A novel eicosanoid is the major arachidonic acid metabolite of cultured human monocytes

Human peripheral blood monocyte-macrophages (M phi) generate a novel eicosanoid during in vitro culture. The metabolite is generated during incubation of the cells with 14C - arachidonic acid (AA). Lack of prior recognition of this metabolite probably results from the facts that: 1) on thin-layer chromatography (TLC) in two standard solvent systems, the novel metabolite co-chromatographed with either prostaglandin D2 or thromboxane B2, and 2) its generation, under the conditions studied, does not occur until between 90 and 180 minutes after culture initiation which is a time period beyond that used for most leukocyte studies. The generation of the metabolite is inhibited by nordihydroguaiaretic acid (NDGA) but not by indomethacin. Base hydrolysis did not alter its migration on TLC. On both reversed phase and straight phase high pressure liquid chromatography (HPLC), the novel peak isolated by TLC elutes as a single major peak of radioactivity with a retention time different from the known leukotrienes, hydroxy acids, or their metabolites. Furthermore, the peak isolated on HPLC has a single ultraviolet absorption maximum at 270 nm. M phi cultured for 1 week prior to a 24 hour incubation with 14C-AA generated proportionally less of the novel eicosanoid (roughly 68% of total radiolabeled product) than did M phi cultured for 3 weeks prior to a similar incubation with 14C-AA (roughly 86% of total radiolabeled product). Under the conditions studied, the novel eicosanoid is the major AA metabolite generated from exogenous AA by cultured M phi and it appears to be generated in increasing quantity as the M phi differentiate.     

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.     

Metabolism of exogenous arachidonic acid by murine macrophage-like tumor cell lines

Murine macrophage-like cell lines, J774.2, P388D1, RAW264.7 and PU-5-1R, were incubated with exogenous arachidonic acid (AA). The major metabolites were identified by comigration with known standards in TLC and HPLC and by characteristic behavior following reduction. During a 30 min incubation J774.2 cells metabolized exogenous ¹⁴C-AA (10 μM) to PGE₂ (14.8%), 12-hydroxy-5,8,10-heptadecatrienoic acid (HTT)_ (13.0%), thromboxane B₂ (TXB₂) (7.4%), PGD₂ (4.4%) and PGF_2α (3.0%). The remainder was incorporated into phospholipids (39.0%), triglycerides (6.1%), and as yet unidentified metabolites (8.2%). No PGF_1α was found. Metabolism of exogenous AA was rapid, being >90% completed at 3.5 min. Metabolism of exogenous AA is not increased by the simultaneous addition of macrophage stimuli including the cation ionophore A-23187, particulate phagocytic stimuli and endotoxin. The synthesis of cyclooxygenase products was inhibited by low doses of indomethacin (ID₅₀=0.6 μM) while the synthesis of TXB₂ and HHT was selectively inhibited by benzylimidazole (ID₅₀=9.5 μM). Identification of a probable lipoxygenase product is being pursued. The synthesis of this product is not inhibited by indomethacin and migrates with an R_f value close to 5,12-diHETE in TLC. P388D1 and RAW264.7 cells metabolize exogenous AA to the same products as J774.2, in different proportions, while PU-5-1R does not produce cylooxygenase metabolites to any appreciable extent.     

The effects of alcoholism and smoking on platelet eicosanoid production in vitro

Ethanol induces changes in eicosanoid synthesis in blood platelets and brain tissue. Cigarette smoking also causes alterations in eicosanoid formation. This preliminary report examined in vitro platelet sonicate eicosanoid production using 14C-arachidonic acid (14C-AA) and in separate experiments, 14C-PGH2, as substrates. Radiometric thin layer chromatography (TLC) was used to identify the products formed. Eicosanoid product formation in platelet sonicates collected from 28 abstinent male alcoholics were compared to those from 11 male control subjects. All but one of the alcoholics were chronic smokers and all control subjects were non-smokers. All smokers abstained from smoking for 12 h prior to the blood collection to control for any acute effects of cigarette smoke on eicosanoid production. Significant reductions in platelet sonicate production of PGD2 and PGE2 in vitro were observed in alcoholic smokers when 14C-PGH2, but not 14C-AA, was the substrate. These reductions were predicted equally well by two variables, smoking and alcoholism, using several statistical models. This is the first investigation that controlled for the acute effects of smoking and accounted for the potential effects of cigarette smoking on platelet eicosanoid production in alcoholics. Because cigarette smoking is prevalent among alcoholics, future studies on the role of eicosanoids in alcoholism should control for smoking.     

