Modulation of human colonic arachidonic acid metabolism by sulfasalazine

The effects of sulfasalazine and its moieties on synthesis of individual products of arachidonic acid metabolism by human colonic mucosa have been investigated. Sulfasalazine inhibited synthesis of the lipoxygenase products. Sulfasalazine and sulfapyridine also inhibited synthesis of thromboxane B2 while enhancing synthesis of prostaglandin (PG) F2 alpha or PGE2, respectively. Inhibition of synthesis of lipoxygenase products and modulation of the profile of cyclooxygenase products could reduce inflammation and enhance mucosal resistance to damage in ulcerative colitis.     

Role of arachidonic acid metabolism in human platelet activation and irreversible aggregation

Previous studies from our laboratory have demonstrated that the aggregation response of platelets inhibited by agents blocking cyclooxygenase activity could be restored to a normal state of sensitivity by prior stimulation of alpha-adrenergic receptors. Since cyclooxygenase activity and thromboxane synthesis are not absolutely required for irreversible platelet aggregation, it is important to define precisely what role this pathway serves in platelet physiology. The present study has evaluated the influence of agents that selectively block arachidonic acid conversion at different steps of synthesis. Inhibition of peroxidase, cyclooxygenase, lipoxygenase, and thromboxane synthetase blocked the second wave response of platelets to several agonists, but did not cause dissociation of aggregates preformed by prior exposure to arachidonate (AA) or adenosine diphosphate. Phospholipase (A2/C) inhibitors, similar to prostaglandin inhibitors, blocked the second wave response of platelets to the action of agonists and, in addition, caused dissociation of aggregates induced by aggregating agents. Results of our study demonstrate that when single agonists are tested at threshold concentrations, products of arachidonate metabolism may play a role in the activation process. However, continued generation of these metabolites does not appear to be essential for the maintenance of irreversible aggregation. When a combination of agents or high concentration of physiological agonists are used, both activation and irreversible aggregation can be secured independent of prostaglandin synthesis or the release reaction.     

The influence of albumin and calcium on human platelet arachidonic acid metabolism

The effects of in vitro changes in calcium and albumin on human platelet arachidonic acid metabolism were evaluated. Hypoalbuminemia enhanced the conversion of released 14C-arachidonic acid from prelabeled platelet phospholipids to the metabolites of the platelet cyclooxygenase and lipoxygenase pathways. This effect was, however, associated with a decreased release of arachidonic acid in the presence of hypoalbuminemia, such that the overall conversion of released 14C- arachidonic acid to platelet thromboxane B2 was similar in the presence of physiologic albumin concentration (3.5 g/dl) or at decreased albumin concentrations of 0.7 and 0.0 g/dl. External calcium was shown to be important for optimal platelet arachidonic acid release, with maximal release occurring at 1 mM calcium.     

Melatonin effect on arachidonic acid metabolism to cyclooxygenase derivatives in human platelets

The effect of melatonin on thrombin-induced [3H]-arachidonic acid (AA) metabolism to cyclooxygenase derivatives was determined in platelets obtained from normal volunteers at 0830 and 2030 h. Percent conversion of radioactive AA was generally greater at 2030 h than at 0830 h for every cyclooxygenase derivative analyzed. Micromolar or greater concentrations of melatonin decreased significantly the conversion of [3H]-AA to prostaglandin (PG) F2 and thromboxane (Tx) B2, and inhibited slightly the conversion to PGE2 and PGD2. After preincubation of platelets with 1 mM imidazole, the melatonin inhibitory effect was significant for PGF2 only. Melatonin (10(-6) M) showed a significant inhibitory influence on platelet ATP release induced by phorbol-12 myristate-13 acetate (PMA) at 2030 h, an effect inhibited by 1 mM aspirin. These results indicate that at pharmacological concentrations melatonin inhibits human platelet cyclooxygenase.     

The endoplasmic reticulum-sarcoplasmic reticulum connection: distribution of endoplasmic reticulum markers in the sarcoplasmic reticulum of skeletal muscle fibers.

