Edema from cyclooxygenase products of endogenous arachidonic acid in isolated lung

We infused A23187, a calcium ionophore, into the pulmonary circulation of dextran-salt-perfused isolated rabbit lungs to release endogenous arachidonic acid. This led to elevations in pulmonary arterial pressure and to pulmonary edema as measured by extravascular wet-to-dry weight ratios. The increase in pressure and edema was prevented by indomethacin, a cyclooxygenase enzyme inhibitor, and by 1-benzylimidazole, a selective inhibitor of thromboxane (Tx) A2 synthesis. Transvascular flux of 125I-albumin from vascular to extravascular spaces of the lung was not elevated by A23187 but was elevated by infusion of oleic acid, an agent known to produce permeability pulmonary edema. We confirmed that A23187 leads to elevations in cyclooxygenase products and that indomethacin and 1-benzylimidazole inhibit synthesis of all cyclooxygenase products and TxA2, respectively, by measuring perfusate levels of prostaglandin (PG) I2 as 6-ketoprostaglandin F1 alpha, PGE2, and PGF2 alpha and TxA2 as TxB2. We conclude that release of endogenous pulmonary arachidonic acid can lead to pulmonary edema from conversion of such arachidonic acid to cyclooxygenase products, most notably TxA2. This edema was most likely from a net hydrostatic accumulation of extravascular lung water with an unchanged permeability of the vascular space, since an index of permeability-surface area product (i.e., transvascular albumin flux) was not increased.     

Metabolic products of arachidonic acid in rats

The pattern of eicosanoid metabolites appearing in urine and feces following oral administration of radioactive arachidonic acid was investigated using rats deficient in essential fatty acids. About 70-80% of the radioactivity in the urine during the first day after feeding was adsorbed to XAD-2 resin and represented eicosanoid metabolites, whereas the rest of the radioactivity was mainly 3H2O. The eicosanoid metabolites were fractionated into different polarity classes using reverse phase Sep-Pak C18 cartridges. Gas chromatographic analysis of the urinary metabolites following their derivatization into methyl ester-methoxime-tert-butyl-dimethylsilyl ethers revealed that nearly one-half of the metabolites had ECL values less than 22 and represented metabolites more oxidized than commonly described. Only 30% of the metabolites had ECL values between 26 to 32, corresponding to the values for the metabolites that originate from exogenously infused prostaglandins. A large portion of the eicosanoid metabolites was also excreted with the feces. The isotopic patterns from the reverse phase chromatography indicated that many of the fecal metabolites may be similar to those in urine although some metabolites in feces were not present in urine. Based on the specific radioactivity of the administered arachidonic acid, it appeared that at least 6 to 8 mg of eicosanoid metabolites were excreted through urine and feces within 24 hrs following an oral bolus of 60 mg arachidonic acid. The rapid increase and subsequent decrease in eicosanoid metabolite excretion after oral administration of arachidonate indicates that the dietary intake of polyunsaturated fatty acids may have a more rapid effect upon the endogenous production of eicosanoids than is generally recognized.     

Metabolic products of arachidonic acid in rats

The pattern of eicosanoid metabolites appearing in urine and feces following oral administration of radioactive arachidonic acid was investigated using rats deficient in essential fatty acids. About 70–80% of the radioactivity in the urine during the first day after feeding was adsorbed to XAD-2 resin and he represented eicosanoid metabolites, whereas the rest of the radioactivity was mainly ³H₂O. The eicosanoid metabolites were fractioned into different polarity classes using reverse phase Sep-Pak C₁₈ cartridges. Gas chromatographic analysis of the urinary metabolites following their derivatization into methyl ester-methoxime- -butyl-dimethylsilyl ethers revealed that nearly one-half of the metabolites had ECL values less than 22 and represented metabolites more oxidized than commonly described. Only 30% of the metabolites had ECL values between 26 to 32, corresponding to the values for the metabolites that originate from exogenously infused prostaglandins. A large portion of the eicosanoid metabolites was also excreted with the feces. The isotropic patterns from the reverse phase chromatography indicated that many of the fecal metabolites may be similar to those in urine although some metabolites in feces were not present in urine. Based on the specific radioactivity of the administered arachidonic acid, it appeared that at least 6 to 8 mg of eicosanoid metabolites were excreted through urine and feces within 24 hrs following an oral bolus of 60 mg arachidonic acid. The rapid increase and subsequent decrease in eicosanoid metabolite excretion after oral administration of arachidonate indicates that the dietary intake of polyunsaturated fatty acids may have a more rapid effect upon the endogenous production of eicosanoids than is generally recognized.     

