12-Hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-HPETE stimulate melatonin synthesis in rat pineals

The role of arachidonic acid metabolites in norepinephrine (NE)-induced N-acetyltransferase (NAT) activity and melatonin release was examined from 6 h-incubations of rat pineal glands. A cyclooxygenase inhibitor, indomethacin (5 x 10(-8) - 5 x 10(-6) M) was ineffective on melatonin release, in the presence of absence of NE (5 x 10(-6) M) while a lipoxygenase inhibitor, nordihydroguaiaretic acid (5 x 10(-7) -5 x 10(-5) M) had an inhibitory effect. Among the lipoxygenase metabolites, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-HPETE stimulated both NAT activity and melatonin release in a dose-dependent manner, with a maximal effect occurring at 10(-6) M, while 5-HPETE or hydroxy derivatives of these compounds (12-HETE, 15-HETE and 5-HETE) were ineffective. These results indicate that 12-HPETE and 15-HPETE can be involved in NE-induced melatonin release.     

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.     

Regeneration of vitamin E in human platelets

Human platelets possess active lipoxygenase and cyclooxygenase which convert arachidonic acid to (12S)-12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) plus (12S)-12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and thromboxane B2 plus 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), respectively. When platelet homogenates were incubated with arachidonate, there was a rapid consumption of platelet tocopherol. Time course analysis revealed that within 0.5 min, over half of arachidonate and tocopherol were metabolized. Mass formation of 12-HPETE and 12-HETE or thromboxane B2 and HHT exceeded that of the mass of tocopherol oxidized. Preincubation with the lipoxygenase inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) completely abolished this arachidonate-induced tocopherol oxidation whereas cyclooxygenase inhibitors (indomethacin and aspirin) further potentiated tocopherol oxidation, indicating that this oxidation is closely linked with platelet 12-lipoxygenase activity. Incubation with lipoxygenase metabolites of arachidonic acid showed that only 12-HPETE caused a rapid tocopherol oxidation which was followed by a gradual tocopherol regeneration. By using nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor which is also a strong reductant, over 60% of the arachidonate-induced oxidized tocopherol was regenerated. Tocopherol regeneration declined with increasing oxidation time induced by arachidonate, and after 30-60 min virtually no regeneration could be observed, suggesting that the precursor molecule was unstable. We postulate that the precursor molecule is the tocopheroxyl radical. In the presence of ETYA, a lipoxygenase inhibitor without antioxidant properties, either ascorbate or GSH provided significant tocopherol regeneration. Kinetic studies showed that tocopherol regeneration after the addition of ascorbate was essentially completed by 1 min. By contrast, GSH addition caused a steady increase in tocopherol which peaked after 10 min of its addition. To determine whether this rapid regeneration is chemical or enzymic, regeneration was studied in the presence of chloroform and methanol. Comparison of various reductants in this denaturing condition for enzymes showed that ascorbate and NDGA afforded significant regeneration whereas GSH was ineffective, indicating that there are distinct enzymic and non-enzymic mechanisms for tocopherol regeneration. This study provides direct evidence from mass analysis that tocopherol can be regenerated in human cell homogenates. This finding implies that maintenance of membrane tocopherol status may be an essential function of ascorbate and GSH which operate in concert to ensure maximum membrane protection against oxidative damage.     

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.     

Arachidonic acid metabolim 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 chromtography 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 arachidonic derivative was stimulated by the calcium ionophore ionomycin. Stimulation of lipoxygenase pathway by ionomycin was confirmed by thin layer chromatography. In addition, 6-keto-PGF_1α, PGF_2α, 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.     

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.     

Novel membrane target proteins for lipoxygenase-derived mono(S)hydroxy fatty acids.

Hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecadienoic acids (HODEs) are major bioactive lipids formed via the lipoxygenase oxygenation of arachidonic and linoleic acid, respectively. These metabolites appear to be involved in various cellular actions including cell proliferation, migration and regulation of enzyme activities such as phospholipases and kinases. In view of the diversity of biological effects of these hydroxy fatty acids, it seems likely that multiple mechanisms are involved. Previous reports showed that 15(S)-HETE inhibited the 5-lipoxygenase in rat basophilic leukemia (RBL-1) cell homogenates and established the presence of specific cellular HETE binding sites in these and other cells. The present study used 15(S)-HETE biotin hydrazide and 15(S)-HETE biotin pentyl amide as probes to identify membrane target proteins present in RBL-1 cells that specifically interact with HETEs and HODEs. Two membrane-associated proteins, with apparent molecular weights of 43 and 58 kDa, were identified that specifically interact with these probes and competition experiments indicated that 13(S)-HODE and 15(S)-HETE were the most effective competitors for the hydrazide probe, followed in decreasing effectiveness by 5(S)-HETE, arachidonic acid, 15(R)-HETE, stearic acid and 12(S)-HHT, a cyclooxygenase product. The two proteins were isolated and microsequencing analysis established their identities as actin and the alpha-subunit of mitochondrial ATP synthase, respectively. In vitro binding studies confirmed that purified actin is a potential 15-HETE binding protein. Subcellular cytosolic fractions exhibited fewer protein-probe complexes than membrane fractions. The association of HETEs and HODEs with these cytoskeletal and mitochondrial proteins, respectively, represents a new development in the potential actions of these hydroxy fatty acids.     

Lipoxygenase metabolites of arachidonic and linoleic acids modulate the adhesion of tumor cells to endothelium via regulation of protein kinase C.

12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE) and 13(S)-hydroxyoctadecadienoic acid (13[S]-HODE), lipoxygenase metabolites of arachidonic acid and linoleic acid, respectively, previously have been suggested to regulate tumor cell adhesion to endothelium during metastasis. Adhesion of rat Walker carcinosarcoma (W256) cells to a rat endothelial cell monolayer was enhanced after treatment with 12(S)-HETE and this 12(S)-HETE enhanced adhesion was blocked by 13(S)-HODE. Protein kinase inhibitors, staurosporine, calphostin C, and 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, inhibited the 12(S)-HETE enhanced W256 cell adhesion. Depleting W256 cells of protein kinase C (PKC) with phorbol 12-myristate-13-acetate abolished their ability to respond to 12(S)-HETE. Treatment of W256 cells with 12(S)-HETE induced a 100% increase in membrane-associated PKC activity whereas 13(S)-HODE inhibited the effect of 12(S)-HETE on PKC translocation. High-performance liquid chromatographic analysis revealed that in W256 cells 12-HETE and 13-HODE were two of the major lipoxygenase metabilites of arachidonic acid and linoleic acid, respectively. Therefore, these two metabolites may provide an alternative signaling pathway for the regulation of PKC. Further, these findings suggest that the regulation of tumor cell adhesion to endothelium by 12(S)-HETE and 13(S)-HODE may be a PKC-dependent process.     

Lipoxygenase Pathway of Arachidonic Acid Metabolism in Growth Control of Tumor Cells of Different Type

The influence of inhibitors of different lipoxygenases (LOX) on the growth of human tumor cells with different profiles of synthesized eicosanoids was studied. The studied LOX inhibitors had virtually no influence on the growth of A549 cells actively synthesizing cyclooxygenase and lipoxygenase metabolites of arachidonic acid (AA). The inhibitor of 12-LOX, baicalein, significantly inhibited proliferation in cultures of A431 epidermoid carcinoma cells with a characteristic domination of the major lipoxygenase metabolite of AA, 12-hydroxyeicosatetraenoic acid (12-HETE), in the profile of synthesized eicosanoids and reduced to 70% the incorporation of [3H]thymidine into DNA. Treatment of these cultures with 12-HETE virtually restored the growth potential of the tumor cells. The findings suggest that the lipoxygenase metabolite of AA, 12-HETE, is a growth-limiting factor for tumor cells of definite type.     

Effects of lipoxygenase metabolites of arachidonic acid on the growth of human mononuclear marrow cells and marrow stromal cell cultures.

