Lipoxygenase Metabolites Of Arachidonic Acid Research Articles

ОСОБЛИВОСТІ ВМІСТУ ДЕЯКИХ МЕТАБОЛІЧНИХ ПОКАЗНИКІВ ТА ТУМОРНЕКРОТИЧНОГО ФАКТОРУ У ХВОРИХ НА КОМОРБІДНУ ПАТОЛОГІЮ

РЕЗЮМЕ. Відомо, що в регуляції трофічних та репаративних процесів тканин значну роль відіграють метаболіти арахідонової кислоти та ліпоперекисні похідні, що підкреслює їх важливу роль у рецидивуванні та/ або хронізації запального процесу. Окрім того, не виключається їх значно більший вплив на перебіг захворювань в умовах коморбідності.Мета дослідження – вивчення вмісту ліпооксигеназних метаболітів арахідонової кислоти (лейкотрієни (LT) С4 і В4; радіоімунний метод), проміжної та кінцевої ліпоперекисних сполук (дієнові кон'югати (DC) та малоновий діальдегід (MDA); спектрофотометричний метод), а також сироваткового туморнекротичного фактора α (TNFα; імуноферментний метод) у ході лікування 40 хворих (вік 37 – 49 років, 25 чоловіків та 15 жінок) на пептичну виразку (ПВ), у 23 з яких вона була коморбідною із хронічним бронхітом; референтною нормою були показники 20 практично здорових осіб (групи не відрізнялись за віком і статтю).Виявилось, що у коморбідних хворих після лікування рівень LTВ4, порівняно з хворими на ПВ ((108,5±20,6) пг/ мл), був підвищеним у 1,3 раза (у здорових – ((53,2±11,4) пг/мл; р<0,001) і більш ніж в 1,4 раза перевищував рівень LTС4 (у хворих на ПВ – (98,7±18,9) пг/мл; референтна норма – (39,7±10,4) пг/мл; р<0,001). З’ясовано, що підвищення вмісту ліпооксигеназних метаболітів АК у хворих на коморбідну патологію супроводжувалось також суттєвим підвищенням рівня проміжних і кінцевих ліпоперекисних сполук, при цьому рівень DC у хворих з коморбідною патологією перевищував такий у хворих на ПВ майже на третину ((6,81±0,33) мкмоль/л, р<0,001), а вміст MDA – більш ніж на чверть ((9,53±0,41) мкмоль/л, р<0,001). Вказані зміни метаболічних показників у коморбідних хворих після лікування супроводжував більш ніж удвічі підвищений вміст сироваткового TNFα ((51,0±2,8) пг/мл; у здорових – (24,5±3,2) пг/мл; р<0,001), що було також у 1,34 раза вище, ніж у хворих на ПВ після лікування. Виявлені особливості вмісту ліпооксигеназних метаболітів арахідонової кислоти, проміжного та кінцевого ліпоперекисних сполук та прозапального TNFα після лікування у хворих на коморбідну патологію свідчать про збереження у них високої прозапальної активності крові, що передбачає розробку заходів з оптимізації лікування та профілактики означеної патології у вказаної категорії хворих.

Lipidomic analysis of patients with microbial invasion of the amniotic cavity reveals up-regulation of leukotriene B4.