Substance P and astrocytes: stimulation of the cyclooxygenase pathway of arachidonic acid metabolism

Substance P (SP) may play an important role in the interactions between the nervous system and the immune system. Astrocytes carry receptors for SP on their surfaces. We examined whether ligand-induced receptor activation would lead to the release of arachidonic acid metabolites. SP (10(-10)-10(-8) M) evokes the formation of prostaglandin E and thromboxane B2 in a dose-dependent manner. Structure-activity studies disclosed that the COOH-terminal peptide sequence of SP is primarily responsible for this biological activity. The generation by astrocytes of arachidonate-derived proinflammatory and immunoregulatory compounds in response to SP receptor activation may be relevant to immunoinflammatory responses within the central nervous system and emphasizes the concept of neuroimmunomodulation.     

Thyroid autoregulation. Inhibitory effects of iodinated derivatives of arachidonic acid on iodine metabolism

Thyroid autoregulation has been linked to an organified iodocompound. Since several iodolipids are produced by the gland their possible role in thyroid autoregulation was examined. The following pure synthetic compounds were prepared: 1) 14-iodo-15-hydroxy-5,8,11-eicosatrienoic acid (I-OH-A); 2) its omega lactone (IL-omega); 3) 5-hydroxy-6-iodo-8,11,14-eicosatrienoic acid delta lactone (IL-delta). Their action on iodine metabolism was studied. Iodine uptake was measured in calf thyroid slices. At 10(-4)M I-OH-A caused a 64% decrease in the T/M ratio, while IL-omega inhibited it by 36% and IL-delta was without effect. At 10(-5)M the inhibition was 44% for I-OH-A and 19% for IL-omega, while T3 was without action. A possible isotopic dilution effect was excluded, and no change in iodine efflux was observed. The inhibition by I-OH-A of iodide uptake was observed after only 15 min preincubation. This compound also decreased 125I accumulation in rats. In calf thyroid slices, I-OH-A at 10(-4)M, inhibited PB125I formation by 80%, IL-omega by 62% and IL-delta by 37%. T3 and arachidonic acid were without action. I-OH-A also caused a dose-dependent inhibition of TSH-stimulated iodide organification. The present results demonstrate, for the first time, that iodinated derivatives of arachidonic acid inhibit thyroid function and mimic the effect of iodide on thyroid autoregulation.     

Thyroid autoregulation. Inhibitory effects of iodinated derivatives of arachidonic acid on iodine metabolism

Thyroid autoregulation has been linked to an organified iodocompound. Since several iodolipids are produced by the gland their possible role in thyroid autoregulation was examined. The following pure synthetic compounds were prepared: 1) 14-iodo-15-hydroxy-5,8,11-eicosatrienoic acid (I-OH-A); 2) its omega lactone (IL-ω);3)5-hydroxy-6-iodo-8,11,14-eicosatrienoic acid delta lactone (IL-δ). Their action on iodine metabolism was studied. Iodine uptake was measured in calf thyroid slices. At 10-⁴M I-OH-A caused a 64% decrease in the T/M ratio while IL-ω inhibited it by 36% and IL-δ was without effect. At 10⁻⁵M the inhibition was 44% for I-OH-A and 19% for IL-ω, while T₃ was without action. A possible isotopic dilution effect was excluded, and no change in iodine efflux was observed. The inhibition by I-OH-A of iodine uptake was observed after only 15 min preincubation. This compound alse decreased ¹²⁵I accumulation in rats.In calf thyroid slice, I-OH-A at 10⁻⁴M, inhibited PB¹²⁵I formation by 80%, IL-ω 62% and IL-ω by 37% and arachidonic acid were without action. I-OH-A also caused a dose-dependent inhibition of TSH-stimulated iodide organification.The present results demonstrate, for the first time, that iodinated derivatives of arachidonic acid inhibition thyroid function and mimic the effect of iodine on thyroid autoregulation.     