Effect of radiation on the biosynthesis of L-Tryptophan from 14C-Anthranilic acid in kohlrabi (Brassica oleracea L., var.gongylodes L.)

The metabolism of¹⁴C-anthranilic acid (¹⁴C-AA) in kohlrabi (Brassica oleracea L. var.gongylodes L.) and the effect of radiation gamma⁶⁰Co on this metabolism was investigated. In hypocotylar segmnents of seven days old etiolated seedlings¹⁴C-AA was metabolised par, tially to its detoxication product¹⁴C-β-glucoside of AA. Simultaneously L-tryptophan was also formed, which in these plants is a precursor of indolic glucosinolates glucobrassicin and neoglucobrassicin. The metabolism of¹⁴C-AA was followed for 97 h. Radiation, applied both to seeds and to seven days old plants did not affect the metabolism of¹⁴C-AA substantially. The intermediary reaction AA → L-tryptophan in the biosynthesis of L-tryptophan is not a radiosensitive part of the synthesis of this amino acid. A not too high radiation sensitivity (max. 45%) was observed in the metabolic pathway leading from L-tryptophan to glucobrassicin.     

Lipoxygenase- and cyclooxygenase-reaction products and incorporation into glycerolipids of radiolabeled arachidonic acid in the bovine retina

The metabolism of radiolabeled arachidonic acid (AA) by the intact bovine retina has been studied. Synthesis of prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs), and incorporation of AA into glycerolipids has been measured by reverse-phase and straight-phase high performance liquid chromatography with flow scintillation detection, and by thin-layer chromatography. AA was actively acylated into glycerolipids, particularly triglycerides, phosphatidylcholine and phosphatidylinositol. AA was also converted to the major PGs, PGF_2α, PGE₂, PGD₂, 6-keto-PGF_1α and TXB₂, and to the lipoxygenase reaction products, 12-HETE, 5-HETE, and other monohydroxy isomers. Approximately 6% of the radiolabeled AA was converted to eicosanoids. The synthesis of HETEs was inhibited in a concentration-dependent manner (IC₅₀ = 8.3 NM) by nordihydroguaiaretic acid (NDGA). PG synthesis was inhibited by aspirin (10 μM), indomethacin (1 μM) and NDGA (IC₅₀ = 380 nM). Metabolism of AA via lipoxygenase, cyclooxygenase and activation-acylation was inhibited by boiling retinal tissue prior to incubation. These studies demonstrate an active system for the uptake and utilization of AA in the bovine retina, and provide the first evidence of lipoxygenase-mediated metabolism of AA, resulting in the synthesis of mono-hydroxyeicosatetraenoic acids, in the retina.     

Effects of extracellular Ca2+ and the intracellular Ca2+ antagonist 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate on rabbit platelet conversion of arachidonic acid into thromboxane

The regulatory role of Ca²⁺ on the conversion of arachidonic acid (AA) into thromboxane B₂ (TXB₂) was examined in washed rabbit platelets, whose secretoary processes are known to have requirements for extracellular CA²⁺. Varying the extracellular free Ca²⁺ [Ca_f²⁺] concentration from < 10^?8 to 10^?3 M had no significant effect on the synthesis of immunoreactive TXB₂ by rabbit platelets incubated with 1–4 μM AA. On the other hand, 8-(N,N-diethylamino) octyl-3,4,5- trimethoxybenzoate (TMB-8), a putative antagonist of intracellular Ca²⁺ movement, inhibited AA-stimulated synthesis of TXB₂ in a concentration dependent manner--an effect which could be partially overcome by increasing the AA concentration. The TMB-8 inhibition could not be reversed by increasing the [Ca²⁺_f]. Studies examining platelet metabolism of ¹⁴C-AA and ¹⁴C-prostaglandin H₂ demonstrated that TMB-8 inhibited platelet cyclooxygenase, but not thromboxane synthetase. These studies demonstrate the absence of a requirement for [Ca²⁺_f] but suggest the presence of a TMB-8 sensitive intracellular Ca²⁺ pool in the rabbit platelet synthesis of TXB₂ from AA.     

