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Biomedical subjects

A A Spector

Publications and source records attributed to A A Spector.

At least 19 recordsLinked to original sources
PubMed1992

Myristic acid utilization in Chinese hamster ovary cells and peroxisome-deficient mutants.

Chinese hamster ovary (CHO) cells convert [9,10-3H]myristic acid ([3H]14:0) to several lipid-soluble, radioactive metabolites that are released into the medium. The main products are lauric (12:0) and decanoic (10:0) acids. Some of the 12:0 formed also is retained in cell lipids. Similar metabolites are not synthesized from palmitic (16:0), oleic (18:1), or arachidonic (20:4) acids, and the addition of these fatty acids does not reduce the conversion of [3H]14:0 to 12:0. Two peroxisome-deficient CHO cell lines do not convert [3H] 14:0 to any polar metabolites, but, they elongate, desaturate, and incorporate [3H]14:0 into intracellular lipids and proteins normally. While BC3H1 muscle cells convert some [3H]14:0 to 12:0, they also produce at least nine lipid-soluble polar products from [3H]12:0. These findings suggest that a previously unrecognized function of myristic acid is to serve as a substrate for the synthesis of 12:0, which can be either secreted into the medium or converted to other oxidized metabolites. The absence of this peroxisomal oxidation pathway, however, does not interfere with other aspects of myristic acid metabolism, including protein myristoylation.

Animals↗
PubMed1992

Detection of lipid radicals by electron paramagnetic resonance spin trapping using intact cells enriched with polyunsaturated fatty acid.

Electron paramagnetic resonance (EPR) spin trapping was used to detect lipid-derived free radicals generated by iron-induced oxidative stress in intact cells. Using the spin trap alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), carbon-centered radical adducts were detected. These lipid-derived free radicals were formed during incubation of ferrous iron with U937 cells that were enriched with docosahexaenoic acid (22:6n-3). The EPR spectra exhibited apparent hyperfine splittings characteristic of a POBN/alkyl radical, aN = 15.63 +/- 0.06 G and aH = 2.66 +/- 0.03 G, generated as a result of beta-scission of alkoxyl radicals. Spin adduct formation depended on the FeSO4 content of the incubation medium and the number of 22:6-enriched cells present; when the cells were enriched with oleic acid (18:1n-9), spin adducts were not detected. This is the first direct demonstration, using EPR, of a lipid-derived radical formed in intact cells in response to oxidant stress.

Cell Line↗
PubMed1992

Interaction of lysophosphatidylcholine with aortic endothelial cells.

To better understand the vascular actions of lysophosphatidylcholine (lysoPC), we studied the interaction of [1-14C]palmitate-labeled lysoPC with bovine aortic endothelial cells. These cells took up lysoPC from media containing albumin, low-density lipoproteins (LDL), or acetyl-LDL. Uptake occurred faster than conversion to phosphatidylcholine (PC), leading to some lysoPC accumulation in endothelial lipids. Endothelial cell monolayers grown on micropore filters took up lysoPC from both apical and basolateral surfaces, preventing substantial amounts from passage across the endothelial monolayer. However, lysoPC present in the interstitial medium of an endothelial-smooth muscle coculture was incorporated primarily by the smooth muscle cells. Endothelial cells grown on filters released lysoPC into both the apical and basolateral medium in the presence of albumin or lipoproteins. Exposure to 50 microM lysoPC produced no evidence of endothelial cytotoxicity, but prostaglandin (PG)I2 production was reduced. These studies suggest that the endothelium can participate in the processing of circulating lysoPC and, through basolateral uptake, can facilitate the removal of lysoPC formed within the arterial wall. By decreasing PGI2 output, however, exposure to high concentrations of lysoPC may reduce the antithrombotic and vasodilator capacity of the endothelium.

Animals↗
PubMed1991

Cytotoxic effect of cis-parinaric acid in cultured malignant cells.

