Formation Of Arachidonic Acid Metabolites Research Articles

Fluconazole Inhibits Cytochrome P450 1B1 and its Associated Arachidonic Acid Metabolites in the Heart and Protects against Angiotensin II‐induced Cardiac Hypertrophy

Cytochrome P450 1B1 (CYP1B1) has been reported to have a major role in metabolizing arachidonic acid (AA) into cardiotoxic metabolites, mid‐chain hydroxyeicosatetraenoic acid (HETEs). Of particular interest, several studies have demonstrated the role of mid‐chain HETEs in the development of cardiac hypertrophy. Recently, we have shown that fluconazole inhibits the formation of mid‐chain HETEs metabolite, however, whether fluconazole would be able to modulate CYP1B1‐mediated AA metabolism in an Ang II‐induced cellular hypertrophy model and in rats' heart has never been investigated before. Therefore, the objectives of this study were; 1) to investigate the effect of fluconazole on CYP1B1 mediated AA metabolites and to explore the potential protective effect against Ang II‐induced cellular hypertrophy, and 2) to study the effect of fluconazole on CYP1B1 and its associated AA metabolites in vivo in Sprague Dawley rats. For this purpose, both H9c2 and RL‐14 cells were treated with 10 μM Ang II in the absence and presence of 50 μM fluconazole for 24 hours. Also, Sprague Dawley rats (n=6) were injected intraperitoneally with a single dose of fluconazole (20 mg/kg) or saline and the heart tissues were harvested 24 hours post‐treatment. Thereafter, the formation of AA metabolites was measured using liquid chromatography‐electron spray ionization‐mass spectrometry (LC‐ESI‐MS). Whereas, the expression of cardiac hypertrophic markers and CYP1B1 were determined by real time‐polymerase chain reaction and Western blot analysis, respectively. Moreover, enzymatic activity and determination of CYP1B1 inhibition kinetics by fluconazole were assessed using 7‐ethoxyresorufin O‐deethylase (EROD) and recombinant human CYP1B1 assays. Our results demonstrated that fluconazole was able to attenuate Ang‐II‐induced cellular hypertrophy as evidenced by a significant inhibition of hypertrophic markers, β‐myosin heavy chain (MHC)/ α‐MHC, BNP as well as cell surface area. The protective effect of fluconazole was associated with a significant decrease in the level of CYP1B1 gene, protein, activity levels and its associated mid‐chain HETEs metabolite induced by Ang II. Furthermore, treatment of rats with fluconazole significantly decreased the expression of CYP1B1 enzyme and the formation level of cardiotoxic mid‐chain HETEs metabolites in the heart. In conclusion, our results showed that fluconazole protects against Ang II‐induced cellular hypertrophy by inhibiting CYP1B1 and its associated mid‐chain HETEs metabolites. Also, fluconazole may be repurposed as a mid‐chain HETEs formation inhibitor for the treatment of cardiac hypertrophy and heart failure.Support or Funding InformationThis work was supported by a grant from the Canadian Institutes of Health Research [Grant 106665] to A.O.S.E. A.H.A is the recipient of Saudi Arabian government scholarship provided by Qassim University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lipoxygenase-allene oxide synthase pathway in octocoral thermal stress response

Marine ecosystems are sensitive to elevated seawater temperature, with stony corals serving as model organisms for temperature-imposed declines in population viability and diversity. Several stress markers, including heat shock proteins, have been used for the detection and prediction of stress responses in stony corals. However, the stress indicators in soft corals remain elusive. In higher animals and plants, oxylipins synthesized by fatty acid di- and monooxygenases contribute to stress-induced signaling; however, the role of eicosanoid pathways in corals remains unclear. The eicosanoid gene specific to corals encodes for a natural fusion protein of allene oxide synthase and lipoxygenase (AOS-LOX). In this work, using the easily cultivated soft coral Capnella imbricata as the stress response model, we monitored the expression of the AOS-LOX and the formation of arachidonic acid metabolites in response to an acute rise in water temperature. Gene expression profiles of two 70 kDa heat shock proteins (Hsps: Hsp70 and Grp78) were used as a positive control for the stress response. In comparison with normal seawater temperature (23 °C), AOS-LOXa and Hsps were all up-regulated after modest (28 °C) and severe (31 °C) temperature elevation. While the up-regulation of AOS-LOXa and Grp78 was more sensitive to moderate temperature changes, Hsp70s were more responsive to severe heat shock. Concurrently, endogenous and exogenous AOS-LOXa-derived eicosanoids were up-regulated. Thus, together with the up-regulation of AOS-LOX by other abiotic and biotic stress stimuli, these data implicate AOS-LOX as part of the general stress response pathway in corals.