The effects of various lipoxygenase metabolites of arachidonic acid (AA) were investigated on the growth of freshly isolated human bone marrow mononuclear cells and marrow stromal cell cultures. LTB4, LXA4, LXB4, 12-HETE and 15-HETE (1 microM) decreased [3H]-thymidine incorporation on marrow stromal cell cultures without affecting cell number. Only 12-HETE showed a dose-response effect on [3H]-thymidine incorporation. While LTB4 (1 microM) decreased thymidine incorporation on marrow mononuclear cells, LTC4, LXA4, LXB4, 12-HETE and 15-HETE had no effect. The lipoxygenase inhibitor NDGA had no effect on both cell types suggesting no role of endogenous lipoxygenase metabolites on cell growth. These results suggest no important role of lipoxygenase metabolites of AA on the proliferation of human marrow mononuclear cells and marrow stromal cell cultures.     

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

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

12-Hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-HPETE stimulate melatonin synthesis in rat pineals

The role of arachidonic acid metabolites in norepinephrine (NE)-induced N-acetyltransferase (NAT) activity and melatonin release was examined from 6 h-incubations of rat pineal glands. A cyclooxygenase inhibitor, indomethacin (5×10⁻⁸ − 5×10⁻⁶ M) was ineffective on melatonin release, in the presence of absence of NE (5×10⁻⁶ M) while a lipoxygenase inhibitor, nordihydroguaiaretic acid (5×10⁻⁷ −5×10⁻⁵ M) had an inhibitory effect. Among the lipoxygenase metabolites, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-HPETE stimulated both NAT activity and melatonin release in a dose-dependent manner, with a maximal effect occuring at 10⁻⁶ M, while 5-HPETE or hydroxy derivatives of these compounds (12-HETE, 15-HETE and 5-HETE) were ineffective. These results indicate that 12-HPETE and 15-HPETE can be involved in NE-induced melatonin release.     

Arachidonic acid metabolism in growth control of A549 human lung adenocarcinoma cells

The role of individual eicosanoids of the arachidonic acid (AA) cascade in the growth control of A549 human lung adenocarcinoma cells has been studied. Cyclooxygenase and lipoxygenase metabolites of [¹⁴C]AA incorporated were actively synthesized in the cultures of tumor cells with full confluence unaccomplished. In such cultures inhibitors of AA metabolism (indomethacin and esculetin) and also a lipoxygenase metabolite of AA, 15-hydroxyeicosatetraenoic acid (15-HETE), significantly suppressed the incorporation of [³H]thymidine and biosynthesis of prostaglandin E₂(PGE₂). Other lipoxygenase metabolites of AA (5-HETE and 12-HETE) had no effect on these parameters. The basic fibroblast growth factor (bFGF) had practically no affect on the growth of A549 cells and the PGE₂ production in cultures with 5% fetal calf serum (FCS); however, in the presence of 0.5% FCS this factor significantly increased the number of tumor cells. The growth-stimulating effect of bFGF was completely abolished by a cyclooxygenase inhibitor indomethacin. The data suggest a key role of PGE₂ in the growth control of A549 cells with an active synthesis of cyclooxygenase and lipoxygenase metabolites of AA, its importance in realization of the mitogenic effect of bFGF, and specific features of 15-HETE as a down-regulator of the PGE₂-dependent proliferation.     

Release of prostaglandins and monohydroxy and trihydroxy metabolites of linoleic and arachidonic acids by adult and fetal aortae and ductus arteriosus

