Eicosapentaenoic acid metabolism in human and rabbit anterior uvea

Eicosapentaenoic acid (EPA) metabolism into 3 series cyclooxygenase and 5 series lipoxygenase products was assessed in human and rabbit anterior uvea. Both tissues synthesized 3 series cyclooxygenase products such as delta17 6-keto-PGF1 (PGI3 metabolite), PGE3 alpha, PGE3, PGD3 and TxB3 (a stable product of TxA3) and lipoxygenase products 12-hydroxyeicosapentaenoic acid (HEPE), 5-HEPE and 5,12-diHEPE from 14C-EPA. EPA-derived cyclooxygenase product synthesis was considerably greater than the formation of lipoxygenase products from EPA in both tissues.     

The effect of dietary eicosapentaenoic acid on arachidonic acid incorporation and metabolism in rat leukocytes

Arachidonic Acid (AA) released from membrane phospholipids by phospholipase A₂ during cell activation is the major polyunsaturated fatty acid precursor in mammals for the cyclooxygenase and lipoxygenase pathways. Eicosaspentaenoic acid (EPA), a major polyunsaturated fatty acid in fish oils competes with AA for these enzymes. The resulting products from EPa are generally less potent than the corresponding AA metabolites which may explain the beneficial effects of this oil in reducing thrombotic and inflammatory responses. This study compares the incorporation of ¹⁴C-AA into leukocyte phospholipids and its release and metabolism by the cyclooxygenase and lipoxygenase pathways in rats fed a ‘Max EPA’ fish oil rich diet (EPA group) and a hydrogenated coconut/safflower oil control diet. More than 75% of radiolabel was incorporated into leukocytes with no difference seen between dietary groups. Upon stimulation with calcium ionophore, the EPA group released significantly more radiolabelled AA than the control group. The EPA diet showed a significant increase in the formation of 5-hydroxyeicosatetraenoic acid and 6-keto-prostaglandin F_1α but no difference was seen in leukotriene B₄ formation. The majority of radiolabel released was free AA, this being significantly higher in the EPA grou than in the control. The percentage of radiolabel remaining after stimulation in phosphatidylglycerol, phosphatidylethanolamine and neutral lipids was significantly less in EPA fed rats. As the release and metabolism of endogenous AA may not be the same as ¹⁴C-AA, these results do not necessarily indicate that the mass of AA available for eicosanoid biosynthesis has been altered by the EPA diet.     

Effects of organic and inorganic Ca antagonists on rat platelet arachidonic acid metabolism in the presence of Ca2+, Sr2+ and Ba2+

The effects of Ca-antagonists on the thrombin-induced mobilization of radiolabeled arachidonate preincorporated into rat platelets as well as the subsequent formation of labeled cyclooxygenase and lipoxygenase products were analyzed in the presence of either Call or Ca²⁺-substitutes, Sr²⁺ and Ba²⁺. Results indicate that following thrombin stimulation (0.2 U/ml) in the presence of Ca²⁺, nitrendipine (Nit), Cd²⁺ or Mn²⁺ reduced the release of arachidonate and the biosynthesis of thromboxane 132- Inhibition of arachidonic acid release and metabolism were also obtained by both Nit and Cd2+ in the presence of Sr2+ and Bat+. Results from studies with a semi-purified phospholipase A₂ fraction prepared from rat platelets indicated that the activity was almost unaffected by Nit or Cd²⁺. From these findings, we concluded that inhibition of platelet-induced release and metabolism of arachidonic acid by the Ca-antagonists tested require intact platelets. These data support the hypothesis of an interaction of these agents at an unknown surface membrane level.Abbreviations AA Arachidonic Acid - 5-HT 5-hydroxy-tryptamine - HETE 12-hydroxyeicosatetraenoic acid - HHT 12-hydroxy-heptadecanoic acid - HHT Nitrendipine - TXB₂ Thromboxane B₂     