The skeletal muscle sarcoplasmic reticulum (SR) was investigated for the presence of well-known endoplasmic reticulum (ER) markers: the lumenal protein BiP and a group of membrane proteins recognized by an antibody raised against ER membrane vesicles. Western blots of SR fractions revealed the presence of BiP in fast- and slow-twitch muscles of the rabbit as well as in rat and chicken muscles. Analyses of purified SR subfractions, together with cryosection immunofluorescence and immunogold labeling, revealed BiP evenly distributed within the longitudinal SR and the terminal cisternae. Within the terminal cisternae BiP appeared not to be mixed with calsequestrin but to be distributed around the aggregates of the latter Ca2+ binding protein. Of the various membrane markers only calnexin (91 kDa) was found to be distributed within both SR subfractions, whereas the other markers (apparent molecular masses of 64 kDa and 58 kDa and a doublet around 28 kDa) were concentrated in the terminal cisternae. These results suggest that the SR is a specialized ER subcompartment in which general markers, such as the ones we have investigated, coexist with the major SR proteins specifically responsible for Ca2+ uptake, storage, and release. The differential distribution of the ER markers reveals new aspects of the SR molecular structure that might be of importance for the functioning of the endomembrane system.     

Effects of oestrogen on progesterone synthesis and arachidonic acid metabolism in human luteal cells

OBJECTIVE Locally produced oestrogens and prostaglandins (PGs) are implicated in the regulation of luteal lifespan in the human ovary. This study (1) assesses direct effects of these factors on progesterone synthesis in isolated luteal cells, and (2) explores interactions between luteal age and treatment with gonadotrophin or oestrogen on the metabolism of arachidonic acid (prostaglandin precursor) by steroidogenic luteal cells in vitro. DESIGN Primary monolayer cultures of human luteal cells obtained at different stages of the luteal phase were used to investigate the effect of oestradiol, catechol oestrogens (2- and 4-hydroxyoestradiol), diethylstilboestrol, PGE₂ and PGF_2x on basal and human chorionic gonadotrophin (hCG) stimulated progesterone production in vitro. The role of PGs as modulators of luteal cell function was further investigated by studying the metabolic fate of radioactively labelled arachidonic acid in hormone treated (oestradiol and hCG) and control cultures, assessed by high performance liquid chromatography. ATIENTS Corpora lutea were enucleated from nine women with regular ovulatory cycles undergoing microsurgical reversal of tubal sterilization. Granulosa cell aspirates were obtained from three patients undergoing in-vitro fertilization treatment. RESULTS PGE₂ and PGF_2α, at various concentrations did not have a consistent effect, whereas oestradiol, diethylstilboestrol (and 2-hydroxyoestradiol in early luteal cell cultures) significantly inhibited basal and hCG stimulated progesterone biosynthesis. Evidence for direct inhibition of 3β-hydroxysteroid dehydrogenase enzymic activity by oestradiol was obtained. Both major metabolic pathways of arachidonic acid (lipoxygenase and cyclo-oxygenase) were operative in steroidogenic luteal cells recovered throughout the luteal phase. The ratio of PGE₂ to PGF₂ synthesis in vitro by human luteal cells from endogenously incorporated arachidonic acid did not change significantly with corpus luteum age, with PGE₂ tending to predominate. Oestradiol treatment shifted arachidonic acid metabolism from the lipoxygenase towards the cyclo-oxygenase pathway in cells isolated from ageing corpora lutea. CONCLUSIONS Oestradiol, at relatively high concentrations, is a potent inhibitor of basal and hCG induced luteal cell steroidogenesis in vitro. No support is provided for the concept that luteolysis is mediated by local production of PGF_2α. The putative luteolytic effect of oestradiol may entail reduced metabolism of arachidonic acid to lipoxygenase derived products by luteal cells rather than direct stimulation of prostaglandin production by itself.     

