Major Arachidonic Acid Metabolite Research Articles

Microsomal prostaglandin E synthase-1 and blood pressure regulation

Prostaglandin E (PGE)(2) is a major arachidonic acid metabolite in a wide variety of tissues and is implicated in the control of inflammatory as well as physiological responses. At least three major forms of PGE synthase (PGES) have recently been cloned and characterized: membrane-associated PGES (mPGES)-1, mPGES-2, and cytosolic PGES (cPGES). Among them, mPGES-1 is highly inducible by cytokine and is critically involved in pain and inflammatory responses. Emerging evidence suggests that mPGES-1 may also participate in blood pressure (BP) regulation through an impact on renal and vascular functions. Within the kidney, mPGES-1 predominates in the distal nephron where its expression is highly inducible by salt loading. Mice lacking mPGES-1 exhibit blunted natriuretic response paralleled with remarkably suppressed nitric oxide production, leading to salt-sensitive hypertension. These mice also exhibit an exaggerated hypertensive response to angiotensin II infusion. Together, these results suggest that mPGES-1 may be an important physiological regulator of BP.

Expression of 15-lipoxygenase type-1 in human mast cells

Mast cells play a key role in the pathophysiology of asthma. These cells exert their effector functions by releasing a variety of proinflammatory and immunoregulatory compounds. Mast cells infiltrate the bronchial epithelium and smooth muscle to a higher degree in patients with asthma compared to control subjects. 15-Lipoxygenase type-1 (15-LO-1) is a prooxidant enzyme which is expressed in asthmatic lungs leading to formation of pro- and anti-inflammatory mediators. Here we report that interleukin-4 (IL-4) induced the expression of 15-LO-1 in human cord blood derived mast cells (CBMC) as demonstrated by RT-PCR, western blot and immunocytochemistry. The major metabolite of arachidonic acid formed via the 15-LO pathway in IL-4 treated CBMC was identified as 15-ketoeicosatetraenoic acid (15-KETE, also named 15-oxo-ETE) with smaller amounts of 15-hydroxyeicosatetraenoic acid (15-HETE) as identified by HPLC and mass spectrometry (MS/MS). Furthermore, immunohistochemical stainings demonstrated the expression of 15-LO-1 in mast cells in lung and skin in vivo. Osmotic activation of CBMC with mannitol resulted in activation of the 15-LO-1 pathway. In conclusion, the expression of 15-LO-1 and release of 15-LO-1 derived products by mast cells may contribute to the role of these cells in asthma and other inflammatory diseases.

Role of 20-HETE in D1/D2 dopamine receptor synergism resulting in the inhibition of Na+-K+-ATPase activity in the proximal tubule

Previous studies propose 20-hydroxyeicosatetraenoic acid (20-HETE), a major arachidonic acid metabolite of cytochrome P-450 (CYP), as a possible mediator of Na(+)-K(+)-ATPase inhibition by dopamine (DA). The aim of this study was to investigate the intracellular mechanisms involved in this effect and to elucidate the DA receptor associated with the 20-HETE pathway in the rat kidney. DA (10(-5) M) inhibited Na(+)-K(+)-ATPase activity in microdissected tubular segments to 59.4 +/- 3.8% of control activity. This response was suppressed by the CYP4A inhibitor 17-octadecynoic acid (10(-6) M), which had no effect per se, thus confirming the participation of CYP arachidonic acid metabolites in DA-induced Na(+)-K(+)-ATPase inhibition. We next examined whether 20-HETE is involved in the signaling pathways triggered by either D(1) or D(2) receptors. Neither fenoldopam nor quinpirole (D(1) and D(2) agonists, respectively, both 10(-5) M) modified Na(+)-K(+)-ATPase activity when tried alone. However, coincubation of a threshold concentration of 20-HETE (10(-9) M) with fenoldopam resulted in a synergistic inhibition of Na(+)-K(+)-ATPase activity (66 +/- 2% of control activity), while 20-HETE plus quinpirole had no effect. Furthermore, 20-HETE (10(-9) M) synergized with forskolin (10(-5) M) and with the diacylglycerol analog 1-oleoyl-2-acetoyl-sn-glycerol (OAG; 10(-11) M; 62.0 +/- 5.3 and 69.9 +/- 2.0% of control activity, respectively), indicating a cooperative role of 20-HETE with the D(1)-triggered pathways. In line with these results, no additive effect was observed when OAG and 20-HETE were combined at concentrations which per se produced maximal inhibition (10(-6) M). These results demonstrate that the inhibition of Na(+)-K(+)-ATPase activity by DA in the proximal tubule may be the result of the synergism between 20-HETE and the D(1) signaling pathway.