Gestational diabetes mellitus decreases placental uptake of long-chain polyunsaturated fatty acids: involvement of long-chain acyl-CoA synthetase

The long-chain polyunsaturated fatty acids (LC-PUFAs) arachidonic (AA) and docosahexaenoic (DHA) acids are essential for fetal development. Gestational diabetes mellitus (GDM) is a pregnancy disorder associated with perinatal and lifelong risk complications for both the mother and the newborn. Our aim was to investigate the influence of GDM, and some of its associated conditions, upon the placental uptake of AA and DHA. Uptake of ¹⁴C-AA and ¹⁴C-DHA by human trophoblasts obtained from normal pregnancies (NTB cells) was mediated by both saturable (for lower substrate concentrations) and non-saturable (for higher substrate concentrations) mechanisms. Uptake of both fatty acids was inhibited by other LC-PUFAs and, markedly, by the long-chain acyl-CoA synthetase (ACSL) inhibitor, triacsin C. Human trophoblasts obtained from GDM pregnancies (DTB cells) showed a significantly lower ¹⁴C-AA and ¹⁴C-DHA accumulation, through a decrease in both the saturable and the non-saturable components of uptake, which was associated with a decrease in ACSL1 mRNA levels. Uptake of LC-PUFAs by NTB cells increased (by 20–25%) after short-term exposure to TNF-α (¹⁴C-AA and ¹⁴C-DHA) and insulin (¹⁴C-DHA). In conclusion, GDM, distinctly from its associated conditions, markedly decreases placental uptake of LC-PUFAs, which probably contributes to the deleterious effects of this disease for the newborn.     

Isolation and purification of anticardiolipin antibody from plasma of a patient with antiphospholipid syndrome: induced generation of platelet thromboxane A2 synthesis

Antiphospholipid antibodies, particularly anticardiolipin antibodies (aCL) are autoantibodies frequently detected in the serum of patients with systemic lupus erythematosus (SLE) and the primary antiphospholipid antibody syndrome (PAPS). These patients commonly suffer from thrombosis, recurrent fetal loss and thrombocytopenia. Since platelet aggregation is pivotal in the genesis of thrombosis, we tested the hypothesis that perturbation of platelet membrane by aCL/β₂-glycoprotein (aCL/β₂GP) complex could trigger the biosynthesis of TXA₂, a proaggregatory metabolite of AA. The preincubation of ¹⁴C-arachidonic acid (¹⁴C-AA)-labeled platelet pellets (¹⁴C-PP) from normal individuals with aCL alone followed by incubation with thrombin, resulted in a moderate increase in platelet thromboxane B₂ (¹⁴C-TXB₂) biosynthesis when compared to controls (without aCL). Similar incubations with β₂GP-I alone resulted in negligible ¹⁴C-TXB₂ biosynthesis. In contrast, the preincubations of normal ¹⁴C-PP with aCL/β₂GP-I complex resulted in marked thrombin-induced TXB₂ biosynthesis, underscoring the requirement of β₂GP-I in aCL-induced platelet TXB₂ biosynthesis. Taken together, these results are consistent with the view that aCL/β₂GP-I platelet interactions do play a role, at least in part, in platelet hyperactivity and thrombosis in antiphospholipid antibody syndrome.     

The effect of cigarette smoke on the metabolism of arachidonic acid in isolated hamster lungs

The effects of cigarette smoke on the metabolism of exogenous arachidonic acid (AA) were investigated in isolated hamster lungs. Arachidonate was injected into the pulmonary circulation and the metabolites were analysed from the nonrecirculating perfusion effluent by thin layer chromatography. After the pulmonary injection of 66 nmol of ¹⁴C-AA about 20 % of the injected radioactivity appreated in the perfusion effluent mostly as metabolites in six minutes. When isolated lungs were ventilated with cigarette smoke during the perfusion, the amounts of PGF_2α, PGE₂ and two unidentified metabolite groups increased in the lung effluent. In two other experimental series hamsters were exposed to cigarette smoke before the lung perfusion either once for 30 min or during one hour daily for ten consecutive days. Neither pre-exposures caused any changes in the amounts of arachidonate metabolites in the lung effluent.     