Bioactive lipids derived from the metabolism of polyunsaturated fatty acids are important mediators of the inflammatory response. Labor per se is considered a sterile inflammatory process. Intra-amniotic inflammation (IAI) due to microorganisms (i.e., intra-amniotic infection) or danger signals (i.e., sterile IAI) has been implicated in the pathogenesis of preterm labor and clinical chorioamnionitis at term. Early and accurate diagnosis of microbial invasion of the amniotic cavity (MIAC) requires analysis of amniotic fluid (AF). It is possible that IAI caused by microorganisms is associated with a stereotypic lipidomic profile, and that analysis of AF may help in the identification of patients with this condition. To test this hypothesis, we analyzed the fatty acyl lipidome of AF by liquid chromatography-mass spectrometry from patients in spontaneous labor at term and preterm gestations. We report that the AF concentrations of proinflammatory lipid mediators of the 5-lipoxygenase pathway are significantly higher in MIAC than in cases of sterile IAI. These results suggest that the concentrations of 5-lipoxygenase metabolites of arachidonic acid, 5-hydroxyeicosatetraenoic acid, and leukotriene B4 in particular could serve as potential biomarkers of MIAC. This finding could have important implications for the rapid identification of patients who may benefit from anti-microbial treatment.-Maddipati, K. R., Romero, R., Chaiworapongsa ,T., Chaemsaithong, P., Zhou, S.-L., Xu, Z., Tarca, A. L., Kusanovic, J. P., Gomez, R., Chaiyasit, N., Honn, K. V. Lipidomic analysis of patients with microbial invasion of the amniotic cavity reveals up-regulation of leukotriene B4.

Leukotriene B4 Inhibits L-Type Calcium Channels via p38 Signaling Pathway in Vascular Smooth Muscle Cells

Background/Aims: Arachidonic acid (AA) and its metabolites are important endogenous lipid messengers. In this study, we test the effect of Leukotriene B₄ (LTB₄), a 5-lipoxygenase metabolite of AA, on L-type calcium channels in A7r5 rat aortic vascular smooth muscle cells. Methods: L-type calcium channel currents were recorded by a patch-clamp technique. The mRNA expression of Ca_V1.2 was determined by Real-time RT-PCR. The protein expression of Ca_V1.2 and p38 activity was determined by Western blot analysis. Results: LTB₄ inhibits L-type channel currents in A7r5 cells in a dose-and time- dependent manner. LTB₄ reduced the mRNA/protein expression of Ca_V1.2 channels in A7r5 cells. BLT1 receptor antagonist LY29311 abrogated the inhibitory effect of LTB₄, while BLT2 receptor antagonist LY255283 had no effect. 5Z-7-oxozeaenol and SB203580, which block TAK1 and p38 kinase respectively, abrogated the LTB₄ inhibitory effect on L-type calcium channels. LTB₄ increased p38 activity in A7r5 cells. Blockage of Src, PI3K, JNK and NF-κB kinase had no effects on LTB₄ inhibition of L-type calcium channel currents in A7r5 cells. Conclusion: We conclude that LTB₄ inhibits L-type calcium channels through BLT1-TAk1-p38 signaling pathway. The LTB₄ inhibitory effect on L-type calcium channels may be involved in its pathological processes such as atherosclerosis.

Role of Lipoxygenase Metabolites of Arachidonic Acid in Enhanced Pulmonary Artery Contractions of Female Rabbits

Pulmonary arterial hypertension is characterized by elevated pulmonary artery pressure and vascular resistance. In women the incidence is 4-fold greater than that in men. Studies suggest that sustained vasoconstriction is a factor in increased vascular resistance. Possible vasoconstrictor mediators include arachidonic acid-derived lipoxygenase (LO) metabolites. Our studies in rabbits showed enhanced endothelium-dependent contractions to arachidonic acid in pulmonary arteries from females compared with males. Because treatment with a nonspecific LO inhibitor reduced contractions in females but not males, the present study identified which LO isoform contributes to sex-specific pulmonary artery vasoconstriction. The 15- and 5- but not 12-LO protein expressions were greater in females. Basal and A23187-stimulated release of 15-, 5-, and 12-hydroxyeicosatetraenoic acids (HETEs) from females and males were measured by liquid chromatography/mass spectrometry. Only 15-HETE synthesis was greater in females compared with males under both basal and stimulated conditions. Vascular contractions to 15-HETE were enhanced in females compared with males (maximal contraction: 44±6%versus 25±3%). The specific 15-LO inhibitor PD146176 (12 μmol/L) decreased arachidonic acid-induced contractions in females (maximal contraction: 93±4% versus 57±10%). If male pulmonary arteries were incubated with estrogen (1 μmol/L, 18 hours), protein expression of 15-LO and 15-HETE production increased. Mechanisms to explain the increased incidence of pulmonary hypertension in women are not known. Results suggest that the 15-LO pathway is different between females and males and is regulated by estrogen. Understanding this novel sex-specific mechanism may provide insight into the increased incidence of pulmonary hypertension in females.

