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A A Spector

Publications and source records attributed to A A Spector.

At least 19 recordsLinked to original sources
PubMed1996

Arachidonic acid diols produced by cytochrome P-450 monooxygenases are incorporated into phospholipids of vascular endothelial cells.

Epoxyeicosatrienoic acids (EETs) are synthesized by cytochrome P-450 monooxygenases and released into the blood. When taken up by vascular endothelial and smooth muscle cells, the EETs are primarily esterified to phospholipids or converted to dihydroxyeicosatetraenoic acids (DHETs) and released. In the present studies, radiolabeled 8,9-, 11,12-, and 14,15-DHETs released into the medium from vascular smooth muscle cells were isolated and incubated for 4-16 h with cultured bovine aortic endothelial cells. The uptake ranged from 2 to 50% for the three regioisomers. Hydrolysis of the endothelial lipids and gas chromatographic-mass spectral analyses of the products indicated that all three DHET regioisomers were incorporated intact into phosphatidylcholine and phosphatidylinositol. Similar incubations with EETs confirmed that small amounts of DHETs were also esterified to endothelial phospholipids. These studies indicate that DHETs are incorporated into phospholipids either at the time of EET conversion to DHET or upon release and re-uptake of DHETs. Beside demonstrating for the first time that fatty acid diols are incorporated intact into endothelial lipids, these studies raise the possibility that both EETs and DHETs remain long enough in the vascular wall to produce chronic vasoactive effects.

Animals↗
PubMed1995

Functional and ultrastructural effects of essential fatty acid deficiency in kidney epithelial cells.

Madin-Darby canine kidney (MDCK) epithelial cells were grown in culture medium supplemented with 1% fetal bovine serum (FBS) to provide a cell culture model of essential fatty acid deficiency (EFAD). 5,8,11-Eicosatrienoic acid (20:3n-9) accumulated in cellular phospholipids, and arachidonic acid (20:4) decreased. A large increase in cellular cholesterol/phospholipid ratio was observed. Hemicyst formation was greatly reduced from normal levels in the EFAD-MDCK cells. Scanning and transmission electron microscopy revealed that EFAD-MDCK were much flatter than their normal counterparts. They had much less dense surface microvilli, mitochondria and other organelles were very sparse, except in the perinuclear area, and much of the peripheral cytoplasm was amorphous. The EFAD was rapidly reversed by the addition of as little as 10 microM linoleic or arachidonic acid to the medium. Cells supplemented with 10% FBS, the usual culture condition, displayed borderline EFAD, with intermediate levels of 20:3n-9 and 20:4 and hemicyst formation. These studies suggest that EFAD reduces water and electrolyte transport in renal tubular epithelium.

8,11,14-Eicosatrienoic Acid↗
PubMed1995

Epoxyeicosatrienoic acid metabolism in arterial smooth muscle cells.

Epoxyeicosatrienoic acids (EETs) are eicosanoids synthesized from arachidonic acid by the cytochrome P450 eposygenase pathway. The present studies demonstrate that 8,9-, 11,12-, and 14,15-EET are rapidly taken up by porcine aortic smooth muscle cells. About half of the uptake is incorporated into phospholipids, and saponification indicates that most of this remains in the form of EET. The EETs also are converted to the corresponding dihydroxyeicosatrienoic acids (DHETs) and during prolonged incubations, additional metabolites that do not retain the EET carboxyl group are formed. Most of these products are released into the medium. However, some DHET and metabolites less polar than EET are incorporated into the phospholipids, and a small amount of unesterified EET is also present in the cells. The incorporation of 14,15-EET and its conversion to DHET did not approach saturation until the concentration exceeded 10-20 microM, indicating that vascular smooth muscle has a large capacity to utilize this EET. These findings suggest that certain vasoactive effects of EETs may be due to their incorporation by smooth muscle cells. Furthermore, through conversion to DHET and other oxidized metabolites, smooth muscle apparently has the capacity to inactivate EETs that are either formed in or penetrate into the vascular wall.

8,11,14-Eicosatrienoic Acid↗
PubMed1995

Docosahexaenoic acid synthesis in human skin fibroblasts involves peroxisomal retroconversion of tetracosahexaenoic acid.