Lactacystin stimulates arachidonic acid metabolism in rat liver cells: effects of cell density on arachidonic acid release, PGI2 production and cyclooxygenase activity

Lactacystin, an inhibitor of proteasome activity, amplifies prostaglandin I2 production by rat liver cells stimulated by 12-O-tetradecanoylphorbol-13-acetate, transforming growth factor-alpha or interleukin-1. Lactacystin also stimulates the cell's release of arachidonic acid (AA) and increases the cyclooxygenase activity in these cells. In serum deprived cells, the enhanced AA release is reduced, cyclooxygenase activity on exogenous AA is increased and endogenous production of prostaglandin I2 is unchanged. These findings suggest that, in vivo, the ratio of dividing to quiescent cells in a tissue may influence eicosanoid production. The increases in prostaglandin I2 production, AA release and cyclooxygenase activity on exogenous AA resulting from the combined lactacystin and 12-O-tetradecanoylphorbol-13-acetate treatment are inhibited by actinomycin or cycloheximide.     

Identification of an unusual cyclooxygenase metabolite of arachidonic acid in rabbit renal medulla

A renal medulla 100,000g pellet metabolized arachidonic acid, C_20:4, to the previously described prostaglandins prostaglandin E₂, 6-ketoprostaglandin F_1α, thromboxane B₂, 12-hydroxyheptadecatrienoic acid, and 11-hydroxyeicosatetraenoic acid. In addition, under conditions of low enzyme to substrate ratios, the renal medulla also produced an unusual metabolite from arachidonic acid. This metabolite was inhibited by indomethacin, and thus suggested that it was a product of the cyclooxygenase. Addition of GSH to the incubation inhibited its formation, while p-hydroxymercuri-benzoate enhanced its formation. This compound was identified by HPLC purification, uv absorption, and gas chromatography-mass spectroscopy. The compound was 9,15 dioxo,11-hydroxyprosta-5,13-dienoic acid.     

The cyclooxygenase isoforms: structural insights into the conversion of arachidonic acid to prostaglandins

Despite the marked differences in their physiological roles, the structures and catalytic functions of the cyclooxygenase isozymes COX-1 and -2 are virtually identical. Nevertheless, a handful of amino acid substitutions give rise to subtle differences in ligand binding between the two isoforms. These 'small' alterations of isozyme structure are sufficient to allow the design of new, isoform-selective drugs.     

Liquid chromatographic-mass spectrometric determination of cyclooxygenase metabolites of arachidonic acid in cultured cells

A liquid chromatographic-electrospray ionization-mass spectrometric (LC-ESI-MS) technique was developed to simultaneously determine the cyclooxygenase metabolites of arachidonic acid (6-keto-PGF(1alpha), PGD(2), PGE(2), PGF(2alpha), and PGJ(2)) produced by cultured cells. Samples were separated on a C(18) column with water-acetonitrile mobile phase, ionized by electrospray, and detected in the positive mode. Selected ion monitoring (SIM) of m/z 353, 335, 335, 319, and 317 were used for quantifying 6-keto-PGF(1alpha), PGD(2), PGE(2), PGF(2alpha), and PGJ(2), respectively. Prostaglandins were detected at concentrations as low as 1 pg (S/N=3) on the column. The method was used to determine the production of PGs from bovine coronary artery endothelial cells (ECs) and human prostate cancer cells (PC-3) with different degree of invasiveness. Bradykinin (10(-6) M) stimulated a marked increase in the production of 6-keto-PGF(1alpha), PGE(2), and PGF(2alpha) and a small increase of PGD(2) by ECs. 6-Keto-PGF(1alpha) was the major metabolite in these cells. The production of PGE(2) was threefold higher and PGD(2) was twofold higher in PC-3-S (invasive) cells than in PC-3-U (non-invasive) cells.     