Organotypical vascular model for characterization of radioprotective compounds: studies on antioxidant 2,3-diaryl-substituted indole-based cyclooxygenase-2 inhibitors.

Radiotherapy of various cancers is closely associated with increased cardiovascular morbidity and mortality. Arachidonic acid metabolites are supposed to play a key role in radiation-induced vascular dysfunction. This study was designed to evaluate the effects of novel, antioxidative 2,3-diaryl-substituted indole-based selective cyclooxygenase-2 (COX-2) inhibitors (2,3-diaryl-indole coxibs) on radiation-induced formation of arachidonic acid metabolites via COX-2 and oxidant stress pathways in an organotypical vascular model of rat aortic rings. Acute and subacute effects of X-ray radiation (4 and 10 Gy; 1 and 3 days post irradiation) with or without the presence of 1 μM of the 2,3-diaryl-indole coxib 2-[4-(aminosulfonyl)phenyl]-3-(4-methoxyphenyl)-1H-indole (C1) or celecoxib as reference compared to sham-irradiated controls were assessed. The following parameters were measured: metabolic activity of the aortic rings; induction and regulation of COX-2 expression; release of prostaglandin E2 and F2α-isoprostane. Irradiation without presence of coxibs resulted in a dose-dependent augmentation of all parameters studied. When aortic rings were exposed to the 2,3-diaryl-indole coxib 1 h before irradiation, metabolic activity was restored and the release of both prostaglandin and isoprostane was inhibited. The latter indicates a direct interaction with oxidant stress pathways. By contrast, celecoxib exhibited only slight effects on the formation of isoprostane. The reduction of radiation-induced vascular dysfunction by antioxidative coxibs may widen the therapeutic window of COX-2 targeted treatment.

JJK694, a Synthesized Obovatol Derivative, Inhibits Platelet Activation by Suppressing Cyclooxygenase and Lipoxygenase Activities

Obovatol has various biological activities, including anti-proliferative, neurotrophic, anti-fibrillogenic, anti-platelet, anti-fungal and anti-inflammatory activities. In this study, we investigated the effects of JJK694, a synthesized obovatol derivative, on rabbit platelet activation and its molecular mechanisms. JJK694 significantly inhibited washed rabbit platelet aggregation and serotonin secretion induced by collagen and arachidonic acid, but had little effect on thrombin- or U46619-induced aggregation. These results suggest that JJK694 selectively inhibits collagen- and arachidonic acid-mediated signaling. JJK694 also showed a concentration-dependent decrease in cytosolic Ca(2+) mobilization, but it had no effect on arachidonic acid liberation. On the other hand, it significantly inhibited the formation of arachidonic acid metabolites, including thromboxane A(2) (TXA(2)), prostaglandin D(2), and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), by suppression of cyclooxygenase (COX)-1 and lipoxygenase (LOX) activities. These results indicate that JJK694 hasanti-platelet activities through inhibition of arachidonic acid metabolite production by suppression of COX-1 and LOX activities.

W1726 Lansoprazole, a Proton Pump Inhibitor, Mediated Anti-Inflammatory Effect in Gastric Mucosal Cells Through the Induction of Heme Oxygenase-1 Via the Activation of NRF2 and the Oxidation of Keap1

Dietary fats, which increase the risk of prostate cancer, stimulate release of intestinal neurotensin (NT), a growth-promoting peptide that enhances the formation of arachidonic acid metabolites in animal blood. This led us to use PC3 cells to examine the involvement of lipoxygenase (LOX) and cyclooxygenase (COX) in the growth effects of NT, including activation of EGF receptor (EGFR) and downstream kinases (ERK, AKT), and stimulation of DNA synthesis. NT and EGF enhanced [3H]-AA release, which was diminished by inhibitors of PLA2 (quinacrine), EGFR (AG1478) and MEK (U0126). NT and EGF phosphorylated EGFR, ERK and AKT, and stimulated DNA synthesis. These effects were diminished by PLA2 inhibitor (quinacrine), general LOX inhibitors (NDGA, ETYA), 5-LOX inhibitors (Rev 5901, AA861), 12-LOX inhibitor (baicalein) and FLAP inhibitor (MK886), while COX inhibitor (indomethacin) was without effect. Cells treated with NT and EGF showed an increase in 5-HETE levels by HPLC. PKC inhibitor (bisindolylmaleimide) blocked the stimulatory effects of NT, EGF and 5-HETE on DNA synthesis. We propose that 5-LOX activity is required for NT to stimulate growth via EGFR and its downstream kinases. The mechanism may involve an effect of 5-HETE on PKC, which is known to facilitate MEK-ERK activation. NT may enhance 5-HETE formation by Ca2+-mediated and ERK-mediated activation of DAG lipase and cPLA2. NT also upregulates cPLA2 and 5-LOX protein expression. Thus, the growth effects of NT and EGF involve a feed-forward system that requires cooperative interactions of the 5-LOX, ERK and AKT pathways.