The purpose of this study was to determine whether the formation of docosahexaenoic acid in human cells occurs through a pathway that involves 24-carbon n-3 fatty acid intermediates and retroconversion. Normal human skin fibroblasts synthesized radiolabeled docosahexaenoic acid from [1-(14)C]18:3n-3, [3-(14)C]22:5n-3, [3-(14)C]24:5n-3, and [3-(14)C]24:6n-3. The amount of docosahexaenoate formed was reduced in fibroblasts defective in peroxisomal biogenesis, by 90-100% in Zellweger's syndrome and by 50-75% in infantile Refsum's disease. Fatty acid elongation and desaturation were intact in these mutant cells. No decrease in radiolabeled docosahexaenoic acid production occurred in mutant fibroblasts defective in peroxisomal alpha-oxidation or mitochondrial beta-oxidation, or in normal fibroblasts treated with methyl palmoxirate to inhibit mitochondrial beta-oxidation. Therefore, the retroconversion step in docosahexaenoic acid formation occurs through peroxisomal beta-oxidation in normal human cells. These results demonstrate that the pathway for docosahexaenoic acid synthesis in human cells involves 24-carbon intermediates. The limited ability to synthesize docosahexaenoic acid may underlie some of the pathology that occurs in genetic diseases involving peroxisomal beta-oxidation.

Cells, Cultured↗
PubMed1994

Formation and release of a peroxisome-dependent arachidonic acid metabolite by human skin fibroblasts.

Human skin fibroblasts labeled with [5,6,8,9,11,12,-14,15-3H]arachidonic acid produce a radioactive metabolite that has a shorter retention time on reverse-phase high-performance liquid chromatography than arachidonic acid. This product is not retained in the cells; it is released entirely into the extracellular fluid in a time-dependent manner. The metabolite does not cochromatograph with any of the eicosanoid standards, and its formation is not prevented by the addition of cyclooxygenase, lipoxygenase, or cytochrome P-450 inhibitors. The compound is not produced by fibroblasts labeled with [1-14C]arachidonic acid, suggesting that it is formed through an oxidative process. Chemical analyses indicated that the metabolite is 4,7,10-hexadecatrienoic acid (16:3). Peroxisome-deficient human skin fibroblasts did not produce 16:3, indicating that it probably is formed through peroxisomal beta-oxidation. Human umbilical vein endothelial cells and porcine pulmonary artery smooth muscle cells also release radioactive 16:3 following labeling with [3H]arachidonic acid. Therefore, the production of this metabolite is not limited only to fibroblasts. The fact that 16:3 is released into the extra-cellular fluid suggests that it may be a new type of lipid mediator derived from arachidonic acid, formed through a peroxisome-dependent oxidative process.

Animals↗
PubMed1994

15-HETE: selective incorporation into inositol phospholipids of MDCK cells.

The interaction of 15-hydroxyeicosatetraenoic acid (15-HETE) and cultured MDCK renal tubular epithelial cells was investigated to determine whether incorporation of this lipoxygenase product will affect polyphosphoinositide formation. MDCK cells were incubated with 1 microM [3H]-15-HETE for 15 to 120 minutes. Maximum uptake occurred between 15 and 30 minutes, and after 60 minutes, 70% of the incorporated 15-HETE was present in the phosphatidylinositol (PI) fraction. Some 15-HETE was also incorporated into phosphatidylinositol-4-monophosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2). However, even though more 15-HETE than arachidonic acid was incorporated into PI, the fractional amount of 15-HETE present in the polyphosphoinositides was smaller than arachidonic acid. Therefore, although 15-HETE is selectively channeled into PI, conversion of PI species containing 15-HETE to PIP and PIP2 is relatively impaired. This suggests that either PI containing 15-HETE is a less effective substrate for phosphorylation, or PI containing arachidonic acid is a preferred substrate. MDCK cells converted 15-HETE to polar metabolites that were released into the extracellular fluid. This process may constitute a renal tubular mechanism for the clearance of 15-HETE and related lipoxygenase products.

Animals↗
PubMed1994

13-HODE increases intracellular calcium in vascular smooth muscle cells.

