Stimulation of bone resorption by lipoxygenase metabolites of arachidonic acid

We have studied the effect of leukotrienes, (LT): B₄, C₄, D₄ and E₄ and the hydroxyeicosatetraenoic acids (HETEs) 5-HETE and 12-HETE on bone respiration . Resorption was measured by colorimetric assay of calcium released from neonatal mouse calvaria maintained in organ culture for 72h. All the LTs and HETEs stimulated bone resorption, with optimum responses at picomolar or nanomolar concentrations. The responses were biphasic, with a decreasing effect at higher concentrations. In contrast, prostaglandin E₂ (PGE₂) stimulated resorption only at 10nM and above. Indomethacin partially inhibited resorption by LTB4, LTC4 and LTD4, but did not affect resorption stimulated by LTE4, 5-HETE nd 12-HETE. These results indicate that liposygenase products of arachidonic acid are highly potent bone resorbing factors and may play an important role in the localised bone loss associated with inflammatory lesions.     

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.     

Stimulation of gonadotropin release by arachidonic acid and its lipoxygenase metabolites in superfused pituitary cells

Luteinizing hormone and follicle stimulating hormone secretion was stimulated by 4 min pulses of arachidonic acid (3 X 10(-5) to 10(-4)M) in superfused rat pituitary cells. The effect of its lipoxygenase metabolites, 5-hydroxy-6,8,11,14-eicosatetranoic acid (5-HETE) and 15-hydroxy-5,8,10,14-eicosatetranoic acid (15-HETE) was more potent on hormone release when added in the same dose. Using 3 X 10(-5)M 5-HETE, its releasing activity on gonadotropins was comparable to that of GnRH (10(-9)M). 15-HETE (3 X 10(-5)M) was even more potent on LH and FSH secretion than 5-HETE. The secretory profile induced by 5-HETE and 15-HETE was also similar to that shown for GnRH, resulting in a rapid increase and a more prolonged decline of the hormone release. The addition of these fatty acids to superfused pituitary cells did not alter the response of the cells to their physiological ligand. These findings give further support to the proposal that metabolites of arachidonic acid may be involved in receptor-mediated mechanisms of gonadotropin release in pituitary cells.     

Preparation of oxygen-18-labeled lipoxygenase metabolites of arachidonic acid

Plasma pseudocholinesterase and porcine liver esterase were used to catalyse the incorporation of the stable isotope oxygen-18 into the carboxyl moiety of lipoxygenase metabolites of arachidonic acid. This simple method produces eicosanoid products containing two oxygen-18 atoms; but the enzymes studied were found to display large substrate specificity in the efficiencies at which oxygen-18 could be incorporated into the lipoxygenase metabolites. Furthermore, [18O2]LTB4 was found not to back exchange during in vitro incubation with human neutrophils. The methods involved for stable isotope incorporation are simple, efficient and produce highly enriched species in a short time. By varying the type of esterase, the amount of esterase or the length of incubation highly enriched species of all eicosanoids tested could be prepared.     

A role for lipoxygenase metabolites of arachidonic acid in porcine ovulation

Prostaglandins, products of arachidonic acid via the cyclooxygenase pathway, are essential to the porcine ovulatory process in that inhibition of their synthesis results in ovulation failure. Studies in the rat have shown that ovulation is also preceded by a rise in three ovarian hydroxyeicosatetraenoic acids, products of the lipoxygenase pathway, and inhibition of this pathway also inhibits ovulation. Experiments were designed, using a pregnant mare serum gonadotropin/human chorionic gonadotropin (hCG)-treated prepuberal gilt model, to measure pre-ovulatory changes in follicular fluid concentrations of 15-hydroxyeicosatetraenoic acid (15-HETE), and to compare the effects of indomethacin and nordihydroguaiaretic acid (NDGA) on ovulation in the pig and on 15-HETE and prostaglandin F₂α synthesis both in vivo and in vitro. Follicular fluid concentrations of 15-HETE were elevated significantly just prior to the expected time of ovulation (40 h after hCG). When indomethacin (10 mg) was injected into the ovarian stalk at 24 h after hCG, follicular fluid concentrations of both 15-HETE and prostaglandin F₂α were lower (P<0.01) than controls at 40 h and ovulation rate was suppressed (P<0.01). When NDGA (5 mg) was administered in the same manner, ovulation rate was suppressed (P<0.01), but the levels of 15-HETE and prostaglandin F₂α were not altered. Synthesis of 15-HETE by cultured granulosa and theca interna cells was reduced by the presence of NDGA (1 mg/ml), whereas indomethacin (100 ng/ml) lowered 15-HETE production in theca interna cells only. These results clearly demonstrate that indomethacin can block the lipoxygenase as well as the cyclooxygenase pathways, depending on the dose used, and suggest that lipoxygenase metabolites of arachidonic acid are involved in the ovulatory process in the pig.     