Effects of thromboxane synthase and cyclooxygenase inhibition on PAF-induced changes in lung function and arachidonic acid metabolism.

PAF was administered as an intravenous bolus (0.1 micrograms/kg) to eight chronically instrumented awake sheep. The effects of pretreatment with an inhibitor of cyclooxygenase (meclofenamate) on PAF-induced changes in lung function were compared to those observed with a specific inhibitor of thromboxane synthase (DP1904). Each animal was studied four times in varied order: PAF alone, PAF + DP1904, PAF + meclofenamate, and DP1904 alone. Saline alone (control), DP1904 alone, and meclofenamate alone did not cause changes in any of the measured variables. DP1904 and meclofenamate significantly attenuated the PAF-induced fall in lung compliance, elevation in peak pulmonary artery pressure, and increased lung lymph flow. Both drugs abolished the PAF-induced increases in lung lymph thromboxane B2 concentrations. Meclofenamate, but not DP1904, blocked the rise in lymph 6-keto-PGF1 alpha. Although meclofenamate blocked the rise in lymph PGE2, DP1904 resulted in levels 2.7 times higher than PAF alone. We conclude that: (1) inhibition of thromboxane synthase is as effective as inhibition of cyclooxygenase in attenuating PAF-induced changes in lung function, and (2) thromboxane synthase inhibition results in augmented production of PGE2 following PAF administration in vivo.     

Reversed-phase high-pressure liquid chromatography of arachidonic acid metabolites formed by cyclooxygenase and lipoxygenases

High-pressure liquid chromatography is required to resolve the complex mixtures of arachidonic acid metabolites synthesized by many tissues. We have investigated some of the factors which affect the retention times of these substances in reversed-phase HPLC on columns of 5-micron octadecylsilyl silica. There are considerable differences in selectivity between mobile phases based on methanol and those based on acetonitrile, the latter being much better for cyclooxygenase products. The chromatographic behavior of peptidoleukotrienes (LTC4, LTD4, and LTE4) is quite different from that of other arachidonic acid metabolites which do not contain amino acids. Addition of phosphoric acid to the mobile phase results in very long retention times for peptidoleukotrienes. Very low concentrations of trifluoroacetic acid have effects similar to that of phosphoric acid, but as its concentration is raised, the retention times of peptidoleukotrienes decrease, whereas those of other arachidonic acid metabolites are unaffected. Changing the concentration of acetonitrile in the mobile phase also affects the retention times of peptidoleukotrienes differently from those of other metabolites. This information has been used to devise simple linear gradients which separate most of the major cyclooxygenase and lipoxygenase products of arachidonic acid metabolism.     

Conversion of arachidonic acid to cyclooxygenase and lypoxygenase products,and incorporation into phospholipids in the mouse neuroblastoma clone,Neuro-2A

Arachidonic acid (AA) incorporation into phospholipids and cyclooxygenase and lipoxygenase mediated metabolism of arachidonic acid were studied in homogenized and intact Neuro-2A cells. When 3H8-AA was added to homogenized cells and incubated 20 minutes, 39% of the label was converted to prostaglandins (PGs), 10% to hydroxy-eicosatetraenoic acid (HETE) and 26% was incorporated into phospholipids. PGE2 and PGF2a were the major PGs produced. Synthesis of PGs was blocked by 10 microM indomethacin and synthesis of PGs and HETE was blocked by 10 microM eicosatetraynoic acid (ETYA). The cell homogenate produced the 13,14-dihydro-15-keto metabolites of PGE2 and PGF2a from 3H8-AA and also converted exogenous 3H7-PGE2 and 3H8-PGF2a to metabolites. When intact cells were labeled for 24 hours with 14C1-AA and the cells and media then analyzed, 75% of the radioactivity was incorporated into cellular phospholipids, 0.8% was converted to PGs and metabolites and 0.7% converted to HETE. Cells prelabeled for 24 hours were washed and incubated for 30 minutes in fatty acid free media. There was a 23% release of AA from phospholipids. One-fifth of the released AA was converted to HETE. PG synthesis in the intact resting cells was low. In summary, the Neuro-2A cell provides a good model system for studying arachidonic acid metabolism and incorporation into phospholipids in cells of neuronal origin.     

