETE with MDCK cells was investigated to determine whether this lipoxygenase product might influence tubular epithelial function. MDCK cells took up 12-HETE from both the luminal and interstitial surfaces. Much of the 12-HETE was incorporated into phospholipids but, unlike arachidonic acid, there was no specificity for the sn-2 position. 12-HETE was incorporated without chemical modification, suggesting that it might perturb tight packing in the lipid bilayer. 12-HETE uptake was reduced by the presence of arachidonic acid, but not by oleic acid. 12-HETE was removed from the cell lipids more rapidly than arachidonic acid and it was released into the medium in the form of both more and less polar metabolites. When MDCK cells were incubated with 12-HETE, their capacity to produce prostaglandin E2 was reduced. Since 12-HETE enters epithelial phospholipids and is converted to metabolic products and reduces prostaglandin production, it apparently has the capacity to modulate renal function if it is released in the proximity of the tubular epithelium.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1987

Polarity of arachidonic acid metabolism by bovine aortic endothelial cell monolayers.

Monolayers of bovine aortic endothelial cells cultured on micropore filters were used to determine the polarity of endothelial uptake, release, and transfer of arachidonic acid and some of its metabolites. Uptake and spontaneous release of arachidonic acid were more rapid at the luminal than at the interstitial surface. Transfer of arachidonic acid was more rapid from the luminal to the interstitial compartment than from the interstitial to the luminal compartment. After stimulation with the ionophore A23187, monolayers released arachidonate metabolites, including prostacyclin, to both the luminal and the interstitial compartments. The ability of the endothelium to rapidly take up and release arachidonic acid from the luminal surface and the ability to release biologically active eicosanoids to both the lumen and interstitium could be important for endothelial modulation of vascular events.

Animals↗
PubMed1986

Composition and incorporation of [3H]arachidonic acid into molecular species of phospholipid classes by cultured human endothelial cells.

Based on quantitative high-performance liquid chromatographic analyses of molecular species in selected phospholipid subclasses from culture human umbilical vein endothelial cells, the relative degree of unsaturation was ethanolamine plasmalogens greater than phosphatidylethanolamine greater than phosphatidylcholine. A total of 36 different molecular species were identified in the phosphatidylcholine fraction. Interestingly, the phosphatidylcholine contained a significant amount (11.7%) of the dipalmitoyl species, a lipid normally associated with lung surfactant. The arachidonoyl-containing molecular species of phosphatidylserine/inositol were labeled to the highest extent and the ethanolamine plasmalogens contained the lowest specific radioactivity after incubating [3H]arachidonic acid with human endothelial cells for 4 h. Within each phospholipid subclass the arachidonoyl species where both acyl groups of the phospholipid are unsaturated (20:4-20:4, 18:2-20:4 + 16:1-20:4, and 18:1-20:4) had higher specific radioactivities, after labeling with [3H]arachidonic acid, than those that contained saturated aliphatic chains (16:0-20:4 and 18:0-20:4). This indicates that the unsaturated species have higher turnover rates.

Arachidonic Acid↗
PubMed1986

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.

Animals↗
PubMed1986

Inositol phospholipid arachidonic acid metabolism in GH3 pituitary cells.

Inositol phospholipids in cultured GH3 cells, a prolactin secreting, thyrotropin-releasing hormone (TRH) sensitive rat pituitary cell line, exhibit a preferential selectivity for incorporating arachidonic acid. Fatty acid composition data show that all inositol phospholipids are enriched in stearic and arachidonic acids to a much greater degree than other cellular phospholipids. Incubation of GH3 cells with radioactive stearate, oleate, arachidonate, eicosapentaenoate or docosahexaenoate also showed that much more stearate and arachidonate were incorporated into inositol phospholipids. In short term incubations with tracer amounts of radioactive arachidonate, incorporation was initially into phosphatidylinositol (PtdIns), with phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] being labelled at later times. During longer incubations, all of the inositol phospholipids reach equilibrium at about 10 h, and the resulting specific activities of the three fractions were similar. These findings suggest that arachidonate is incorporated initially into PtdIns and that PtdIns is then phosphorylated. There was no release of either arachidonate or eicosanoid products when GH3 cells were incubated with TRH. However, TRH stimulation of 32P-labelled GH3 cells resulted in rapid breakdown of PtdIns(4,5)P2 and PtdIns4P, with concomitant increases in [32P]phosphatidic acid and [32P]PtdIns. When the [32P]PtdIns was further analysed by argentation chromatography to separate PtdIns molecular species, it was found that tetraenoic (stearate/arachidonate) species accounted for 80% of the stimulated labelling. The selectivity for arachidonate incorporation into inositol phospholipids coupled with turnover of the arachidonate-containing molecular species suggests that inositol phospholipids containing arachidonic acid or the diacylglycerol resulting therefrom may play a vital cellular role in GH3 cells. This role may involve the operation of the PtdIns cycle itself rather than a stimulated release of arachidonate for eicosanoid formation.

