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Biomedical subjects

A A Spector

Publications and source records attributed to A A Spector.

At least 91 records · Page 5Linked to original sources
PubMed1991

Effect of differentiation on platelet-activating factor metabolism in HL-60 cells.

The formation and metabolism of 1-O-alkyl-2-acetyl-sn-glycerol (AAG), a protein kinase C (PKC) activator formed from platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3- phosphocholine; PAF), was studied in HL-60 cells to determine whether differentiation may influence this process. HL-60 cells differentiated to macrophages (HL-60/M phi) with a phorbol ester convert added [3H]PAF to AAG; 22% of the incorporated radioactivity is converted to AAG within 15s. By contrast, neither undifferentiated HL-60 cells (HL-60/U) nor HL-60 cells differentiated to granulocytes (HL-60/GN) with retinoic acid produce AAG from PAF. The HL-60/M phi rapidly convert radiolabeled AAG to 1-O-alkyl-sn-glycerol and, subsequently, to two other unidentified metabolites. However, some apparently unmodified AAG persists in the cell lipids for at least 6 h. The HL-60 subtypes which do not convert PAF to AAG can nevertheless catabolize AAG; HL-60/U and HL-60/GN produce alkylglycerol and the other AAG metabolites. These findings demonstrate that differentiation can alter the processing of PAF in a human leukocyte cell line. Furthermore, they suggest that PAF may produce at least some of its biological effects in macrophages by conversion to AAG.

Cell Differentiation↗
PubMed1990

Role of the blood-brain barrier in the formation of long-chain omega-3 and omega-6 fatty acids from essential fatty acid precursors.

Elongated, more highly polyunsaturated derivatives of linoleic acid (18:2 omega-6) and linolenic acid (18:3 omega-3) accumulate in brain, but their sites of synthesis and mechanism of entry are not well characterized. To investigate the role of the blood-brain barrier in this process, cultured murine cerebromicrovascular endothelia were incubated with [1-14C]18:2 omega-6 or [1-14C]18:3 omega-3 and their elongation/desaturation products determined. The major metabolite of 18:2 omega-6 was 20:4 omega-6, whereas the primary product from 18:3 omega-3 was 20:5 omega-3. Although these products were found primarily in cell lipids, they were also released from the cells and gradually accumulated in the extracellular fluid. Eicosanoid production was observed from the 20:4 omega-6 and 20:5 omega-3 that were formed. No 22:5 omega-6 or 22:6 omega-3 fatty acids were detected, suggesting that these endothelial cells are not the site of the final desaturation step. Although the uptake of 18:3 omega-3 and 18:2 omega-6 was nearly identical, 18:3 omega-3 was more extensively elongated and desaturated. Competition experiments demonstrated a preference for 18:3 omega-3 by the elongation/desaturation pathway. These findings suggest that the blood-brain barrier can play an important role in the elongation and desaturation of omega-3 and omega-6 essential fatty acids during their transfer from the circulation into the brain.

Acetyltransferases↗
PubMed1990

Effects of lipids on cancer therapy.

The fatty acid composition of cancer cell membranes can change substantially when the cells are exposed to different types of fat. Such change occurs when tumors are grown in animals fed high-fat diets that differ in degree of unsaturation or during culture in media supplemented with various fatty acids. Certain physical and functional properties of the membrane are modified when the polyunsaturated fatty acid content is increased, and the cells become more sensitive to hyperthermia or treatment with doxorubicin. These findings suggest a potential role for lipid nutrition in cancer therapy. By altering the properties of the membrane lipids, changes in the dietary fat intake may provide a new approach for enhancing the effectiveness of certain antineoplastic therapies.

Animals↗
PubMed1990

Reduced prostacyclin formation after reoxygenation of anoxic endothelium.