Involvement of arachidonic acid metabolism and EGF receptor in neurotensin-induced prostate cancer PC3 cell growth

Dietary fats, which increase the risk of prostate cancer, stimulate release of intestinal neurotensin (NT), a growth-promoting peptide that enhances the formation of arachidonic acid metabolites in animal blood. This led us to use PC3 cells to examine the involvement of lipoxygenase (LOX) and cyclooxygenase (COX) in the growth effects of NT, including activation of EGF receptor (EGFR) and downstream kinases (ERK, AKT), and stimulation of DNA synthesis. NT and EGF enhanced [ 3H]-AA release, which was diminished by inhibitors of PLA2 (quinacrine), EGFR (AG1478) and MEK (U0126). NT and EGF phosphorylated EGFR, ERK and AKT, and stimulated DNA synthesis. These effects were diminished by PLA2 inhibitor (quinacrine), general LOX inhibitors (NDGA, ETYA), 5-LOX inhibitors (Rev 5901, AA861), 12-LOX inhibitor (baicalein) and FLAP inhibitor (MK886), while COX inhibitor (indomethacin) was without effect. Cells treated with NT and EGF showed an increase in 5-HETE levels by HPLC. PKC inhibitor (bisindolylmaleimide) blocked the stimulatory effects of NT, EGF and 5-HETE on DNA synthesis. We propose that 5-LOX activity is required for NT to stimulate growth via EGFR and its downstream kinases. The mechanism may involve an effect of 5-HETE on PKC, which is known to facilitate MEK-ERK activation. NT may enhance 5-HETE formation by Ca 2+-mediated and ERK-mediated activation of DAG lipase and cPLA2. NT also upregulates cPLA2 and 5-LOX protein expression. Thus, the growth effects of NT and EGF involve a feed-forward system that requires cooperative interactions of the 5-LOX, ERK and AKT pathways.

Role of cyclooxygenase-2, but not cyclooxygenase-1, on type II collagen-induced arthritis in DBA/1J mice

The purpose of this paper is to explore the contribution of isoforms of cyclooxygenase (COX) to chronic inflammation in DBA/1J mice with type II collagen-induced arthritis (CIA). To address this question pharmacologically, we tested the effects of selective inhibitors of COX-1 and COX-2 on paw edema and the formation of arachidonic acid metabolites in the inflamed paws immunized with type II collagen (CII). Oral administration of FR140423 (3-(difluoromethyl)-1-(4-methoxyphenyl)-5-[4-(methylsulfinyl)phenyl]pyrazole), a selective inhibitor of COX-2, showed a dose-dependent anti-inflammatory effect in mouse CIA with ed 50 value of 0.20 mg/kg. Indomethacin, a non-selective inhibitor of COX, also inhibited paw edema in this arthritic model. In contrast, the selective COX-1 inhibitors, FR122047 (1-[(4,5-bis(4-methoxyphenyl)-2-thiazoyl)carbonyl]-4-methylpiperazine hydrochloride) and SC-560 (5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole), had no effect in mouse CIA model. The increase of prostaglandin (PG) E 2 and thromboxane (TX) B 2 in the mouse inflamed paws was associated with the development of paw edema induced by CII. FR140423 dose dependently inhibited the levels of PGE 2 and TXB 2 in the CIA mouse paws with ed 50 values of 0.20 and 0.12 mg/kg, respectively, similar to indomethacin. In contrast, FR122047 and SC-560 had no effect. These results suggest that COX-2, but not COX-1, contributes to the edema and the formation of PGE 2 and TXB 2 in mouse CIA model.

Bacterial challenge stimulates formation of arachidonic acid metabolites by human keratinocytes and neutrophils in vitro.