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.

Endothelin-1 induced vascular smooth muscle cell proliferation is mediated by cytochrome p-450 arachidonic acid metabolites.

Endothelins (ETs) are a family of three peptides (ET-1, ET-2, ET-3) that are implicated in the physiological control of vascular smooth muscle cell (VSMC) and myocardial contractility and growth. ET-1 is vasoactive peptide that acts via ET-A receptors coupling inducing vascular smooth muscle cell contraction. ET-1 is involved in the development and maintenance of hypertension. Aim of this study was to investigate whether ET-1 can induce vascular smooth muscle cell proliferation through arachidonic acid (AA) metabolites formed via cytochrome P¬450 (CYP-450). VSMC proliferation was measured by [3H]thymidine incorporation in cultured cells treated by ET-1 (10 to l00 nmol/L) in presence of different inhibitors of CYP-450 (17-ODYA 5 μmol/L), lipoxygenase (LO) (baicalein 20 μmol/L) and cyclooxygenase (COX) (indomethacin 5 μmol/L). ET-1 (10 to 100 nmol/L) induced VSMC proliferation and this effect was attenuated by CYP-450 inhibitor (17-ODYA) and lipoxygenase (LO) inhibitor (baicalein) but not by cyclooxygenase (COX) inhibitor (indomethacin). CYP-450 and LO metabolites of AA, 20-hydroxyeicosatetraenoic acid (HETE) and 12-HETE increased [3H]thymidine incorporation in VSMC. Inhibitors of MAP kinase (PD-98059 50 μmol/L) and cPLA2 (MAFP 50 μmol/L) attenuated ET-1 as well as 20-HETE induced VSMC proliferation. These results suggest AA metabolites via CYP-450 mediates ET-1 induce VSMC proliferation.

Differential Oxylipid Metabolism in Response to EPA and DHA in IgA Nephropathy

Immunoglobulin A nephropathy (IgAN) is associated with renal hypertension and inflammation. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in fish oil (FO) improve kidney function in animal models but are inconsistent in humans. Plasma oxylipid profiles from IgAN patients (n=10) and healthy controls (n=12) pre vs. post 2 g/d EPA + 1.5 g/d DHA and IgAN patients pre vs. post corn oil (CO) (n=9) were analyzed by LC/MS/MS. At baseline, patients had significantly higher EPA, DHA, linoleic acid (LA), α‐linolenic acid, and arachidonic acid (ARA) epoxides and diols and higher prostaglandins and leukotrienes. There were isomer‐specific differences between patients and controls pre and post FO. Several EPA and DHA metabolites were increased in both patients and controls post FO, but unchanged in patients post CO. The ARA lipoxygenase metabolites 5‐, 8‐, 9‐, and 15‐HETE and the LA lipoxygenase metabolites 9‐HODE, 9‐oxo‐ODE, and 13‐HODE were decreased in controls but unchanged in patients post FO. There were significant differences between controls and patients pre and post FO supplementation, which are indicative of differences in ω3 and oxylipid metabolism in IgAN.

Role of arachidonic acid lipoxygenase metabolites in the regulation of vascular tone