Human umbilical vein endothelial cells subjected to 24 h of anoxia followed by reoxygenation released less prostacyclin (PGI2) in response to thrombin, calcium ionophore A23187, or arachidonic acid. This was associated with a substantial increase in stimulated platelet adherence. Increased lactate dehydrogenase and 51Cr release occurred after 1 h of reoxygenation, but the high rate of release did not persist during the subsequent 23 h of reoxygenation. The changes in platelet adherence and PGI2 release partially resolved over 24 h. PGI2 formation from prostaglandin H2 was not reduced, suggesting that cyclooxygenase activity, but not prostacyclin synthase, is affected by reoxygenation. A decrease in arachidonic acid release from cellular lipids also occurred. The reduction in cyclooxygenase activity, but not arachidonic acid release, was prevented by the presence of ibuprofen during reoxygenation. Addition of catalase or superoxide dismutase during reoxygenation increased PGI2 release but did not completely overcome the reduction relative to control cultures. These findings suggest that the increase in platelet adherence during reoxygenation may be mediated in part by a change in cyclooxygenase activity. This is only partly overcome by extracellular oxygen species scavengers but is prevented by the presence of a reversible cyclooxygenase inhibitor during reoxygenation.

Aerobiosis↗
PubMed1990

Hydroxyeicosatetraenoic acid metabolism in cultured human skin fibroblasts. Evidence for peroxisomal beta-oxidation.

To determine whether the peroxisome is responsible for hydroxyeicosatetraenoic acid (HETE) oxidation, 12- and 15-HETE oxidation was measured in normal and peroxisomal deficient skin fibroblasts from patients with Zellweger's (cerebrohepatorenal) syndrome. When incubated for 1 h with normal fibroblasts, reverse phase HPLC indicated that 24% of the 12-HETE radioactivity was converted to one major polar metabolite. Chemical derivatization followed by reverse phase HPLC and TLC indicated that this metabolite is 8-hydroxyhexadecatrienoic acid [16:3(8-OH)]. Similarly, 33% of the added 15-HETE was also converted to a more polar metabolite. Neither 12- nor 15-HETE were converted to any metabolites by the peroxisomal deficient (Zellweger) cells. No defect in HETE oxidation was found in other human fibroblast cell lines with diverse metabolic abnormalities. Zellweger fibroblasts accumulated increased amounts of 12-HETE, compared with normal fibroblasts. As in the normal cells, most of the 12-HETE incorporated into Zellweger fibroblasts was present in the choline and ethanolamine phosphoglycerides. Protein synthesis, lysosomal acid lipase activity, and mitochondrial butyrate oxidation were not impaired in the Zellweger fibroblasts. Since the Zellweger cells do not convert 12- and 15-HETE to oxidative metabolites, peroxisomes appear to be the cellular organelle responsible for HETE oxidation.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1990

Localization of 12-hydroxyeicosatetraenoic acid in endothelial cells.

Bovine aortic endothelial cells take up 12-hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase product formed from arachidonic acid. The uptake of [3H]12-HETE reached a maximum in 2 to 4 h. At this time, from 75 to 80% of the incorporated radioactivity was contained in phospholipids, about 85% of the esterified radioactivity remained in the form of 12-HETE, and at least 90% of the phospholipid radioactivity was present in the sn-2-position. Subcellular fractionation on Percoll and sucrose gradients demonstrated that 65 to 74% of the radioactivity was present in membranes enriched in NADPH-cytochrome c reductase and UDP-galactosyl transferase. The specific radioactivity relative to protein of these intracellular membranes was 2.9-times higher than in a plasma membrane fraction enriched in 5'-nucleotidase. A similar intracellular localization was observed when [3H]5-HETE or [3H]arachidonic acid were taken up. The 12-HETE was contained primarily in the choline glycerophospholipids of the microsomal membranes. After incorporation, [3H]12-HETE was removed from the cell lipids much more rapidly than [3H]arachidonic acid, and 80% of the radioactivity released into the medium during the first hour remained as 12-HETE. Because it accumulates in microsomal membranes, 12-HETE uptake may perturb certain intracellular processes and thereby lead to endothelial dysfunction. The relatively rapid removal of the newly incorporated 12-HETE may be an important protective mechanism that prevents excessive accumulation and more extensive endothelial damage.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1989

Formation of 9-hydroxyoctadecadienoic acid from linoleic acid in endothelial cells.