Although the interactions of bacteria with keratinocytes induce the synthesis of various mediators, the capability of epithelial cells to form arachidonic acid mediators has not been studied, and therefore the first part of this study was initiated. The complex mixture of epithelium-derived mediators suggests that chemoattraction is not their only effect on neutrophils and that they may also affect neutrophil mediator synthesis. The effect of epithelium-derived mediators on neutrophil eicosanoide synthesis was evaluated in the second part of this study. We incubated human keratinocytes with human-pathogenic bacteria for 2 h and harvested the supernatants after 4, 6, 10, and 18 h of culture. Subsequently, the supernatants were coincubated for 5 min with human neutrophils with or without arachidonic acid. The formation of the arachidonic acid metabolites prostaglandin E(2) (PGE(2)), leukotriene B(4) (LTB(4)), 12-hydroxyeicosatetraenoic acid (12-HETE), and 15-HETE in keratinocytes and neutrophils was measured by reverse-phase high-pressure liquid chromatography. We demonstrated for the first time that keratinocytes produced significant amounts of LTB(4) and 12-HETE 4 to 6 h after bacterial challenge. Upon stimulation with epithelial supernatants, neutrophils produced significant amounts of PGE(2), LTB(4), 12-HETE, and 15-HETE throughout the observation period of 18 h, with a maximum synthesis by supernatants harvested 4 to 10 h after bacterial infection. The results of the study suggest that arachidonic acid mediator formation by epithelial cells following bacterial challenge may act as an early inflammatory signal for the initiation of the immune response. The epithelial supernatants were capable of inducing the formation of arachidonic acid mediators by neutrophils, which may have further regulatory effects on the immune response.

Anti-inflammatory activity of a novel selective cyclooxygenase-2 inhibitor, FR140423, on type II collagen-induced arthritis in Lewis rats

The mechanism of action of FR140423 (3-(difluoromethyl)-1-(4-methoxyphenyl)-5-[4-(methylsulfinyl)-phenyl]pyrazole), a novel and selective cyclooxygenase (COX)-2 inhibitor, in rat type II collagen-induced arthritis was investigated and compared with that of indomethacin. We tested the inhibitory effects of FR140423 on paw edema and the formation of arachidonic acid metabolites in inflamed paws immunized with type II collagen. Oral administration of FR140423 showed a dose-dependent anti-inflammatory effect and was two-fold more potent than indomethacin. The increase of prostaglandin (PG) E 2 and thromboxane (TX) B 2 but not leukotriene B 4 in inflamed paws was associated with the development of paw edema. FR140423 and indomethacin dose-dependently suppressed the levels of PGE 2 and TXB 2 in arthritic rat paws. Unlike indomethacin, FR140423 did not induce gastric lesions in arthritic rats. These results suggest that FR140423 shows a potent anti-inflammatory effect mediated by inhibition of prostanoids produced by COX-2 in inflamed tissues immunized with type II collagen, with a greatly improved safety profile compared to indomethacin.

Separation of hydroxyeicosatetraenoic acids and leukotrienes originating from kidney tissue by C-18 reverse-phase high-pressure liquid chromatography.

AbstractMetabolism of arachidonic acid results in formation of eicosanoids including prostaglandins (PGs), thromboxanes (Txs), monohydroxyeicosatetraenocinoids (HETEs), leukotrienes (LTs), lipoxins (Lxs), epoxy metabolites, and hepoxilins (1). Most of these compounds are formed in minute quantities in response to specific stimuli and participate in modulation of the vascular tone or of the inflammatory process. We have been studying the formation of arachidonic acid metabolites in kidney tissue (mainly glomeruli) following immune injury. Normal glomeruli synthesize very small quantities of HETEs and LTs. Following immune injury, glomerular synthesis and levels of these eicosanoids increase (2). Leukotrienes (B4, C4, and D4), 5-HETE, and 12-HETE can be separated, detected, and quantitated by several analytical techniques, including immunoassay, bioassay, gas chromatography-mass spectrometry (GC-MS), thin-layer chromatography (TLC), ultraviolet spectroscopy, and high pressure liquid chromatography (HPLC).KeywordsHigh Pressure Liquid ChromatographyReverse Phase ColumnInjection PortHydroxyeicosatetraenoic AcidImmune InjuryThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Some perspectives on dietary inhibition of carcinogenesis: studies with curcumin and tea.