Manipulation of platelet aggregation by prostaglandins and their fatty acid precursors: Pharmacological basis for a therapeutic approach

Addition of the one-, two- or three- series endoperoxide to human platelet-rich plasma tend to supress aggregation, through the action of their respective non-enzymatic breakdown products PGE₁, PGD₂, or PGD₃ all of which elevate cyclic AMP levels. On the other hand, these stable primary products do not arise in appreciable amounts from intrinsic endoperoxides generated from either endogenous or exogenous free fatty acids. 5,8,11,14,17-Eicosapentaenoic acid (EPA) suppresses arachidonic acid (5,8,11,14-eicosatetraenoic acid) conversion by cycloogygenase (as well as lipoxygenase) to aggregatory metabolites in platelets. Exogenously added EPA was capable of inhibiting PRP aggregation induced either by exogenous or endogenous (released by ADP or collagen) arachidonate. The hypothetical combination of an EPA-rich diet and a thromboxane synthetase inhibitor might abolish production of the pro-aggregatory species, thromboxane A₂, and enhance formation of the anti-aggregatory metabolite, prostacyclin.Whereas EPA is not detectably metabolized by platelets, dihomo-γ-linolenic acid (8,11,14,-eicosatrienoic acid) is primariley converted by cyclooxygenase and thromboxane synthetase into the inactive metabolite, 12-hydroxyheptadecadienoic (HHD) acid. Pretreatment of human platelet suspensions with the thromboxane synthetase inhibitor imidazole unmasks the aggregatory property of PGH₁ and DLL which was partially compromised by the PGE₁ formed. The combination of the thromboxane synthetase inhibitor and an adenylate cyclase inhibitor unmasks a complete irreversible aggregation by DLL or PGH₁. The basis of a dietary strategy that replaces AA with DLL must rely on the production by the platelet of an inactive metabolite (HHD) rather than thromboxane A₂.     

Dual inhibitory effect of Glycyrrhiza glabra (GutGard™) on COX and LOX products

Glycyrrhiza glabra and its phytoconstituents have been known to possess widespread pharmacological properties as an anti-inflammatory, anti-viral, antitumour and hepatoprotective drug. In this study, we examined the inhibitory potential of extract of G. glabra (GutGard™) root and its phytoconstituents (glabridin, glycyrrhizin, and isoliquiritigenin) on both cyclooxygenase (COX) and lipoxygenase (LOX) products in order to understand the mechanism of its anti-inflammatory action. Inhibitory effect of GutGard™ and its phytoconstituents on lipopolysaccharide (LPS) induced prostaglandin E₂ (PGE₂), calcimycin (A23187) induced thromboxane (TXB₂), and leukotriene (LTB₄) release was studied using murine macrophages (J774A.1) and human neutrophil (HL-60) cells. Results revealed that, G. glabra and glabridin significantly inhibited PGE₂, TXB₂ (COX) and LTB₄ (LOX), while, isoliquiritigenin exerted inhibitory effect only against COX products but failed to suppress LOX product. However, glycyrrhizin at the tested concentrations failed to exhibit inhibitory effect on both COX and LOX products. Here, we report for the first time that G. glabra (almost devoid of glycyrrhizin) exhibits anti-inflammatory property likely through the inhibition of PGE₂, TXB₂ and LTB₄ in mammalian cell assay system, which could be influenced in part by glabridin and isoliquiritigenin.     

