14C-labeled arachidonic acid bioconversion in guinea pig placenta during the last third of gestation

In the present study we investigated the arachidonic acid metabolism in guinea pig placenta during the last third of gestation. Homogenates were incubated with 14C-labeled substrate, and eicosanoid formation was determined using rp HPLC. Arachidonic acid was substantially converted to cyclooxygenase products i.e 6-keto-PGF1 alpha, TxB2, PGF2 alpha, PGE2, PGD2 and 12-HHT. Lipoxygenase activity was also found but of a much lower degree and represented by the mono-hydroxy acids 12-HETE and 15-HETE. The total conversion of arachidonic acid exhibited a progressive rise from day 50 to term, due principally to the increasing part of TxB2, PGE2 and 12-HHT throughout this gestational period and in addition, near term, of 6-keto-PGF1 alpha and PGF2 alpha. These results suggest that there is an increasing concentration and/or activity of cyclooxygenase system enzymes with placental development in guinea pig, which may contribute to the augmented intrauterine availability of prostanoids near parturition. Additional experiments were performed to compare the metabolism of exogenously added 14C-arachidonic acid and endogenously present 12C-arachidonic acid during placental homogenate incubation by means of isotope dilution GC-MS. Although the 14C- and 12C-prostanoid patterns were comparable, the 14C/12C ratios of the prostanoids formed during incubation were significantly different. These data indicate that exogenous arachidonic acid and endogenous arachidonic acid in placental homogenate do not follow up exactly the same metabolic pathway so that the assumption of biochemical identity between exogenous radio-tracer and studied endogenous substrate is not quite true.     

Metabolism of arachidonic acid in sheep uterus: in vitro studies

Arachidonic acid (AA) metabolism in the non-pregnant sheep uterus was studied in vitro using conventional chromatographic and HPLC techniques. High expression of both lipoxygenase (LOX) as well as cyclooxygenase (COX) enzymes and their activities was found in the uterine tissues. On incubation of uterine enymes with AA, the LOX products formed were identified as 5-hydroperoxyeicosatetraenoic acid (5-HPETE), 12- and 15-hydroxyeicosatetraenoic acids (12- and 15-HETEs), based on their separation on TLC and HPLC. By employing differential salt precipitation techniques, the LOXs generating products 5-HPETE (5-LOX), 12-HETE and 15-HETE (12- and 15-dual LOX) were isolated. Based on their analysis on TLC, the COX products formed were identified as prostaglandins - PGF2alpha and prostacyclin derivative 6-keto PGF1alpha. The study forms the first report on the comprehensive analysis on the metabolism of AA in sheep uterus in vitro via the LOX and COX pathways.     

Arachidonic acid metabolism by perfused ram testis

1. Arachidonic acid was metabolized by lipoxygenase and prostaglandin synthetase enzymes systems in the perfused ram testis. 2. The major product of the prostaglandin synthetase was 6-keto-PGF1 alpha (6KF). 3. Addition of testosterone resulted in a significant increase in the 6KF. 4. Arachidonic acid (AA) as well as testosterone penetrated the perfused testis. 5. Both 15-HPETE and 15-HETE, the products of the 15-lipoxygenase enzyme, were detected. 6. Addition of 0.1% BSA changed the pattern of the oxidized arachidonic acid metabolism.     

Formation of 11-hydroxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid in human umbilical arteries is catalyzed by cyclooxygenase