Specific high affinity binding of lipoxygenase metabolites of arachidonic acid by liver fatty acid binding protein

Liver fatty acid binding protein (L-FABP) binds avidly the arachidonic acid metabolites, hydroperoxyeicosatetraenoic acids (HPETEs) and hydroxyeicosatetraenoic acids (HETEs). Binding of 15-[3H]HPETE was specific, saturable, reversible, and rapid. Protein specificity was indicated by the following order: L-FABP greater than bovine serum albumin greater than ovalbumin = beta-lactoglobulin greater than ribonuclease. Ligand specificity was evidenced by the following order of apparent competition: 15-HPETE greater than or equal to 5-HETE greater than or equal to 5-HPETE = oleic acid greater than 12-HETE greater than 12-HPETE greater than or equal to 15-HETE greater than prostaglandin E1 much greater than leukotriene C4 greater than prostaglandin E2 much greater than thromboxane B2 = leukotriene B4. Once bound, 15-HPETE was reversibly displaced. Ligand was recovered from the protein complex and confirmed to be 15-[3H]HPETE by TLC. L-FABP bound HPETE with a dissociation constant of 76 nM,5-HETE at 175 nM, and 15-HETE at 1.8 microM, and the reference fatty acids oleic acid at 1.2 microM and arachidonic acid at 1.7 microM. Thus, the affinity was approximately 16-fold greater for 15-HPETE, and 7-fold higher for 5-HETE, than for oleic acid. The need exists for studies of complexes of L-FABP with the HPETEs and HETEs in hepatocytes, especially since L-FABP has previously been associated with mitosis in normal hepatocytes, and shown to be the target protein of two liver carcinogens, and these arachidonic acid metabolites have been found to be able to modulate activities related to cell growth.     

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 . 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 (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 characterized by a switch in arachidonic acid metabolism in intra-uterine tissues from lipoxygenase to cyclo-oxygenase products.     

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.     

Role of arachidonic acid lipoxygenase metabolites in acetylcholine-induced relaxations of mouse arteries

Arachidonic acid (AA) metabolites function as EDHFs in arteries of many species. They mediate cyclooxygenase (COX)- and nitric oxide (NO)-independent relaxations to acetylcholine (ACh). However, the role of AA metabolites as relaxing factors in mouse arteries remains incompletely defined. ACh caused concentration-dependent relaxations of the mouse thoracic and abdominal aorta and carotid, femoral, and mesentery arteries (maximal relaxation: 57 ± 4%, 72 ± 4%, 82 ± 3%, 80 ± 3%, and 85 ± 3%, respectively). The NO synthase inhibitor nitro-L-arginine (L-NA; 30 μM) blocked relaxations in the thoracic aorta, and L-NA plus the COX inhibitor indomethacin (10 μM) inhibited relaxations in the abdominal aorta and carotid, femoral, and mesenteric arteries (maximal relaxation: 31 ± 10%, 33 ± 5%, 41 ± 8%, and 73 ± 3%, respectively). In mesenteric arteries, NO- and COX-independent relaxations to ACh were inhibited by the lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA; 10 μM) and BW-755C (200 μM), the K(+) channel inhibitor apamin (1 μM), and 60 mM KCl and eliminated by endothelium removal. They were not altered by the cytochrome P-450 inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (20 μM) or the epoxyeicosatrienoic acid antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 μM). AA relaxations were attenuated by NDGA or apamin and eliminated by 60 mM KCl. Reverse-phase HPLC analysis revealed arterial [(14)C]AA metabolites that comigrated with prostaglandins, trihydroxyeicosatrienoic acids (THETAs), hydroxyepoxyeicosatrienoic acids (HEETAs), and hydroxyeicosatetraenoic acids (HETEs). Epoxyeicosatrienoic acids were not observed. Mass spectrometry confirmed the identity of 6-keto-PGF(1α), PGE(2), 12-HETE, 15-HETE, HEETAs, 11,12,15-THETA, and 11,14,15-THETA. AA metabolism was blocked by NDGA and endothelium removal. 11(R),12(S),15(S)-THETA relaxations (maximal relaxation: 73 ± 3%) were endothelium independent and blocked by 60 mM KCl. Western immunoblot analysis and RT-PCR of the aorta and mesenteric arteries demonstrated protein and mRNA expression of leukocyte-type 12/15-LO. Thus, in mouse resistance arteries, 12/15-LO AA metabolites mediate endothelium-dependent relaxations to ACh and AA.     