Hydrogen-peroxide-induced arachidonic acid metabolism in the rat alveolar macrophage

Mounting evidence suggests that reactive oxygen metabolites can initiate the release and metabolism of arachidonic acid (AA). We therefore examined the effects of hydrogen peroxide (H2O2), a biologically relevant oxygen metabolite, on AA release and cyclooxygenase metabolism by the rat alveolar macrophage (AM). At concentrations between 10(-4) and 10(-3) M, which were largely noncytotoxic as assessed by chromium release, H2O2 exposure for 30 min caused a steep dose-dependent increase in AA release that peaked at approximately 5-fold stimulation at 10(-3) M H2O2. AA release induced by H2O2 was inhibited by the H2O2 scavenger catalase, but not by inactivated catalase or by scavengers of superoxide anion, hydroxyl radical, or ferric iron. An evaluation of cyclooxygenase metabolite formation by specific radioimmunoassays and high performance liquid chromatography demonstrated a greater than 2-fold increment in thromboxane (Tx)A2 (measured as TxB2) synthesis at 10(-4) M H2O2, but no increment in prostaglandin (PG) E2 synthesis. H2O2-induced TxB2 synthesis was cyclooxygenase-dependent, since it was inhibited by indomethacin (1 microM). There was no significant degradation of either PGE2 or TxB2 in AM cultures by H2O2 at concentrations to 10(-2) M. The effect of H2O2 on agonist-induced cyclooxygenase metabolism was also examined. H2O2 at 10(-4) M inhibited PGE2 synthesis induced by zymosan and A23187, whereas agonist-induced TxB2 synthesis was either unaffected (zymosan) or augmented (A23187) by H2O2. These findings suggest inhibition by H2O2 of PGE2 synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclooxygenase metabolism of endogenous arachidonic acid by cultured human tracheal epithelial cells

The epithelial cell may contribute to the regulation of pulmonary function during inflammatory diseases of the airways by producing metabolites of arachidonic acid (AA). We have used human tracheal epithelial cells (HTE), grown in serum-free medium, to examine cyclooxygenase metabolism of endogenous AA by these cells. Gas chromatography-negative ion mass spectrometry demonstrated that, regardless of stimulus (buffer, bradykinin, or the calcium ionophore A23187), epithelial cells produce PGE2 and PGF2 alpha but no detectable levels of PGD2, thromboxane B2, 6-keto-PGF1 alpha, or 9 alpha, 11 beta-PGF2. Preincubation of cultures with medium containing 5% human serum led to striking increases in the production of PGE2 and PGF2 alpha, regardless of stimulus. Concomitant with these increases in prostanoids, serum exposure caused a 3.6-fold increase in total cellular arachidonate. Arachidonate levels increased in all phosphoglyceride classes, with the greatest increases in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol. In serum-pretreated cells, PGE2 production was 1.46 +/- 0.12, 4.74 +/- 0.6, and 6.35 +/- 0.93 ng/10(6) cells (mean +/- SEM; n = 7) upon exposure to buffer, 10(-6) M bradykinin, and 1 micrograms/ml A23187, respectively, whereas PGF2 alpha levels were 1.53 +/- 0.22, 4.44 +/- 0.36, and 5.77 +/- 0.78 ng/10(6) cells, respectively. The response of HTE to bradykinin was dose-dependent (10(-8) to 10(-6) M) and was maximal within 5 min. We conclude that cyclooxygenase metabolism of endogenous arachidonate in HTE results in the specific production of PGE2 and PGF2 alpha. HTE in culture retain receptors for bradykinin and can be used to study lipid metabolism independent of other cell types.     

Decreased arachidonic acid release in peripheral blood monocytes of patients with systemic lupus erythematosus

OBJECTIVE: To investigate the release of arachidonic acid (AA) in unfractionated peripheral blood mononuclear cells (PBMC), separated monocytes and T lymphocytes of patients with systemic lupus erythematosus (SLE). METHODS: AA release was measured in cells from 56 patients with SLE and from 48 controls. Of the 56 patients with SLE, 38 were receiving glucocorticosteroids and 18 were not. [3H]AA was incorporated into the membranes of PBMC and purified subsets of monocytes and T lymphocytes. The release of [3H]AA was measured both in nonstimulated cells cultured for 24 h and in cell cultures stimulated by phorbol ester (PMA) and Ca2+ ionophore for 4 h. RESULTS: In the PBMC of SLE patients not taking glucocorticosteroids, the release of AA was decreased in both stimulated and nonstimulated cells. There was a decrease of AA production in monocytes but not in T lymphocytes. This phenomenon could be observed in the active and inactive phases of the disease. CONCLUSION: A defect in AA production may exist in the peripheral monocytes of patients with SLE, resulting in decreased release of AA in patients not receiving glucocorticosteroid therapy.     