Topical application of curcumin inhibits chemically induced carcinogenesis on mouse skin, and oral administration of curcumin inhibits chemically induced oral, forestomach, duodenal, and colon carcinogenesis. Curcumin and other inhibitors of cyclooxygenase and lipoxygenase are thought to inhibit carcinogenesis by preventing the formation of arachidonic acid metabolites. In contrast to our expectation of a tumorigenic effect of arachidonic acid, we found that treatment of 7,12-dimethylbenz[a]anthracene-initiated mouse skin with very high doses of arachidonic acid twice daily, 5 days a week for 26 weeks, failed to result in tumors. We considered the possibility that some of the cancer chemopreventive effects of curcumin may be related to an effect of this compound on cellular differentiation, and we investigated the effect of curcumin on differentiation in the human promyelocytic HL-60 leukemia cell model system. Although curcumin alone had little or no effect on cellular differentiation, when it was combined with all-trans retinoic acid or 1alpha,25-dihydroxyvitamin D3 a synergistic effect was observed. It is possible that many dietary chemicals in fruits, vegetables, and other edible plants can prevent cancer by synergizing with endogenously produced stimulators of differentiation such as all-trans retinoic acid, 1alpha,25-dihydroxyvitamin D3, and butyrate. More research is needed to test this hypothesis. Administration of green or black tea inhibits carcinogenesis in several animal models, and tumor growth is also inhibited. Several examples were presented of chemopreventive agents that inhibit carcinogenesis in one animal model but enhance carcinogenesis in a different animal model. Greater efforts should be made to understand mechanisms of cancer chemoprevention and to determine whether a potential chemopreventive agent is useful in many experimental settings or whether it is useful in only a limited number of experimental settings.

PRODUCTION OF ARACHIDONIC ACID LIPOXYGENASE METABOLITES BY THE INTESTINAL EPITHELIAL CELL LINE HT29 CL. 19A AND THEIR EFFECT ON CHLORIDE SECRETION.

Arachidonic acid (AA) lipoxygenase products may be important in the pathogenesis of diarrhoea associated with intestinal inflammation, because they may stimulate electrogenic chloride secretion in the epithelium. However, the role of the epithelial cells as producers or as targets for lipoxygenase products remains controversial. To clarify this role, we measured the following on the same clone of a chloride-secreting intestinal cell line of human origin (HT29 cl. 19A): 1) the expression of the mRNAs of 5-lipoxygenase, platelet and leukocyte type 12-lipoxygenase, 15-lipoxygenase, LTA4 hydrolase, Five-Lipoxygenase-Activating-Protein (FLAP), prostaglandin H (PGH) synthase-1 and PGH synthase-2 after total RNA extraction, reverse transcription and cDNA amplification by PCR, using specific primers; 2) the formation of AA metabolites by high pressure liquid chromatography (HPLC) and 3) the effect of such metabolites on the stimulation of short-circuit current (Isc). an index of chloride secretion, in filter-grown HT29 cl. 19A cells mounted in Ussing chambers. Results: The mRNAs of 5-lipoxygenase, 15-lipoxygenase, LTA4 hydrolase, PGH synthase-1 and PGH synthase-2 were equally expressed in HT29 cl. 19A cells at different stages of culture, but FLAP, platelet and leukocyte type 12-lipoxygenase mRNAs were not expressed. When dispersed HT29 cl. 19A cells were incubated with [1-14C] arachidonic acid, calcium ionophore A23187, Mg2+ and Ca2+, they produced 5-hydroxyeicosatetraenoic acid (5-HETE), 15-hydroxyeicosatetraenoic acid (15-HETE), and a peak containing leukotriene B4 (LTB4) unresolved from its 6-trans-stereoisomers, and 5(S)-12(S)-di-HETEs. No peptidoleukotriene were detected. Unlabelled AA metabolites, analyzed by HPLC and enzyme immunoassay, were mainly 6-trans-stereoisomers of LTB4 and unidentified di-HETEs, but only a small amount of LTB4 was detected. In addition to prostaglandin E2 (PGE2), which was the most potent compound, several hydroperoxyeicosatetraenoic acids (HPETEs) produced by autoxidation of AA (5-, 9-, 11-, 12- and 15-HPETEs) induced bumetanide-sensitive chloride secretion by HT29 cl. 19A cells (Δ Isc: 3.2 ± 0.4 to 12.1 ± 3.7 μA cm-2). The 9-, 12- and 15-HETEs obtained by triphenylphosphine reduction of the corresponding HPETEs, displayed the same activity as the parent compound, but the 5- and 11-HETEs lose their potencies. LTB4 had no effect on chloride secretion. The present results suggest that the enterocyte may participate in the production of AA metabolites through the lipoxygenase and prostaglandin synthase pathways. In addition, the enterocytes are a target for the lipoxygenase pathway metabolites they and other cells produce.