The conversion of arachidonic acid (20:4) to prostaglandins by vascular tissue is important in the adult because of the antithrombotic effect of prostacyclin and in the fetus because of the vasodilatory effect of prostaglandin (PG) E2 on the ductus arteriosus. We have shown that vascular tissue converts various polyunsaturated fatty acids to monohydroxy and trihydroxy metabolites derived from hydroperoxides, which may be involved in regulating prostaglandin synthesis. We have now measured the amounts of these hydroperoxide metabolites, as well as those of prostaglandins, released from slices of rat, rabbit and bovine aortae, as well as from fetal calf aorta and ductus arteriosus. The major oxygenated polyunsaturated fatty acid metabolite formed by rat and bovine blood vessels was 6-oxo-PGF1 alpha. Fetal calf aorta and ductus arteriosus produced about five times as much 6-oxo-PGF1 alpha as adult bovine aorta. Much smaller amounts of the cyclooxygenase products, PGE2, 12-hydroxy-5,8,10-heptadecatrienoic acid, 11-hydroxy-5,8,12,14-icosatetraenoic acid (11-hydroxy-20:4), and 15-hydroxy-20:4, were released by aortae. Small amounts of the lipoxygenase product, 12-hydroxy-20:4, were also detected. Substantial amounts of free and esterified monohydroxy and trihydroxy metabolites of linoleic acid (18:2) were detected, especially in rat and rabbit aortae. Rabbit aorta, which had low cyclooxygenase activity, formed more oxygenated 18:2 metabolites than 20:4 metabolites. Indomethacin did not inhibit the formation of the 18:2 metabolites, indicating that cyclooxygenase was not involved. Neither exogenous 13-hydroxy-18:2 nor trihydroxyoctadecenoic acid was incorporated to a large extent into lipids from vascular endothelial or smooth muscle cells, suggesting that the esterified 18:2 oxygenation products had arisen mainly via direct oxygenation of lipids.     

Alterations of intrauterine eicosanoid production in pregnancy-induced hypertension: Decreased production of 12-hydroxyeicosatetraenoic acid in the placenta

The important role of eicosanoids in pregnancy-induced hypertension is generally accepted. Because of the lack of innervation of the uteroplacental vessels, humoral vasoactive factors are important for the regulation of vascular tone. Until now, mainly the balance of vasodilatative and vasoconstrictive prostaglandins has been studied. We were able to confirm their intrauterine imbalance in hypertensive pregnancies. In addition, the placental production of less known lipoxygenase metabolites has been analyzed in this study. Intrauterine tissues (30–100mg wet weight) were examined for their release of eicosanoids. Short term tissue cultures were performed in Hanks balanced salts solution (HBSS) at 37°C in an atmosphere of 95% air/5% CO₂ with and without incorporation of tritiated arachidonic acid. The arachidonate metabolites in culture media were analyzed by High Performance Liquid Chromatography (HPLC) with radioactivity detection or by enzymeimmunoassays or radioimmunoassays, respectively. All intrauterine tissues released more lipoxygenase metabolites than cyclooxygenase metabolites with 12-hydroxyeicosatetraenic acid (12-HETE) as their main metabolite. The placental release of 12-HETE was significantly decreased in hypertensive pregnancies. In hypertensive pregnancies the ratio TXB₂/6-keto-PGF_1α synthesis was increased.Lipoxygenase metabolites, especially 12-HETE, seem to have impotant physiological and pathophysiological functions in the intrauterine compartment. Their biological role in this context needs further investigation.     

Lipoxygenase products regulate IRGpIIb/IIIa receptor mediated adhesion of tumor cells to endothelial cells, subendothelial matrix and fibronectin

Tumor cell adhesion to endothelial cells, subendothelial matrix, and fibronectin is stimulated by the lipoxygenase metabolite of arachidonic acid, 12(S)-HETE, but not by 12(R)-HETE, 5-HETE or 15-HETE. Adhesion is also stimulated by the phorbol ester TPA, an effect inhibited by lipoxygenase but not cyclooxygenase inhibitors. TPA and 12(S)-HETE mediated adhesion is due, in part, to an integrin receptor (i.e., IRGpIIb/IIIa) related to the platelet glycoprotein IIb/IIIa complex and is inhibited by specific monoclonal and polyclonal antibodies against platelet IIb/IIIa. TPA and 12(S)-HETE stimulated adhesion is also inhibited by a lipoxygenase product of linoleic acid; i.e., 13-HODE. These results suggest bidirectional control of tumor cell adhesion by lipoxygenase products of arachidonic acid (increase) and linoleic acid (decrease).     

