Molecular evolution of cyclooxygenase and lipoxygenase.

Four oxygenases of the arachidonic acid cascade (cyclooxygenase, 5-lipoxygenase, 12-lipoxygenase and 15-lipoxygenase) were investigated by the method of computer-assisted sequence comparison. From the calculations, some aspects of evolution and function of these enzymes were revealed. (1) The evolutionary origin of cyclooxygenase was different from that of lipoxygenases. (2) Cyclooxygenase was a distantly related member of a peroxidase family. (3) Enzymes with 12-lipoxygenase activity were created independently twice by gene duplication.     

Arachidonic acid-induced human platelet aggregation independent of cyclooxygenase and lipoxygenase

It is generally agreed that arachidonic acid (20: 4 omega 6) can stimulate platelet aggregation after conversion to prostaglandin G2 and H2 and thence to thromboxane A2. This action is prevented by cyclooxygenase inhibitors. Washed platelets were isolated on metrizamide gradient and resuspended in a Ca2+-free buffer. Their stimulation by C 20: 4 6 was followed by 14C serotonin (5HT) release, thromboxane (TX) synthesis and an increase of light transmission, not dependent on aggregation, accompanied by slight lysis (14%). The addition of extrinsic Ca2+ suppressed lysis and allowed the formation of aggregates. Under these conditions, cyclooxygenase inhibitors such as acetyl salicylic acid, indomethacin or flurbiprofen totally suppressed TX synthesis without preventing platelet aggregation or [14C]-5HT release. Other C 20 polyunsaturated fatty acids could not substitute for C 20: 4 omega 6 in inducing aggregation, and Ca2+ was found to be a prerequisite for protection of the cell against lysis as well as for aggregation in the absence or TX formation. The use of the lipoxygenase inhibitor BW 755 C did not prevent C 20: 4 omega 6-induced aggregation of aspirin-treated platelets, suggesting that the phenomenon was independent of this pathway also. The total suppression of oxidative metabolism with these inhibitors was verified by the analysis of icosanoids using glass capillary column gas chromatography. It is suggested that under these conditions, C 20: 4 omega 6-induced platelet aggregation might be due to an increased membrane permeability to Ca2+ induced by this fatty acid in the absence of oxidation.     

Arachidonic acid-induced human platelet aggregation independent of cyclooxygenase and lipoxygenase

It is generally agreed that arachidonic acid (20:4ω6) can stimulate platelet aggregation after conversion to prostaglandin G₂ and H₂ and thence to thromboxane A₂. This action is prevented by cyclooxygenase inhibitors. Washed platelets were isolated on metrizamide gradient and resuspended in a Ca²⁺-free buffer. Their stimulation by C 20:4 6 was followed by ¹⁴C serotonin (5HT) release, thromboxane (TX) synthesis and an increase of light transmission, not dependent on aggregation, accompanied by slight lysis (14%). The addition of extrinsic Ca²⁺ suppressed lysis and allowed the formation of aggregates. Under these conditions, cyclooxygenase inhibitors such as acetyl salicylic acid, indomethacin or flurbiprofen totally suppressed TX synthesis without preventing platelet aggregation or [¹⁴C]-5HT release. Other C 20 polyunsaturated fatty acids could not substituted for C 20:4ω6 in inducing aggregation, and Ca²⁺ was found to be a prerequesite for protection of the cell against lysis as well as for aggregation in the absence or TX formation. The use fo the lipoxygenase inhibitor BW 755 C did not prevent C 20:4ω6-induced aggregation of aspirin-treated platelets, suggesting that the phenomenon was independent of this pathway also. The total suppression of oxidative metabolism with these inhibitors was verified by the analysis of icosanoids using glass capillary column gas chromatography. It is suggested that under these condition, C 20:4ω6-induced platelet aggregation might be due to an increased membrane permeability to Ca²⁺ induced by this fatty acid in the absence of oxidation.     

Metabolism of 8,11,14,17-eicosatetraenoic acid by human platelet lipoxygenase and cyclooxygenase

Human platelets metabolize 8,11,14,17-eicosatetraenoic acid primarily into 12-hydroxy-8,10,14,17-eicosatetraenoic acid. Several other hydroxy acids were also produced in small amounts via an indomethacin insensitive pathway. Platelet cyclooxygenase metabolized this acid only into 12-hydroxy-8,10,14-heptadecatrienoic acid. It was not possible to detect any cyclic products even though vesicular gland cyclooxygenase metabolizes this (n-3) acid to 17,18-dehydroprostaglandin E1 (Oliw, E.H., Sprecher, H. and Hamberg, M. (1986) J. Biol. Chem. 261, 2675-2683).     

