Arachidonic acid metabolism by isolated epidermal basal and differentiated keratinocytes from the hairless mouse

The metabolism of arachidonic acid was studied using basal and differentiated keratinocytes as well as sebaceous cells isolated from hairless mice. These disassociated cells metabolized arachidonic acid predominantly to the prostaglandin H synthase products prostaglandins E2 and D2. 12-Hydroxyheptadecatrienoic acid (HHT), prostaglandin F2 alpha, thromboxane B2 and 6-ketoprostaglandin F1 alpha were also detected. Smaller amounts of the lipoxygenase products 5-, 12- and 15-hydroxyeicosatetraenoic acids (HETEs) were also detected. The major lipoxygenase product observed was 12-HETE. No leukotrienes or dihydroxy fatty acids were observed. The identity of the metabolites was established using several high-pressure liquid chromatography solvent systems. The biosynthesis of prostaglandins E2 and D2 was very rapid and was inhibited by the addition of indomethacin to the cells. The mixed population of keratinocytes and sebaceous cells were separated into enriched fractions by metrizamide gradients and elutriation techniques. The small, undifferentiated cells had high prostaglandin H synthase and 12-lipoxygenase activity. The basal cell-enriched fractions had the highest activity. With increasing differentiation of the cells, decreased biosynthetic activity was observed. These results indicate that undifferentiated keratinocytes, that is, the basal cells, may be an important source of prostaglandins and 12-HETE but are not a source of leukotrienes for the hairless mouse. It also suggests a role for keratinocyte-derived eicosanoids in the normal physiology of epidermal differentiation.     

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.     

Opposite effects of adrenalectomy on eicosanoid release in rat peritoneal macrophages and spleen

The effect of adrenalectomy on the formation of cyclo-oxygenase and lipoxygenase products by activated peritoneal rat macrophages was determined and compared with that of the spleen. After isolation, the cells and tissues were incubated with [1-14C] arachidonic acid and the Ca-ionophore A23187 and the metabolites isolated by HPLC chromatography. The main components formed in the macrophages of the controls are 6-keto-PGF1 alpha, TxB2 and 12-HETE. One peak represents 5, 12 di HETE. Smaller amounts of PGF2 alpha, PGE2, PGD2, LTB4 and 15-HETE are also present. After adrenalectomy, a considerable increase occurs in the amounts of LTB4, 15-HETE and 12-HETE. The increase in the PG is smaller. The compounds formed from endogenous arachidonic acid are also determined. In the cells of the controls, the formation of LTB4 is considerably increased after adrenalectomy. In the spleen, PGD2 and 12-HETE are decreased after adrenalectomy. The effect of the macrophages is most probably related to a diminished amount or inactivation of lipocortin, a glucocorticosteroid induced peptide with PlA2 inhibitory activity in adrenalectomized animals. In the decrease in formation in the spleen, the absence of the permissive effect of glucocorticosteroids on the hormone-induced lipolysis may play a role.     

Uptake, release and novel species-dependent oxygenation of arachidonic acid in human and animal airway epithelial cells

To determine identities of mediators and mechanisms for their release from pulmonary airway epithelial cells, we examined the capacities of epithelial cells from human, dog and sheep airways to incorporate, release and oxygenate arachidonic acid. Purified cell suspensions were incubated with radiolabeled arachidonic acid and/or ionophore A23187; fatty acid esterification and hydrolysis were traced chromatographically, and oxygenated metabolites were identified using high-pressure liquid chromatography and mass-spectrometry. In each species, cellular uptake of 10 nM arachidonic acid was concentrated in the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine fractions, and subsequent incubation with 5 microM A23187 caused release of 10-12% of the radiolabeled pool selectively from phosphatidylcholine and phosphatidylinositol. By contrast, the products of arachidonic acid oxygenation were species-dependent and in the case of human cells were also novel: A23187-stimulated human epithelial cells converted arachidonic acid predominantly to 15-hydroxyeicosatetraenoic acid (15-HETE) and two distinct 8,15-diols in addition to prostaglandin (PG) E2 and PGF2 alpha. Cell incubation with exogenous arachidonic acid (2.0-300 microM) led to progressively larger amounts of 15-HETE and the dihydroxy, epoxyhydroxy and keto acids characteristic of arachidonate 15-lipoxygenase. Both dog and sheep cells converted exogenous or endogenous arachidonic acid to low levels of 5-lipoxygenase products, including leukotriene B4 without significant 15-lipoxygenase activity. In the cyclooxygenase series, sheep cells selectively released PGE2, while dog cells generated predominantly PGD2. The findings demonstrate that stereotyped esterification and phospholipase activities are expressed at uniform levels among airway epithelial cells from these species, but pathways for oxygenating arachidonic acid allow mediator diversity depending greatly on species and little on arachidonic acid presentation.     

