Acylation Of Arachidonic Acid Research Articles

Valnoctamide, a non‐teratogenic amide derivative of valproic acid, inhibits arachidonic acid activationin vitroby recombinant acyl‐CoA synthetase‐4

Valproic acid (VPA), a mood stabilizer used for treating bipolar disorder (BD), uncompetitively inhibits acylation of arachidonic acid (AA) by recombinant AA-selective acyl-CoA synthetase 4 (Acsl4) at an enzyme inhibition constant (Ki ) of 25 mM. Inhibition may account for VPA's ability to reduce AA turnover in brain phospholipids of unanesthetized rats and to be therapeutic in BD. However, VPA is teratogenic. We tested whether valnoctamide (VCD), a non-teratogenic amide derivative of a VPA chiral isomer, which had antimanic potency in a phase III BD trial, also inhibits recombinant Acsl4. Rat Acsl4-flag protein was expressed in Escherichia coli. We used Michaelis-Menten kinetics to characterize and quantify the ability of VCD to inhibit conversion of AA to AA-CoA by recombinant Acsl4 in vitro. Acsl4-mediated activation of AA to AA-CoA by Acsl4 was inhibited uncompetitively by VCD, with a Ki of 6.38 mM. VCD's ability to uncompetitively inhibit AA activation to AA-CoA by Acsl4, at a lower Ki than VPA, suggests that, like VPA, VCD may reduce AA turnover in rat brain phospholipids. If so, VCD and other non-teratogenic Acsl4 inhibitors might be considered further for treating BD.

Propylisopropylacetic acid (PIA), a constitutional isomer of valproic acid, uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: A potential drug for bipolar disorder

BackgroundMood stabilizers used for treating bipolar disorder (BD) selectively downregulate arachidonic acid (AA) turnover (deacylation–reacylation) in brain phospholipids, when given chronically to rats. In vitro studies suggest that one of these, valproic acid (VPA), which is teratogenic, reduces AA turnover by inhibiting the brain long-chain acyl-CoA synthetase (Acsl)4 mediated acylation of AA to AA-CoA. We tested whether non-teratogenic VPA analogues might also inhibit Acsl4 catalyzed acylation, and thus have a potential anti-BD action. MethodsRat Acsl4-flag protein was expressed in Escherichia coli, and the ability of three VPA analogues, propylisopropylacetic acid (PIA), propylisopropylacetamide (PID) and N-methyl-2,2,3,3-tetramethylcyclopropanecarboxamide (MTMCD), and of sodium butyrate, to inhibit conversion of AA to AA-CoA by Acsl4 was quantified using Michaelis–Menten kinetics. ResultsAcsl4-mediated conversion of AA to AA-CoA in vitro was inhibited uncompetitively by PIA, with a Ki of 11.4mM compared to a published Ki of 25mM for VPA, while PID, MTMCD and sodium butyrate had no inhibitory effect. ConclusionsPIA's ability to inhibit conversion of AA to AA-CoA by Acsl4 in vitro suggests that, like VPA, PIA may reduce AA turnover in brain phospholipids in unanesthetized rats, and if so, may be effective as a non-teratogenic mood stabilizer in BD patients.

Propylisopropylacetic acid (PIA), a constitutional isomer of valproic acid, uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: A potential drug for bipolar disorder

Propylisopropylacetic acid (PIA), a constitutional isomer of valproic acid, uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: A potential drug for bipolar disorder

Cyclosporin Treatment Alters Prostanoid and Thromboxane Production by Rat Isolated Kidney Mitochondria

This study was designed to investigate the effects of chronic treatment with cyclosporin A (CSA) on the endogenous synthesis of prostanoids (PGs) and thromboxane (Tx) by renal isolated medullary and cortical mitochondria. The administration of CSA, dissolved in 10% ethanol in olive oil, to male Wistar rats (20 mg kg-1 day-1 i.p.) for 14 days resulted in alterations in mitochondrial biosynthesis of immunoreactive PGs. The endogenous synthesis of thromboxane by medullary and cortical mitochondria isolated from CSA-treated rats was significantly enhanced by 120 and 55%, respectively, whereas the synthesis of prostaglandin E2 by medullary mitochondria was reduced by 35%. The synthesis of prostaglandin F2 alpha and prostacyclin was not affected by CSA treatment. The conversion of exogenous arachidonic acid to PGs and Tx by cortical mitochondria isolated from CSA-treated rats was significantly increased. In addition, CSA treatment resulted in i) a reduced acylation of arachidonic acid into medullary phospholipids by 25% and into medullary and cortical triglycerides by 33 and 27%, respectively, and ii) an increase in cortical and medullary triglycerides. We suggest that the alterations in the endogenous mitochondrial production of PGs and Tx caused by CSA, may play a role in the impairment of membrane mediated functions.