Human umbilical vein endothelial cells convert linoleic acid to two monohydroxyoctadecadienoic (HODE) acids, 9- and 13-HODE. More 9-HODE than 13-HODE is formed under most conditions. The production of these metabolites is reduced substantially by acetylsalicylic acid, ibuprofen, or arachidonic acid, suggesting that cyclooxygenase may be involved in endothelial HODE synthesis. Incubations lasting up to 4 h indicate that the endothelial cells can convert [U-14C] linoleic acid into at least four additional products, some of which may be derived from the HODE that is formed initially. Radioactive 9- and 13-HODE are produced when the endothelial cells are labeled with linoleic acid and then exposed to thrombin, suggesting that these metabolites also may be formed when the endothelium is activated. If endothelial monolayers grown on micropore filters are incubated with linoleic acid, a substantial amount of the HODE formed accumulates in the basolateral fluid. This suggests that HODE may have extracellular effects, especially within the vascular wall. Furthermore, when 9- or 13-HODE are added, endothelial cultures produce less prostaglandin I2 and convert less 12-hydroxyeicosatetraenoic acid to its main metabolite, 8-hydroxyhexadecatrienoic acid. Therefore, in addition to extracellular actions, HODE also may have functional effects within the endothelium.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1989

Interaction of platelet-activating factor with endothelial and vascular smooth muscle cells in coculture.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine [PAF]) is a vasoactive ether lipid produced by activated blood cells. To examine the molecular traffic and sites of metabolism of PAF released in the vascular wall, we used a coculture system in which endothelial cells are grown on micropore filters suspended over confluent cultures of vascular smooth muscle cells. The endothelial cells took up PAF 5-7 times more readily from the apical than from the basolateral surface, converting it to 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (2-acyl-PAF) and other minor metabolites. Intact endothelial monolayers effectively shielded the underlying smooth muscle cells from PAF present in the apical fluid; after a 30-min incubation with [3H]-PAF, only 1% of the radioactivity was transferred to the interstitial fluid. By contrast, PAF readily entered the interstitial fluid when the endothelial monolayers were injured by exposure to xanthine and xanthine oxidase. PAF did not significantly increase the permeability of endothelial monolayers to albumin. Smooth muscle cells took up and metabolized interstitial PAF more quickly and more completely than did endothelial cells; 65% was converted to 2-acyl-PAF in 15 min by the smooth muscle cells. PAF enhanced the proliferative effect of PDGF on smooth muscle cells, as assessed by [3H]-thymidine incorporation. These findings suggest that endothelial cells form a barrier to PAF released at the luminal surface, but PAF released in the vascular intima interacts primarily with smooth muscle cells, possibly stimulating proliferation in these cells.

Albumins↗
PubMed1989

Effects of omega-3 fatty acids on vascular smooth muscle cells: reduction in arachidonic acid incorporation into inositol phospholipids.