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.     

Release of prostaglandins and thromboxanes by guinea pig isolated type II pneumocytes

Lung cells have been isolated by enzymatic digestion of guinea pig lungs and mechanical dispersion to obtain a suspension of viable cells (approximately 500 X 10(6) cells). Type II pneumocytes have been purified to approximately 92% by centrifugal elutriation (2000 rpm, 15 ml/min) followed by a plating in plastic dishes coated with guinea pig IgG (500 micrograms/ml). We have investigated the arachidonic acid metabolism through the cyclooxygenase pathway in this freshly isolated type II cells (2 x 10(6) cells/ml). Purified type II pneumocytes produced thromboxane B2 (TxB2) predominantly and to a smaller extent the 6-keto prostaglandin PGF1 alpha (6-keto-PGF1 alpha) and prostaglandin E2 (PGE2) after incubation with 10 microM arachidonic acid. The stimulation of pneumocytes with 2 microM calcium ionophore A23187 released less eicosanoids than were produced when cells were incubated with 10 microM arachidonic acid. There was no additive effect when the cells were treated with both arachidonic acid and the ionophore A23187. Guinea pig type II pneumocytes failed to release significant amounts of TxB2, 6-keto-PGF1 alpha and PGE2 after stimulation with 10 nM leukotriene B4, 10 nM leukotriene D4, 10 nM platelet-activating factor, 5 microM formyl-methionyl-leucyl-phenylalanine, 0.2 microM bradykinin and 10 nM phorbol myristate acetate. Our findings indicate that guinea pig type II pneunomocytes possess the enzymatic machinery necessary to convert arachidonic acid to specific cyclooxygenase products, which may suggest a role for these cells in lung inflammatory processes.     

Ultrastructural demonstration of endogeneous peroxidase activity in mammalian epidermis

With the diaminobenzidine method, endogenous peroxidase activity was demonstrated in the nuclear envelope and in the endoplasmic reticulum of non-keratinized keratinocytes and Langerhans cells of the epidermis of the newborn mouse, adult guinea pig and man. In the guinea pig all non-keratinized layers of keratinocytes showed this enzyme activity, whereas in the two other species examined peroxidase activity was limited to the suprabasal layers. The most pronounced activity was found in the Langerhans cells. The melanocytes were negative. With the same method, cytochrome c/cytochrome oxidase activity could be localized in the mitochondria of all epidermal cells of mouse and man, but not in the guinea pig.     

Effect of prostaglandins on cyclic nucleotide levels in cultured keratinocytes.

Ginea pig ear epidermal cells (keratinocytes) were established in primary cultures using trypsin, and treated in their proliferative phase of growth with prostaglandins E1, D1, F1 alpha, E2, D2, or F2 alpha. This phase is induced by the addition of retinoic acid during cell plating. Intracellular content of cAMP and cGMP was measured by radioimmunoassay at various times after treatment. Maximum stimulation of cAMP levels was observed with PGD2, smaller increases with PGE2 and relatively transient rises with PGF2 alpha which were of low significance, but confirm earlier data. Similar results were observed with PGD1, PGE1, and PGF1 alpha with smaller increases. The effects of D and E PGs were biphasic. Significant increases in cGMP were immediately observed with PGD2 and PGE2. With PGF2 alpha, maximum cGMP levels were noted after some delay. All PGs tested showed some effect in elevating cyclic nucleotides in keratinocytes. The most striking result was the increase in cAMP on PGD2 treatment.     