Effect of various lipoxygenase metabolites of arachidonic acid on degranulation of polymorphonuclear leukocytes

Lipoxygenase metabolites of guinea pig peritoneal polymorphonuclear leukocytes stimulated with 10 microM A23187 plus arachidonic acid were isolated and identified. These metabolites were compared with each other and to chemically synthesized arachidonate metabolites for their ability to stimulate leukocyte degranulation. 5(S),12(R)-Dihydroxy-6,8,10-(cis/trans/trans)14-cis-eicosatetraenoic acid (leukotriene B4) produced a significant release of lysozyme, but not beta-glucuronidase or beta-N-acetylglucosaminidase at low concentrations (EC50 = 6.5 x 10(-9) M), while the leukocyte nonenzymatically generated 5,12-or 5,6-dihydroxyeicosatetraenoic acids had no effect at these concentrations. Higher concentrations (1--10 microM) of all the dihydroxyeicosatetraenoic acids, 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and its hydroperoxy precursor stimulated significant lysozyme release which was greater than that produced by 15-hydroxy-5,8,11-13-eicosatetraenoic acid, arachidonic acid, or its acetylene analogue, 5,8,11,14-eicosatetraynoic acid. Micromolar concentrations of leukotriene B4 and 5-HETE also stimulated significant release of beta-N-acetylglucosaminidase above controls, but not beta-glucuronidase. These results suggest that leukotriene B4 may play a role in regulating the release of certain granule-bound enzymes from polymorphonuclear leukocytes.     

Effect of lipoxygenase metabolites of arachidonic acid on the surface charge of the erythrocyte membrane

The influence of metabolites of arachidonic acids on the electrophoretic mobility of the rat erythrocytes has been investigated. It is found that they can increase or decrease the surface charge of the erythrocyte membranes.     

Mechanism of action of glucocorticoid-induced immunoglobulin production: role of lipoxygenase metabolites of arachidonic acid

Glucocorticoids stimulate polyclonal immunoglobulin (Ig) production in cultures of human peripheral blood lymphocytes. The mechanism of action of glucocorticoids in this system, and indeed in any physiologic system, is unknown. Because glucocorticoids stimulate the production of phospholipase A2-inhibitory glycoproteins, we investigated whether glucocorticoids stimulate polyclonal Ig production by inhibition of arachidonic acid metabolism. Nonspecific lipoxygenase/cyclooxygenase inhibitors stimulate polyclonal Ig production in a manner similar to the effect of glucocorticoids, whereas specific cyclooxygenase inhibitors actually inhibit Ig production. Two specific 5-lipoxygenase inhibitors, with little or no activity against cyclooxygenase or other lipoxygenases, also stimulate Ig production. The dose-response effect of all of these drugs on Ig production was similar to the dose response of inhibition of 5-lipoxygenase. Leukotriene B4 (LTB4) added in low concentrations (10(-10)M) on days 1, 2, and 3 of a culture eliminated the stimulatory effect of glucocorticoids or 5-lipoxygenase inhibitors, whereas LTC4, LTD4, prostaglandin E, or 5-hydroxyeicosatetraenoic acid had no effect. These results suggest that the relevant action of glucocorticoids in stimulating Ig production might be in preventing endogenous arachidonic acid metabolism, perhaps the endogenous production of LTB4.     