A rapid increase in arachidonic acid incorporation into phosphatidylinositol (PI) occurred following exposure of cultured porcine pulmonary artery smooth muscle cells to calcium ionophore A23187. This response was specific for PI and phosphatidic acid; none of the other phosphoglycerides showed any increase in arachidonic acid incorporation. The incorporation of [3H]inositol also was increased, indicating that complete synthesis of PI rather than only fatty acylation occurred in response to the ionophore. The presence of omega-3 fatty acids, especially eicosapentaenoic acid (EPA), reduced arachidonic acid but not inositol incorporation into PI. Stimulated incorporation of EPA also occurred under these conditions, suggesting that EPA replaces arachidonic acid in the newly synthesized pool of PI. Although much less arachidonic acid was incorporated into the polyphosphoinositides following exposure to the ionophore, arachidonic acid incorporation into these phosphorylated derivatives also decreased when EPA was present. These findings suggest that when omega-3 fatty acids are available, less arachidonic acid is channeled into the inositol phospholipids of activated smooth muscle cells because of replacement by EPA. This may represent a mechanism whereby omega-3 fatty acids, especially EPA, can accumulate in the metabolically active pools of inositol phospholipids and thereby possibly influence the properties or responsiveness of vascular smooth muscle.

Animals↗
PubMed1989

1-O-alkyl-2-acetyl-sn-glycerol: a platelet-activating factor metabolite with biological activity in vascular smooth muscle cells.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a potent vasoactive ether lipid produced by activated blood cells and endothelial cells. Vascular smooth muscle cells partially convert exogenous PAF to 1-O-alkyl-2-acetyl-sn-glycerol (AAG), a biologically active diacylglycerol analogue. AAG is formed rapidly (less than 15 s) after exposure of the smooth muscle cells and does not appear to be a substrate for diacylglycerol kinase in these cells. Although most of the compound is metabolized to 1-O-alkyl-sn-glycerol, a small quantity remains as AAG for greater than or equal to 6 h. AAG inhibits phorbol ester binding, and it is as effective an activator of protein kinase C as diolein in an in vitro assay. Furthermore, AAG and PAF produce the same pattern of effects on smooth muscle cell proliferation. These observations suggest that at least some of the actions of PAF in vascular smooth muscle may be mediated through the formation of AAG, a stable, bioactive metabolite that appears to function as a diacylglycerol analogue.

Animals↗
PubMed1989

Brain microvessels produce 12-hydroxyeicosatetraenoic acid.

Cerebral microvessels isolated from perfused, adult murine brain produce a compound with the chromatographic properties of a monohydroxyeicosatetraenoic acid when incubated with arachidonic acid or stimulated with calcium ionophore A23187. The formation of this arachidonic acid metabolite is not reduced in the presence of the cyclooxygenase inhibitor ibuprofen, but it is abolished by the lipoxygenase inhibitor nordihydroguaiaretic acid. Analysis by gas chromatography combined with chemical ionization and electron impact mass spectrometry of reduced and nonreduced derivatives of the metabolite, indicate that the compound is 12-hydroxyeicosatetraenoic acid. Fractions of isolated microvessels enriched with capillaries produce 2.1 times more 12-hydroxyeicosatetraenoic acid per microgram of protein than do fractions of microvessels enriched with arterioles. These studies confirm that brain microvessels can produce 12-hydroxyeicosatetraenoic acid and strongly suggest that cerebral endothelia are the primary source of microvessel-derived 12-hydroxyeicosatetraenoic acid. They further suggest that in brain injury, the liberation and accumulation of arachidonic acid in cerebral tissues may lead to the production of 12-hydroxyeicosatetraenoic acid within microvessels. The 12-hydroxyeicosatetraenoic acid formed in this way may mediate some of the blood-brain barrier and cerebrovascular dysfunction that occurs following stroke, brain trauma, or seizures.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1989

5-HETE: uptake, distribution, and metabolism in MDCK cells.