Metabolism of arachidonic acid in isolated glomeruli from pig kidney

Arachidonic acid metabolism in isolated glomeruli from pig kidney was investigated. Arachidonic acid metabolism via cyclooxygenase was studied by three different methodological approaches: radioimmunoassay (RIA), high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). By all these techniques, the major prostaglandins (PG) formed by pig glomeruli appeared to be 6-keto-PGF1 alpha and PGF2 alpha, the former being the most abundant. RIA and GC-MS also detected lower amounts of thromboxane B2 (TxB2) and PGE2. This emphasises the similarity with human glomeruli, in which the main cyclooxygenase product has indeed been reported to be 6-keto-PGF1 alpha. The lipoxygenase activity in isolated pig glomeruli, as studied by HPLC, generated 15-HETE, 12-HETE and 5-HETE. These data demonstrate that isolated glomeruli from pig kidney possess cyclooxygenase as well as lipoxygenase activity. Since a marked functional similarity exists between human and pig kidney, the pig can be regarded as a good model for studying the influence of arachidonic acid metabolites on glomerular pathophysiology.     

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.     

Evidence for inhibition of leukotriene A4 synthesis by 5,8,11,14-eicosatetraynoic acid in guinea pig polymorphonuclear leukocytes

The sensitivity of the 5-lipoxygenase to inhibition by 5,8,11,14-eicosatetraynoic acid (ETYA) is species- and/or tissue-dependent. Guinea pig peritoneal polymorphonuclear leukocytes prelabeled with [3H]arachidonic acid and stimulated with ionophore A23187 formed 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), as well as several dihydroxy fatty acids, including 5(S),12(R)-dihydroxy-6,8,10-(cis/trans/trans)-14-(cis)-eicosatetraenoic acid. ETYA (40 microM) did not inhibit, but, rather, increased the incorporation of 3H label into 5-HETE. In contrast, ETYA markedly inhibited the formation of radiolabeled dihydroxy acid metabolites by the A23187-stimulated cells. Assay of products from polymorphonuclear leukocytes incubated with exogenous arachidonic acid plus A23187, by reverse phase high performance liquid chromatography combined with ultraviolet absorption, showed a concentration-dependent inhibition of the formation of dihydroxy acid metabolite by ETYA (1-50 microM) and an increase in 5-HETE levels (maximum of 2- to 3-fold). The latter finding was verified by stable isotope dilution assay with deuterated 5-HETE as the internal standard. Another lipoxygenase inhibitor, nordihydroguaiaretic acid, potently inhibited the formation of both 5-HETE and dihydroxy acids, with an IC50 of 2 microM. The data suggest that ETYA can inhibit the enzymatic step whereby 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid is converted to leukotriene A4 in guinea pig polymorphonuclear leukocytes.     

The differences in 12-hydroxyeicosatetraenoic acid and thromboxane B2 release from guinea pig platelets.

Anti-12(S)-hydroxyeicosatetraenoic acid (12-HETE)-antibody and anti-thromboxane B2 (TXB2)-antibody were generated and applied to the radioimmunoassay. The detection limit for 12-HETE was 16 pg. The cross-reactivities of anti-12-HETE-antibody were 4.6% for 15-HETE, 0.18% for 5-HETE and below 0.15% for leukotrienes and prostaglandins (PGs). 12-HETE and TXB2 released from guinea pig platelets were measured by radioimmunoassay. Platelet activating factor (PAF) at 10(-9) M induced the aggregation of platelets, the releases of immunoreactive-12-HETE (1.8 +/- 1.2 ng/10(8) platelets, mean +/- S.D.) and immunoreactive-TXB2 (18.5 +/- 17.3 ng/10(8) platelets). Collagen at 1 microgram/ml also evoked platelet aggregation, the releases of immunoreactive-12-HETE (2.7 +/- 1.1 ng/10(8) platelets) and immunoreactive-TXB2 (11.8 +/- 4.6 ng/10(8) platelets). By the stimulation with these compounds, TXB2 was produced in a greater amount than 12-HETE from guinea pig platelets. Although 10(-7) M and 10(-6) M U46619, a TXA2 mimetic, caused platelet aggregation, arachidonic acid metabolites were not released. These data suggest the presence of different mechanisms of platelet activation depending on each stimulus.     