13-Hydroxyoctadecadienoic acid (HODE) (2 microM) consistently increased porcine aortic and pulmonary artery smooth muscle cell calcium concentrations ([Ca2+]i), whereas 9-HODE and linoleic acid had no significant effect in the aortic cells and a much lesser effect in the pulmonary artery cells. A transient increase in [Ca2+]i occurred with as little as 50 nM 13-HODE. Structural specificity for elevation of [Ca2+]i also was seen with the monohydroxyeicosatetraenoic acids (HETEs), with 12-HETE but not 5- or 15-HETE increasing [Ca2+]i. 13-HODE, but not 9-HODE, increased smooth muscle cell guanosine 3',5'-cyclic monophosphate (cGMP) levels. The [Ca2+]i increase produced by 13-HODE was dependent on extracellular calcium and was inhibited by the calcium channel blockers verapamil and nifedipine and by KT-5823, a cGMP-dependent kinase inhibitor. A similar increase in [Ca2+]i was produced by 8-bromo-cGMP. These results suggest that 13-HODE, a 15-lipoxygenase product formed from linoleic acid, can act as a lipid mediator in vascular smooth muscle. It raises smooth muscle cGMP, causing a secondary increase in [Ca2+]i due to Ca2+ influx through a cGMP kinase-dependent L-type channel.

Animals↗
PubMed1994

Cell fatty acid composition affects free radical formation during lipid peroxidation.

Lipid-derived free radicals generated from intact human U937 monocytes exposed to iron-induced oxidative stress were detected by electron paramagnetic resonance (EPR) with the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). Lipid radical formation was enhanced when the cells were enriched with n-3 or n-6 polyunsaturated fatty acids. Computer simulation indicated that at least two POBN spin adducts were formed, having spectral characteristics consistent with carbon-centered radicals (aN = 15.9 G and aH = 2.6 G; aN = 15.1 G and aH = 2.8 G). These alkyl radicals are probably formed by beta-scission of alkoxyl radicals. POBN spin adduct formation correlated with ethane generation. Addition of ascorbate to the assay medium greatly increased the radical signal intensity. Although radical generation was cell dependent and POBN spin adducts were observed in cell homogenates, the adducts formed by the intact cells were detected only in the extracellular medium. These findings indicate that the extent of lipid radical formation in response to oxidative stress can be influenced by changes in the polyunsaturated fatty acid composition of the cell lipids and suggest the possibility that carbon-centered lipi radicals may interact with extracellular structures.

Ascorbic Acid↗
PubMed1994

Regulation of CTP:choline-phosphate cytidylyltransferase by polyunsaturated n-3 fatty acids.

Disaturated phosphatidylcholine (DSPC) is the most distinctive surface-active lipid in pulmonary surfactant. The feeding of docosahexanoic acid (DHA) 22:6 n-3 has recently been described to elevate the levels of DSPC in rodent lung. The purpose of the present study was to determine the mechanisms by which this n-3 fatty acid might regulate CTP:choline-phosphate cytidylyltransferase, a key enzyme required for phosphatidylcholine (PC) synthesis. Cytidylyltransferase exists in lung cytosol as a large lipid-associated aggregate (H form) which is active, and as an inactive, low-molecular-weight species (L form). Fatty acids in vitro stimulate and aggregate the inactive L form to the active H form. Short-term (2-h) and long-term (24-h) exposure of fetal lung explants to DHA (150 microM) stimulated choline incorporation into PC by 54 and 64%, respectively. The fatty acid also enhanced DSPC synthesis by 88%. These changes were associated with an increase in the activity of cytidylyltransferase by 63% after addition of DHA to the explant medium. In vitro, DHA (50 microM) stimulated L form nearly 15-fold and appeared to be a more potent activator and aggregator of the enzyme than either linoleic 18:2 n-6 or arachidonic 20:4 n-6 acids. The effect of DHA on L-form activation was comparable, however, with other members of the n-3 family. Kinetic studies revealed that DHA increased the maximum velocity of enzyme reaction for cytidylyltransferase, although it did not alter the Michaelis constant of the enzyme for CTP. These observations provide in vitro evidence that n-3 fatty acids may play an important role in the regulation of surfactant PC biosynthesis.

Animals↗
PubMed1994

Polyunsaturated fatty acids increase lipid radical formation induced by oxidant stress in endothelial cells.