Parinaric acid, a naturally occurring 18-carbon fatty acid containing 4 conjugated double bonds, is toxic to human monocytic leukemia cells at concentrations of 5 microM or less. Conditioning of the medium reduces the cytotoxic effect, suggesting that parinaric acid and not a metabolite is the active agent. The mechanism of parinaric acid toxicity appears to involve lipid peroxidation because the toxic action can be blocked by the addition of butylated hydroxytoluene. When U-937 cells are differentiated to the monocytic form, they become resistant to as much as 30 microM parinaric acid. This difference in sensitivity may be explained in part by the fact that the undifferentiated cells take up 3 to 4 times more parinaric acid. Concentrations of parinaric acid less than 5 microM are also toxic to human THP-1 monocytic leukemia, HL-60 human promyelocytic leukemia, and Y-79 human retinoblastoma cells. Measurements of protein synthesis indicate that differentiated U-937 cells, confluent cultures of human fibroblasts, bovine aortic endothelial cells, and CaCo-2 colonic mucosal cells are much less sensitive to parinaric acid than the malignant cell lines tested, suggesting that the cytotoxic action may be selective for rapidly growing malignant tumors. Thus, parinaric acid may be the prototype of a new class of lipid chemotherapeutic agents that contain a conjugated system of double bonds and act by sensitizing tumor cells to peroxidation.

Butylated Hydroxytoluene↗
PubMed1991

Hydroxyeicosatetraenoic acid oxidation in Chinese hamster ovary cells: a peroxisomal metabolic pathway.

To evaluate the peroxisomal requirement for beta-oxidation of hydroxyeicosatetraenoic acids (HETES), we tested 5-, 12- and 15-HETE oxidation in wild-type and mutant Chinese hamster ovary (CHO) cells. Mutant CHO cells contain peroxisomal ghosts, have random cytosolic localization of catalase and lack two of the enzymes necessary for peroxisomal beta-oxidation. Reverse-phase HPLC indicated that 33% of 12-HETE radioactivity was converted by wild-type CHO cells during a 2 h incubation to one major and several minor polar metabolites. Wild-type CHO cells also converted 15-HETE to one major and several minor polar metabolites. Neither 12- nor 15-HETE were converted to any metabolites by the mutant CHO cell lines, despite appreciable cellular uptake of these hydroxyeicosanoids. 5-HETE was not converted to any metabolic products by either the wild-type or the mutant CHO cells. Docosahexaenoic acid beta-oxidation was substantially reduced in the mutants as compared to the wild-type cells, palmitic acid beta-oxidation was reduced to an intermediate extent in the mutants, but octanoate beta-oxidation and citrate synthase activity were not impaired. Protein immunoblotting for mitochondrial manganese superoxide dismutase indicated a single band of identity at 20 kDa in both wild-type and mutant CHO cells. Since mutant CHO cells fail to convert 12- and 15-HETE to oxidative metabolites but contain normal mitochondrial enzymatic activities, intact peroxisomes appear to be the organelle responsible for HETE oxidation.

Animals↗
PubMed1991

Myristic acid utilization and processing in BC3H1 muscle cells.

Because myristic acid (14:0) is important in regulating cell function, we have studied its utilization in BC3H1 muscle cells. Phosphatidylcholine contained 70-80% of the [9,10-3H]14:0 radioactivity incorporated into the cell phospholipids. In both myoblasts and myocytes, however, large amounts of radioactivity also accumulated in a labile neutral lipid pool consisting mostly of triacylglycerol. Therefore, radioactive lipid products formed when BC3H1 cells labeled with 14:0 are stimulated are not necessarily derived only from phosphatidylcholine. Elongation of [9,10-3H]14:0 occurred rapidly in the myoblasts and myocytes, and extensive desaturation also occurred in the myoblasts. Thus, even after short periods of labeling, substantial amounts of radioactivity are contained in fatty acids other than 14:0. The labeling of proteins with [9,10-3H]myristic acid was generally similar in the myoblasts and myocytes. A number of lipid-soluble, polar radioactive metabolites were released into the medium during incubation of [9,10-3H]14:0 with the cells. [1-14C] 14:0 was not converted to these compounds, indicating that they are chain-shortened 14:0 derivatives. Based on chemical analysis, two of the major products appear to be hydroxylated fatty acids. This oxidation process shows some specificity for 14:0 because similar compounds were not produced from palmitic, oleic, or linoleic acids. The myocytes formed larger amounts of the metabolites than the myoblasts, suggesting that differentiation may increase the activity of this 14:0 oxidative pathway.

Acylation↗
PubMed1991

Astrocytes, not neurons, produce docosahexaenoic acid (22:6 omega-3) and arachidonic acid (20:4 omega-6).