Regulation of macrophage eicosanoid production by hydroperoxy-and hydroxy-eicosatetraenoic acids.

Resident mouse peritoneal macrophages when exposed to zymosan during the first day of cell culture synthesize and secrete large amounts of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4), the respective products of cyclo-oxygenase- and 5-lipoxygenase-catalysed oxygenations of arachidonic acid. Under these conditions of cell stimulation only small amounts of hydroxyeicosatetraenoic acids (HETEs) are concomitantly produced. However, exogenously added arachidonic acid is metabolized to large amounts of 12- and 15-HETE and only relatively small amounts of PGE2. No LTC4 is formed under these conditions. In contrast, resident mouse peritoneal macrophages in cell culture for 4 days synthesized less PGE2 and LTC4 when exposed to zymosan. However, these macrophage populations continue to synthesize 12-HETE from exogenously added arachidonic acid. Zymosan induced the secretion of a lysosomal enzyme, N-acetyl-beta-glucosaminidase, equally in both 1- and 4-day cultures. Both 12- and 15-hydroperoxyeicosatetraenoic acids (HPETEs), the precursors of 12- and 15-HETE, were found to be irreversible inhibitors of the cyclo-oxygenase pathway and reversible inhibitors of the 5-lipoxygenase pathway in macrophages. 15-HETE were found to be reversible inhibitors of both pathways. Thus the oxidation of arachidonic oxidation of arachidonic acid to both prostaglandins and leukotrienes may be under intracellular regulation by products of 12- and 15-lipoxygenases.     

Characterization of 11-HETE and 15-HETE, together with prostacyclin, as major products of the cyclooxygenase pathway in cultured rat aorta smooth muscle cells

Arachidonic acid is the precursor of several potent derivatives that regulate physiological functions in the cardiovascular system. Thromboxane (TXA2) and prostacyclin (PGI2) are synthesized by the cyclooxygenase enzyme. The proaggregatory and vasoconstrictive TXA2 produced by platelets is opposed in vivo by the antiaggregatory and vasodilating activity of PGI2 synthesized by blood vessels. Arachidonic acid is also converted via a 5-lipoxygenase to leukotrienes, the vasoconstrictive components of SRSA. We have shown that this latter pathway is regulated by 15-HETE, a product of the 15-lipoxygenase present in lymphocytes. Confluent cultures of rat aorta smooth muscle cells (RSM) were superfused briefly with [14C]arachidonic acid. The products were isolated and analyzed by thin-layer chromatography-radioautography, high performance liquid chromatography, and gas-liquid chromatography-mass spectrometry. Prostacyclin (PGI2) was identified as the major product both by its biological properties in a platelet aggregation assay and by the mass spectrum of its tetra-trimethylsilylether-methyl ester derivative. Minor quantities of PGE2, PGD2, and PGF2 alpha were also synthesized. Three other compounds with chromatographic properties of mono-hydroxy eicosanoic acids were also formed in major amounts. These were shown to be cyclooxygenase products since their synthesis, together with that of prostacyclin, was blocked by the cyclooxygenase inhibitors aspirin (0.2 mM) and indomethacin (10 microM). Quantities of the hydroxy-eicosanoids were isolated from large scale incubations by silicic acid chromatography. Following methylation and reduction with platinum oxide/H2, the compounds were converted to their trimethylsilylether derivatives and analyzed by gas-liquid chromatography-mass spectrometry. The compounds were identified as 11-hydroxy-5,8,12,14-eicosatetraenoic acid (11-HETE), 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), and hydroxy-5,8,10-heptadeca-trienoic acid (HHT) by simultaneous ion monitoring of characteristic ions at M/e ratios of 287, 258, 229 for 11-HETE and 343, 314, 173 for 15-HETE, and by comparison with the mass spectra of authentic samples. Rat smooth muscle cells, prelabeled by 24-hour incubation with [14C]arachidonic acid, released large amounts of prostacyclin together with enhanced amounts of 11- and 15-HETE in response to physiological levels of thrombin (0.5-5 units/ml). These experiments demonstrate that, in addition to the thromboxane antagonist prostacyclin, vascular smooth muscle cells produce significant quantities of the leukotriene inhibitor 15-HETE via the cyclooxygenase pathway in response to physiological stimuli such as thrombin. The release of both prostacyclin and 15-HETE by vascular smooth muscle cells may thus play an important role in vascular homeostasis.     

