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.     

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.     

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.     

The effect of vitreous humour on prostaglandin production by cultured rabbit chorioretinal fibroblasts

Factors in vitreous humour which regulate prostaglandin production were investigated using cultured rabbit chorioretinal fibroblasts. These cells produced predominantly prostaglandin E2, 6-ketoprostaglandin F1 alpha, a compound likely to be a metabolite of prostaglandin E2 and 5-hydroxyeicosatetraenoic acid. The synthesis of 6-ketoprostaglandin F1 alpha was nearly completely inhibited by the cyclooxygenase inhibitor aspirin and partially inhibited by 10(-6) M dexamethasone (49%) and 10(-5) M forskolin (68%). Addition of 10% rabbit vitreous humour to subconfluent cells maintained in Dulbecco's modified Eagle's medium plus 1% fetal bovine serum resulted in stimulation of 6-ketoprostaglandin F1 alpha production by as much as 246% as measured by radioimmunoassay. Chorioretinal fibroblasts labelled by [3H]arachidonic acid incorporation into cellular phospholipids synthesised greater amounts of all labelled arachidonic acid metabolites in response to vitreous humour. It was concluded, therefore, that there are factors present in vitreous humour of molecular weight above 10 kDa which are capable of stimulating cellular cyclooxygenase activity. Confluent cells also responded to a factor(s) present in vitreous humour. The fraction of less than 10 kDa inhibited 6-ketoprostaglandin F1 alpha production by 50% when used at a concentration of 10%. Furthermore, 6-ketoprostaglandin F1 alpha production in confluent cells (but not subconfluent cells) was inhibited to 40% of control levels by vitamin C at a concentration of 1 mg/100 ml. The latter result points to an inhibitory role for vitamin C in vitreous humour. We conclude, therefore, that vitreous humour contains factors important for the regulation of prostaglandin metabolism in the eye.     

Lipoxygenases in rat embryo tissue

It has previously been reported that rat embryonic tissue produces various prostanoids. This report demonstrates that rat embryo homogenates synthesized various lipoxygenase metabolites, including 12-hydroxyeicosatetraenoic acid (12-HETE) as the major metabolite, 5-HETE, and 15-HETE. The cyclooxygenase product 11-HETE was also formed. Product identification was based on radioimmunoassay and comparison of reverse-phase- and straight-phase-high-pressure liquid chromatography retention times with authentic standards. Additional evidence was the observation that the lipoxygenase inhibitor nordihydroguaiaretic acid inhibited HETE formation. It appears that, under the same (though not necessarily optimal) experimental conditions, lipoxygenase metabolites predominate quantitatively over cyclooxygenase pathway products and that 11-day embryonic tissue produces more HETEs than either 12-day or 13-day embryo homogenates.     

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.     

Arachidonic and linoleic acid metabolism in mouse intestinal tissue: evidence for novel lipoxygenase activity.

Previous studies in our laboratory revealed a high expression of 15-lipoxygenase-1 in human colorectal carcinomas, suggesting the importance of lipoxygenase in colorectal tumor development. In this report, we have investigated the metabolism of arachidonic and linoleic acid by intestinal tissues of Min mice, an animal model for intestinal neoplasia. The polyp and normal tissues from Min mice intestine were homogenized, incubated with arachidonic or linoleic acid, and analyzed by reverse-, straight-, and chiral-phase HPLC. Arachidonic acid was converted to prostaglandins E2 and F2alpha. Little 12- or 15-hydroxyeicosatetraenoic acid was detected. Cyclooxygenase (COX)-2 was detected in polyps and the adjacent normal tissues by Western immunoblotting, but neither COX-1 nor leukocyte-type 12-lipoxygenase, the murine ortholog to human 15-lipoxygenase-1, was detected. These tissue homogenates converted linoleic acid to an equal mixture of 9(S)- and 13(S)-hydroxyoctadecadienoic acid (HODE). Inhibition of lipoxygenase activity with nordihydroguaiaretic acid blocked HODEs formation, but the COX inhibitor indomethacin did not. Degenerative-nested PCR analyses using primers encoded by highly conserved sequences in lipoxygenases detected 5-lipoxygenase, leukocyte-type 12-lipoxygenase, platelet-type 12-lipoxygenase, 8-lipoxygenase, and epidermis-type lipoxygenase-3 in mouse intestinal tissue. All of these PCR products represent known lipoxygenase that are not reported to utilize linoleic acid preferentially as substrate and do not metabolize linoleic acid to an equal mixture of 9(S)- and 13(S)-HODE. This somewhat unique profile of linoleate product formation in Min mice intestinal tissue suggests the presence of an uncharacterized and potentially novel lipoxygenase(s) that may play a role in intestinal epithelial cell differentiation and tumor development.     