Stimulation of vascular endothelial cells with agonists such as acetylcholine (ACh) or bradykinin or with shear stress activates phospholipases and releases arachidonic acid (AA). AA is metabolized by cyclooxygenases, cytochrome P-450s, and lipoxygenases (LOs) to vasoactive products. In some arteries, a substantial component of the vasodilator response is dependent on LO metabolites of AA. Nitric oxide (NO)- and prostaglandin (PG)-independent vasodilatory responses to ACh and AA are reduced by inhibitors of LO and by antisense oligonucleotides specifically against 15-LO-1. Vasoactive 15-LO metabolites derived from the vascular endothelium include 15-hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-HEETA) that is hydrolyzed by soluble epoxide hydrolase to 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA). HEETA and THETA are endothelium-derived hyperpolarizing factors that induce vascular relaxations by activation of smooth muscle apamin-sensitive, calcium-activated, small-conductance K(+) channels causing hyperpolarization. In other arteries, the 12-LO metabolite 12-hydroxyeicosatetraenoic acid is synthesized by the vascular endothelium and relaxes smooth muscle by large-conductance, calcium-activated K(+) channel activation. Thus formation of vasodilator eicosanoids derived from LO pathways contributes to the regulation of vascular tone, local blood flow, and blood pressure.

Whole-body synthesis-secretion rates of long-chain n-3 PUFAs from circulating unesterified α-linolenic acid in unanesthetized rats

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), long-chain n-3 PUFAs important for brain and heart function, can be obtained from dietary fish products or by liver synthesis from alpha-linolenic acid (alpha-LNA). Their daily human dietary requirements are not clear, and their liver synthesis rates in humans and nonhumans are unknown. We estimated whole-body (presumably liver) synthesis rates in unanesthetized rats by infusing [U-(13)C]alpha-LNA intravenously for 2 h and measuring labeled and unlabeled n-3 PUFA in arterial plasma using negative chemical ionization GC-MS. Newly synthesized esterified [(13)C]DHA, [(13)C]EPA, and [(13)C]docosapentaenoic acid (DPA) appeared in arterial plasma after 60 min of infusion, then their concentrations rose in an S-shaped manner. Esterified concentration x plasma volume data were fit with a sigmoidal equation, whose peak first derivatives provided synthesis rates of unlabeled EPA, DPA, and DHA equal to 8.40, 6.27, and 9.84 mumol/day, respectively. The DHA synthesis rate exceeded the published daily rat brain DHA consumption rate by 30-fold, suggesting that liver synthesis from alpha-LNA could maintain brain DHA homeostasis were DHA absent from the diet. This stable isotope infusion method could be used to quantify whole-body DHA synthesis rates in human subjects.

5-LOX, 12-LOX and 15-LOX in immature forms of human leukemic blasts

Several reports have demonstrated an important role of leukotriene B 4 (LTB 4) in the immune system. We investigated whether leukemic blasts from acute myeloid leukemic (AML) and acute lymphoid leukemic (ALL) patients produced LTB 4, 12- and 15-hydroxyeicosatetraenoic acids (12-HETE and 15-HETE) and whether these compounds affected blast proliferation and apoptosis. Leukemic blasts from AML M 0-2 and ALL patients expressed 5-LOX, 12-LOX and 15-LOX transcripts. Quantitative polymerase chain reaction indicated that 5-LOX transcripts were far more abundant than 12-LOX and 15-LOX ones. Leukemic blasts expressed 5-LOX activating protein (FLAP) transcripts and produced LTB 4 in response to calcium ionophore. In contrast no 15-HETE production was found. Calcium ionophore-stimulated leukemic blasts produced 12-HETE but also released thromboxane A 2 suggesting that contaminating platelets accounted for the release of these compounds. No significant effect of LTB 4, 12-HETE or 15-HETE could be documented on leukemic blast growth and on their apoptose rate. Results of the present study indicate that immature form of leukemic blasts produce LTB 4. However, the three major lipoxygenase metabolites of arachidonic acid; i.e., LTB 4, 12-HETE or 15-HETE, had no evident effect on their growth and apoptosis. We may speculate that LTB 4-derived blast cells might initiate, augment or prolong tissue inflammation and damages by affecting the marrow and blood cytokine network.