Analysis of leukotrienes, prostaglandins, and other oxygenated metabolites of arachidonic acid by high-performance liquid chromatography

High-performance liquid chromatography procedures were developed which separate leukotrienes (LTs), hydroxy-fatty acids (HETEs), prostaglandins (PGs), the stable metabolite of prostacyclin (6-keto-PGF1 alpha), the stable metabolite of thromboxane A2 (TXB2), 12-hydroxyheptadecatrienoic acid (HHT), and arachidonic acid (AA). Two methods employing reverse-phase columns are described. One method uses a radial compression system, the other a conventional steel column. Both systems employ methanol and buffered water as solvents. The radial compression system requires 60 min for separation of the AA metabolites, while the conventional system requires 100 min. Both methods provide good separation and recovery of 6-keto-PGF1 alpha, TXB2, PGE2, PGF2 alpha, PGD2, LTC4, LTB4, LTD4, LTE4, HHT, 15-, 12-, and 5-HETE; and AA. The 5S,12S-dihydroxy-6-trans, 8-cis, 10-trans, 14-cis-eicosatetraenoic acid (5S,12S-diHETE), a stereoisomer of LTB4, coelutes with LTB4. To determine the applicability of the methods to biologic systems, AA metabolism was studied in two models, guinea pig lung microsomes and rat alveolar macrophages. Both HPLC systems demonstrated good recovery and resolution of eicosanoids from the two biological systems. A simple evaporation technique for HPLC sample preparation, which avoids the use of chromatographic and other time-consuming methodology, is also described.     

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.     

Expression of ia antigens by murine keratinizing epithelial langerhans cells

Immunofluorescent and immunoelectron-microscopic staining methods were utilized to investigate the localization of Ia antigens in murine keratinizing epithelia. Approximately 3-5% of epidermal cells were shown to be Ia positive. Only dendritic Langerhans cells in the interfollicular epidermis and outer root sheaths were found to express Ia antigens. These Ia determinants were shown to be controlled by both theI- A andI- EC subregions of theH-2 complex. The results were confirmed by identifying positively stained cells containing Langerhans cell granules at the ultrastructural level. No staining was noted on the surface of keratinocytes, melanocytes, or immigrant lymphocytes. The results presented are in close agreement with those previously reported for Ia-bearing Langerhans cells in human and guinea pig epidermis.     