Evidence for the generation of hydroxyl radical during arachidonic acid metabolism by human platelets

Reactive oxygen species, probably hydroxyl radicals (OH.), have been suggested to be generated during arachidonic acid (AA) metabolism and, once released, these species can modify the rate and extent of various reactions involved in AA metabolism. We have studied this phenomenon in washed human platelets. OH. generation was quantitated using 14C-benzoic acid as a specific trap in a continuous ionization chamber system. Resting platelets did not produce any detectable signal, whereas addition of AA resulted in gradual OH. production with peak values detected at approximately 20 min. Similar studies conducted under nitrogen or after boiling the platelets almost abolished OH. generation. Aspirin had no significant effect, whereas 5,8,11,14-eicosatetraynoic acid decreased the signal by greater than 90%, thus suggesting that OH. is produced primarily through the lipoxygenase pathway. Superoxide dismutase (SOD) and catalase had no effect and, as expected, phenol and mannitol decreased OH. production considerably, by greater than 50% and 90%, respectively. Azide and cyanide also reduced the OH. generation by about two thirds. We conclude that OH. is generated during AA metabolism by human platelets. It is primarily produced via the lipoxygenase pathway and may require a heme-dependent peroxidase. This highly reactive oxidant may play an important role in normal and abnormal hemostasis.     

Altered renal and platelet arachidonic acid metabolism in cirrhosis

Urinary excretion rates of prostaglandin (PG) E2, PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane (TX) B2 were evaluated in three groups of cirrhotic patients [without ascites (group 1, 13 cases), with ascites and normal renal function (group 2, 15 cases), and with ascites and renal failure (group 3, 5 cases)] and in 14 healthy controls. All urinary arachidonate metabolites were significantly increased in group 2 patients. Patients with renal failure showed lower PGE2, PGF2 alpha, and TXB2 values than those from group 2; PGF2 alpha values were also lower than controls. Platelet TXA2 production during whole blood clotting was significantly reduced in all groups of patients. Administration of low-dose aspirin and sulindac, two cyclooxygenase inhibitors selectively sparing renal cyclooxygenase activity, effectively inhibited platelet TXA2 production without affecting urinary TXB2 excretion, thus ruling out platelets as a possible source of urinary TXB2. We conclude that patients with ascites and normal renal function show an overall activation of the renal PG system. Renal production of vasodilating PGE2 and PGI2 may be involved in supporting renal function in these patients. A reduced platelet synthesis of proaggregatory TXA2 also occurs in cirrhotic patients. This may play a role in the bleeding tendency of cirrhosis.     

Production of prostanoids by rheumatic synovial cells in vitro: effects of anti-inflammatory drugs on arachidonic acid metabolism

Summary To evaluate the role of prostanoids in rheumatoid arthritis the effects of anti-inflammatory drugs on prostanoid concentrations and their ratios were studied in a primary culture of adherent synovial cells from patients with rheumatoid arthritis. Cells from rheumatoid synovium have a great capacity for prostanoid production. PGE₂ is the main prostanoid but synovial cells are also capable of producing 6-keto-PGF₁ and PGF₂. There were also very low TxB₂ concentrations in the culture medium after incubation. All nonsteroidal anti-inflammatory drugs used (diclofenac, indomethacin and tolfenamic acid) reduced markedly in concentrations achieved therapeutically (0.13 mol/l) the production of all the prostanoids from endogenous substrate. There were no differences in the efficacity of the drugs. Hydrocortisone was needed for higher concentrations to inhibit PGE₂, 6-keto-PGF₁ and PGF₂ production. TxB₂ formation remained almost unaltered. After the drug incubation there were also clear alterations in the ratios between these prostanoids, which may have therapeutic importance. It is suggested that this kind of synovial cell culture can be used for testing the effects and mechanisms of different anti-inflammatory drygs in standardized cell culture conditions.     