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.     

Brain Microvessels Produce 12-Hydroxyeicosatetraenoic Acid

Cerebral microvessels isolated from perfused, adult murine brain produce a compound with the chromatographic properties of a monohydroxyeicosatetraenoic acid when incubated with arachidonic acid or stimulated with calcium ionophore A23187. The formation of this arachidonic acid metabolite is not reduced in the presence of the cyclooxygenase inhibitor ibuprofen, but it is abolished by the lipoxygenase inhibitor nordihydroguaiaretic acid. Analysis by gas chromatography combined with chemical ionization and electron impact mass spectrometry of reduced and nonreduced derivatives of the metabolite, indicate that the compound is 12-hydroxyeicosatetraenoic acid. Fractions of isolated microvessels enriched with capillaries produce 2.1 times more 12-hydroxyeicosatetraenoic acid per microgram of protein than do fractions of microvessels enriched with arterioles. These studies confirm that brain microvessels can produce 12-hydroxyeicosatetraenoic acid and strongly suggest that cerebral endothelia are the primary source of microvessel-derived 12-hydroxyeicosatetraenoic acid. They further suggest that in brain injury, the liberation and accumulation of arachidonic acid in cerebral tissues may lead to the production of 12-hydroxyeicosatetraenoic acid within microvessels. The 12-hydroxyeicosatetraenoic acid formed in this way may mediate some of the blood-brain barrier and cerebrovascular dysfunction that occurs following stroke, brain trauma, or seizures.     

The effects of products and inhibitors of arachidonic acid metabolism on the hamster sperm acrosome reaction

The mammalian sperm acrosome reaction (AR) is a fusion and fenestration of sperm head membranes which is essential for fertilization. Our earlier work demonstrated that arachidonic acid could stimulate the AR 15 min after addition to hamster sperm capacitated by incubation for 4.5 h. The present study was undertaken to determine whether inhibitors of arachidonic acid metabolism could affect the stimulation of the AR by arachidonic acid and whether products of its metabolism could stimulate the AR. Phenidone or nordihydroguaiaretic acid, inhibitors of both the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism, and docosahexaenoic acid, a cyclo-oxygenase pathway inhibitor, inhibited the AR induced by arachidonic acid. PGE2, a product of the cyclo-oxygenase pathway of arachidonic acid metabolism and 5- or 12-hydroxyeicosatetraenoic acid (HETEs) products of the lipoxygenase pathway, stimulated the AR when added to sperm capacitated by incubation for 4.5 h. Prostaglandins not derived from arachidonic were also tested: PGE1 stimulated the AR, but PGF1 alpha and PGA2 did not. We suggest that arachidonic acid metabolites produced by the sperm and by the female reproductive tract are important for the mammalian sperm AR.     

Evidence for a novel pathway of leukotriene formation in human platelets

The metabolism of arachidonic acid via lipoxygenase-catalyzed reactions in washed human platelets was investigated. In addition to the previously discovered lipoxygenase metabolites, 12-hydroxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid, 8,15-dihydroxyeicosatetraenoic acid and 14,15-dihydroxyeicosatetraenoic acid, several other products were formed. The compounds were all dihydroxylated metabolites of arachidonic acid, containing a conjugated triene structure, and identified as 11,12-dihydroxyeicosatetraenoic acid (two isomers) and 5,12-dihydroxyeicosatetraenoic acid (four isomers). The identification was based on ultraviolet spectroscopy and gas chromatography-mass spectrometry of native and hydrogenated compounds. Stereochemical analysis of the hydroxyl groups of the 5,12-dihydroxyeicosatetraenoic acids and experiments with 18O2 indicated that the compounds were formed by the 12-lipoxygenase pathway, probably via an unstable epoxide.     

Diacylglycerol provides arachidonic acid for lipoxygenase products that mediate angiotensin II-induced aldosterone synthesis

The lipoxygenase products of arachidonic acid metabolism have been shown to be important mediators of stimulus secretion coupling in various endocrine tissues. We have recently shown that the 12-lipoxygenase product, 12-hydroxyeicosatetraenoic acid plays a key role as a new specific mediator of angiotensin II-induced aldosterone secretion in the adrenal. In view of the several pathways by which cellular arachidonate can be generated and the important role of diacylglycerol in angiotensin II-responses, we studied the role of diacylglycerol as the source of arachidonic acid for 12-hydroxyeicosatetraenoic formation. Treatment of normal human adrenal glomerulosa cells with the selective diacylglycerol-lipase inhibitor, RHC 80267, resulted in a dose-dependent inhibition of angiotensin II-induced aldosterone as well as 12-hydroxyeicosatetraenoic formation. These results suggest that AA derived from diacylglycerol is the precursor of 12-hydroxyeicosatetraenoic involved in angiotensin II-induced aldosterone secretion. These results reveal a new second messenger role for diacylglycerol in addition to activation of protein kinase C.     