Gout: Diagnosis and management

Goutis a heterogeneous group of diseases resulting from monosodium urate (MSU) crystal deposition in tissues or from supersaturation of uric acid in extracellular fluids. Clinical manifestations include 1) Recurrent attacks of articular and periarticular inflammation, also called gouty arthritis; 2) Accumulation of articular, osseous, soft tissue, and cartilaginous crystalline deposits, called tophi; 3) Uric acid calculi in the urinary tract; and 4) Interstitial nephropathy with renal function impairment, called gouty nephropathy. Gout predominantly is a disease of adult men, with a peak incidence in the fifth decade. In women usually found after menopause.The metabolic disorder underlying gout is hyperuricaemia. The duration and magnitude of hyperuricemia directly correlate with the likelihood of developing gouty arthritis and uric acid urolithiasis, and with age at onset of initial clinical gouty manifestations. The urate crystals induce phagocytes and synovial cells to generate and release such mediators as cyclooxygenase and lipoxygenase metabolites of arachidonic acid, phospholipase A2-activating protein, lysosomal proteases, tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, and IL-8. Definitive diagnosis of gout needs the demonstration of MSU crystals in synovial fluid or tophus. Gout is frequently associated with comorbidity such as obesity, hypertension, renal disease and dyslipidaemia. Therapeutic goals include terminating acute attacks; providing rapid, safe relief of pain and inflammation; averting future attacks; and preventing such complications as formation of tophi, kidney stones, and destructive arthropathy. Colchicine, nonsteroidal anti-inflammatory drugs and corticosteroid are drugs used for treating acute gouty arthritis. Colchicine is also used for prophylaxis. Urate lowering drugs also play a role in prophylactic management of gout. With early intervention, careful monitoring, and patient education, the prognosis is excellent. (Med J Indones 2007; 16:47-54) Keywords: Hyperuricemia, tophus, arthritis, inflammatory mediators, dietary and lifestyle, urate lowering drugs

Fibroblast Growth Factor-2 Is a Downstream Mediator of Phosphatidylinositol 3-Kinase-Akt Signaling in 14,15-Epoxyeicosatrienoic Acid-induced Angiogenesis

To determine the efficacy of cytochrome P450 2C9 metabolites of arachidonic acid, viz. 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), in inducing angiogenesis, we have studied their effects on human dermal microvascular endothelial cell (HDMVEC) tube formation and migration. All four EETs stimulated HDMVEC tube formation and migration in a dose-dependent manner. Because 14,15-EET was found to be slightly more efficacious than 5,6-, 8,9-, and 11,12-EETs in stimulating HDMVEC tube formation and migration, we next focused on elucidation of the signaling mechanisms underlying its angiogenic activity. 14,15-EET stimulated Akt and S6K1 phosphorylation in Src- and phosphatidylinositol 3-kinase (PI3K)-dependent manner in HDMVECs. Inhibition of Src and PI3K-Akt-mTOR signaling by both pharmacological and dominant-negative mutant approaches suppressed 14,15-EET-induced HDMVEC tube formation and migration in vitro and Matrigel plug angiogenesis in vivo. In addition, 14,15-EET induced the expression of fibroblast growth factor-2 (FGF-2) in Src- and PI3K-Akt-dependent and mTOR-independent manner in HDMVECs. Neutralizing anti-FGF-2 antibodies completely suppressed 14,15-EET-induced HDMVEC tube formation and migration in vitro and Matrigel plug angiogenesis in vivo. Together, these results show for the first time that Src and PI3K-Akt signaling via targeting in parallel with FGF-2 expression and mTOR-S6K1 activation plays an indispensable role in 14,15-EET-induced angiogenesis.

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.

15(S)-Lipoxygenase-2 Mediates Arachidonic Acid-stimulated Adhesion of Human Breast Carcinoma Cells through the Activation of TAK1, MKK6, and p38 MAPK