Inhibition of 5-lipoxygenase pathway of arachidonic acid metabolism in human neutrophils by sulfasalazine and 5-aminosalicylic acid

The possible effect of sulfasalazine, 5-aminosalicylic acid, and acetyl-5-aminosalicylic acid on endogenous arachidonic acid release and metabolism was studied in human polymorphonuclear leukocytes (PMNs). A new in vitro assay was used by which [1-14C]arachidonic acid is incorporated by purified peripheral PMNs until steady state was obtained (5 hr). After preincubation with the test drugs prior to activation with calcium ionophore A23187, the released eicosanoids were isolated by extraction and thin-layer chromatography (TLC) and quantitated by autoradiography and laser densitometry. Median drug concentrations needed for 50% inhibition of leukotriene B4 and 5-hydroxyeicosatetraenoic acid (5-HETE) release was 4-5 mM (range 1-9 mM) for both sulfasalazine and 5-aminosalicylic acid. The acetylated derivative of 5-aminosalicylic acid was ineffective. The present data suggest that inhibition of arachidonic acid lipoxygenation may be an essential action of sulfasalazine and its active metabolite, 5-aminosalicylic acid. Interference with lipoxygenase enzymes, rather than a steroid-like inhibition of arachidonic acid release from intracellular phospholipids, seems to be the mode of action.     

Stimulation of arachidonic acid metabolism in silica-exposed alveolar macrophages

The molecular events involved in both the initiation and development of silicosis are at present poorly defined, although mediators released from macrophages exposed to silica particles are believed to play a role. We have investigated the in vitro production of arachidonic acid (AA) metabolites in adherent bovine alveolar macrophages (BAM) incubated with crystalline silica. BAM were prelabeled with 3H-AA and incubated with 0.5-5.0 mg silica. Lipid metabolites released into the culture medium were analyzed by high-performance liquid chromatography. Simultaneously, lactate dehydrogenase (LDH) was assayed to provide an indication of cell injury. No 5-lipoxygenase metabolites were detected at the lowest silica dose tested (0.5 mg/well), but 5-hydroxyeicosatetraenoic acid (5-HETE) was the major AA metabolite detected between 1.5 and 5.0 mg of silica. A fivefold increase in the production of leukotriene B4 (LTB4) and its two nonenzymatic diastereomers (Isomers I and II) was observed as the silica concentration was increased from 1.0 to 5.0 mg. In contrast, the release of cyclooxygenase products declined with increasing concentrations of silica. LDH release increased in a linear, dose-dependent fashion in the range of silica doses used. The kinetics of eicosanoid release was investigated over a 3-h interval and LDH release was assayed for each time point. Within 15 min following silica addition, a shift to the production of 5-lipoxygenase metabolites was observed, accompanied by a reduction in cyclooxygenase products. This rapid alteration in AA metabolism preceded cell injury as measured by LDH release. These results demonstrate that silica is a powerful stimulator of arachidonic acid metabolism in BAM. Moreover, silica selectively stimulates the 5-lipoxygenase pathway as the dose of silica increases. Our results suggest that dysfunction in arachidonate metabolism could contribute to the pathogenesis of silicosis.     

Endogenous biosynthesis of arachidonic acid epoxides in humans: increased formation in pregnancy-induced hypertension.