Modulation of the beta-adrenergic response in cultured rat heart cells

Incubation of rocker-cultured neonatal rat heart cells with 3 mM L(+)-lactate led to a sharp increase in the sensitivity of cardiomyocytes to the beta-adrenergic agonist isoprenaline, as measured by their chronotropic response. This effect was accompanied by a reduction in the arachidonic acid content of the total phospholipids. The phospholipase A₂-activator melittin as well as free arachidonic acid induced this supersensitivity to the same degree. On the other hand, the L(+)-lactate-evoked supersensitivity could be blocked by the phospholipase A₂ inhibitors mepacrine and n-bromophenacyl-bromide, suggesting an involvement of phospholipase A₂ in the process of beta-adrenergic sensitization. The sensitizing action of arachidonic acid was blocked by the lipoxygenase inhibitors esculetin and nordihydroguaiaretic acid, but not by the cyclooxygenase inhibitor indomethacin. Supersensitivity was likewise evoked by 15-S-hydroxyeicosatetraenoic acid (15-S-HETE), but not by 5-S-HPETE or 5-S-HETE. These findings suggest that the phospholipase A₂-15-lipoxygenase pathway plays a role in the induction of beta-adrenergic supersensitivity in the cultured cardiomyocytes and point to a new physiological role of the lipoxygenase product 15-S-HETE.Abbreviations NDGA nordihydroguaiaretic acid - HETE hydroeicosatetraenoic acid - HPETE hydroperoxyeicosatetraenoic acid     

Lipid and fatty acid yield of nine stationary-phase microalgae: Applications and unusual C<Subscript>24</Subscript>–C<Subscript>28</Subscript> polyunsaturated fatty acids

Nine microalgal species from the classes Bacillariophyceae, Cryptophyceae, Prymnesiophyceae and Dinophyceae were isolated from Australian waters, cultured to stationary phase and analyzed for their lipid and fatty acid composition and yield. Five species (Pavlova pinguis, Heterocapsa niei, Proteomonas sulcata, Navicula jeffreyi and Thalassiosira pseudonana) produced high proportions of triacylglycerol (TAG: 22–57% total lipid). An unidentified Navicula-like diatom (CS-786), despite having a low TAG content, had the highest EPA yield (5.8 mg L⁻¹), due to high biomass and a high relative proportion of EPA. Heterocapsa niei had the highest DHA yield (2.9 mg L⁻¹), due to a high cellular lipid and DHA content (171 pg cell⁻¹ and 13.7 pg cell⁻¹, respectively) despite its relatively low biomass. The desirable PUFA composition and yield of both diatom CS-786 and H. niei make them potential candidates for optimization of biomass and PUFA production for use as live-feeds in aquaculture. In addition, H. niei may have potential as a source of DHA for other uses. Low proportions (< 1.2%) of 24:6(n−3) accompanied by trace proportions of 24:5(n−6) were detected in most strains, while 28:8(n−3) was found in dinoflagellates and also in the prymnesiophyte P. pinguis. All non-diatomaceous species contained 26:7(n−3) in minor quantities. This is the first time these unusual C₂₄ and C₂₆ PUFA have been reported in microalgae and the first report of C₂₈ PUFA in a microalga other than dinoflagellates. Possible biosynthetic reasons why these might occur in stationary phase cultures are considered and the likely dietary transfer of these PUFA to higher aquatic life is discussed.     

Specific enhancement of LTD4-induced contractions of isolated guinea pig trachea by 5-hydroxyeicosatetraenoic acid (5-HETE)

In view of the likely production of monohydroxyeicosatetraenoic acid (HETE's) in bronchial asthma, the role of these lipoxygenase products in the development of a classical clinical element of airway disease, namely airway hyperreactivity, has been investigated. Tracheas removed from guinea-pigs actively sensitized to ovalbumin produced, upon antigenic challenge (0.01 μg/ml), a 17-fold increase (0.97 ± 0.34 ng/ml to 16.73 ± 1.58 ng/ml) in the amount of 5-hydroxyeicosatetraenoic acid (5-HETE) as measured by radioimmunoassay of the tissue-bath fluid, indicating that this tissue is capable of producing 5-HETE. While 5-HETE alone, at concentrations equal to or greater than those found during the above antigenic response (0.001 to 1.0 μM), failed to produce intrinsic contractions of normal, nonsensitized guinea-pig trachea, a 30 min pretreatment with 5-HETE (1.0 μM) enhanced subsequent LTD₄-induced contractions. Pretreatment with either 12- or 15-HETE, at similar concentrations and conditions, failed to potentiate LTD₄ concentration-response curves. The effect of 5-HETE was time-dependent, since pretreatment for either 15 or 60 min had little or no effect on subsequent LTD₄ responses. Also, the 5-HETE-induced enhancement seemed specific fot LTD₄, since contractions to LTC₄ (in the presence of l-serine borate), acetylcholine, histamine, PGD₂ or U-46619 were unaffected by 5-HETE. Therefore, 5-HETE may have a role in the development of airway hyperreactivity by interacting with released LTD₄ to exacerbate airway smooth muscle contraction in asthma.     