Valproate uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: Relevance to valproate's efficacy against bipolar disorder

Valproate uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: Relevance to valproate's efficacy against bipolar disorder

Valproate uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: Relevance to valproate's efficacy against bipolar disorder

Background: The ability of chronic valproate (VPA) to reduce arachidonic acid (AA) turnover in brain phospholipids of unanesthetized rats has been ascribed to its inhibition of acyl-CoA synthetase (Acsl)-mediated activation of AA to AA-CoA. Our aim was to identify a rat Acsl isoenzyme that could be inhibited by VPA in vitro. Methods: Rat Acsl3-, Acsl6v1- and Acsl6v2-, and Acsl4-flag proteins were expressed in E. coli, and the ability of VPA to inhibit their activation of long-chain fatty acids to acyl-CoA was estimated using Michaelis–Menten kinetics. Results: VPA uncompetitively inhibited Acsl4-mediated conversion of AA and of docosahexaenoic (DHA) but not of palmitic acid to acyl-CoA, but did not affect AA conversion by Acsl3, Acsl6v1 or Acsl6v2. Acsl4-mediated conversion of AA to AA-CoA showed substrate inhibition and had a 10-times higher catalytic efficiency than did conversion of DHA to DHA-CoA. Butyrate, octanoate, or lithium did not inhibit AA activation by Acsl4. Conclusions: VPA's ability to inhibit Acsl4 activation of AA and of DHA to their respective acyl-CoAs, when related to the higher catalytic efficiency of AA than DHA conversion, may account for VPA's selective reduction of AA turnover in rat brain phospholipids, and contribute to VPA's efficacy against bipolar disorder.

Perturbations in the control of cellular arachidonic acid levels block cell growth and induce apoptosis in HL-60 cells.

Our previous studies demonstrated that inhibitors of arachidonate-phospholipid remodeling [i.e. the enzyme CoA-independent transacylase (CoA-IT)] decrease cell proliferation and induce apoptosis in neoplastic cells. The goal of the current study was to elucidate the molecular events associated with arachidonate-phospholipid remodeling that influence cell proliferation and survival. Initial experiments revealed the essential nature of cellular arachidonate to the signaling process by demonstrating that HL-60 cells depleted of arachidonate were more resistant to apoptosis induced by CoA-IT inhibition. In cells treated with CoA-IT inhibitors a marked increase in free arachidonic acid and AA-containing triglycerides were measured. TG enrichment was likely due to acylation of arachidonic acid into diglycerides and triglycerides via de novo glycerolipid biosynthesis. To determine the potential of free fatty acids to affect cell proliferation, HL-60 cells were incubated with varying concentrations of free fatty acids; exogenously provided 20-carbon polyunsaturated fatty acids caused a dose-dependent inhibition of cell proliferation, whereas oleic acid was without effect. Blocking 5-lipoxygenase or cyclooxygenases had no effect on the inhibition of cell proliferation induced by arachidonic acid or CoA-IT inhibitors. An increase in cell-associated ceramides (mainly in the 16:0-ceramide fraction) was measured in cells exposed to free arachidonic acid or to CoA-IT inhibitors. This study, in conjunction with other recent studies, suggests that perturbations in the control of cellular arachidonic acid levels affect cell proliferation and survival.

Oxidant-induced arachidonic acid release and impairment of fatty acid acylation in vascular smooth muscle cells.

Oxidative damage, which plays a major role in the early stages of atherosclerosis, is associated with arachidonic acid (AA) release in vascular smooth muscle cells (VSMC) as in other cell types. In this study, H2O2 was used to investigate mechanisms of AA release from VSMC on oxidative stress. Cell treatment with H2O2 inhibited AA incorporation in an inverse relationship to prolonged H2O2-induced AA release. Identical kinetics of inhibition of AA incorporation and AA release were observed after cell treatment with AlF4-, a process not involving phospholipase A2 (PLA2) activation as recently described (A. Cane, M. Breton, G. Béréziat, and O. Colard. Biochem. Pharmacol. 53: 327-337, 1997). AA release was not specific, since oleic acid also increased in the extracellular medium of cells treated with H2O2 or AlF4- as measured by gas chromatography-mass spectrometry. In contrast, AA and oleic acid cell content decreased after cell treatment. Oleoyl and arachidonoyl acyl-CoA synthases and acyltransferases, assayed using a cell-free system, were not significantly modified. In contrast, a good correlation was observed between decreases in AA acylation and cell ATP content. The decrease in ATP content is only partially accounted for by mitochondrial damage as assayed by rhodamine 123 assay. We conclude that oxidant-induced arachidonate release results from impairment of fatty acid esterification and that ATP availability is probably responsible for free AA accumulation on oxidative stress by preventing its reesterification and/or transmembrane transport.