Lipid-derived free radicals were detected by electron paramagnetic resonance (EPR) spectrometry when cultured endothelial cells attached to Cytodex beads were exposed to iron-induced oxidant stress in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). Radical adduct formation was enhanced greatly when the cells were supplemented during growth with polyunsaturated fatty acids. The largest EPR signal intensity was observed in cells enriched with docosahexaenoic acid (DHA) or eicosapentaenoic acid, but enhanced radical adduct production also occurred after exposure to arachidonic, alpha-linolenic, gamma-linolenic, or linoleic acids. Radical adduct formation increased as the DHA content of the cells increased and approached a maximum after only 6 h of exposure to DHA. Ascorbic acid, acting as a pro-oxidant, enhanced radical adduct formation in cells enriched with DHA. The EPR signal intensity was reduced when the cells were tested 6 h after replacement of the DHA-enriched medium with a medium containing 5-20 microM oleic acid, indicating that the increased endothelial responsiveness to oxidant stress is reversible. Likewise, when U937 monocytes enriched with DHA were exposed subsequently to 20 microM oleic acid, a 35-45% decrease in radical adduct formation also occurred. These findings suggest that the endothelium may become more susceptible to oxidative injury when it is exposed to elevated amounts of polyunsaturated fatty acids. However, the effect appears to be temporary. The protective action of oleic acid against oxidant stress is not confined to the endothelium; it applies to monocytes as well.

Animals↗
PubMed1994

Mitochondrial metabolism of 12- and 15-hydroxyeicosatetraenoic acids.

We have previously demonstrated that peroxisomal-deficient human skin fibroblasts and mutant Chinese hamster ovary cells do not convert 12- and 15-hydroxyeicosatetraenoic acids (HETEs) to chain-shortened, polar metabolites, suggesting that peroxisomes are the intracellular location for beta-oxidation of these compounds. This implies that mitochondria do not beta-oxidize HETEs. To test this hypothesis we incubated highly purified rat liver mitochondria with [3H]12-(S)- and [3H]15-(S)-HETE in the presence of carnitine and an acylcoenzyme A-generating system. Extracts obtained from these incubations were analyzed for radiolabeled polar metabolites. Both HETEs were converted to apparent products of beta-oxidation, although the 12-HETE compound was a markedly better substrate. The presence of 50 microM 2-tetradecyloxirane carboxylate, a potent inhibitor of carnitine palmitoyl transferase, completely blocked 12- and 15-HETE conversion to these metabolites as did omission of carnitine from the medium. These data demonstrate carnitine-dependent beta-oxidation of HETEs in isolated mitochondria and suggest that mitochondria are competent to carry out this metabolic process in eukaryotic cells. Prevailing metabolic conditions in subcellular compartments may have precluded observation of mitochondrial activity in our earlier work with cultured cells. Alternatively, transport mechanisms may exist in the cell types studied that distribute 12-(S)- and 15-(S)-HETEs specifically to peroxisomes.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1993

Lysophosphatidylcholine causes cGMP-dependent verapamil-sensitive Ca2+ influx in vascular smooth muscle cells.

Lysophosphatidylcholine (lyso-PC) is a vasoactive phospholipid present in oxidized low-density lipoprotein. We used a coculture model of the vascular wall to study its interaction with endothelial cells (EC) and vascular smooth muscle cells (SMC). Lyso-PC was taken up readily by SMC and gradually acylated to phosphatidylcholine. Low concentrations (< or = 1 microM) of lyso-PC present in the interstitial medium of an EC-SMC coculture system were taken up primarily by the SMC. Lyso-PC produced a rapid two- to three-fold increase in SMC guanosine 3',5'-cyclic monophosphate (cGMP) levels, reaching a maximum in 1 min. This increase was associated with decreased SMC proliferation and increased calcium influx. The increase in intracellular calcium was inhibited by verapamil and KT5823, a specific cGMP-dependent kinase inhibitor, while a similar increase was produced by the membrane-permeant cGMP analogue 8-bromoguanosine 3',5'-cyclic monophosphate. These studies suggest that SMC are the primary target for the biological effects of lyso-PC present in the vessel wall and that the responses are mediated by calcium influx, possibly due to opening of a verapamil-sensitive cGMP kinase-dependent channel.