Disturbed brain phospholipid and docosahexaenoic acid metabolism in calcium-independent phospholipase A2-VIA (iPLA2β)-knockout mice

Calcium-independent phospholipase A₂ group VIA (iPLA₂β) releases docosahexaenoic acid (DHA) from phospholipids in vitro. Mutations in the iPLA₂β gene, PLA2G6, are associated with dystonia-parkinsonism and infantile neuroaxonal dystrophy. To understand the role of iPLA₂β in brain, we applied our in vivo kinetic method using radiolabeled DHA in 4 to 5-month-old wild type (iPLA₂β^+/+) and knockout (iPLA₂β^−/−) mice, and measured brain DHA kinetics, lipid concentrations, and expression of PLA₂, cyclooxygenase (COX), and lipoxygenase (LOX) enzymes. Compared to iPLA₂β^+/+ mice, iPLA₂β^−/− mice showed decreased rates of incorporation of unesterified DHA from plasma into brain phospholipids, reduced concentrations of several fatty acids (including DHA) esterified in ethanolamine- and serine-glycerophospholipids, and increased lysophospholipid fatty acid concentrations. DHA turnover in brain phospholipids did not differ between genotypes. In iPLA₂β^−/− mice, brain levels of iPLA₂β mRNA, protein, and activity were decreased, as was the iPLA₂γ (Group VIB PLA₂) mRNA level, while levels of secretory sPLA₂-V mRNA, protein, and activity and cytosolic cPLA₂-IVA mRNA were increased. Levels of COX-1 protein were decreased in brain, while COX-2 protein and mRNA were increased. Levels of 5-, 12-, and 15-LOX proteins did not differ significantly between genotypes. Thus, a genetic iPLA₂β deficiency in mice is associated with reduced DHA metabolism, profound changes in lipid-metabolizing enzyme expression (demonstrating lack of redundancy) and of phospholipid fatty acid content of brain (particularly of DHA), which may be relevant to neurologic abnormalities in humans with PLA2G6 mutations.     

15-Hydroxyeicosatetraenoic Acid Inhibits Superoxide Anion Generation by Human Neutrophils: Relationship to Lipoxin Production

Human neutrophils can aggregate, degranulate, and release mediators of inflammation including oxygen radicals and lipoxygenase (LO)-derived products of arachidonic acid. The regulation of 5– and 15-lipoxy-genases appears to be important since their products (e.g. leukotrienes and lipoxins) display unique spectra of bioactions. Addition of 15-HETE. a product of the 15-LO, to neutrophils in suspension dramatically shifted the LO products generated and led to a dose-dependent increase in lipoxins, while the production of leukotriene B₄ and its μ-oxidation products (i.e. 20-COOH-LTB₄ and 20-OH-LTB₄) was inhibited. Exogenous 15-HETE also dose-dependently inhibited the generation of superoxide anions induced by either the chemotactic peptide f-met-leu-phe or the divalent cation ionophore A23187. Neither lipoxin A, nor lipoxin B₄ (10^?8?10^?6M) inhibited O₂^?_? generation induced by either f-met-leu-phe or A23187. These results indicate that in addition to serving as a substrate for lipoxin generation, 15-HETE also inhibits superoxide anion generation by human neutrophils. Together they provide further evidence to suggest that products of the 15-lipoxygenase may serve a regulatory role at inflammatory loci.     