The dietary cis-polyunsaturated fatty acid, arachidonic acid, stimulates adhesion of metastatic human breast carcinoma cells (MDA-MB-435) to the extracellular matrix, but the molecular mechanisms by which fatty acids modify the behavior of these cells are unclear. Exposure to arachidonic acid activates multiple signaling pathways. Activation of p38 mitogen-activated protein kinase (p38 MAPK) is required for increased cell adhesion to type IV collagen, and this activation is sensitive to inhibitors of lipoxygenases, suggesting a requirement for arachidonic acid metabolism. The goals of the current study were to identify the one or more key metabolites of arachidonic acid that are responsible for activation of p38 MAPK and to elucidate the upstream kinases that lead to p38 MAPK activation. High performance liquid chromatographic analysis revealed that MDA-MB-435 cells metabolize exogenous arachidonic acid predominantly to 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE). Immunoblot analysis with antibodies specific to 15(S)-lipoxygenase-1 (LOX-1) and 15(S)-lipoxygenase-2 (LOX-2) demonstrated the expression of 15-LOX-2, but not 15-LOX-1, in these tumor cells. A LOX inhibitor, nordihydroguaiaretic acid, attenuated production of 15(S)-HETE and inhibited the phosphorylation of p38 MAPK following exposure to arachidonic acid. In contrast, overexpression of LOX-2 sensitized the cells to the addition of arachidonic acid, leading to increased activation of p38 MAPK. Addition of exogenous 15(S)-HETE to MDA-MB-435 cells stimulated cell adhesion to type IV collagen and activated the p38 MAPK pathway, including the upstream kinases transforming growth factor-beta1-activated protein kinase-1 (TAK1) and MAPK kinase 6. Transfection of these cells with a dominant negative form of TAK1 blocked arachidonic acid-stimulated p38 MAPK phosphorylation. These data demonstrate that 15(S)-LOX-2 generation of 15(S)-HETE activates specific growth factor receptor-related signaling pathways, thereby initiating signal transduction events leading to increased cell adhesion to the extracellular matrix.

15(S)-Hydroxyeicosatetraenoic Acid Induces Angiogenesis via Activation of PI3K-Akt-mTOR-S6K1 Signaling

To determine whether the lipoxygenase metabolites of arachidonic acid, 5(S)-, 12(S)-, and 15(S)-hydroxyeicosatetraenoic acids [5(S)-HETE, 12(S)-HETE, and 15(S)-HETE, respectively] are angiogenic, we have studied their effects on human dermal microvascular endothelial cell (HDMVEC) tube formation and migration. All three HETEs stimulated HDMVEC tube formation and migration. Because 15(S)-HETE was found to be more potent than 5(S)-HETE and 12(S)-HETE in HDMVEC tube formation, we next focused on elucidation of the signaling mechanisms underlying its angiogenic activity. 15(S)-HETE stimulated Akt and S6K1 phosphorylation in HDMVEC in a time-dependent manner. Wortmannin and LY294002, two specific inhibitors of phosphatidylinositol 3-kinase (PI3K), blocked both Akt and S6K1 phosphorylation, whereas rapamycin, a specific inhibitor of Akt downstream effector, mammalian target of rapamycin (mTOR), suppressed only S6K1 phosphorylation induced by 15(S)-HETE suggesting that this eicosanoid activates the PI3K-Akt-mTOR-S6K1 signaling in HDMVEC. Wortmannin, LY294002, and rapamycin also inhibited 15(S)-HETE-induced HDMVEC tube formation and migration. In addition, all three HETEs stimulated angiogenesis as measured by in vivo Matrigel plug assay with 15(S)-HETE being more potent. Pharmacologic inhibition of PI3K-Akt-mTOR-S6K1 signaling completely suppressed 15(S)-HETE-induced in vivo angiogenesis. Consistent with these observations, adenoviral-mediated expression of dominant-negative Akt also blocked 15(S)-HETE-induced HDMVEC tube formation and migration and in vivo angiogenesis. Together, these results show for the first time that 15(S)-HETE stimulates angiogenesis via activation of PI3K-Akt-mTOR-S6K1 signaling.