Metabolism of 4,7,10,13,16-docosapentaenoic acid by human platelet cyclooxygenase and lipoxygenase

Washed human platelets are shown to metabolize 4,7,10,13,16-docosapentaenoic acid into three major metabolites which were purified by reverse-phase HPLC. The mass spectra of the methyl ester-trimethylsilyl ether and ethyl ester-trimethylsilyl ether of compound A established it as delta 4-dihomo-thromboxane B2. Compound B was shown to be 14-hydroxy-4,7,10,12-nonadecatetraenoic acid, which is analogous to 12-hydroxy-5,8,10-heptadecatrienoic acid from arachidonic acid. Compound C was produced via an indomethacin-insensitive pathway and was identified as 14-hydroxy-4,7,10,12,16-docosapentaenoic acid. Time- and substrate-dependent studies showed that compounds A,B and C were produced approximately 10,15 and 65% of the extent to which thromboxane B2, 12-hydroxy-5,8,10-heptadecatrienoic acid and 12-hydroxy-5,8,10,14-eicosatetraenoic acid were produced, respectively, from arachidonic acid.     

Cell adhesion property affected by cyclooxygenase and lipoxygenase: Opto-electric approach

Expression of cyclooxygenases (COX) and lipoxygenases (LOX) has been linked to many pathophysiological phenotypes, including cell adhesion. However, many current approaches to measure cellular changes are performed only in a fixed-time point. Since cells dynamically move in conjunction with the cell matrix, there is a pressing need for dynamic or time-dependent methods for the investigation of cell properties. In the presented study, we used stable human colorectal cancer cell lines ectopically expressing COX-1, COX-2, and 15LOX-1, to investigate whether expression of COX-1, COX-2, or 15LOX-1 would affect cell adhesion using our opto-electric methodology. In a fixed-time point experiment, only COX-1- and COX-2-expressing cells enhanced phosphorylation of focal adhesion kinase, but all the transfected cells showed invasion activity. However, in a real-time experiment using opto-electric approaches, transmitted cellular morphology was much different with tight adhesion being shown in COX-2 expressing cells, as imaged by differential interference contrast microscopy (DICM) and interference reflection contrast microscopy (IRCM). Furthermore, micro-impedance measurements showed a continued increase in both resistance and reactance of COX- and LOX-transfected cells, consistent with the imaging data. Our data indicate that both COX- and LOX-expressing cells have strong cell-to-cell and cell-to-substrate adhesions, and that cell imaging analysis with cell impedance data generates fully reliable results on cell adhesion measurement.     

Cigarette smoking stimulates lipoxygenase but not cyclooxygenase pathway in platelets

Male rats were exposed to freshly generated cigarette smoke once daily for 4 to 8 weeks. Inhalation of smoke was verified by elevated level of carboxyhemoglobin. Arachidonate metabolism through lipoxygenase and cyclooxygenase pathways in platelets was determined. Cigarette smoking increased 12-lipoxygenase activity significantly without affecting the cyclooxygenase pathway. In view of platelet-leukocyte interactions and potent chemotactic activity of 12-HETE for aortic smooth muscle cell migration, increased 12-lipoxygenase activity may predispose individuals to atherosclerosis, thromboembolism and emphysema commonly found in smokers.     

Effects of chalcone derivatives on lipoxygenase and cyclooxygenase activities of mouse epidermis

The effects of chalcone derivatives on 12-lipoxygenase and cyclooxygenase of mouse epidermis were investigated. The chalcone derivatives which have 3,4-dihydroxycinnamoyl structure in the molecule, such as 3,4-dihydroxychalcone, 3,4,2'-trihydroxychalcone, 3,4,4'-trihydroxychalcone and 3,4,2'4'-tetrahydroxychalcone, potently inhibited epidermal 12-lipoxygenase activity. Although some of them also inhibited cyclooxygenase activity at relatively high concentrations, the inhibitory effects of these chalcone derivatives on 12-lipoxygenase were 10 times or more potent than their effects on cyclooxygenase. The chalcone derivatives which have cinnamoyl or 4-hydroxycinnamoyl structure, instead of 3,4-dihydroxycinnamoyl structure, in the molecule, showed little or no inhibitory effects on either 12-lipoxygenase or cyclooxygenase activities. The inhibitory effects of chalcone derivatives on 12-lipoxygenase and cyclooxygenase of mouse epidermis are dependent on the particular structure, i.e. 3,4-dihydroxycinnamoyl structure, of the chalcone derivatives.     

Do inhibitors of lipoxygenase and cyclooxygenase block neonatal hypoxic pulmonary vasoconstriction?