Elongated, 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 are not fully characterized. To investigate whether neurons themselves are capable of essential fatty acid elongation and desaturation or are dependent upon the support of other brain cells, primary cultures of rat neurons and astrocytes were incubated with [1-14C] 18:2 omega-6, [1-14C]20:4 omega-6, [1-14C]18:3 omega-3, or [1-14C]20:5 omega-3 and their elongation/desaturation products determined. Neuronal cultures were routinely incapable of producing significant amounts of delta 4-desaturase products. They desaturated fatty acids very poorly at every step of the pathway, producing primarily elongation products of the 18- and 20-carbon precursors. In contrast, astrocytes actively elongated and desaturated the 18- and 20-carbon precursors. The major metabolite of 18:2 omega-6 was 20:4 omega-6, whereas the primary products from 18:3 omega-3 were 20:5 omega-3, 22:5 omega-3, and 22:6 omega-3. The majority of the long-chain fatty acids formed by astrocyte cultures, particularly 20:4 omega-6 and 22:6 omega-3, was released into the extracellular fluid. Although incapable of producing 20:4 omega-6 and 22:6 omega-3 from precursor fatty acids, neuronal cultures readily took up these fatty acids from the medium. These findings suggest that astrocytes play an important supportive role in the brain by elongating and desaturating omega-6 and omega-3 essential fatty acid precursors to 20:4 omega-6 and 22:6 omega-3, then releasing the long-chain polyunsaturated fatty acids for uptake by neurons.

Animals↗
PubMed1991

Brain microvessel 12-hydroxyeicosatetraenoic acid is the (S) enantiomer and is lipoxygenase derived.

12-Hydroxyeicosatetraenoic acid (12-HETE) production from arachidonic acid by cerebral microvessels isolated from perfused adult murine brain was reduced by the lipoxygenase inhibitors baicalein, esculetin, gossypol, nordihydroguaiaretic acid, and quercetin. Except for quercetin and gossypol, the IC50 did not exceed 10 microM. Each inhibitor, except baicalein, also decreased microvessel prostaglandin production when present in concentrations above their IC50 value for 12-HETE. In contrast, inhibitors of the cytochrome P450 monooxygenase system, clotrimazole, metyrapone, and proadifen (SKF-525A), had little effect on microvessel 12-HETE production. Chiral phase HPLC analysis revealed that only the (S) enantiomer of 12-HETE was formed. The major microvessel metabolite of eicosapentaenoic acid co-eluted with 12-hydroxyeicosapentaenoic acid (12-HEPE) on reverse-phase HPLC and the (S) enantiomer of 12-HEPE on chiral phase HPLC. Furthermore, like 12-HETE, 12-HEPE production was blocked by lipoxygenase inhibitors. These studies demonstrate that brain microvessels produce only the (S) enantiomeric 12-hydroxy derivatives of both arachidonic acid and eicosapentaenoic acid by the action of a lipoxygenase that can be selectively inhibited by baicalein. Since arachidonic acid and eicosapentaenoic acid are available to cerebral blood vessels in certain pathological settings, these 12-hydroxy acid lipoxygenase products may mediate some of the cerebrovascular dysfunction that occurs following stroke, brain trauma, or seizures.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1991

HETEs and coronary artery endothelial cells: metabolic and functional interactions.

Porcine coronary artery endothelial cells have been established in culture. These cells produce prostaglandin (PG) I2, PGF2 alpha, and PGE2 when exposed to either arachidonic acid or ionophore A23187. PG formation was reduced when the cells were exposed to monohydroxy and dihydroxy unsaturated fatty acids. Although all of the hydroxyeicosatetraenoic acids (HETEs) produced reductions, 5-HETE caused the largest decrease in PGI2 formation. Therefore, these lipoxygenase products, especially 5-HETE, may impair the nonthrombogenic surface and some vasodilator responses of coronary endothelium. The cells took up each of the HETEs and incorporated them into phospholipids. Uptake was not affected by equimolar amounts of oleic or linoleic acids; even arachidonic acid reduced 12- and 15-HETE uptake by only 50-60%. Like other cells, the coronary endothelium converted 12- and 15-HETE to polar metabolites. As opposed to other cells, however, these cultures also converted 5-HETE to a more polar metabolite. Thus coronary artery endothelium can take up and metabolize all of the major HETEs, including 5-HETE, and thereby reduce their potentially injurious effects in the coronary circulation.

Animals↗
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↗