Synthesis of hydroxyeicosatetraenoic (HETEs) and epoxyeicosatrienoic acids (EETs) by cultured bovine coronary artery endothelial cells

Endothelial cells release several factors which influence vascular tone, leukocyte function and platelet aggregation. Some of these factors are metabolites of arachidonic acid, most notably prostacyclin. However, many of the endothelial metabolites of arachidonic acid have not been positively identified. The purpose of these studies is to identify the arachidonic acid metabolites synthesized by bovine coronary endothelial cells. Cultured bovine coronary artery endothelial cells were incubated with [ ¹⁴C]arachidonic acid. The incubation media was extracted and the radioactive metabolites resolved by a combination of reverse phase- and normal phase-high pressure liquid chromatography (HPLC). The cells synthesized 6-keto prostaglandin (PG)F_1α, PGE₂, 12-hydroxyheptadecatrienoic acid (HHT), 12-, 15-, and 11- hydroxyeicosatetraenoic acids (HETE), and 14,15-, 11,12-, 8,9-, and 5,6-epoxyeicosatrienoic acids (EET). Several of the HETEs were further analyzed by chiral-phase HPLC. The cells synthesized predominately 12(S)-, 15(S)-, and 11(R)-HETE. The synthesis of the S optical isomers of 12- and 15-HETE suggested that the 12- and 15-lipoxygenases were present in these cells. 11(R)-HETE is probably derived from cyclooxygenase. They also synthesized smaller amounts of 9-, 8- and 5-HETEs. The structures of the HETEs and EETs were confirmed by mass spectrometry. The release of 6-keto PGF_1α and 15-HETE was measured by specific radioimmunoassays. Melittin, thrombin, arachidonic acid and A23187 stimulated the release of both eicosanoids in a concentration-related matter. Under all conditions, the release of 6-keto PGF_1α exceed the release of 15-HETE. Therefore, cultured bovine coronary artery endothelial cells synthesize cyclooxygenase, lipoxygenase and cytochrome P-450 metabolites of arachidonic acid.     

Lipoxygenation in rat brain?

It has been previously claimed that rodent brain possesses lipoxygenase activity, based upon the structure of products which were formed from arachidonic acid and the inhibition of this activity by "lipoxygenase inhibitors." Our studies confirm that various positional isomers of hydroxyeicosatetraenoic acids (HETE) are formed (e.g., 15-, 12-, 11-, 9-, 8- and 5-HETE) by brain homogenate and that their production is inhibited by certain lipoxygenase inhibitors, such as nordihydroguaiaretic acid (NDGA) but not by cyclooxygenase or cytochrome P-450 inhibitors. However, stereochemical analysis indicated racemic distributions of these products suggesting that they were not formed by a lipoxygenase enzyme but rather by a peroxidative process. It should also be noted that the presence of 12(S)-lipoxygenase activity could be demonstrated by stereochemical analysis only when the brain was not perfused properly, indicating this activity was due to blood cell contamination. It is known that many lipoxygenase inhibitors are also capable of inhibiting peroxidative reactions apparently due to their free radical scavenging properties. For these reasons, it is essential that the stereochemical purity of purported lipoxygenase products be determined and that previous claims of lipoxygenase activity in mammalian brain be reexamined.     

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.     

Stimulation of progesterone and prostaglandin E2 production by lipoxygenase metabolites of arachidonic acid

The role of several lipoxygenase metabolites of arachidonic acid in the action of luteinizing hormone-releasing hormone (LHRH) on ovarian hormone production was investigated. Like LHRH, treatment of rat granulosa cells with 5-HETE, 5-HPETE, 12-HETE, 15-HETE or 15-HPETE stimulated progesterone (P) and prostaglandin E2 (PGE2) production. 12-HEPE was most potent and stimulated P and PGE2 equally well. By contrast, 5-HETE stimulated P better than PGE2, while 15-HETE was a potent stimulator of PGE2 but not of P. Stimulation of P and PGE2 by LHRH or 12-O-tetradecanoylphorbol 13-acetate (TPA) was further augmented by several HETEs and HPETEs. Like protein kinase C, arachidonic acid metabolites appear to mediate the multiple actions of LHRH in the ovary.