Inhibition of GSH-dependent PGH2 isomerase in mammary adenocarcinoma cells by allicin.

We have reported tha allicin, a constituent of garlic oil, has no effect on the activities of platelet cyclooxygenase or thromboxane synthase, or vascular PGI₂ synthase. The effect of allicin on glutathione (GSH) dependent PGH₂ to PGE₂ isomerase is unknown. We therefore studied the effect of allicin on PGE₂ biosynthesis in a murine mammary adenocarcinoma cell line (No 4526). Intact or sonicated cells were incubated with either ¹⁴C-arachidonic acid (AA) or ¹⁴C-PHG₂, respectively. Following metabolism, products were extracted, separated by TLC and analyzed by radiochromatographic scan. PGE₂ was predominantly formed with minimal amounts of PGF_2α and PGD₂. Formation of 6-keto-PGF_1α or TXB₂ was not detected indicating the absence of TXA₂ and PGI₂ synthase activity. Indomethacin and ibuprofen inhibited the PGE₂ formation (p < 0.05). The enzymatic PGE₂ formation in sonicates was blocked by depletion of the cellular non-protein thiols by buthionine sulfoximine and was shown to be dependent on GSH. Allicin, over the range of 10–1000 μM, inhibited the formation of PGE₂ in cells exposed to 2.0 μM ¹⁴C-AA for 20 min. and in sonicated cells incubated with 20.0 μM ¹⁴C-PGH₂ for 2 min (p < 0.05). Allicin did not alter cyclooexygenase-mediated oxygen utilization in ram seminal vessicle microsomes, suggesting that allicin selectively inhibits the GSH-dependent PGH₂ to PGE₂ isomerase in this adenocarcinoma cell line.     

The effects of alpha1-adrenergic and muscarinic cholinergic stimulation on prostaglandin release by rabbit iris

We have investigated the effects of norepinephrine (NE) and acetylcholine (ACh) on prostaglandin (PGE₂ and 6 keto-PGF_1α) production by rabbit iris, measured by radioimmunoassay (RIA), and the type of phospholipase activated by NE in irides in which phosphatidylinositol (PI) was doubly prelabeled with [³H] myo-inositol and [1-¹⁴C] arachidonic acid (¹⁴C-AA), quantitated by radiometric and chromatographic methods. PGE₂ output in 60 min (3.6 μg/g tissue) was 2.6 times greater than 6 keto-PGF_1α. PG production is time-dependent and it is stimulated by NE and ACh in a dose-dependent manner. The Ne- and ACh-induced release of PGE₂, measured by RIA, is mediated through α₁-adrenergic and muscarinic cholinergic receptors, respectively, and it requeires Ca²⁺ for maximal stimulation. Studies on the mechanism of AA release PI in irides doubly prelabeled with ¹⁴C-AA and [³H] myo-inositol revelased the following: (a) Both Ne and ACh increased the breakdown of PI, and this was accompanied by a significant increase in the release of AA and consequently PGE₂. The stimulatory effects of NE and ACh are mediated through α₁-adrenergic and muscainic cholinergic receptors respectively. (b) The NE-induced formation of ³H-lyso PI and the NE-induced metabolism of ¹⁴C-1,2-diacyl-glycerol (DG) are time-dependent. Two pathways for AA release from PI are probably operaitve in the iris: (a) An indirect release by PI-specific phospholipase C which producers DG, followed by the actions of DG- and monoacylglycerol lipases on DG to release AA. (b) A direct release by phospholipase A₂. Whether lyso PI is a product of the polypholipids such as prosphatidylcholine and phosphatidylethanolamine could also serve as a source for AA in PG synthesis. In conclusion, the data presented provide evidence that in the iris the neurotransmitter-stimulated release of PG and AA, from phosphoinositides, for PG synthesis is coupled to the activation of α₁-adrenergic and muscarinic cholinergic receptors.     