Human umbilical arteries convert arachidonic acid into three hydroxy-eicosatetraenoic acids as well as 6-ketoprostaglandin F1 alpha, prostaglandins E2, F2 alpha and D2 and thromboxane B2. Two of these hydroxy derivatives of arachidonic acid were purified by reverse-phase HPLC and identified by GC-MS as 11-hydroxyeicosatetraenoic acid (11-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) while a third, presumed dihydroxy derivative has not yet been identified. Both the cyclooxygenase and HETE synthesizing activities were found to be localized mainly in the microsomal fraction (100 000 X g pellet) (51 and 61% of total, respectively), and approx. 25% of both activities was found in the 10 000 X g pellet. The formation of these HETEs was inhibited by the cyclooxygenase inhibitors indomethacin and aspirin but not by the lipoxygenase inhibitor nordihydroguaiaretic acid. Production of immunoreactive 15-HETE as well as 6-ketoprostaglandin F1 alpha were also decreased significantly when arterial segments were incubated in the presence of either indomethacin or aspirin. Indomethacin inhibited the formation of both prostanoids and HETEs by microsomes in a concentration-dependent and time-dependent manner. The ID50 values for indomethacin against HETE synthesizing activity and against cyclooxygenase were 4.5 and 3.8 microM, respectively. The inactivation constants were found to be 0.09 and 0.08 min-1 for HETE synthesizing activity and cyclooxygenase, respectively. These two microsomal activities were solubilized in parallel with Tween-20. Incubation with three distinct monoclonal antibodies against different epitopes on cyclooxygenase precipitated both cyclooxygenase and HETE synthesizing activity. Each of these activities was recovered in the immune pellets. These studies demonstrate that in human umbilical arteries 11-HETE, 15-HETE and a presumed di-HETE are the products of cyclooxygenase.     

Possible inhibitory function of endogenous 15-hydroperoxyeicosatetraenoic acid on prostacyclin formation in bovine aortic endothelial cells

Arachidonic acid is metabolized via the cyclooxygenase pathway to several potent compounds that regulate important physiological functions in the cardiovascular system. The proaggregatory and vasoconstrictive thromboxane A2 produced by platelets is opposed in vivo by the antiaggregatory and vasodilating activity of prostacyclin (prostaglandin I2) synthesized by blood vessels. Furthermore, arachidonic acid is metabolized by lipoxygenase enzymes to different isomeric hydroxyeicosatetraenoic acids (HETE's). This metabolic pathway of arachidonic acid was studied in detail in endothelial cells obtained from bovine aortae. It was found that this tissue produced 6-ketoprostaglandin F1 alpha as a major cyclooxygenase metabolite of arachidonic acid, whereas prostaglandins F2 alpha and E2 were synthesized only in small amounts. The monohydroxy fatty acids formed were identified as 15-HETE, 5-HETE, 11-HETE and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The latter two compounds were produced by cyclooxygenase activity. Nordihydroguaiaretic acid (NDGA), a rather selective lipoxygenase inhibitor and antioxidant blocked the synthesis of 15- and 5-HETE. It also strongly stimulated the cyclooxygenase pathway, and particularly the formation of prostacyclin. This could indicate that NDGA might exert its effect on prostacyclin levels by preventing the synthesis of 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a potent inhibitor of prostacyclin synthetase. 15-HPETE could therefore act as an endogenous inhibitor of prostacyclin production in the vessel wall.     

Profiles of eicosanoid production from 14C-arachidonic acid and prostaglandin metabolism by rabbit esophageal mucosa and muscularis

In an attempt to elucidate the possible involvements of eicosanoids in esophageal functions and disorders, we have investigated the formation of both cyclooxygenase and lipoxygenase metabolites from 14C-arachidonic acid by rabbit esophageal tissues. Homogenates of rabbit esophageal mucosa and muscularis were incubated with 14C-arachidonic acid and after ether extraction eicosanoids were separated and quantified by reverse phase high performance liquid chromatography. The predominant cyclooxygenase products were 6-keto-PGF1 alpha, PGF2 alpha, and PGE2 for mucosa and 6-keto-PGF1 alpha, and PGE2 for muscularis. The formation of these products was inhibited both by indomethacin and the dual pathway inhibitor, nordihydrogualaretic acid (NDGA). In mucosa the major eicosanoid was 12-HETE (12-hydroxyeicosatetraenoic acid) which was inhibited by NDGA but not by indomethacin which on the contrary enhanced its formation. Additionally four polar products were synthesized which appeared to be lipoxygenase-dependent as their formation was inhibited by NDGA but not by indomethacin. Muscularis produced as a minor lipoxygenase product only 12-HETE, which was inhibited by NDGA but unchanged in the presence of indomethacin. In addition, both tissues, but mucosa more than muscularis, possessed large prostaglandin catabolizing capacity. The present findings indicate that rabbit esophageal tissues can convert 14C-arachidonic acid into lipoxygenase as well cyclo-oxygenase products which may have a role in esophageal physiology and pathophysiology.     