Dietary Effects of Eicosapentaenoic and Docosahexaenoic Acid Esters on Lipid Metabolism and Immune Parameters in Sprague-Dawley Rats

Sprague-Dawley rats were fed eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) ethyl esters at the 2% level for 3 weeks to clarify their effects on immune functions. In the rats fed EPA or DHA, serum cholesterol, triglyceride, and phospholipid (PL) levels were significantly lower than those in the rats fed safflower oil. In PL fractions of serum, liver, lung, splenocytes, and peritoneal exudate cells (PEC), increases in linoleic and dihomo-γ-linolenic acid contents and a decrease in arachidonic acid (AA) content were observed in the rats fed EPA or DHA. In addition, the EPA content increased in the rats fed EPA and DHA. In the rats fed EPA or DHA, a decrease of LTB₄ productivity and an increase of LTB₅ productivity were observed in the PEC, in response to the treatment with 5 μM calcium ionophore A23187 for 20 min. The changes in leukotriene production were more marked in EPA-fed rats than in DHA-fed rats. These results suggest that dietary EPA affects lipid metabolism and leukotriene synthesis more strongly than DHA.     

Lignin Deposition and Effect of Postharvest Treatment on Lignification of Green Asparagus (Asparagus officinalis L.)

To understand how lignin synthesis is regulated after harvest, detached green asparagus stalks (Asparagus officinalis L.) were treated with 1 μl l⁻¹ of 1-methylcyclopropene (1-MCP), 50 μg l⁻¹ gibberellic acid (GA₃), 2% (v:v) ethanol or 1 μl l⁻¹ ethylene. The results showed that lignin concentration in asparagus stalks stored at room temperature rapidly increased. Three conventional precursors of lignin, 4-hydroxycinnamic acid (coumaric acid), 3,4-dihydroxycinnamic acid (caffeic acid) and 4-hydroxy-3-mythoxycinnamic acid (ferulic acid), were found to be the major phenolics in the asparagus stalks. Furthermore, the concentrations of O₂⁻ in asparagus stalks steadily increased during the storage. Deposition of lignin in harvested asparagus was significantly reduced by treating the stalks with GA₃, 1-MCP or ethanol. The concentration of lignin in stalks treated with GA₃, 1-MCP or ethanol was 32, 20 or 27% lower, respectively, than in controls 3 days after treatment. Treating stalks with ethylene enhanced lignin synthesis (p<0.05). The concentration of total phenol in stalks was also significantly reduced by GA₃, 1-MCP and ethanol, but was enhanced by ethylene treatment. However, the concentration of active oxygen (O₂^−⋅) in stalks was significantly reduced by treatment with GA₃, 1-MCP and ethanol, but was enhanced by treatment with ethylene. Our study show that postharvest treatment with 1-MCP, GA₃ or ethanol may be applied to improve the quality of green asparagus.     

The effects of polyunsaturated fatty acids and their metabolites on osteoclastogenesis in vitro