Arachidonic acid is metabolized by means of P450 isoenzyme(s) to form epoxyeicosatrienoic acids (EETs) and their corresponding dihydroxy derivatives (DHETs). In the present study, we established the presence in human urine of 8,9-, 11,12-, and 14,15-EETs and their corresponding DHETs by developing quantitative assays and using negative ion, chemical ionization GC/MS and octadeuterated internal standards. Urinary excretion of 8,9- and 11,12-DHET increased in healthy pregnant women compared with nonpregnant female volunteers. By contrast, excretion of 11,12-DHET and 14,15-DHET, but not the 8,9-DHET regioisomer, increased even further in patients with pregnancy-induced hypertension. Intravenous administration of [3H]14,15-EET to three dogs markedly increased its DHET in plasma. The terminal half-life ranged from 7.9-12.3 min and the volume of distribution (3.5-5.3 liters) suggested limited distribution outside the plasma compartment. Negligible radioactivity was detected in urine; this fact infers that under physiological circumstances, urinary DHETs largely derive from the kidney. That P450 metabolites of arachidonic acid are formed in humans supports the hypothesis that these metabolites contribute to the physiological response to normal pregnancy and the pathophysiology of pregnancy-induced hypertension.     

The association of age with the induction of drug-metabolizing enzymes in human monocytes

In vivo studies in man suggest that the enzyme-inducing effect of environmental influences such as drugs or smoking may be reduced in elderly people. We have investigated the basal activity and response to induction of the oxidative enzyme, aryl hydrocarbon hydroxylase (AHH) in human monocytes. Three groups were studied: ten fit young, ten fit elderly and eight frail elderly subjects. Significant induction of AHH activity in response to the hydrocarbon benz(a)anthracene was achieved in all three groups. No impairment of basal AHH activity or in the synthesis of new enzyme protein was noted with age or frailty. There is still no direct evidence of an age-associated fall in drug metabolizing enzyme activities in man.     

Biochemical, immunological, and immunocytochemical evidence for the association of chalcone synthase with endoplasmic reticulum membranes.

Chalcone synthase [naringenin-chalcone synthase; malonyl-CoA:4-coumaroyl-CoA malonyltransferase (cyclizing), E.C. 2.3.1.74], the key enzyme of flavonoid pathways that was believed to be soluble, has been localized on ribosome-bearing endoplasmic reticulum membranes in the epidermis of buckwheat (Fagopyrum esculentum M.) hypocotyls. Enzyme activity measurement and immunoblots of buckwheat hypocotyl homogenates that were fractionated on linear sucrose density gradients and developed with a specific chalcone synthase antibody and a 20-nm ImmunoGold conjugate showed the presence of chalcone synthase in fractions enriched in endoplasmic reticulum membranes. The presence of chalcone synthase on these membranes was not caused by nonspecific adsorption or entrapment of proteins. Immunocytochemical investigations with both a 5-nm and a 20-nm ImmunoGold conjugate showed that chalcone synthase was associated with the cytoplasmic face of rough (ribosome bearing) endoplasmic reticulum membranes. Plasma membrane, nucleus, plastids, mitochondria, golgi, and the tonoplast were not labeled. These data are consistent with our earlier described model suggesting that the synthesis of phenylpropanoids and flavonoids takes place partially or fully on membrane-associated enzyme complexes.     

Phorbol ester-stimulated secretion in chicken ileum: role of arachidonic acid metabolism

Phorbol 12,13-dibutyrate, an activator of protein kinase C, stimulates electrogenic anion secretion in chicken ileum. This effect can be inhibited by the cyclooxygenase inhibitor piroxicam, suggesting arachidonic acid metabolites as mediators. Phorbol 12,13-dibutyrate stimulates increases in prostaglandin E2 release and mucosal 3',5'-cyclic adenosine monophosphate content in intact mucosa. Stimulation of prostaglandin E2 release appears to come mainly from the subepithelial compartment and not from epithelial cells. Unlike its effect on anion secretion, phorbol 12,13-dibutyrate inhibition of sodium uptake by sodium-hydrogen exchange in isolated mature villus enterocytes is not inhibited by piroxicam. This study concludes that phorbol ester effects on anion secretion in chickens appear to be mediated by arachidonic acid metabolites most likely produced by cells of the lamina propria and submucosa, whereas phorbol ester effects on inhibiting upper villus cell sodium-hydrogen exchange may be a direct effect of phorbol esters.