Indomethacin increases the formation of lipoxygenase products in calcium ionophore stimulated human neutrophils

Arachidonic acid metabolism in human neutrophils stimulated in vitro with the calcium ionophore A23187 was studied using combined HPLC and radioimmunoassays. Indomethacin (0.1 and 1.0 microM) caused a 300% increase in LTB4 formation in neutrophils stimulated with A23187. 5-, 12- and 15-HETE levels were also increased. In the presence of exogenous arachidonic acid 1.0 microM Indomethacin caused a 37% increase in LTB4 formation. Acetyl Salicylic Acid and Ibuprofen had no effect on the formation of lipoxygenase metabolites. The effect of indomethacin on LTB4 formation does not appear to be due to a simple redirection of substrate arachidonic acid from the cyclooxygenase to the lipoxygenase pathways.     

Glomeruli cooperate with macrophages in converting arachidonic acid to prostaglandins and hydroxyeicosatetraenoic acids

The interaction of proliferating macrophages with the glomerulus may play an important role in certain forms of glomerulonephritis. This interaction could involve metabolites of arachidonic acid (C20:4) such as prostaglandins (PG) and lipoxygenase products. We therefore investigated the conversion of exogenous |³H| C20:4 into hydroxyeicosatetraenoic acids (HETE) and PG by isolated glomeruli and macrophages from rats, alone and in combination. As demonstrated by HPLC, glomeruli converted C20:4 predominantly to lipoxygenase products -mainly 12-HETE- and, to a lesser extent, to PG. Resident macrophages converted C20:4 to equivalent amounts of HETE and PG, mainly 12-HETE and 6 keto-PGF_1α. When macrophages and glomeruli were studied in combination, a striking interaction was detected in both pathways of C20:4 metabolism. Production of 6 keto-PGF_1α was stimulated and considerable amounts of TXB₂, PGD₂ and hydroxyheptadecatrienoic acid (HHT) were also produced Total 12-HETE production was unchanged. When a lipid extract of glomeruli, containing oxygenated metabolites of C20:4, was added to macrophages, stimulation of 12-HETE occurred without any change in HHT or PG formation. When, on the contrary, a lipid extract from macrophages was added to glomeruli, 12-HETE production by the glomeruli was nearly completely abolished. Thus the unchanged total 12-HETE production by coincubated glomeruli and macrophages resulted from its increased production by macrophages and its decreased production by glomeruli. These data suggest that interaction between glomeruli and macrophages results in activation of C20:4 metabolism by macrophages followed by inhibition of C20:4 metabolism by glomeruli. Such a regulatory process could play a role in the inflammatory response to immunological injuries during macrophage-dependent human and experimental glomerulonephritis.     

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.     

Arachidonic acid metabolism 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 chromatography 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 arachidonate derivative was stimulated by the calcium ionophore ionomycin. Stimulation of the lipoxygenase pathway by ionomycin was confirmed by thin layer chromatography. In addition, 6-keto-PGF1 alpha, PGF2 alpha, 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.     

Distribution of cyclooxygenase products with cyclooxygenase inhibition in isolated dog lung