Analysis by high performance liquid chromatography of cyclooxygenase products of arachidonic acid metabolism in plasma of rabbits bearing the VX2 carcinoma

Cyclooxygenase products of arachidonic acid metabolism in the plasma of normal rabbits and animals bearing the VX₂ carcinoma were separated by high performance liquid chromatography and the effluent fractions assayed by serologic methods. The products measured were 6-keto-PGF_lα, thromboxane B₂, PGE₂, PGF_2α, 13,14-dihydro-PGE₂, 13,14-dihydro-15-keto-PGE₂, 15-keto-PGE₂, and 13,4-dihydro-15-keto-PGF_2α. In hypercalcemic, tumor-bearing rabbts, the plasma concentrations o 13,14-dihydro-15-keto-PGE₂ and 13,14-dihydro-15-keto-PGF_2α were markedly elevated (in the range of 0.5 to 16 ng/ml). Previously unmeasured 6-keto-PGF_1α, thromboxape B₂, 13,14-dihydro-PGE₂ and 15-keto-PGE₂ were not found in high concentrations in the plasma of tumor-bearing rabbits. These results add further support to our conclusion that the VX₂ tumor produces hypercalcemia in the host by a mechanism which utilizes PGE₂, rather than a subsequent metabolite of this prostaglandin, as the mediator between the neoplasm and bone.     

Effect of tert-butyl hydroperoxide on cyclooxygenase and lipoxygenase metabolism of arachidonic acid in rabbit platelets

The effect of tert-butyl hydroperoxide (t-BOOH) on the formation of thromboxane (TX) B2, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from exogenous arachidonic acid (AA) in washed rabbit platelets was examined. t-BOOH enhanced TXB2 and HHT formation at concentrations of 8 microM and below, and at 50 microM it inhibited the formation, suggesting that platelet cyclooxygenase activity can be enhanced or inhibited by t-BOOH depending on the concentration. t-BOOH inhibited 12-HETE production in a dose-dependent manner. When the platelets were incubated with 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) instead of AA, t-BOOH failed to inhibit the conversion of 12-HPETE to 12-HETE, indicating that the inhibition of 12-HETE formation by t-BOOH occurs at the lipoxygenase step. Studies utilizing indomethacin (a selective cyclooxygenase inhibitor) and desferrioxamine (an iron-chelating agent) revealed that the inhibitory effect of t-BOOH on the lipoxygenase is not mediated through the activation of the cyclooxygenase and that this effect of t-BOOH is due to the hydroperoxy moiety. These results suggest that hydroperoxides play an important role in the control of platelet cyclooxygenase and lipoxygenase activities.     

Endothelium-derived hyperpolarizing factor in coronary microcirculation: responses to arachidonic acid

In coronary resistance vessels, endothelium-derived hyperpolarizing factor (EDHF) plays an important role in endothelium-dependent vasodilation. EDHF has been proposed to be formed through cytochrome P-450 monooxygenase metabolism of arachidonic acid (AA). Our hypothesis was that AA-induced coronary microvascular dilation is mediated in part through a cytochrome P-450 pathway. The canine coronary microcirculation was studied in vivo (beating heart preparation) and in vitro (isolated microvessels). Nitric oxide synthase (NOS) (N(omega)-nitro-L-arginine, 100 microM) and cyclooxygenase (indomethacin, 10 microM) or cytochrome P-450 (clotrimazole, 2 microM) inhibition did not alter AA-induced dilation. However, when a Ca(2+)-activated K(+) channel channel or cytochrome P-450 antagonist was used in combination with NOS and cyclooxygenase inhibitors, AA-induced dilation was attenuated. We also show a negative feedback by NO on NOS-cyclooxygenase-resistant AA-induced dilation. We conclude that AA-induced dilation is attenuated by cytochrome P-450 inhibitors, but only when combined with inhibitors of cyclooxygenase and NOS. Therefore, redundant pathways appear to mediate the AA response in the canine coronary microcirculation.