The interaction of (S)-5-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HETE) with Madin-Darby canine kidney (MDCK) cells was investigated to determine whether this lipoxygenase product might influence tubular epithelial function. When incubated with arachidonic acid, MDCK cells failed to synthesize any 5-HETE. However, MDCK cells can take up 5-HETE to a much greater extent than either 12- or 15-HETE. 5-HETE uptake occurred from both the apical and basolateral surfaces and was not saturated at concentrations up to 10 microM. Much of the 5-HETE was incorporated into phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine. After a 1-h incubation 5-HETE was found to be localized in either the microsomal and/or plasma membrane of MDCK cells. After pulse labeling for 1 h, MDCK cells released 35% of 5-HETE compared with 10% of the incorporated arachidonate during the next 24 h, indicating a much more rapid turnover of newly incorporated 5-HETE. When MDCK cells were incubated with 5.0 microM 5-HETE, their capacity to produce prostaglandin E2 was reduced greater than 50% in as little as 5.0 min. Since 5-HETE enters epithelial phospholipids and reduces prostaglandin production, it apparently has the capacity to modulate renal function if it is released in the proximity of the tubular epithelium during inflammatory reactions.

Animals↗
PubMed1989

Eicosapentaenoic acid metabolism in brain microvessel endothelium: effect on prostaglandin formation.

Mouse brain microvessel endothelial cells convert eicosapentaenoic acid (EPA) to prostaglandin (PG) E3, PGI3, and several hydroxy fatty acid derivatives. Similar types of products are formed by these microvessel endothelial cells from arachidonic acid. The formation of PGI2 and PGE2 is reduced, however, when the brain microvessel endothelial cultures are incubated initially with EPA. Exposure to linolenic or docosahexaenoic acid also decreased the capacity of these microvessel endothelial cells to form PGI2 and PGE2, but the reductions were smaller than those produced by EPA. Like the endothelial cultures, intact mouse brain microvessels convert EPA into eicosanoids, and incubation with EPA reduces the subsequent capacity of the microvessels to produce PGI2 and PGE2. Brain microvessel endothelial cells took up less EPA than arachidonic acid, primarily due to lesser incorporation into the inositol, ethanolamine, and serine glycerophospholipids. By contrast, considerably more EPA than arachidonic acid was incorporated into triglycerides. These findings suggest that the microvessel endothelium may be a site of conversion of EPA to eicosanoids in the brain and that EPA availability can influence the amount of dienoic prostaglandins released by the brain microvasculature. Furthermore, the substantial incorporation of EPA into triglyceride suggests that this neutral lipid may play an important role in the processing and metabolism of EPA in brain microvessels.

Animals↗
PubMed1989

Identification of the major metabolite of 12-HETE produced by renal tubular epithelial cells.

The identification and polarity of release of the major metabolite of 12-HETE produced by cultured canine renal tubular epithelial cells was determined. When incubated with 1.0 microM [3H]12-HETE for 1 h, cultured Madin Darby Canine Kidney (MDCK) cells converted 35% of the radiolabeled 12-HETE to a more polar metabolite. Following high performance liquid chromatography isolation and chemical derivatization, gas-liquid chromatography combined with mass spectrometry was used to identify the compound as 8-hydroxyhexadecatrienoic acid [16:3(8-OH)]. The electron impact mass spectrum of the hydrogenated derivative contained major ions at m/z = 215 and 245, corresponding to cleavage on either side of the trimethylsilyl group, and chemical ionization with NH3 yielded a major ion at m/z = 359, corresponding to the protonated molecular weight of the methyl ester. Incubation with 25 mM alpha-naphthoflavone, 20 microM nordihydroguaiaretic acid, and 0.1 mM 4-pentenoic acid failed to inhibit the formation 16:3 (8-OH), suggesting that the formation of 16:3 (8-OH) is not mediated by the cytochrome P-450, lipoxygenase, or mitochondrial beta-oxidation pathways. When grown on fibronectin-treated polycarbonate filters, MDCK cells released the 16:3 (8-OH) in both the apical and basolateral directions, irrespective of which side the 12-HETE was encountered. These results demonstrate the conversion of 12-HETE to a 16-carbon monohydroxy derivative by renal tubular epithelium and suggest that this product can be released to either the potential urinary space or the kidney parenchyma and renal microcirculation.

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

Conversion of 15-hydroxyeicosatetraenoic acid to 11-hydroxyhexadecatrienoic acid by endothelial cells.