Interaction of substance P with epidermal growth factor and fibroblast growth factor in cyclooxygenase‐dependent proliferation of human skin fibroblasts

Substance P (SP), fibroblast growth factor (FGF), and epidermal growth factor (EGF) are mitogens for fibroblasts. EGF acts as a progression factor, whereas FGF and SP have competence factor activity. The ability of eicosanoids to regulate proliferation of fibroblasts and the increased production of prostaglandins by fibroblasts in response to the growth factors, led us to investigate the involvement of cyclooxygenase-dependent arachidonic acid metabolites in the mitogenic response of serum-starved human skin fibroblasts to SP, FGF, and EGF. We tested the interaction of a submaximal concentration of SP(10−9 M) with baFGF (40 μg/ml) and EGF(0.01 μg/ml) both on fibroblast proliferation and release of arachidonic acid metabolites. A combination of SP and EGF synergistically stimulated fibroblast proliferation and prostaglandin E2 release, whereas addition of SP to FGF-containing cultures did not affect cell growth. Inhibition of cyclooxygenase by acetylsalicylic acid augmented the growth response of fibroblasts to all: SP, FGF, and EGF. In the presence of acetylsalicylic acid, SP combined with FGF enhanced fibroblast proliferation, whereas a combination with EGF inhibited cellular growth with respect to growth induced by EGF alone. Thus, interactions of SP with FGF and EGF differently affected the mitogenic response depending on the formation of arachidonic acid metabolites. The findings indicate that eicosanoids may be important mediators of competence and progression factor activities that may determine the effects of substance P on fibroblast proliferation in a cytokine network. © 1996 Wiley-Liss, Inc.

Interaction of substance P with epidermal growth factor and fibroblast growth factor in cyclooxygenase-dependent proliferation of human skin fibroblasts.

Substance P (SP), fibroblast growth factor (FGF), and epidermal growth factor (EGF) are mitogens for fibroblasts. EGF acts as a progression factor, whereas FGF and SP have competence factor activity. The ability of eicosanoids to regulate proliferation of fibroblasts and the increased production of prostaglandins by fibroblasts in response to the growth factors, led us to investigate the involvement of cyclooxygenase-dependent arachidonic acid metabolites in the mitogenic response of serum-starved human skin fibroblasts to SP, FGF, and EGF. We tested the interaction of a submaximal concentration of SP(10(-9)M) with baFGF(40 micrograms/ml) and EGF(0.01 microgram/ml) both on fibroblast proliferation and release of arachidonic acid metabolites. A combination of SP and EGF synergistically stimulated fibroblast proliferation and prostaglandin E2 release, whereas addition of SP to FGF-containing cultures did not affect cell growth. Inhibition of cyclooxygenase by acetylsalicylic acid augmented the growth response of fibroblasts to all: SP, FGF, and EGF. In the presence of acetylsalicylic acid, SP combined with FGF enhanced fibroblasts proliferation, whereas a combination with EGF inhibited cellular growth with respect to growth induced by EGF alone. Thus, interactions of SP with FGF and EGF differently affected the mitogenic response depending on the formation of arachidonic acid metabolites. The findings indicate that eicosanoids may be important mediators of competence and progression factor activities that may determine the effects of substance P on fibroblast proliferation in a cytokine network.

Effects of linoleic acid, eicosapentaenoic acid, and docosahexaenoic acid on the growth and metastasis of MM48 mammary tumor transplants in mice

In this study, we investigated the effects of linoleic acid (LA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) on the growth, metastasis and cell proliferation of a murine mammary tumor (MM48) transplanted into mice. We also examined the effect of these treatments on survival and tumor fatty acids. The growth of the primary tumor, number of metastatic tumors in the lung, and cell proliferation of the tumor were significantly inhibited in the EPA and DHA groups compared with the control and/or LA groups. Mice in the EPA group survived significantly longer than those in the control and LA groups, while mice in the DHA group survived longer than those in the control group. While the ratio of arachidonic acid (AA)/higher PUFAs (polyunsaturated fatty acids) in tumors correlated with tumor growth and lung metastases, the ratio of AA/LA did not. Therefore, it appears that both EPA and DHA exert their effects by competing with AA, thereby lowering the formation of AA metabolites.