Bone homeostasis is maintained by active remodeling through the balance between resorption (by osteoclasts) and synthesis (by osteoblasts). In this study, we examined the effects of polyunsaturated fatty acids (PUFAs) and their metabolites on sRANKL-induced differentiation of bone marrow-derived macrophages (BMMs) into osteoclasts in vitro. Docosahexaenoic acid (DHA) strongly inhibited osteoclastogenesis; however, dihomo-γ-linolenic acid (DGLA), arachidonic acid (AA) and eicosapentaenoic acid (EPA) enhanced it. The enhancement effect of PUFAs on osteoclastogenesis was mediated predominantly by cyclooxygenase (COX) products, because the effect was inhibited by a COX inhibitor. It was also found that COX products of PUFAs, prostaglandin E₁, E₂, and E₃, clearly increased in osteoclastogenesis. The inhibitory effect of DHA on osteoclastogenesis was reversed by treatment with a lipoxygenase (LOX) inhibitor. Furthermore, resolvin D1, a LOX product of DHA, significantly inhibited osteoclastogenesis. Quantitative analysis of specific mRNA levels revealed that DHA-mediated attenuation of osteoclastogenesis might be due to a decrease in DC-STAMP expression. These results suggested that the effect of DHA on osteoclastogenesis is, at least in part, mediated by lipoxygenase products. This study showed a distinct mechanism of the effect of PUFAs on osteoclastogenesis and will provide evidence for therapeutic treatment with DHA in osteoporotic patients.     

Arachidonic acid metabolism in rat basophilic leukemia (RBL-1) cells

Rat basophilic leukemia (RBL-1) cells metabolized arachidonic acid through more than one enzymatic pathway. The major cyclooxygenase product was prostaglandin (PG) D₂ as established by chromatographic and chemical behavior and the effect on platelet aggregation. PGD₂ formation from exogenous arachidonic acid was inhibited by indomethacin, 1 μg/ml. RBL-1 incubated with exogenous arachidonic acid also formed SRS-A the synthesis of which was not inhibited by indomethacin. However, the SRS-A activity was blocked by the specific receptor antagonist FPL 55712. [¹⁴C]arachidonic acid was effectively incorporated into the phospholipids of RBL-1 cells. Challenge of such prelabelled cells or unlabelled cells with A 23187 caused release of PGD₂, SRS-A and another presently unidentified product. However, with A 23187 as a stimulus, the RBL-1 cyclo-oxygenase could not be blocked by low concentrations of indomethacin. This work further substantiates our earlier findings that SRS-A formed from arachidontic acid is not a cyclooxegenase product.     

Effect of Bicuculline-Induced Status Epilepticus on Prostaglandins and Hydroxyeicosatetraenoic Acids in Rat Brain Subcellular Fractions

Abstract: Rat cerebrum, prelabeled in vivo by intraventric-ular injection of [1-¹⁴C]arachidonic acid, was used to assess cyclooxygenase and lipoxygenase reaction products in total homogenates, cytosol, synaptosomes, and microsomes. Effects of bicuculline-induced status epilepticus on arachi-donic acid metabolism in synaptosomes and microsomes were also measured. Lipoxygenase activity, resulting in the synthesis of hydroxyeicosatetraenoic acids (HETEs), and cyclooxygenase activity, resulting in the synthesis of prostaglandins (PGs), were measured by reverse-phase and normal-phase HPLC with flow scintillation detection. Endogenous lipoxygenase products in synaptosomes were identified by capillary gas chromatography-mass spectrometry. PGs and HETEs were detected in all subcellular fractions. The synaptosomal fraction showed the highest lipoxygenase activity, with 5-HETE, 12-HETE, and leukotriene B₄ as the major products. Following bicuculline-induced status epilepticus, endogenous free arachidonic acid and other fatty acids accumulated in synaptosomes, but not in microsomes. Incorporation of [1-^l4C]arachidonic acid into synaptosomal and microsomal phospholipids was decreased after bicuculline treatment. Bicuculline-induced status epilepticus resulted in increased synthesis of HETEs in synaptosomes. PG synthesis increased in the microsomal fraction. When [1-¹⁴C]arachidonic acid-labeled synaptosomes and microsomes were incubated for 1 h at 37°C the synthesis of eicosa-noids, particularly PGD₂, was increased significantly in bi-cuculline-treated rats, as compared with untreated rats. Depolarization (45 mM K⁺) of synaptosomes induced a loss of [1-¹⁴C]arachidonic acid from phosphatidylinositol, and increased the synthesis of PGD₂ and HETEs, an effect that was enhanced in bicuculline-treated rats. This study localizes changes in arachidonic acid metabolism and lipoxygenase activity resulting from bicuculline-induced status epilepticus in the brain subcellular fraction enriched in nerve endings.     