Arachidonic acid metabolism can lead to synthesis of cyclooxygenase products in the lung as indicated by measurement of such products in the perfusate of isolated lungs perfused with a salt solution. However, a reduction in levels of cyclooxygenase products in the perfusate may not accurately reflect the inhibition of levels of such products as measured in lung parenchyma. We infused sodium arachidonate into the pulmonary circulation of isolated dog lungs perfused with a salt solution and measured parenchymal, as well as perfusate, levels of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 (PGE2), and thromboxane B2 (TxB2). These studies were repeated with indomethacin (a cyclooxygenase enzyme inhibitor) in the perfusate. We found that indomethacin leads to a marked reduction in perfusate levels of PGF2 alpha, PGE2, 6-keto-PGF1 alpha, and TxB2, as well as a marked reduction in parenchymal levels of 6-keto-PGF1 alpha and TxB2 when parenchymal levels of PGF2 alpha and PGE2 are not reduced. We conclude that, with some cyclooxygenase products, a reduction in levels of these products in the perfusate of isolated lungs may not indicate inhibition of levels of these products in the lung parenchyma and that a reduction in one parenchymal product may not predict the reduction of other parenchymal products. It can be speculated that some of the physiological actions of indomethacin in isolated lungs may result from incomplete or selective inhibition of synthesis of pulmonary cyclooxygenase products.     

Eicosanoid synthesis by alveolar macrophages in rats with malignant mammary tumors: differences in rats treated with and without carrageenan implants

Eicosanoid synthesis by alveolar macrophages (AM), harvested from tumor bearing animals, was measured after tumor inoculation in rats treated with or without carrageenan (carra), an immunomodulating agent. After incubation of the cells with [14]C-arachidonic acid and the Ca-ionophore A23187, samples were measured by high pressure liquid chromatography (HPLC). From the HPLC profiles the lypoxygenase products, 12-hydroxyeicosatetraenoic acid (12-HETE), 15-HETE, and leukotriene-B4 (LTB4) were determined as well as the cyclooxygenase products, prostaglandin (PG)E2, PGF2 alpha and TXB2. After tumor inoculation AM-synthesis of lipoxygenase products tended to increase to values twice those of the base line values, whereas cyclooxygenase products showed subnormal values. In the non treated animals, 10 days after tumor inoculation, statistically significant increases in 12- and 15-HETE, LTB4 and PGE2 were observed when compared with carra treated animals. Later measurements did not show these differences in AM metabolism. AM metabolism was (negatively) correlated with the number of macrophages, which was particularly evident in the correlation with 12-HETE synthesis.     

Synthesis and degradation of eicosanoids in primary rat hepatocyte cultures

Arachidonic acid metabolites may play an important role in liver physiology, yet hepatocyte prostaglandin synthesis has not been characterized extensively. We used RIA to study production and clearance of several eicosanoids in confluent primary cultures of rat hepatocytes in serum-free, hormonally-defined medium. Under basal, unstimulated conditions 6-keto-PGF1 alpha (spontaneous breakdown product of prostacyclin) and 13,14-dihydro-15-keto-PGE (DHK-PGE, a metabolite of PGE) accumulated in the culture medium. Hepatocytes cleared 6-keto-PGF1 alpha, thromboxane B2, and DHK-PGE from the medium. Production of eicosanoids by primary cultures appeared resistant to indomethacin and several other cyclooxygenase inhibitors. This apparent resistance to indomethacin was not caused by rapid metabolism of indomethacin, by failure of the drug to enter hepatocytes, or by insensitivity of hepatocyte cyclooxygenase to the drug. Metabolism of PGE to DHK-PGE may be saturated under in vitro conditions. Hepatocytes can synthesize significant amounts of eicosanoids, although they are probably less active in this regard than are non-parenchymal cells.     

14C-labeled arachidonic acid bioconversion in guinea pig placenta during the last third of gestation