Aspirin inhibits arachidonic acid metabolism via lipoxygenase and cyclo-oxygenase in hamster isolated lungs

The effect of aspirin on the fate of exogenous arachidonic acid (AA) was investigated in isolated perfused lungs of female hamsters. During pulmonary infusion of aspirin (10 μM, 100 μM or 1 mM) 45 nmol of ¹⁴C-AA was infused in two minutes into the pulmonary circulation. The nonrecirculating perfusion effluent was collected for 6 minutes after the beginning of the AA infusion. Arachidonate infusion increased the perfusion pressure. This pressor response was completely abolished by 1 mM aspirin. When aspirin was infused into the pulmonary circulation, the amount of radioactivity was increased in the perfused lungs and decreased dose dependently in the nonrecirculating perfusion effluent. The amount of unmetabolized free arachidonate was not changed significantly by aspirin in the perfused lungs or in the perfusion effluent. In the effluent the amounts of all arachidonate metabolites, which were extracted with ethyl acetate first at pH 7.4 and then at pH 3.5 and analysed by thin layer chromatography, were decreased quite similarly by aspirin. The formation of arachidonate metabolites was completely inhibited by 1 mM aspirin. In the perfused lung tissue the amount of ¹⁴C-AA was increased by aspirin in phospholipids and neutral lipids. The present study indicates that the metabolism of arachidonic acid is inhibited by aspirin in hamster lungs not only via cyclo-oxygenase but also via other lipoxygenases.     

Arachidonic acid stimulates interleukin-6 release from rat peritoneal macrophages in vitro: Evidence for a prostacyclin-dependent mechanism

Interleukin-6 (IL-6) is a cytokine involved in the differentiation of B-cells to antibody secreting plasma cells, the activation of T-cells, and the stimulation of hepatocyte production of acute phase proteins. Because of the pro-inflammatory effects of this cytokine, we investigated the ability of the fatty acid arachidonic acid (AA) to regulate the release of IL-6 from rat resident peritoneal macrophages (Mø) in vitro. AA (0.5–16 μM) stimulated IL-6 release during a 4 h incubation period in a biphasic manner, with 4 μM AA generating a peak of IL-6 release (3-5-fold). AA (0.5–16 μM) also induced an increasing release of the AA metabolite thromboxane B₂ (TXB₂). The AA-induced release of IL-6 occurred within 1–2 h of incubation, whereas TXB₂ concentrations were elevated within 5 min of AA treatment. The TX synthetase inhibitor CGS 12970 (4.0 μM and 40.0 μM) effectively blocked the generation of TXB₂, but increased prostacyclin (PGI₂) generation and potentiated the release of IL-6. In addition, PGI₂, as well as the PGI₂ agonists iloprost and cicaprost, stimulated IL-6 release from Mø by greater than 5-fold over vehicle-treated basal levels. These data suggest that PGI₂ (but not TXA₂) is involved in AA-induced IL-6 release from peritoneal Mø.     

An inhibitory effect of arachidonic acid on subsequent responses of canine cerebral arteries to serotonin and other agonists

Arachidonic acid (AA) at 10^?4M and 10^?3M produced a phasic contraction in isolated canine basilar arteries that peaked rapidly and then slowly declined. This contraction was evidently due to the conversion of AA to prostanoids because it was blocked by cyclooxygenase inhibitors and because 11, 14, 17 eicosatrienoic acid (10^?3M), which is not a cyclooxygenase substrate, failed to produce a contraction. When the artery was exposed to 10^?3M AA for 20 min and washed, subsequent contractile responses to 10^?6M serotonin (5-HT) were only 10% of control. Contractions produced by prostaglandin E₂ (10^?5M), uridine triphosphate (10^?4M) and potassium (5.5×10^?4M) were inhibited to a lesser degree than 5-HT, the response to potassium being the least affected (66% of control). This damaging effect of 10^?3M AA did not occur if the artery was washed at peak contraction nor with 10^?4M AA. Autooxidation products were evidently not responsible for the damage because prior oxygenation (90 min) of 10^?4M AA had no such effect. Pretreatment with superoxide dismutase or ascorbate did not prevent the inhibition, suggesting that free radical reactions were not involved. Pretreatment with indomethacin (3×10^?4M) or meclofenamate (10^?4M) also failed to prevent the inhibitory phenomenon. Saponin, a detergent, produced similar inhibitory effects but 11, 14, 17 eicosatrienoic acid or oleate (10^?3M) did not. The arteries partially recovered from the inhibition with time. In conclusion, AA produced contraction in basilar arteries by inducing prostaglandin synthesis but can produce secondarily by an unidentified mechanism an inhibition of the contractile responses evoked by various agonists that is both time and concentration dependent.     