Arachidonic acid metabolim in rat pancreatic acinar cells: Calcium-mediated stimulation of the lipoxygenase system

Isolated rat pancreatic acini were employed to demonstrate that the exocrine pancreas can metabolize [¹⁴C]-arachidonic acid by way of the lipoxygenase pathway as well as the cyclooxygenase pathway. Analysis by high performance liquid chromtography delineated a monohydroxy acid, presumably 12-L-hydroxy-5,8–10,14-eicosatetraenoic acid (12-HETE) as the major lipoxygenase product. The formation of this hydroxy arachidonic derivative was stimulated by the calcium ionophore ionomycin. Stimulation of lipoxygenase pathway by ionomycin was confirmed by thin layer chromatography. In addition, 6-keto-PGF_1α, PGF_2α, and PGE₂ were identified; and ionomycin, carbamylcholine, and caerulein enhanced the formation of these metabolites of the cyclooxygenase pathway. Ionomycin induced stimulation of HETE formation was inhibited by ETYA and nordihydroguaiaretic acid, but spontaneous and evoked enzyme secretion was unaffected. Thus, although ionomycin, a pancreatic secretagogue, stimulates the lipoxygenase pathway, the precise role of these arachidonate metabolites in the physiology of the exocrine pancreas is still obscure.     

Metabolism of Arachidonic Acid to Epoxyeicosatrienoic Acids. Hydroxyeicosatetraenoic Acids, and Prostaglandins in Cultured Rat Hippocampal Astrocytes

Abstract: We have recently shown that brain slices are capable of metabolizing arachidonic acid by the epoxy-genase pathway. The purpose of this study was to begin to determine the ability of individual brain cell types to form epoxygenase metabolites. We have examined the astrocyte epoxygenase pathway and have also confirmed metabolism by the cyclooxygenase and lipoxygenase enzyme systems. Cultured rat hippocampal astrocyte homogenate, when incubated with radiolabeled [³H]-arachidonic acid, formed products that eluted in four major groups designated as R_17–30, R_42–50, R_51–82, and R_83–90 based on their retention times in reverse-phase HPLC. These fractions were further segregated into as many as 13 peaks by normal-phase HPLC and a second reverse-phase HPLC system. The principal components in each peak were structurally characterized by gas chromatography/electron impact-mass spectrometry. Based on HPLC retention times and gas chromatography/electron impactmass spectrometry analysis, the more polar fractions (R_17–30) contained prostaglandin D₂ as the major cyclooxygenase product. Minor products included 6-keto prostaglandin F_1α, prostaglandin E₂, prostaglandin F_2α, and thromboxane B₂. Fractions R_42–50, R_51–82. and R_83–90 contained epoxygenase and lipoxygenase-like products. The major metabolite in fractions R_83–90 was 5, 6-epoxyeicosatrienoic acid (EET). Fractions R_51–82 contained 14, 15-and 8, 9-EETs, 12-and 5-hydroxyeicosatetraenoic acids, and 8, 9-and 5, 6-dihydroxyeicosatrienoic acids (DHETs). In fractions R_42–50, 14, 15-DHET was the major product. When radiolabeled [³H]14, 15-EET was incubated with astrocyte homogenate, it was rapidly metabolized to [³H]14, 15-DHET. The metabolism was inhibited by submicromolar concentration of 4-phenylchalcone oxide, a potent inhibitor of epoxide hydrolase activity. Formation of other polar metabolites such as triols or epoxyalcohols from 14, 15-DHET was not observed. In conclusion, astro-cytes readily metabolize arachidonic acid to 14, 15-EET, 5, 6-EET, and their vicinal-diols. Previous studies suggest these products may affect neuronal function and cerebral blood flow.     