Alkaloids↗
PubMed1993

Effect of glutathione on endothelial prostacyclin synthesis after anoxia.

We previously observed decreased prostacyclin (PGI2) formation after reoxygenation of anoxic endothelium. In the present study, the effects of glutathione on endothelial prostaglandin (PG) H synthase activity after reoxygenation were explored. Intracellular glutathione content decreased 70% after 24 h of anoxia; reoxygenation did not produce any additional decrease in glutathione content. Intracellular glutathione was maintained in the reduced state by the endothelium even during the oxidant stress caused by reoxygenation or the addition of peroxide. Glutathione depletion produced by DL-buthionine-(S,R)-sulfoximine (BSO), 1,3-bis(chloroethyl)1-nitrosourea (BCNU), or incubation in a sulfhydryl-free medium resulted in increased sensitivity of PGH synthase to the effects of added H2O2. However, glutathione depletion resulting from BSO or culture in sulfhydryl-free medium during anoxia did not increase the sensitivity of PGH synthase to reoxygenation. In addition, anoxia did not make the endothelium more sensitive to H2O2. Glutathione peroxidase and glutathione reductase activities were preserved after anoxia-reoxygenation. When glutathione reductase was inhibited with BCNU during reoxygenation, PGI2 release was decreased further. These findings demonstrate that, although anoxia decreases endothelial glutathione content, the endothelium is able to utilize its remaining glutathione to protect against additional oxidant stress because glutathione peroxidase and glutathione reductase retain their activity.

Arachidonic Acid↗
PubMed1993

Attenuated prostaglandin formation in peroxisomal-deficient human skin fibroblasts.

Peroxisomal-deficient skin fibroblasts from patients with Zellweger's syndrome or infantile Refsum's disease produced fewer prostaglandins than normal skin fibroblasts. Radioimmunoassay indicated a 45-55% decrease in prostaglandin E2 (PGE2) production when Zellweger's fibroblasts were incubated with arachidonic acid. This deficiency was not overcome by pretreatment of the Zellweger's fibroblasts with media containing arachidonic acid, and it was not due to channeling of arachidonic acid into other eicosanoid products. Modifications in the peroxide tone of the Zellweger's fibroblasts by addition of H2O2 or catalase failed to increase PGE2 production. Using Northern analysis, we were unable to detect an mRNA transcript for PGH synthase in unstimulated Zellweger fibroblasts but identified a 4.2-kb mRNA transcript after treatment with phorbol myristate acetate (PMA). Treatment for 6 h with 10 nM PMA raised PGE2 production in normal and Zellweger fibroblasts to equivalent levels. These increases were prevented by addition of H-7, staurosporine, cycloheximide, or actinomycin D. Our findings suggest that the reduced PGE2 production in peroxisomal deficient fibroblasts is due to a decrease in PGH synthase mRNA. The reduction in PGH synthase can be overcome by treatment of the cells with agents which enhance gene expression.

Arachidonic Acid↗
PubMed1993

14,15-Epoxyeicosatrienoic acid metabolism in endothelial cells.

Epoxyeicosatrienoic acid (EET) metabolism was studied in endothelial cells to determine whether this tissue may influence their vasoactive properties. Porcine aortic endothelial cells rapidly took up all four EET regioisomers. The uptake of [1-14C]14,15-EET reached a maximum in 15-30 min, and saturation was not observed with concentrations up to 5 microM. More than 70% of the incorporated 14,15-EET was contained in choline and inositol glycerophospholipids, most of it in the form of an EET ester. A metabolite, 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), accumulated in the medium during incubation, and products with similar chromatographic properties also were formed from 5,6-, 8,9-, and 11,12-EET. Much of the 14,15-EET taken up was only temporarily retained by the cells, and in 2 h half was released into the medium as 14,15-DHET. Bovine aortic and human umbilical vein endothelial cells also took up 14,15-EET, incorporated it into choline glycerophospholipids, and converted it to 14,15-DHET. These findings suggest that the endothelium may limit the vascular actions of EETs through rapid uptake, hydration, and release of DHETs into the circulation. Some vasoactive effects of EETs may result from their temporary accumulation in endothelial phospholipids involved in stimulus-response coupling.

8,11,14-Eicosatrienoic Acid↗