A particle beam-liquid chromatography-mass spectrometry method for the determination of lipoxygenase metabolites of arachidonic acid.

An application of the particle beam-liquid chromatography-mass spectrometry technique to the quantification of hydroxyeicosatetraenoic acids (15-, 12-, and 5-HETE) in biological samples is presented. The acids are extracted with Ethyl acetate and then transformed into pentafluorobenzyl esters, thus increasing the sensitivity of their detection by negative chemical ionization mass spectrometry. Reverse-phase HPLC separation of HETEs is performed in about 10 min with a water-methanol gradient. The procedure shows a detection limit of nearly 0.5 pmol, about one order of magnitude lower than that of the widely used HPLC/UV methods. The quantitative determination is linear (r2 greater than 0.998) for all of the HETEs in the range tested (3-1500 pmol) and a CV lower than 8.5% was observed for repeated analysis of samples. As an application of the method, HETEs formed from endogenous arachidonate were evaluated in extracts obtained from coincubates of platelets and neutrophils stimulated with calcium ionophore A23187.     

Selective activation of the gamma-subspecies of protein kinase C from bovine cerebellum by arachidonic acid and its lipoxygenase metabolites

The gamma-subspecies of protein kinase C (PKC) apparently is expressed only in central nervous tissues, and at a high level in the cerebellum and hippocampus. gamma-PKC from bovine cerebellum, but not the alpha- or beta I/beta II-subspecies, is activated by micromolar concentrations of arachidonic acid (AA), in the absence of both phospholipid and diacylglycerol. A significant component of this activation is also calcium independent. Other unsaturated fatty acids are much less active in this respect. Among the AA metabolites tested, lipoxin A (5(S),6(R),15(S)-11-cis-isomer) was a potent, selective activator of the gamma-subspecies, and also, to a lesser extent, 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid could support activation. These results raise the possibility that AA and some of its lipoxygenase metabolites may function as messenger molecules in neurones to activate the gamma-subspecies of PKC.     

Stimulation of osteoclastic bone resorption by hydrogen peroxide.

The molecular mechanisms underlying the pathophysiology of bone destruction still remain poorly understood. We have found that hydrogen peroxide (H2O2), a reactive oxygen species (ROS), is a potent stimulator of osteoclastic bone resorption and cell motility. A marked enhancement of bone resorption was noted when rat osteoclasts, cultured on devitalised bovine cortical bone, were exposed to 10 nM [H2O2]. Apart from exposing osteoclasts to a low extracellular pH, which is known to enhance osteoclastic bone resorption, we provide first evidence for a molecule that stimulates osteoclastic bone resorption in osteoclast cultures that do not respond to parathyroid hormone and 1, 25 dihydroxyvitamin D3. We envisage that both basic biological and practical clinical implications may eventually follow from these studies.     

Conversion of arachidonic acid to lipoxygenase products by human fetal tissues

[14C]Arachidonic acid was converted to several lipoxygenase products by homogenates of human fetal tissues as determined by thin-layer chromatography. The net conversions of [14C]arachidonic acid to radiolabeled lipoxygenase products were high (greater than or equal to 5%) in the case of fetal liver and brain, and low (less than or equal to 2%) in the case of fetal adrenal, heart, and kidney.     

Comparison of the effect of lipoxygenase metabolites of arachidonic acid and eicosapentaenoic acid on human natural killer cell cytotoxicity

We compared in vitro effect of lipoxygenase (LO) products derived from arachidonic acid (AA) and eicosapentaenoic acid (EPA) on cytotoxic activity of human natural killer (NK) cell against human erythroleukemia cell line K-562. Leukotriene B4 (LTB4) derived from AA was found to significantly augment NK cell activity compared to the control level (in the absence of LTB). LTB5 showed a weak, but not significant, enhancing effect on NK cell activity. LTB4 was significantly more potent than LTB5 in the enhancement of NK cell activity. On the other hand, both 5- and 15-hydroperoxy fatty acids derived from AA and EPA significantly enhanced NK cell activity compared to the control level with similar potencies.     