Activation of phospholipase D by prostaglandin F2α in rat luteal cells and effects of inhibitors of arachidonic acid metabolism

In rat luteal cells labeled with (³H]oleic acid, PGF_2α-stimulated phospholipase D (PLD) activation was investigated. The PLD activity was detected by measuring the accumulation of [³H]phosphatidylethanol (PtdEt) in the presence of ethanol. PGF_2α stimulated PtdEt accumulation at concentrations of more than 100 nM in the presence of ethanol. However, PtdEt accumulation did not change in the absence of ethanol. PGF_2α (1 μM) increased PtdEt accumulation after 1 min, and the accumulation reached a plateau by 2–3 min. These results indicate that PGF_2α activates PLD in rat luteal cells. U-73122, a phospholipase C (PLC) inhibitor, and staurosporine, a protein kinase C (PKC) inhibitor, did not inhibit PGF_2α-stimulated [³H]PtdEt accumulation. These results suggest that PGF_2α-induced PLD activation is different from PLC-PKC systems. We reported previously that PGF_2α stimulated the release of arachidonic acid. The effects of indomethacin, nordihydroguaiaretic acid (NDGA), and 5,8,11,14-eicosatetraynoic acid (ETYA), inhibitors of arachidonic acid metabolism, on PGF_2α-stimulated PtdEt accumulation were examined. Pretreatment with indomethacin enhanced PGF_2α-induced PtdEt accumulation. In contrast, pretreatment with NDGA and ETYA inhibited PGF_2α-induced PtdEt accumulation. It is suggested that PGF_2α-stimulated PLD activation is mediated via lipoxygenase products.     

The synthesis and toxicity of tripodal tricarbonyl rhenium complexes as radiopharmaceutical models

We report the synthesis and toxicity of a series of rhenium(I) tricarbonyl complexes incorporating the trisaminomethylethane (TAME) ligand. Compounds with the (TAME)Re(CO)₃⁺ cation were synthesized via several routes, including by use of Re(CO)₅X precursors as well as the aqueous cation Re(CO)₃(H₂O)₃⁺. Salts of the formula [(TAME)Re(CO)₃]X where X = Br⁻, Cl⁻, NO₃⁻, PF₆⁻ and ClO₄⁻ were evaluated using two cell lines: the monoclonal S3 HeLa line and a vascular smooth muscle cell line harvested from mice. All compounds have isostructural cations and differ only in the identity of the non-coordinating anion. None of the complexes exhibited any appreciable toxicity in the HeLa line up to the solubility limit. In the vascular smooth muscle cell line, the bromide salt exhibited some cytotoxicity, but this observation most likely results from the presence of bromide anion, which has been shown to have limited toxicity.     

Production of leukotriene B4 and prostaglandin E2 by turbot (Scophthalmus maximus) leukocytes

Production of two eicosanoids derived from lipoxygenase and cyclooxygenase activities: leukotriene B₄ (LTB₄) and prostaglandin E₂ (PGE₂), respectively, have been simultaneously determined in turbot (Scophthalmus maximus) blood leucocyte and kidney macrophage supernatants by a reverse phase high performance liquid chromatography (HPLC) system coupled with a Diode–Array detector. Levels of LTB₄ after calcium ionophore challenge were 4.08 ng ml⁻¹ in blood leukocyte supernatants and 0.25 ng ml⁻¹ in kidney macrophage supernatants. The levels found for PGE₂ were 428.23 and 606.67 ng ml⁻¹ in blood leukocytes and kidney macrophage supernatants, respectively. When blood leukocytes were treated with the respective inhibitors for the enzymes implicated on the synthesis of both compounds an inhibition of 90.35% was observed for PGE₂ and 76.44% for LTB₄. The detection limit of the method was 0.15 ng ml⁻¹ for LTB₄ and 50 ng ml⁻¹ for PGE₂.