Lipoxygenase products have been suggested as mediators of the hypoxic pulmonary pressor response in newborn animals. Data supporting this suggestion are equivocal, since lipoxygenase and leukotriene receptor antagonists that have been used may produce vasodilation because of phosphodiesterase inhibition. We used a leukotriene receptor antagonist L 649923, which appears not to have smooth muscle relaxant activity. L 649923 blocks pressor responses to leukotriene D4 (LTD4) without diminishing the pressor response to hypoxia. Also, BW 755C did not block the pressor response to hypoxia in newborn sheep and goats, whereas the pressor response to LTD4 (75 ng/kg) was depressed significantly. In newborn sheep there was an augmented response to hypoxia with BW 755C, which is consistent with cyclooxygenase inhibition. Finally, the thromboxane receptor antagonist SQ 29548 was investigated in both species. With this agent the pressor response to LTD4 in contrast to that of hypoxia was completely inhibited. We conclude that thromboxanes are involved in the pressor response to LTD4 in newborn lambs and goats. These data do not support the view that leukotrienes are involved in the ovine or caprine neonatal pulmonary pressor response to hypoxia.     

Expression and function of cyclooxygenase and lipoxygenase enzymes in human islets of Langerhans

Arachidonic acid (AA) is generated in pancreatic beta-cells through the activation of Ca2+-dependent cytosolic phospholipase A2 (cPLA2) and the consequent hydrolysis of membrane phospholipids in the sn-2 position of the glycerophospholipid backbone. AA acts as a second messenger in beta-cells to elevate cytosolic Ca2+ levels and stimulate insulin secretion, but it is not clear whether these are direct effects of AA or are dependent on its metabolism by cyclooxygenase (COX) and/or lipoxygenase (LOX) enzymes. In addition, much of the published data in this area have been generated using insulin-secreting cell lines or rodent islets, with very little information on AA generation and metabolism in human islets of Langerhans. This short review examines cPLA2, COX and LOX expression and function in insulin- secreting cell lines and rodent and human islets.     

Significance of follicular cyclooxygenase and lipoxygenase pathways of metabolism of arachidonate in sheep

Concurrent changes in concentrations of a product of the cyclooxygenase (prostaglandin [PG] F2 alpha) and lipoxygenase (leukotriene [LT] B4) routes of metabolism of arachidonic acid were measured by radioimmunoassay within the wall of periovulatory ovine follicles. Increased concentrations of PGF2 alpha were detected within follicles before, during and following the time of ovulation. A significant rise in LTB4 was not observed until after follicular rupture had occurred. Inhibition of synthesis of PGF2 alpha by indomethacin was associated with a complete blockade of ovulation. Nordihydroguaiaretic acid, an inhibitor of 5-lipoxygenase, had no effect on ovulation. Periovulatory administration of either drug did not alter sera profiles of progesterone during subsequent luteal phases. These results reconfirm the importance of the cyclooxygenase system in the mechanism of ovulation. It does not appear that follicular LTB4 is a key component in the processes of ovulation or luteinization in sheep.     

Platelet synthesis of cyclooxygenase and lipoxygenase products in type I and type II diabetes

In 24 type I and 22 type II diabetic patients without vascular complications and in 25 controls platelet thromboxane A2 (TxA2) and prostaglandin E2 (PGE2) production (by radioimmunoassay-RIA) and 1-14C arachidonic acid (AA) metabolism (by high pressure liquid chromatography-HPLC) after thrombin stimulation were studied. Platelets both from type I and type II diabetics generated larger amounts of TxB2 (p less than 0.001) and PGE2 (p less than 0.005) than controls, independently of the presence of retinopathy. No significant differences in platelet AA uptake or metabolism via the cyclooxygenase (CO) route, after thrombin stimulation (5 NIH U/ml), were observed in diabetic patients: lipoxygenase metabolites were found to be slightly, but significantly decreased. A positive linear relationship (r = 0.64, p less than 0.001) was found between HbA-1c and TxB2 production, but not with fasting plasma glucose. These results indicate that metabolic alterations can affect platelet function independently of vascular complications. The absence of alterations in intraplatelet 1-14C AA metabolism via CO, in the presence of increased TxB2 and PGE2 production from endogenous AA, suggests that the activation of CO is not the only possible mechanism of platelet activation and that probably an increased availability of platelet AA plays an important role in the enhanced platelet aggregation commonly found in diabetics.     

Automated extraction and HPLC resolution of lipoxygenase and cyclooxygenase products utilizing high pressure column switching

An automatable column switching technique for extraction of cyclooxygenase and lipoxygenase products has been developed. The extraction column is a 0.4 cm X 3 cm stainless steel column slurry packed with Polygosil (C18, 25-40 mu particle diameter). Supernatants of incubations terminated by the addition of 1 volume MeOH, bath sonicated and centrifuged were pumped onto the extraction column. The non-adherent polar material was eluted to waste with up to five times the sample volume with a high polarity mobile phase. Following this, the extraction column was then eluted to an analytical column with a mobile phase of decreased polarity.     