The mechanism of arachidonic acid release in collagen-activated human platelets.

The mechanism of arachidonic acid (AA) release in collagen-activated human platelets was studied. An arachidonic acid metabolite, thromboxane B2 (TXB2), was formed in parallel with the formation of phosphatidic acid (PA) without formation of lysophosphatidic acid (lysoPA) or lysophosphatidylinositol (lysoPI) in the absence of extracellular Ca2+, suggesting that AA was released from PI via a PI-specific phospholipase C (PI-PLC)/diacylglycerol (DG) lipase/monoacylglycerol (MG) lipase pathway under the cytosolic low Ca2+ concentrations. Moreover, solubilized DG lipase and MG lipase could hydrolyze the substrates at basal cytosolic free Ca2+ concentrations. Subsequently, the relationship of cytosolic free Ca2+ concentrations and formation of AA metabolites was analyzed using Ca2+ ionophore, A23187. Collagen was able to induce a release of small amounts of AA under basal cytosolic Ca2+ conditions. However, a release of large amounts of AA was induced by phospholipase A2 activated by both collagen-receptor occupancy and elevated Ca2+ levels. A TXA2 mimetic agonist, STA2 induced all the responses except for AA release. From these results, the mechanism of AA release and signal transduction in collagen-activated human platelets is discussed.

Glucocorticoids as cytokine inhibitors: role in neuroendocrine control and therapy of inflammatory diseases

Glucocorticoids are potent inhibitors of inflammation and endotoxic shock. This probably occurs through an inhibition of the synthesis of pro-inflammatory cytokines as well as of many of their toxic activities. Therefore, endogenous glucocorticoids (GC) might represent a major mechanism in the control of cytokine mediated pathologies. GC inhibit the synthesis of cytokines in various experimental models. Adrenalectomy or GC antagonists potentiate TNF, IL-1 and IL-6 production in LPS treated mice. GC inhibit the formation of arachidonic acid metabolites and the induction of NO synthase. They also inhibit various activities of cytokines including toxicity, haemodynamic shock and fever. Adrenalectomy sensitizes to the toxic effects of LPS, TNF and IL-1. On the other hand, GC potentiate the synthesis of several cytokine induced APP by the liver. Since many of these proteins have anti-toxic activities (antioxidant, antiprotease etc.) or bind cytokines, this might well represent a GC mediated protective feedback mechanism involving the liver. Not only do GC inhibit cytokines, but in vivo LPS and various cytokines (TNF, IL-1, IL-6) increase blood GC levels through a central mechanism involving the activation of the HPA. Thus, this neuroendocrine response to cytokines constitutes an important immunoregulatory feedback involving the brain.

Phospholipase A2 modulates different subtypes of excitatory amino acid receptors: autoradiographic evidence.

Exogenous phospholipases have been used extensively as tools to study the role of membrane lipids in receptor mechanisms. We used in vitro quantitative autoradiography to evaluate the effect of phospholipase A2 (PLA2) on N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in rat brain. PLA2 pretreatment induced a significant increase in alpha-[3H]amino-3-hydroxy-5-methyl-isoxazole-4-propionate ([3H]AMPA) binding in the stratum radiatum of the CA1 region of the hippocampus and in the stratum moleculare of the cerebellum. No modification of [3H]AMPA binding was found in the stratum pyramidale of the hippocampus at different ligand concentrations. [3H]-Glutamate binding to the metabotropic glutamate receptor and the non-NMDA-, non-kainate-, non-quisqualate-sensitive [3H]glutamate binding site were also increased by PLA2 pretreatment. [3H]Kainate binding and NMDA-sensitive [3H]glutamate binding were minimally affected by the enzyme pretreatment. The PLA2 effect was reversed by EGTA, the PLA2 inhibitor p-bromophenacyl bromide, and prolonged pretreatment with heat. Bovine serum albumin (1%) prevented the increase in metabotropic binding by PLA2. Arachidonic acid failed to mimic the PLA2 effect on metabotropic binding. These results indicate that PLA2 can selectively modulate certain subtypes of excitatory amino acid receptors. This effect is due to the enzymatic activity but is probably not correlated with the formation of arachidonic acid metabolites. Independent of their possible physiological implications, our results provide the first autoradiographic evidence that an enzymatic treatment can selectively affect the binding properties of excitatory amino acid receptors in different regions of the CNS.