Animals↗
PubMed1986

Eicosapentaenoic acid utilization by bovine aortic endothelial cells: effects on prostacyclin production.

We have investigated whether the presence of other fatty acids in physiologic amounts will influence the effects of eicosapentaenoic acid on cellular lipid metabolism and prostaglandin production. Eicosapentaenoic acid uptake by cultured bovine aortic endothelial cells was time and concentration dependent. At concentrations between 1 and 25 microM, most of the eicosapentaenoic acid was incorporated into phospholipids and of this, 60-90% was present in choline phosphoglycerides. Eicosapentaenoic acid inhibited arachidonic acid uptake and conversion to prostacyclin (prostaglandin I2) but was not itself converted to eicosanoids. Only small effects on the uptake of 10 microM eicosapentaenoic acid occurred when palmitic, stearic or oleic acids were added to the medium in concentrations up to 75 microM. In contrast, eicosapentaenoic acid uptake was reduced considerably by the presence of linoleic, n-6 eicosatrienoic, arachidonic or docosahexaenoic acids. Although a 100 microM mixture of palmitic, stearic, oleic and linoleic acid (25:10:50:15) had little effect on the uptake of 10 or 20 microM eicosapentaenoic acid, less of this acid was channeled into endothelial phospholipids. However, the fatty acid mixture did not prevent the inhibitory effect of eicosapentaenoic acid on prostaglandin I2 formation in response to either arachidonic acid or ionophore A23187. An 8 h exposure to eicosapentaenoic acid was required for the inhibition to become appreciable and, after 16 h, prostaglandin I2 production was reduced by as much as 60%. These findings indicate that the capacity of aortic endothelial cells to produce prostaglandin I2 is decreased by continuous exposure to eicosapentaenoic acid. Even if the eicosapentaenoic acid is present as a small percentage of a physiologic fatty acid mixture, it is still readily incorporated into endothelial phospholipids and retains its inhibitory effect against endothelial prostaglandin I2 formation. Therefore, these actions may be representative of the in vivo effects of eicosapentaenoic acid on the endothelium.

6-Ketoprostaglandin F1 alpha↗
PubMed1986

Docosatetraenoic acid in endothelial cells: formation, retroconversion to arachidonic acid, and effect on prostacyclin production.

Cultured bovine aortic endothelial cells convert arachidonic acid to docosatetraenoic acid and also take up docosatetraenoic acid from the extracellular fluid. After a 24-h incubation with biosynthetically prepared [3H]docosatetraenoic acid, about 20% of the cellular fatty acid radioactivity was converted to arachidonic acid. Furthermore, in pulse-chase experiments, the decrease in phospholipid docosatetraenoic acid content was accompanied by an increase in arachidonic acid, providing additional evidence for retroconversion. These findings suggest that one possible function of docosatetraenoic acid in endothelial cells is to serve as a source of arachidonic acid. The endothelial cells can release docosatetraenoic acid when they are stimulated with ionophore A23187, but they do not form appreciable amounts of eicosanoids from docosatetraenoic acid. Enrichment of the endothelial cells with docosatetraenoic acid reduced their capacity to produce prostacyclin (PGI2) in response to ionophore A23187. This may be related to the fact that docosatetraenoic acid enrichment caused a 40% reduction in the arachidonic acid content of the inositol phosphoglycerides. In addition, less prostacyclin was formed when the enriched cells were incubated with arachidonic acid, suggesting that docosatetraenoic acid also may act as an inhibitor of prostaglandin synthesis in endothelial cells.

Animals↗