Rat hypothalamic corticotropin-releasing hormone secretion in vitro is stimulated by interleukin-1 in an eicosanoid-dependent manner

We studied the effect of interleukin-1 alpha (IL-1) on corticotropin-releasing hormone (CRH) secretion by explanted rat hypothalami in vitro. We also assessed possible mediation of arachidonic acid metabolites on IL-1-stimulated CRH secretion, by preincubating hypothalami with the cyclooxygenase inhibitor indomethacin (INDO, 1 microM), the lipoxygenase and cyclooxygenase inhibitor eicosatetraynoic acid (ETYA, 10 microM), or the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, up to 30 microM). In additional experiments, prostaglandins (PG) E2 and F2 alpha were added to the cultures treated with INDO or ETYA. Finally, we investigated the effect of dexamethasone (DEX) on IL-1-stimulated CRH secretion. IL-1 stimulated immunoreactive CRH (iCRH) secretion by explanted hypothalami in a concentration-dependent fashion. Both INDO and ETYA inhibited IL-1-(10nM)-stimulated iCRH secretion, whereas NDGA did not have any effect. The addition of PGF2 alpha (10 nM) restored the secretion of iCRH inhibited by INDO. DEX treatment significantly inhibited IL-1-stimulated iCRH release. Our results suggest that the stimulatory effect of IL-1 on the hypothalamic CRH neuron is mediated by the cyclooxygenase metabolites of arachidonic acid, and, among others, by PGF2 alpha.     

Arachidonic acid metabolic pathways in the rabbit pericardium

Minced rabbit pericardium actively converts [1-14C]arachidonic acid into the known prostaglandins (6-[1-14C]ketoprostaglandin F1 alpha, [1-14C]prostaglandin E2 and [1-14C]prostaglandin F2 alpha) and into several unidentified metabolites. The major metabolite was separated by C18 reverse-phase high-pressure liquid chromatography (HPLC) and identified by gas chromatography-mass spectrometry (GC-MS) to be 6,15-[1-14C]diketo-13,14-dihydroprostaglandin F1 alpha. The other nonpolar metabolites were 15-[1-14C]hydroxy-5,8,11,13-eicosa-tetraenoic acid (15-HETE), 11-[1-14C]hydroxy-5,8,12,14-eicosatetraenoic acid (11-HETE) and 12-[1-14C]hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). Arachidonic acid metabolites actively produced by the pericardium could influence the tone of surface blood vessels on the myocardium.     

Decreased rate of metabolism induced by a shift of the double bond in prostaglandin F 2alpha from the delta5 to the delta4 position.

The synthesis of an isomer of prostaglandin F 2alpha,9alpha,11alpha,15(S)-trihydroxyprosta-4-cis,13-transdienoic acid is described. The metabolism of this compound in the rat has been investigated. The rate of degradation by beta-oxidation was slowed down considerably. Thus 10-20% of the injected isomer was excreted in the urine unchanged indicating a longer half-life in the circulation than for prostaglandin F 2alpha. More over 2% was excreted as C20 metabolites, 11-18% as C18 metabolites and 8-15% as C16 metabolites. This relative resistance to degradation by beta-oxidation is of considerable biochemical and pharmacological interest.     

The effects of lipoxygenase metabolites of arachidonic acid on human myometrial contractility

The effects of the lipoxygenase products of arachidonic acid, 5- and 12-hydroxyeicosatetraenoic acid (5- and 12-HETE) and leukotriene B4 (LTB4), on the spontaneous contractility of lower uterine segment human myometrial strips obtained prior to labour have been studied in vitro. 5-HETE gave a dose- dependent (10-500ng) increase in both the rate of contractions and overall contractility of myometrial strips while 12-HETE and LTB4 had no effect at the same concentrations. Prostaglandin F2 alpha (50ng) contracted all myometrial strips in a similar pattern to 5-HETE but was approximately 10 times more potent. The effect of 5-HETE may be direct or perhaps indirect via interaction with the cyclo-oxygenase pathway. The findings do not disprove the contention that the onset of parturition may be characterised by a switch in arachidonic acid metabolism in intra-uterine tissues from lipoxygenase to cyclo-oxygenase products.     