Altered phospholipid metabolism in sodium butyrate-induced differentiation of C6 glioma cells.

We examined the changes in phospholipid metabolisms in sodium butyrate-treated C6 glioma cells. Treatment of 2.5 mM sodium butyrate for 24 h induced an increase in the activity of glutamine synthetase, suggesting that these cells were under differentiation. Similar treatment was associated with (i) increased arachidonic acid incorporation into phosphatidylcholine, and (ii) decreased arachidonic acid incorporation into phosphatidylinositol and (iii) phosphatidylethanolamine. These effects were subsequently investigated by examining the acylation process, de novo biosynthesis, and the agonist-stimulated phosphoinositides hydrolysis in these cells. Our results indicated that sodium butyrate stimulated the acylation of arachidonic acid into lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylinositol. The glycerol incorporation into these lipids was not affected, but the inositol incorporation into total chloroform extracts and Pl and phosphatidylinositol 4-phosphate was decreased in the sodium butyrate-treated cells. Moreover, the accumulation of the rapid histamine-stimulated phosphoinositide metabolites, i.e., inositol monophosphate, inositol diphosphate, and inositol triphosphate (IP3) was decreased in these cells. To elucidate whether the decreased inositol phosphates were due to a decrease in the phosphoinositides hydrolysis, we measured the transient IP3 production directly by a receptor-binding assay. Our results indicated that histamine-stimulated transient IP3 formations were decreased. Taken together, these results indicated that multiple changes by multiple mechanisms of phospholipid metabolisms were found in sodium butyrate-treated C6 glioma cells. The decreased IP3 formation and its subsequent action, i.e., Ca2+ mobilization, may play an early but pivotal role by which sodium butyrate induces C6 glioma cell differentiation.

Regulation of cytosolic phospholipase A2 phosphorylation and eicosanoid production by colony-stimulating factor 1.

A colony-stimulating factor 1 (CSF-1)-dependent murine macrophage cell line (BAC1.2F5) and peritoneal macrophages were used to investigate the relationship between growth factor-dependent phosphorylation/activation of the 85-kDa cytosolic phospholipase A2 (cPLA2) and arachidonic acid metabolism. The addition of CSF-1 to quiescent BAC1.2F5 cells was followed by the rapid phosphorylation, electrophoretic gel retardation, and stable increase in the specific activity of cPLA2 that correlated with the activation of ERK kinases. cPLA2 phosphorylation depended on the presence of growth factor and persisted throughout the cell cycle. CSF-1 inhibited prostaglandin E2 production and did not enhance arachidonic acid release or increase the levels of lysophosphatidylcholine or glycerophosphocholine. Treatment of BAC1.2F5 cells with the calcium ionophore A23187 plus CSF-1 did not stimulate eicosanoid release. Instead, CSF-1 enhanced the rate of exogenous arachidonic acid incorporation into phosphatidylcholine and its subsequent transfer to phosphatidylethanolamine suggesting that higher rates of arachidonic acid acylation may contribute to the suppression of prostaglandin production. In peritoneal macrophages, ERK kinase activity was stimulated and cPLA2 was phosphorylated and activated in response to CSF-1. However, CSF-1 did not trigger eicosanoid release but did augment arachidonic acid mobilization and prostaglandin E2 production elicited by zymosan and A23187. Thus, cPLA2 phosphorylation/activation and calcium mobilization are not the only determinants for eicosanoid release, and additional components in differentiated tissue macrophages are also required.

Incorporation of arachidonic acid in a human cancer gastric tumor cell line (HGT) at various stages of cell proliferation

Human cancer gastric cells (HGT) in culture were labeled with [ 14C]arachidonic acid. The kinetic of acylation of arachidonic acid was similar using cells after 3, 5 and 7 days of culture. However, the duration of the proliferation enhanced the acyltransfer to triglycerides and inhibited the transfer to phospholipids. After 8 h of incubation with [ 14C]arachidonic acid the radioactivity was predominantly recovered in the phosphatidylcholine and phosphatidylethanolamine species. Prelabeling-chase experiments indicated a preferential incorporation of arachidonic acid into phosphatidylcholine followed by a transfer of arachidonate to phosphatidylethanolamine.