In the presence study we investigated the arachidonic acid metabolism in guinea pig placenta during the last third of gestation. Homogenates were incubated with 14C-labeled subtrate, and eicosanoid formation was determined using rp HPLC. Arachidonic acid was substantially converted to cyclooxygenase products i.e.-keto-PGF_1α, TxB₂, PGF_2α, PGE₂, PGD₂ and 12-HHT. Lipoxygenase activity was also found but of a much lower degree and represented by the mono-hydroxy acid 12-HETE and 15-HETE. The total conversion of arachiodonic acid exhibited a progressive rise from day 50 to term, due principally to the increasing part of TxB₂, PGE₂ and 12-HHT throughout this gestational perid and in addition, near term, of 6-keto-PGF_1α and PGF_2α. The results suggest that there is an increasing concentration and/or activity of cyclooxygenase system enzymes with placenta development in guinea pig, which may contribute to the augmented intrauterine availability of prostanoids under parturition.Additional experiments were performed to compare the metabolism of exogenously added 14C-arachidonic acid and endogenously present 12C-arachidonic acid during placental homogenate incubation by means of isotopes dilution GC-MS. Although the 14C- and 12-C prostanoid patterns were comparable, the 14C/12C ratios of the prostanoids formed during incubation were significantly different. These data indicate that exogenous arachidonic acid and endogenous arachidonic acid in placental homogenate do not follow up extractly the same metabolic pathway so that assumption of biochemical identity between exogenous radio-tracer and studied endogenous substrate is not quite true.     

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

The metabolism of radiolabeled arachidonic acid (AA) by the intact bovine retina in vitro 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, PGF2 alpha, PGE2, PGD2, 6-keto-PGF1 alpha 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 microM), indomethacin (1 microM) 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.     

The production of arachidonic acid metabolites in rat testis.

There is growing evidence that arachidonic acid is oxygenated enzymatically in every cell type and that the oxygenated metabolites regulate a variety of pathological and physiological processes including reproduction. In the present study, the metabolism of arachidonic acid in the testis via cyclooxygenase and lipoxygenase pathways was analyzed. Testicular microsomes showed substantial cyclooxygenase activity as measured by the polarographic method. Analysis of the products on TLC revealed PGF2 alpha (79.5%) as the main product followed by PGE2 (20.3%) and PGD2 (0.17%). At higher substrate concentrations (150 microM), however, 6-keto-PGF1 alpha, the stable metabolite of prostacyclin, was observed in substantial quantities. Maximum activity of lipoxygenase was observed at pH 6.4 in both microsomes and cytosol, the activity being higher in cytosol. Analysis of lipoxygenase pathway products with arachidonic acid as the substrate, revealed the presence of 12-HPETE as the major product both in cytosol and in microsomes. Besides this, 15- and 5-HPETEs were also observed in substantial quantities.     

Involvement of lipoxygenase products of arachidonic acid metabolism in bovine luteal function

A series of experiments was conducted to determine the effects of lipoxygenase products of arachidonic acid (AA) metabolism on the function of the bovine corpus luteum (CL). In the first experiment, reaction products of soybean lipoxidase-AA were added to dispersed bovine luteal cells in increasing concentrations. These lipoxygenase products resulted in a dose-related reduction in the biosynthesis of progesterone and 6-keto-prostaglandin (PG)F1 alpha, while the synthesis of PGF2 alpha was unaffected. In a second experiment, the addition of 5-hydroxyeicosatetraenoic acid (5-HETE), a specific lipoxygenase product, again resulted in a reduction in progesterone and 6-keto-PGF1 alpha, with no change in PGF2 alpha synthesis. Extremely high endogenous concentrations of 5-HETE were measured in luteal tissues (36 +/- 17 to 46 +/- 13 ng/10(6) cells) in a third experiment. In the fourth experiment, an inhibitor of the lipoxygenase pathways, nordihydroguaiaretic acid (NDGA) infused into the uterine lumen twice daily on Days 14-18 of the estrous cycle delayed luteolysis and resulted in lengthened estrous cycles (27.2 +/- 0.3 vs 21.5 +/- 1.0 days for controls, p less than 0.05). Thus, an inhibitor of the lipoxygenase pathway of arachidonic acid metabolism delays luteolysis, possibly by removing the preferential inhibition of PGF1 alpha biosynthesis caused by 5-HETE and other products of the lipoxygenase system. Collectively, these results suggest that products of the lipoxygenase pathway are involved in luteolysis in normal heifers.     