Effects of cyclooxygenase and lipoxygenase inhibition on basal- and serotonin-induced ion transport in rat colon

The purpose of this study was to determine the effect of a selective cyclooxygenase (COX)-2 inhibitor as compared to non-selective COX and lipoxygenase (LOX) inhibitors in rat colon. Basal- and serotonin (5-hydroxytryptamine, 5-HT)-induced electrogenic ion transport (short circuit current, SCC), prostaglandin E2 (PGE2) release and histological characteristics were measured. Muscle-stripped mucosal sheets of the proximal and distal segment of rat colon were investigated by employing the Ussing chamber technique, radioimmunoassays for PGE2 and light microscopy examinations for control of tissue integrity. 5-HT and PGE2 both induced a concentration-dependent increase in SCC by activation of multiple receptors. The response to 5-HT was bumetanide-sensitive. Neither the non-selective COX inhibitor piroxicam, nor the selective COX-2 inhibitor SC-'236, altered basal- SCC or 5-HT-induced SCC. Indomethacin reduced both basal- and 5-HT-induced SCC in both segments. Nordihydroguaiaretic acid reduced the 5-HT-induced increase in SCC, but did not change basal SCC. 5-HT-induced a concentration-dependent release of PGE2. Only high concentrations of piroxicam and indomethacin reduced basal PGE2 release and 5-HT-induced PGE2 release. Histological examination of the specimens demonstrated only minor changes following mounting in chambers. There were no apparent differences in the morphology following treatment with COX or LOX inhibitors. These results suggest that in rat colon only the COX-1 enzyme is expressed under basal conditions. Furthermore, data suggest neither the COX-1 nor the COX-2 enzyme to be of major importance for 5-HT-induced ion transport in rat colon in vitro. In conclusion, this study supports 5-HT as a mediator of chloride secretion by activating several receptor subtypes and the LOX enzyme, releasing mediators such as leucotrienes.     

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.     

Both cyclooxygenase and lipoxygenase inhibitor partially restore the anorexia by interleukin-1 beta.

Since the peripheral prostaglandin synthetizing system may at least partly involved in the anorexia that follows central interleukin-1 beta (IL-1) administration, this study was undertaken to investigate the effect of ibuprofen (ip), selective cyclooxygenase blocker and AA 861, selective lipoxygenase inhibitor, on changes of food and water intake by a single injection of IL-1 (2 micrograms/rat, ip). We demonstrated that food and water intake were suppressed by peripheral administration of IL-1. Throughout the entire observation periods, suppressed food intake was partially restored to control levels by ibuprofen, while water intake completely restored. In addition, no significant differences about water/food intake were observed in the IL-1 + ibuprofen-treated groups, respectively. In the next experiment, IL-1 induced anorexia was also partially restored to the control level following pretreatment with AA 861. These results may suggest that other mechanism including lipoxygenase blocker besides prostaglandin production may be involved in IL-1 induced anorexia.     

Resveratrol prevents apoptosis in K562 cells by inhibiting lipoxygenase and cyclooxygenase activity.

The natural polyphenolic compound resveratrol (trans-3,4', 5-trihydroxystilbene) is shown to prevent apoptosis (programmed cell death) induced in human erythroleukemia K562 cells by hydrogen peroxide and other unrelated stimuli. Resveratrol reversed the elevation of leukotriene B4 (from 6.40 +/- 0.65 to 2.92 +/- 0.30 pmol.mg protein-1) and prostaglandin E2 (from 11.46 +/- 1.15 to 8.02 +/- 0.80 nmol.mg protein-1), induced by H2O2 challenge in K562 cells. The reduction of leukotriene B4 and prostaglandin E2 correlated with the inhibition of the 5-lipoxygenase activity, and the cyclooxygenase and peroxidase activity of prostaglandin H synthase, respectively. Resveratrol also blocked lipoperoxidation induced by hydrogen peroxide in K562 cell membranes. Resveratrol was found to act as a competitive inhibitor of purified 5-lipoxygenase and 15-lipoxygenase and prostaglandin H synthase, with inhibition constants of 4.5 +/- 0.5 microM (5-lipoxygenase), 40 +/- 5.0 microM (15-lipoxygenase), 35 +/- 4.0 microM (cyclooxygenase activity of prostaglandin H synthase) and 30 +/- 3.0 microM (peroxidase activity of prostaglandin H synthase). Altogether, the results reported here suggest that the anti-apoptotic activity of resveratrol depends on the direct inhibition of the main arachidonate-metabolizing enzymes.