The Acyl-CoA Synthetase Inhibitor Triacsin C Enhanced Eicosanoid Release in Leukocytes

Triacsin C was previously reported to inhibit long-chain acyl-CoA synthetase and to reduce the production of acyl-CoA in rat neutrophils dose- and time-dependently. We found that preincubation with triacsin C inhibited the incorporation of labeled arachidonic acid into rat neutrophils. Furthermore, when triacsin C-treated neutrophils were stimulated with the Ca-ionophore A23187, they released an increased amount of eicosanoids into the culture medium. These results indicate that triacsin C suppressed acylation of arachidonic acid, which resulted in an increase in its metabolites.

The acyl-CoA synthetase inhibitor triacsin C enhanced eicosanoid release in leukocytes.

Triacsin C was previously reported to inhibit long-chain acyl-CoA synthetase and to reduce the production of acyl-CoA in rat neutrophils dose- and time-dependently. We found that preincubation with triacsin C inhibited the incorporation of labeled arachidonic acid into rat neutrophils. Furthermore, when triacsin C-treated neutrophils were stimulated with the Ca-ionophore A23187, they released an increased amount of eicosanoids into the culture medium. These results indicate that triacsin C suppressed acylation of arachidonic acid, which resulted in an increase in its metabolites.

The influence of mono- and divalent cations on the cardiac metabolism of arachidonic acid

Our previous study indicated that, in the isolated rabbit heart, perfusion with Ca 2+ free Krebs Henseleit buffer (KHB) results in increased conversion of exogenous arachidonic acid to PGE 2 and 6-keto-PGF 1α, probably as the result of increased availability of substrate to cuclooxygenase. Since perfusion with Ca 2+ free buffer is known to cause alterations in the cardiac content of various mono- and divalent cations, the present study was performed to determine: a) The relationship between the conversion of exogenous arachidonic acid to prostaglandins and cardiac content of Na +, K +, Ca 2+ and Mg 2+; and b) Whether enhanced arachidonic acid conversion to prostaglandins during Caa 2+ free perfusion is due to reduced incorporation of this fatty acid into tissue lipids. Perfusion of the rabbit heart with Ca 2+ free buffer produced a significant reduction in the tissue content of Na +, K +, Ca 2+ and Mg 2+. However, the production of 6-keto-PGF 1α from exogenous arachidonic acid was linearly correlated with tissue Mg 2+. These observations, together with our finding that perfusion with Ca 2+ free KHB reduced the incorporation of [ 3H] arachidonic acid into tissue lipids, suggests that Ca 2+ free perfusion may, by reducing the activity of arachidonyl CoA synthetase (a Mg 2+ dependent enzyme), decrease the acylation of arachidonic acid into lipids, thus increasing the availability of arachidonic acid to cyclooxygenase.

Enhanced synthesis of prostaglandins and hydroxyeicosatetraenoic acids in retina from a canine model of Batten's disease.

The metabolism of [1-14C]arachidonic acid (20:4, n-6) was studied in intact retina and retinal pigment epithelial cells from normal English setters and English setters affected with hereditary canine ceroid lipofuscinosis. Acylation of arachidonic acid into membrane glycerolipids and oxygenation by lipoxygenase and cyclooxygenase to eicosanoids were measured by radiochromatographic techniques. In addition, the histopathology of accumulated ceroid particles in retinal ganglion cells and pigment epithelial cells was studied by electron microscopy. Synthesis of prostaglandins and hydroxyeicosatetraenoic acids was increased in canine ceroid lipofuscinosis retina, but not in retinal pigment epithelium. Prostaglandin D2, the putative neuronal eicosanoid, was increased nearly eightfold, whereas other eicosanoids increased two- to threefold. Ultrastructural studies revealed accumulation of ceroid and deterioration of neuronal and pigment epithelial cell architecture. These experiments demonstrate that, although lipopigment accumulates in both tissues, alterations of eicosanoid synthesis are specific for the retina, a neuronal tissue. The specific increase in prostaglandin D2 and the specificity of changes for the retina indicate that enhanced eicosanoid synthesis may be a result of an impairment of the control of oxygenation of arachidonic acid in neurons.

Prostaglandin production by methylcholanthrene-transformed mouse BALB/3T3. Requirement for protein synthesis.

Stimulation of prostaglandin synthesis in transformed mouse fibroblasts by serum, thrombin, and bradykinin was blocked by actinomycin D and cycloheximide. These RNA and protein synthesis inhibitors did not affect prostaglandin synthetase in vitro or in vivo; nor did they affect the acylation of arachidonic acid into phospholipids. Serum-stimulated release of arachidonic acid and prostaglandins from [3H]arachidonic acid-labeled cells also was inhibited by actinomycin D and cycloheximide. RNA and protein synthesis appear to be required for expression of phospholipase activity; a prerequisite for prostaglandin synthesis by these cells.