Modulation of erythropoiesis by novel human bone marrow cytochrome P450- dependent metabolites of arachidonic acid

In the hematopoietic system the adherent stromal cells produce cytokines necessary for proliferation and differentiation of hematopoietic cells. In the present study, we showed the ability of adherent stromal cells to generate novel metabolites of arachidonic acid via the NADPH-cytochrome P450-dependent monooxygenase system. These metabolites were recovered in the incubation media, suggesting their release from cells. The formation of arachidonic acid metabolites was inhibited by 7-ethoxyresorufin and SKF-525A, but not by indomethacin or BW-755C. By using two-step high-pressure liquid chromatography (HPLC), bone marrow-adherent stromal cells and incubation media showed the presence of metabolites in a peak eluted at 19 to 20 minutes. The isolated HPLC peak was used to measure its effect on colony-forming unit-erythroid (CFU-E) growth and compare it with that of synthetic cytochrome P450 arachidonate metabolites, 19- and 20- hydroxyeicosatetraenoic (HETE) acid. These bone marrow cytochrome P450 arachidonic acid metabolites at picomolar concentration potentiated erythropoietin (Epo)-induced CFU-E growth by fourfold to sixfold. Addition of 19- and 20-HETE to the bone marrow culture resulted in a potentiating effect on CFU-E number in a dose-dependent manner. 20-HETE was much more potent in stimulating CFU-E growth than 19-HETE at a similar concentration of 10(-11) mol/L. The potentiating effect of 20- HETE resulted in a shifting to the left of the dose-response curve to Epo. To substantiate the finding of an active NADPH-dependent cytochrome P450-metabolizing system, we further examined the ability of adherent cells to metabolize exogenous pharmacologic compounds such as benzo(a)pyrene, a substrate for the heme-cytochrome P450 system, aryl hydrocarbon hydroxylase. The adherent stromal cytochrome P450 metabolizes benzo(a)pyrene at comparable levels to blood vessel endothelial cells. These novel observations underscore the importance of adherent stromal cytochrome P450 to metabolize endogenous substrates, including arachidonic acid, to compounds that may interact in a paracrine manner with Epodependent hematopoietic cells.

Modulation of Erythropoiesis by Novel Human Bone Marrow Cytochrome P450-Dependent Metabolites of Arachidonic Acid

In the hematopoietic system the adherent stromal cells produce cytokines necessary for proliferation and differentiation of hematopoietic cells. In the present study, we showed the ability of adherent stromal cells to generate novel metabolites of arachidonic acid via the NADPH-cytochrome P450-dependent monooxygenase system. These metabolites were recovered in the incubation media, suggesting their release from cells. The formation of arachidonic acid metabolites was inhibited by 7-ethoxyresorufin and SKF-525A, but not by indomethacin or BW-755C. By using two-step high-pressure liquid chromatography (HPLC), bone marrow-adherent stromal cells and incubation media showed the presence of metabolites in a peak eluted at 19 to 20 minutes. The isolated HPLC peak was used to measure its effect on colony-forming unit-erythroid (CFU-E) growth and compare it with that of synthetic cytochrome P450 arachidonate metabolites, 19- and 20-hydroxyeicosatetraenoic (HETE) acid. These bone marrow cytochrome P450 arachidonic acid metabolites at picomolar concentration potentiated erythropoietin (Epo)-induced CFU-E growth by fourfold to sixfold. Addition of 19- and 20-HETE to the bone marrow culture resulted in a potentiating effect on CFU-E number in a dose-dependent manner. 20-HETE was much more potent in stimulating CFU-E growth than 19-HETE at a similar concentration of 10(-11) mol/L. The potentiating effect of 20-HETE resulted in a shifting to the left of the dose-response curve to Epo. To substantiate the finding of an active NADPH-dependent cytochrome P450-metabolizing system, we further examined the ability of adherent cells to metabolize exogenous pharmacologic compounds such as benzo(a)pyrene, a substrate for the heme-cytochrome P450 system, aryl hydrocarbon hydroxylase. The adherent stromal cytochrome P450 metabolizes benzo(a)pyrene at comparable levels to blood vessel endothelial cells. These novel observations underscore the importance of adherent stromal cytochrome P450 to metabolize endogenous substrates, including arachidonic acid, to compounds that may interact in a paracrine manner with Epodependent hematopoietic cells.