The effect of conjugated linoleic acid on arachidonic acid metabolism and eicosanoid production in human saphenous vein endothelial cells

The effects of a conjugated linoleic acid (CLA) mixture of single isomers (50:50, w/w, cis9,trans11:trans10,cis12) and the individual isomers on (a) the production of resting and calcium ionophore stimulated (14)C-eicosanoids and (b) the incorporation of (14)C-arachidonic acid (AA) into membrane phospholipids of human saphenous vein endothelial cells were investigated. The CLA mixture and the individual isomers were found to inhibit resting production of (14)C-prostaglandin F(2a) by 50, 43 and 40%, respectively. A dose dependent inhibition of stimulated (14)C-prostaglandins was observed with the CLA mixture (IC(50) 100 microM). The cis9,trans11 and trans10,cis12 (50 microM) isomers individually inhibited the overall production of stimulated (14)C-prostaglandins (between 35 and 55% and 23 and 42%, respectively). When tested at a high concentration (100 microM), cis9,trans11 was found to inhibit eicosanoid production in contrast to trans10,cis12 that caused stimulation. The overall degree of (14)C-AA incorporation into membrane phospholipids of the CLA (mixture and individual isomers) treated cells was found to be lower than that of control cells and the cis9,trans11 isomer was found to increase the incorporation of (14)C-AA into phosphatidylcholine. Docosahexaenoic acid, eicosapentaenoic acid and linoleic acid did not alter the overall degree of incorporation of (14)C-AA. The results of this study suggest that both isomers inhibit eicosanoid production, and although trans10,cis12 exhibits pro-inflammatory activity at high concentrations, the CLA mixture maintains its beneficial anti-inflammatory action that contributes to its anti-carcinogenic and anti-atherogenic properties.     

Inhibition of eicosanoid release from synovial organ culture by incubation with tepoxalin and its acid metabolite

The pharmacological profile of a novel dual inhibitor, tepoxalin and of its carboxylic acid metabolite on cyclooxygenase and lipoxygenase pathways was evaluated by in vitro incubation with synovial tissue. Tissue specimens obtained at surgery in rheumatoid arthitis (RA, n=10) or osteoarthritis (OA, n=11) patients were incubated. Tepoxalin (10⁻⁷, 10⁻⁶, 10⁻⁵ M) decreased eicosanoid release calculated in % of tyrode control for OA: LTC₄ to 71−33%, 6-keto-PGF_1a to 37−20%, PGE₂ to 29−6%. For RA: LTC₄ to 56−22%, 6-keto-PGF_a to 43−22%, PGE₂ to 57−32%. Similarly, its metabolite (10⁻⁷, 10⁻⁵ M) decreased release in OA: LTC₄ to 99 and 60%, PGE₂ to 42 and 20%, 6-keto-PGF_1a to 54 and 25%. In RA: LTC₄ to 81 and 45%, PGE₂ to 61 and 30%, 6-keto-PGF_1a to 46 and 18%. Significance (p<0.05) was achieved for all but 1 group (LTC₄, metabolite at 10⁻⁷M vs tyrode).In summary a marked and dose dependent decrease of LT and PG release was obtained when incubating the dual inhibitor tepoxalin and its active carboxylic acid metabolite with synovial tissue at doses expected to be reached in the joint during therapy.     