Cultured endothelial cells take up 15-hydroxyeicosatetraenoic acid (15-HETE), a lipoxygenase product formed from arachidonic acid, and incorporate it into cellular phospholipids and glycerides. Uptake can occur from either the apical or basolateral surface. A substantial amount of the 15-HETE incorporated into phospholipids is present in the inositol phosphoglycerides. 15-HETE is converted into several metabolic products that accumulate in teh extracellular fluid; this conversion does not require stimulation by agonists. The main product has been identified as 11-hydroxyhexadecatrienoic acid [16:3(11-OH)], a metabolite of 15-HETE that has not been described previously. Formation of 16:3(11-OH) decreases when 4-pentenoic acid is present, suggesting that it is produced by beta-oxidation. The endothelial cells can take up 16:3(11-OH) only 25% as effectively as 15-HETE, and 16:3(11-OH) is almost entirely excluded from the inositol phosphoglycerides. These results suggest that the endothelial cells can incorporate 15-HETE when it is released into their environment. Through partial oxidation, the endothelium can process 15-HETE to a novel metabolite that is less effectively taken up and, in particular, is excluded from the inositol phosphoglycerides.

Animals↗
PubMed1988

Murine cerebral microvascular endothelium incorporate and metabolize 12-hydroxyeicosatetraenoic acid.

Cultured murine cerebromicrovascular endothelial cells were employed to study the metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) in an in vitro model of the blood-brain barrier. These endothelial cells convert 12-HETE to at least four, more polar compounds. Analysis of the least polar and predominant metabolite by gas chromatography combined with chemical ionization and electron impact mass spectrometry of reduced and nonreduced derivatives indicate that the compound is 8-hydroxyhexadecatrienoic acid (8-HHDTrE). The uptake of 12-HETE into cell phospholipids peaks at 2 hr, and is not saturable up to the highest concentration tested, 5 microM. Seventy-five to 92% of this 12-HETE is incorporated into phosphatidylcholine, while the remainder is divided between the inositol and ethanolamine phospholipids. Incorporation into neutral lipids is slower, with radioactivity gradually accumulating in triglycerides over 24 hr. Saponification of cell lipids demonstrated that not only 12-HETE, but also its major metabolite, 8-HHDTrE, is incorporated into the cell lipids. Prostacyclin and prostaglandin E2 production by the cerebral endothelial cells is inhibited by up to 56% with 1 microM and 90% with 5 microM 12-HETE. These data demonstrate that 12-HETE is actively metabolized by cerebral endothelium and suggest at least two mechanisms through which 12-HETE may alter cerebromicrovascular function: 1) incorporation into cerebral endothelial membranes and 2) inhibition of cerebral endothelial prostaglandin production. Conversion of 12-HETE to more polar compounds, particularly 8-HHDTrE, may be interpreted as either the inactivation of 12-HETE or the production of additional, biological mediators.

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

Lipid and lipoprotein effects on endothelial eicosanoid formation.

An important function of endothelium is to release PGI2, a prostaglandin produced from arachidonic acid that prevents platelet aggregation and causes arterial relaxation. Small amounts of other eicosanoids also are produced, but their role in endothelial function has not been elucidated. Much of the arachidonic acid present in the endothelium is obtained preformed from the plasma, either as FFA or from lipoproteins. Arachidonic acid is efficiently incorporated into endothelial lipids even when only relatively small amounts are available. In response to agonists, arachidonic acid is rapidly released from the endothelial phospholipids and converted to eicosanoids. Small amounts of eicosanoids also are continuously formed due to exposure of the endothelium to free fatty acid and lipoproteins containing arachidonic acid, without the need for any additional stimulus. Although the role of plasma lipid abnormalities has not been systematically investigated, there presently is little indication that hyperlipidemia interferes with endothelial arachidonic acid metabolism or the capacity of the endothelium to produce eicosanoids.

Animals↗