Stimulation of DNA synthesis by tumor promoters in primary rat hepatocytes is not mediated by arachidonic acid metabolites

Studies in vivo using inhibitors of eicosanoid synthesis suggested that prostaglandins may play a role in mediating tumor promotion in liver by agents such as phenobarbital (PB). However, it is not clear whether any stimulation of arachidonic acid metabolism/prostaglandin formation results directly from the action of tumor promoters on hepatocytes or indirectly from effects of promoters on Kupffer cells or other non-hepatocytes. Our laboratory has been utilizing relatively pure populations of rat hepatocytes under the defined conditions of primary cultures, to investigate growth-stimulatory actions of tumor promoters, an important element in the promotion stage of carcinogenesis. It has been shown that most if not all liver tumor promoters tested stimulate hepatocyte DNA synthesis when added in combination with factors such as EGF, insulin, and glucocorticoid. In the present study, we sought evidence for a role of prostaglandins (PGs) in the direct growth-stimulatory actions of tumor promoters on hepatocytes. PGE(2), PGF(2 alpha), and PGD(2) cause concentration-dependent stimulation of hepatocyte DNA synthesis, while arachidonic acid was without any effect. PGE(2) and PGF(2 alpha) required the presence of dexamethasone to exert significant effects. These PGs did not further augment the stimulatory effect of EGF. In contrast, PGD(2) stimulated DNA synthesis in the presence or absence of insulin, dexamethasone, or EGF. The effect of tumor promoters on arachidonic acid metabolism, as measured by [(3)H]arachidonic acid release and PGE(2) production, was determined. The phorbol ester TPA significantly increased [(3)H]arachidonic acid release as well as PGE(2) formation in hepatocytes in line with known effects in other cell types. However, liver tumor promoters phenobarbital (PB), alpha-hexachlorocycohexane (HCH), 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), and pregnenolone-16 alpha-carbonitrile (PCN) were without effects. Finally, inhibitors of arachidonic acid metabolism were tested for effects on the ability of TPA or liver tumor promoters to stimulate DNA synthesis by direct action on cultured hepatocytes. In all cases, lack of selective inhibition was observed. Taken together, the results show that while prostaglandins may directly stimulate DNA synthesis in hepatocytes, they are unlikely to mediate the direct growth-stimulatory actions of liver tumor promoters.     

Cell killing and induction of manganous superoxide dismutase by tumor necrosis factor-alpha is mediated by lipoxygenase metabolites of arachidonic acid.

The signalling pathways utilized by tumor necrosis factor-a (TNF) to elicit its actions have been examined in TA1 adipogenic cells. A role for lipoxygenase metabolites of arachidonic acid as mediators of TNF action in the induction of c-fos has been described. In this paper we report that acute cytotoxicity elicited by TNF, in the presence of cycloheximide (CHX), also utilizes this pathway since inhibitors of lipoxygenase action fully prevent TNF/CHX killing of several cell lines. Our data reveal that TNF induction of manganous superoxide dismutase (MnSOD) is also dependent upon lipoxygenase activity. Radical scavengers such as NAC and PDTC prevent TNF/CHX-induced cell killing and reduce MnSOD induction by TNF. Therefore, cell death by TNF/CHX treatment occurs via a pathway in which lipoxygenase products directly or indirectly operate via the generation of superoxide anions.     

Effect of lipoxygenase metabolites of arachidonic acid on proliferation of human T cells and T cell subsets

The lipoxygenase products LTB4 and 15 HPETE have been reported to stimulate T suppressor cell function and also to inhibit [3H]thymidine incorporation into mitogen-stimulated T cells. This present report documents that although these compounds do indeed inhibit [3H]thymidine incorporation into unfractionated T cells, they significantly enhance [3H]thymidine incorporation into T cell preparation enriched for cells bearing the cytotoxic suppressor cell phenotype identified by the OKT8 monoclonal antibody. The mitogen response of T cells enriched for OKT4+ helper-inducer cells is inhibited in manner similar to the response of unfractionated T cells. Thus, LTB4 and 15 HPETE stimulate both the function and the proliferation of the cytotoxic-suppressor T cell subset.