Phospholipid metabolism in calcium-regulated differentiation in cultured murine keratinocytes

The present findings associate phospholipid alteration, particularly the turnover of phosphatidylinositol, in Ca²⁺ induced differentiation of keratinocytes. These conclusions are based on the hydrolysis of ¹⁴C-AA from prelabeled PI and the accumulation ¹⁴C-DG and ¹⁴C-PA after cells are switched from low to normal concentrations of extracellular Ca²⁺. This novel finding implies that the biological changes which accompany keratinocyte differentiation after switch from low to normal extracellular medium may be due at least in part to increased accumulation of PA and DG which are major deacylation and reacylation products of phosphatidylinositol. A second interesting finding in these studies is the marked transformation of ¹⁴C-AA into lipoxygenase products by proliferating keratinocytes cultured in low Ca²⁺ medium when compared to differentiating cells cultured in normal Ca²⁺. The significance of decreased generation of lipoxygenase products in epidermal differentiation deserve further exploration.     

The EmhABC efflux pump decreases the efficiency of phenanthrene biodegradation by Pseudomonas fluorescens strain LP6a

Pseudomonas fluorescens strain LP6a, designated here as strain WEN (wild-type PAH catabolism, efflux positive), utilizes the polycyclic aromatic hydrocarbon phenanthrene as a carbon source but also extrudes it into the extracellular medium using the efflux pump EmhABC. Because phenanthrene is considered a nontoxic carbon source for P. fluorescens WEP, its energy-dependent efflux seems counter-productive. We hypothesized that the efflux of phenanthrene would decrease the efficiency of its biodegradation. Indeed, an emhB disruptant strain, wild-type PAH catabolism, efflux negative (WEN), biodegraded 44% more phenanthrene than its parent strain WEP during a 6-day incubation. To determine whether efflux affected the degree of oxidation of phenanthrene, we quantified the conversion of ¹⁴C-phenanthrene to radiolabeled polar metabolites and ¹⁴CO₂. The emhB ^? WEN strain produced approximately twice as much ¹⁴CO₂ and radiolabeled water-soluble metabolites as the WEP strain. In contrast, the mineralization of ¹⁴C-glucose, which is not a known EmhB efflux substrate, was equivalent in both strains. An early open-ring metabolite of phenanthrene, trans-4-(1-hydroxynaphth-2-yl)-2-oxo-3-butenoic acid, also was found to be a substrate of the EmhABC pump and accumulated in the supernatant of WEP but not WEN cultures. The analogous open-ring metabolite of dibenzothiophene, a heterocyclic analog of phenanthrene, was extruded by EmhABC plus a putative alternative efflux pump, whereas the end product 3-hydroxy-2-formylbenzothiophene was not actively extruded from either WEP or WEN cells. These results indicate that the active efflux of phenanthrene and its early metabolite(s) decreases the efficiency of phenanthrene degradation by the WEP strain. This activity has implications for the bioremediation and biocatalytic transformation of polycyclic aromatic hydrocarbons and heterocycles.     

The fate of labelled glucose molecules in the rat adipocyte: Dependence on glucose concentration

Isolated rat adipocytes were incubated with 15 nM [3-³H]glucose or 100 nM [U-¹⁴C]glucose with or without insulin and in the absence or presence of unlabelled glucose. Following a 2 h incubation with 15 nM [3-³H]glucose, about two thirds of the cell-associated ³H-labelled metabolic products were hydrophilic largely anionic intermediates and about one third was lipids. The equivalent values were 40 and 60%, respectively, when using 100 nM [U-¹⁴C]glucose. The only ¹⁴C-labelled metabolite escaping to the incubation medium was ¹⁴CO₂, which accounted for about 15% of the rate of metabolism. Therefore, the rate of incorporation of 100 nM [U-¹⁴C]glucose into the cell-associated metabolites was quite a good measure of its net influx rate. The conversion of the two tracers to the sum of the metabolic products in cells treated with a maximally stimulating insulin concentration remained constant with glucose concentrations up to about 100 μM and then decreased progressively. The incorporation of radioactivity into the different metabolites varied markedly over the glucose concentration range 0–100 μM, presumably due to the saturation of different metabolic pools at different glucose concentrations. This variation was much less in cells not stimulated with insulin. Consequently, the maximal effect of insulin on the incorporation of the tracers into a given metabolite (e.g., labelled lipids) varied over the entire glucose concentration range. In addition, the apparent sensitivity (ED₅₀) with respect to the incorporation into a given metabolite was also dependent on the glucose concentration.