Ionophore-stimulated lipoxygenase activity and histamine release in a cloned murine mast cell, MC9

A cloned murine mast cell line designated MC9 expresses a 5-lipoxygenase activity when stimulated with the ionophore A23187. Upon addition of 0.5 microM ionophore, MC9 cells produce 270 +/- 43 pmoles 5-HETE, 74 +/- 40 pmoles 5,12 diHETEs and 65 +/- 31 pmoles LTC4/10(6) cells from 37 microM exogenously added [1-14C]arachidonic acid in two minutes. 5-HETE and 5,12-diHETES, including LTB4 were identified by GC/MS whereas LTC4 was confirmed by HPLC mobility, bio-assay, RIA and enzymatic transformation. The principal cyclooxygenase products were PGD2 and TxB2 (8.5 +/- 2.4 and 5.4 +/- 1.2 pmoles/10(6) cells respectively). Prostanoids were identified by comigration with authentic standards on two-dimensional thin layer chromatograms. Production of arachidonic acid lipoxygenase metabolites stimulated with ionophore proved relatively insensitive to removal of extracellular Ca+2 and chelation by EGTA. In addition, MC9 5-lipoxygenase required only low micromolar amounts of exogenous arachidonic acid for maximal activity. Whereas production of arachidonic acid metabolites lasted only two to five minutes, histamine release stimulated with ionophore was not initiated until 5 minutes (12 +/- 3% cellular histamine) and continued for 30 minutes (37 +/- 7% cellular histamine). Although these cells metabolize arachidonic acid differently from the classic peritoneal-derived mast cell, they resemble subpopulations found in certain tissues (such as mucosa) and should be useful in understanding the biochemistry of mast cell mediator release.     

Development of a radioimmunoassay for 15-HETE and its application to 15-HETE production by reticulocytes

Mono-hydroxy-eicosatetraenoic acids (HETE's) are frequently the principal lipoxygenase-derived products in a number of cell types. This paper describes the development of a selective and sensitive radioimmunoassay procedure for 15-HETE, a metabolite which has previously been shown to be both an activator and inhibitor of leukotriene formation in various cells. Initially, rabbits were immunized with 15-HETE conjugated to bovine serum albumin. After seven months, the anti-plasma showed significant binding of tritiated 15-HETE (40-45% binding with a 1:600 dilution of the anti-plasma) which was displaceable by cold 15-HETE. The sensitivity of the assay was approximately 20 pg. of 15-HETE. The anti-plasma exhibited very little (less than 1%) cross-reactivity with arachidonic acid, 5-, 8-, 9-, 11- and 12-HETE's, HHT, TXB2, PGE2 and 6-Keto-PGF1 alpha. Significant cross-reactivity was observed with 5,15-diHETE (53%), 8, 15-diHETE (6.6%), and several other 15-hydroxy-eicosanoids. Rabbit reticulocytes have a very active 15S-lipoxygenase and converted arachidonic acid (final concentration 7 microM) principally to 15-HETE. Unstimulated reticulocytes were found to release negligible amounts of 15-HETE as determined by radioimmunoassay. Treatment of these cells with the calcium ionophore A23187 (0.16 to 4.0 micrograms/ml) elicited a level of 15-HETE release (8 - 14 ng/ml) that was twenty to forty times less than that obtained with exogenous arachidonic acid (2.5 micrograms/ml). The radioimmunoassay reported here may be useful for identifying factors which stimulate cellular release of 15-HETE and other 15-hydroxy-eicosanoids from endogenous arachidonic acid.     

Arachidonic acid metabolic pathway of the rabbit placenta

Placenta microsomes prepared from animals late in gestation (29 days) efficiently metabolize arachidonic acid into PGE2, PGF2 alpha, PGD2, TxA2 and little or no prostacyclin. In contrast to the late gestation placenta, the early (17 day) placental microsomes synthesize primarily PGE2. The cytosolic (100,000 X g supernatant) fraction from early or late gestation placentae converted arachidonic acid, with a calcium dependent enzyme, into non-polar metabolites whose synthesis was inhibited by ETYA but not indomethacin. These metabolites were purified by HPLC and GC-MS analysis indicated the presence of 12-hydroxy-, 15-hydroxy-, and 11-hydroxy-eicosatetraenoic acid. The mitochondrial (8,000 X g pellet) produced PGE2; PGF2 alpha; 12-, 11-, 15-HETE; the C-17 fragment HHT; and the unusual cyclooxygenase metabolite 15-keto-PGE2. These biologically active metabolites may play a vital role in the reproductive function of the placenta.