The mechanism underlying the contractile effect of a chemotactic peptide, formyl‐Met‐Leu‐Phe on the guinea‐pig Taenia coli

1 The contractile mechanism of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) was investigated in the guinea-pig Taenia coli, by simultaneously monitoring the changes in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) and force. 2 fMLP induced a significant elevation of [Ca(2+)](i) and force at concentrations higher than 10 nM. The maximal response was obtained at a concentration of higher than 1 microM. 3 fMLP (10 microM) augmented the force development induced by a stepwise increment of the extracellular Ca(2+) concentration during 60 mM K(+) depolarization, while it had no effect on the [Ca(2+)](i) elevation, and thus produced a greater force for a given elevation of [Ca(2+)](i) than 60 mM K(+) depolarization. 4 The removal of extracellular Ca(2+) completely abolished the fMLP-induced contraction. The fMLP-induced [Ca(2+)](i) elevation was inhibited substantially but not completely by 10 microM diltiazem, partly by 10 microM SK&F 96365, and completely by their combination. 5 Y27632, a specific inhibitor of rho-kinase, had no significant effect on the fMLP-induced [Ca(2+)](i) elevation and force development. 6 Chenodeoxycholic acid, a formyl peptide receptor antagonist, specifically abolished the fMLP-induced contraction but not high K(+)- or carbachol-induced contractions. 7 A dual lipoxygenase/cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, a nonselective leukotriene receptor antagonist, and a selective type 1 cysteinyl-containing leukotriene receptor antagonist specifically reduced the fMLP-induced contraction. 8 We suggest that the low-affinity-type fMLP receptor and lipoxygenase metabolites of arachidonic acid are involved in the fMLP-induced contraction in the guinea-pig T. coli. This contraction mainly depends on the [Ca(2+)](i) elevation due to Ca(2+) influx and the enhancement of Ca(2+) sensitivity in the contractile apparatus.

LTB4 Can Directly Stimulate Human Osteoclast Formation from PBMC Independent of RANKL

Leukotriene B4, as a kind of 5-lipoxygenase metabolite of arachidonic acid, is known to influence osteoclast formation and bone resorption. In order to determine whether Leukotriene B4 could directly stimulate human osteoclast differentiation and activation independent of RANKL (ODF), three different concentrations of Leukotriene B4 (10(-9)M, 10(-8)M, 10(-7)M) were added to the culture of CD14+ monocyte fraction of peripheral blood mononuclear cell (PBMC) in the presence of macrophage colony-stimulating factor (M-CSF). Under these conditions, Leukotriene B4 could induce multinucleated cells, which were positive for Tartrate-resistant acidic phosphatase (TRAP) staining and capable of bone resorption. Addition of osteoprotegerin (OPG) to PBMC cultures does not abrogate osteoclast formation induced by LTB4. Osteoclastogenesis induced by Leukotriene B4 were dose-dependently increased and weaker than that of RANKL. These results indicated that Leukotriene B4, elevated in many inflammatory diseases, is directly capable of inducing osteoclast formation by a RANKL-independent mechanism.

Strain-related variations of AMPA receptor modulation by calcium-dependent mechanisms in the hippocampus: contribution of lipoxygenase metabolites of arachidonic acid

Several studies have demonstrated that C57 and DBA mice exhibit behavioural differences in diverse learning tasks as well as variations in the expression of long-term potentiation (LTP) in the hippocampus. In the present investigation, we tested the possibility that these differences between the two strains might be attributable to differential regulation of hippocampal α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors by calcium-dependent mechanisms. Using in vitro receptor autoradiography, we found that calcium treatment of C57 mice sections resulted in a marked increase of 3H-AMPA binding in areas CA 3 and CA 1 of the hippocampus and in the dentate gyrus. However, we discovered that the ability of calcium to upregulate 3H-AMPA binding in the DBA strain was much lower than in corresponding regions from the C57 strain. Western blot and immunohistochemical experiments indicated that truncation of AMPA receptor subunits by calcium-dependent mechanisms was possibly not responsible for the binding differences, as no significant variations in glutamate receptor subunit 1 (GluR1) and GluR2/3 immunoreactivity were observed between the two strains after calcium treatment. Interestingly, we found that strain-related variations in the regulation of 3H-AMPA binding by calcium were totally eliminated when brain sections were preincubated with preferential inhibitors of lipoxygenase (LO) pathways of arachidonic acid (AA) metabolism. Taken together, these results suggest that calcium-induced regulation of AMPA receptors varies between the two strains and that this variation might be linked to the production of specific AA metabolites.