Increase in the formation of leukotriene B4 and other lipoxygenase products in peritoneal macrophages of adrenalectomized rats

The effect of adrenalectomy on the formation of cyclooxygenase and lipoxygenase products by activated peritoneal rat macrophages was determined. After isolation, the cells were incubated with [1-14C]arachidonic acid and the calcium ionophore A23187 and the metabolites isolated by HPLC chromatography. The main components formed in the controls are 6-keto-prostaglandin F1 alpha, thromboxane B2 and 12-HETE. One peak represents 5,12-di-HETE. Smaller amounts of prostaglandin F2 alpha, prostaglandin E2, prostaglandin D2, leukotriene B4 and 15-HETE are also present. After adrenalectomy, a considerable increase occurs in the amounts of leukotriene B4, 15-HETE and 12-HETE. The increase in the prostaglandins is smaller. The compounds formed from endogenous arachidonic acid are also determined. In the cells of the controls, 6-keto-prostaglandin F1 alpha and thromboxane B2 are produced in higher amounts than leukotriene B4. After adrenalectomy, the formation of leukotriene B4 is much more increased than that of 6-keto-prostaglandin F1 alpha. These effects are most probably related to a diminished amount or inactivation of lipocortin, a glucocorticosteroid-induced peptide with phospholipase A2 inhibitory activity in adrenalectomized animals.     

Production of arachidonic acid and linoleic acid metabolites by human bronchoalveolar lavage cells.

Fatty acid-derived inflammatory mediators are considered to play an important role in airway hyperresponsiveness of asthmatic patients. The pulmonary macrophage may be an important source for these mediators in airway tissue. We investigated the metabolism of arachidonic acid and linoleic acid by human bronchoalveolar lavage cells, mainly comprising pulmonary macrophages. Arachidonic was mainly metabolized by 5-lipoxygenase, giving rise to the formation of leukotriene B4 and 5-hydroxy-eicosatetraenoic acid (5-HETE). Linoleic acid was converted to 5 major metabolites, including the 9-hydroxy and 13-hydroxy derivatives, 9- and 13-hydroxy-octadecadienoic acid (9- and 13-HODE). The formation of HODEs could be inhibited by cyclooxygenase inhibitors as well as lipoxygenase inhibitors, indicating that both enzymic species play a role in the generation of HODEs.     

Arachidonic acid metabolism in guinea pig Langerhans cells: studies on cyclooxygenase and lipoxygenase pathways

Epidermal Langerhans cells are macrophage-like la+ leukocytes that are critically involved in cutaneous immune reactions. Because macrophages exert their immunoregulatory activity in part by generation of oxygenated arachidonic acid metabolites, we systematically studied arachidonic acid transformations by purified guinea pig Langerhans cells and compared them with mixed epidermal cells and Langerhans cell-depleted keratinocytes. Products formed from arachidonic acid by cell homogenates were measured after thin-layer or reverse-phase high-pressure liquid chromatographic separation. In addition, leukotriene B4 and C4 formation was assessed in supernatants of Ca ionophore A23187-challenged intact cells by radioimmunoassay. Mixed epidermal cells converted arachidonic acid predominantly via cyclooxygenase and 12-lipoxygenase pathways. The main products were prostaglandin D2 (PGD2) and 12-hydroxyeicosatetraenoic acid (12-Hete), although significant amounts of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha were formed as well. PGD2 synthesis was dependent on the presence of reduced glutathione. The product spectrum formed by Langerhans cell-depleted keratinocytes was virtually indistinguishable from mixed epidermal cells. In contrast, Langerhans cells showed a markedly different metabolism of arachidonic acid. They exhibited an exceedingly high PGD2-generating capacity, whereas only minor amounts of 12-HETE and very low amounts of other prostaglandins were synthesized. The PGD2/12-HETE ratio was 1.22 for mixed epidermal cells and 4.37 for Langerhans cells. Leukotriene production from exogenous or endogenous arachidonic acid could not be demonstrated by either radioenzymatic or radioimmunologic detection methods. We conclude that guinea pig Langerhans cells transform arachidonic acid predominantly to PGD2, which might mediate significant immunoregulatory, inflammatory, and antitumoral activity in the skin.