Measurement of prostaglandin G/H synthase and lipoxygenase activity in the stomach wall by HPLC

A method for the determination of prostaglandin G/H synthase and lipoxygenase activities in tissues was developed and employed with rat gastric mucosa samples. Tissues and microsomes were incubated in a buffer containing nonionic detergent and 1.32 mM arachidonic acid for 10 min. Following extraction with ethyl acetate, the oxidation products of arachidonic acid were derivatized with panacyl bromide. A reversed-phase column and a quaternary mobile phase were used to separate and quantitate the panacyl bromide esters of prostaglandin E2 and leukotriene C4/D4. Prostaglandin G/H synthase and lipoxygenase activities were determined in gastric mucosa and were 371 +/- 66 and 173 +/- pg/mg/min, respectively.     

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.     

Leukotriene synthesis by human gastrointestinal tissues

The prostaglandin and leukotriene synthesizing capacity of human gastrointestinal tissues obtained at surgery was investigated using radioimmunoassay for prostaglandin E2, leukotriene B4 and sulfidopeptide leukotrienes. The leukotriene immunoassay data were validated by high-pressure liquid chromatography (HPLC). During incubation at 37 degrees C, fragments of human gastric, jejuno-ileal and colonic mucosa released considerably larger amounts of prostaglandin E2 than of leukotriene B4 and sulfidopeptide leukotrienes. Gastrointestinal smooth muscle tissues released even larger amounts of prostaglandin E2, but smaller amounts of leukotrienes than the corresponding mucosal tissues. Adenocarcinoma tissue released larger amounts of leukotriene B4, sulfidopeptide leukotrienes and prostaglandin E2 than normal colonic mucosa. Ionophore A23187 (5 micrograms/ml) did not stimulate release of prostaglandin E2 from any of the tissues investigated, but enhanced release of leukotriene B4 and sulfidopeptide leukotrienes. HPLC analysis demonstrated that immunoreactive leukotriene B4 co-chromatographed almost exclusively with standard leukotriene B4, while immunoreactive sulfidopeptide leukotrienes consisted of a mixture of leukotrienes C4, D4 and E4. Leukotriene synthesis by human gastrointestinal tissues was inhibited by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) and the dual enzyme inhibitor BW755C (3-amino-1-(trifluoromethylphenyl)-2-pyrazoline hydrochloride). Synthesis of prostaglandin E2 was inhibited by the cyclooxygenase inhibitor indomethacin as well as by BW755C. Incubation of gastrointestinal tissues in the presence of glutathione decreased the amounts of leukotrienes D4 and E4, while release of leukotriene C4 was simultaneously increased. On the other hand, incubation of tritiated leukotriene C4 with incubation media from human gastric or colonic mucosa resulted in conversion of the substrate to [3H]leukotriene D4 and [3H]leukotriene E4. The results indicate the capacity of human gastrointestinal tissues to synthesize the 5-lipoxygenase-derived products of arachidonate metabolism, leukotriene B4 and sulfidopeptide leukotrienes, in addition to larger amounts of prostaglandin E2. Furthermore, considerable activities of the sulfidopeptide leukotriene-metabolizing enzymes gamma-glutamyl transpeptidase and dipeptidase were detected in human gastrointestinal tissues. These enzymes might play an important role in biological inactivation and/or change of biological profile of sulfidopeptide leukotrienes generated in the human gastrointestinal tract.     

12-lipoxygenase in porcine coronary microcirculation: implications for coronary vasoregulation

Noncyclooxygenase metabolites of arachidonic acid (AA) have been proposed to mediate endothelium-dependent vasodilation in the coronary microcirculation. Therefore, we examined the formation and bioactivity of AA metabolites in porcine coronary (PC) microvascular endothelial cells and microvessels, respectively. The major noncyclooxygenase metabolite produced by microvascular endothelial cells was 12(S)-hydroxyeicosatetraenoic acid (HETE), a lipoxygenase product. 12(S)-HETE release was markedly increased by pretreatment with 13(S)-hydroperoxyoctadecadienoic acid but not by the reduced congener 13(S)-hydroxyoctadecadienoic acid, suggesting oxidative upregulation of 12(S)-HETE output. 12(S)-HETE produced potent relaxation and hyperpolarization of PC microvessels (EC(50), expressed as -log[M] = 13.5 +/- 0.5). Moreover, 12(S)-HETE potently activated large-conductance Ca(2+)-activated K(+) currents in PC microvascular smooth muscle cells. In contrast, 12(S)-HETE was not a major product of conduit PC endothelial AA metabolism and did not exhibit potent bioactivity in conduit PC arteries. We suggest that, in the coronary microcirculation, 12(S)-HETE can function as a potent hyperpolarizing vasodilator that may contribute to endothelium-dependent relaxation, particularly in the setting of oxidative stress.