Involvement of 5-lipoxygenase metabolites of arachidonic acid in cyclic AMP-stimulated steroidogenesis and steroidogenic acute regulatory protein gene expression

To understand the mechanism for the role of arachidonic acid (AA) in steroidogenic acute regulatory (StAR) gene transcription, sections of the −1/−966 StAR promoter were deleted to produce constructs of −1/−426, −1/−211, −1/−151, and −1/−110 and inserted into the PGL3 vector to drive luciferase expression. Results indicated that −1/−151 StAR promoter contains the elements that are most responsive to AA. Electrophoretic mobility shift assays using nuclear extracts from AA-treated MA-10 Leydig tumor cells showed that AA enhanced specific binding of the nuclear extract to a 30 bp (−67/−96) sequence of the StAR promoter. Also, HPLC was used to identify AA metabolites involved in StAR gene transcription. It was found that 1 mM N6,2-O-dibutyryladenosine 3:5-cyclic monophosphate (dbcAMP) significantly increased the 5-lipoxygenase metabolites, 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). Moreover, in the presence of 0.2 mM dbcAMP addition of 20 μM 5-HPETE or 5-HETE significantly enhanced StAR protein expression and progesterone production ( P<0.05). Similar results were obtained for StAR gene transcription with StAR mRNA levels and StAR promoter activities being significantly increased ( P<0.05) when 5-HPETE was added to MA-10 cell cultures. In summary, the present studies demonstrated that cyclic AMP (cAMP) stimulated the production of the AA metabolites, 5-HPETE and 5-HETE, and showed that these metabolites enhanced StAR gene expression and steroid hormone production. The results further suggested that the AA-responsive element resides in the −67/−96 region of the StAR promoter.

Norepinephrine-induced stimulation of p38 mitogen-activated protein kinase is mediated by arachidonic acid metabolites generated by activation of cytosolic phospholipase A(2) in vascular smooth muscle cells.

p38 mitogen-activated protein kinase (MAPK) is activated by norepinephrine (NE) in the vasculature and is implicated in vascular smooth muscle hypertrophy, contraction, and cell migration. NE promotes influx of Ca(2+) and activates cytosolic phospholipase A(2) (cPLA(2)) in vascular smooth muscle cells (VSMC). The purpose of this study was to determine the contribution of cPLA(2)-generated arachidonic acid (AA) and its metabolites to the activation of p38 MAPK measured by its phosphorylation, in response to NE in rabbit VSMC. NE-induced p38 MAPK activation was found to be mediated through the stimulation of alpha-1 and alpha-2 adrenergic receptors, was dependent on extracellular Ca(2+), and was attenuated by an inhibitor of cPLA(2) (pyrrolidine-1). Moreover, the cPLA(2) product, AA, activated p38 MAPK in VSMC. p38 MAPK activation elicited by NE was decreased significantly by the lipoxygenase (LO) inhibitor baicalein, and to a lesser extent by the cytochrome P450 inhibitor 17-octadecynoic acid, but was not affected by the cyclooxygenase inhibitor indomethacin. The LO metabolites of AA, namely 5(S)-hydroxyeicosatetraenoic acid (HETE), 12(S)-HETE, and 15(S)-HETE and the cytochrome P450 metabolite 20-HETE, activated p38 MAPK. NE-induced p38 MAPK stimulation was found to be independent of phospholipase D (PLD) activation in rabbit VSMC. Transactivation of the epidermal growth factor receptor (EGFR) by NE also did not contribute to p38 MAPK activation. These data suggest that cPLA(2)-generated AA and its LO metabolites mediate NE-induced p38 MAPK stimulation in rabbit VSMC by a mechanism that is independent of PLD and EGFR activation.