HET0016 inhibits epoxyeicosatrienoic acid‐induced pulmonary vasoconstriction by an ω‐hydroxylase‐independent mechanism

Recently, endogenous epoxyeicosatrienoic acids (EETs) were reported to be required for full expression of hypoxic vasoconstriction in a model of pulmonary hypertension. Previously, a cytochrome P450 (CYP) ω-hydroxylase enzyme, CYP4A, was identified in the rabbit pulmonary artery (PA). The major arachidonic acid (AA) metabolite of CYP4A, 20-HETE, is a pulmonary vasodilator. Endogenous 20-HETE blunts hypoxia- and agonist-induced pulmonary vasoconstriction. Thus, 20-HETE may blunt EET-induced pulmonary vasoconstriction. To test this hypothesis, we determined whether inhibition of ω-hydroxylase activity would augment 5,6-EET-induced PA constriction. PA (1–3 mm od) rings were suspended from force displacement transducers in physiological salt solution gassed with 95% O2, 5% CO2. HET0016, a selective inhibitor of CYP4A activity, reduced 5,6-EET-induced PA contraction at 1–10 μM (P<0.05), whereas a non-selective CYP inhibitor, 17-ODYA (10 μM), did not. HET0016 (10 μM) did not inhibit PGF2α- or 5-HT- induced PA contractions but did inhibit AA- (5 μM) induced contractions. Since 5,6-EET and AA, but not PGF2α and 5-HT -induced PA contractions require cyclooxygenase activity, these results suggest that HET0016 (1–10 μM) inhibits 5,6-EET-induced contractions, not by inhibiting ω-hydroxylase enzyme activity, but by inhibiting cyclooxygenase activity, an often overlooked property HET0016. Support: NIH HL-64180.

Inhibition of PGI 2 signaling by miconazole in vascular smooth muscle cells

Miconazole is widely used clinically as an anti-fungal agent and experimentally as a cytochrome P450 (CYP) inhibitor. In rat coronary arteries that produce PGI 2 as the major arachidonic acid (AA) metabolite, activation of the large-conductance K + (BK) channels in coronary arterial smooth muscle cells by AA was inhibited by miconazole but not by the CYP inhibitor SKF525A. Activation of BK currents in coronary smooth muscle cells by carbacyclin or iloprost also was inhibited by miconazole but not by SKF525A, suggesting that miconazole might have properties other than those of CYP inhibition. In addition, carbacyclin-induced dilation of isolated mesenteric arteries was inhibited by treatment with miconazole (51.9 ± 4.2% dilation in control, n = 7 versus 30.1 ± 4.0% with miconazole, n = 4, p < 0.005) but not SKF525A (52.8 ± 3.6%, n = 8). In contrast, miconazole did not affect BK channel activation and vasodilation produced by the phosphodiesterase inhibitor RO-201724. In cultured coronary smooth muscle cells, carbacyclin (1 μM) stimulated cAMP production by 22-fold (183 ± 29 pmol/mg at baseline, 4062 ± 212 pmol/mg with carbacyclin, n = 3, p < 0.001). The carbacyclin effect was significantly attenuated by treatment with miconazole (1542 ± 201 pmol/mg, n = 3, p < 0.001 versus carbacyclin alone), but not by SKF525A (3460 ± 406 pmol/mg, n = 3, p = NS versus carbacyclin alone). These results indicate that in addition to its CYP inhibition properties, miconazole inhibits PGI 2 signaling. Hence, experiments using miconazole as a CYP inhibitor should be interpreted with caution.

Roles of prostaglandin D2 and prostaglandin E2 in allergic reactions: pro-allergic and anti-allergic modulating pathways

Prostaglandin(PG) D2 and PGE2 are major cyclooxygenase metabolites of arachidonic acid produced during allergic reactions including asthma. However, the role of PGD2 and PGE2 in allergic inflammation has long been ambiguous. This is partly because non-steroidal anti-inflammatory drugs that inhibit prostanoid synthesis are generally ineffective in allergic disorders. Both PGs exert their actions by acting on G-proteincoupled receptors; PGD2 acts at the PGD receptor(DP), PGE2 acts at four subtypes of PGE receptor, EP1 to EP4. To dissect the roles of PGD2-DP pathway and each PGE2-EP pathway in allergic reactions, we subject mice deficient in DP, EP1, EP2, EP3 and EP4 receptor individually to ovalbumin-induced allergic asthma as a model of type I allergy. These studies have revealed that there are opposing actions between two prostanoid pathways in allergic reactions; PGD2-DP pathway and PGE2-EP3 pathway. We found that the PGD2 is an important mediator of allergic responses and that PGE2-EP3 signaling negatively regulates progression of allergic inflammation. This review focuses on the opposite roles of these prostanoid pathways in allergic reactions obtained by our studies and other studies. These findings suggest that selective manipulation of the prostanoid receptors may be beneficial in treatment of allergic diseases, such as bronchial asthma, allergic rhinitis and conjunctivitis.

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.

Impaired Arachidonic Acid–Mediated Activation of Large-Conductance Ca2+-Activated K+ Channels in Coronary Arterial Smooth Muscle Cells in Zucker Diabetic Fatty Rats

We studied the arachidonic acid (AA)-mediated modulation of large-conductance Ca2+-activated K+ (BK) channels in coronary arterial smooth myocytes from lean control and Zucker Diabetic Fatty (ZDF) rats. A total of 1 micromol/l AA enhanced BK current by 274% in lean and by 98% in ZDF rats. After incubation with 10 micromol/l indomethacin, 1 micromol/l AA increased BK currents by 80% in lean and by 70% in ZDF rats. Vasoreactivity studies showed that the dilation of small coronary arteries produced by 1 micromol/l AA was reduced by 44% in ZDF rats. [3H]6-keto-prostagladin F1alpha ([3H]6-keto-PGF1alpha,), the stable metabolite of prostacyclin (PGI2), was the major [3H]AA metabolite produced by coronary arteries of lean vessels, but ZDF vessels produced only 15% as much [3H]6-keto-PGF1alpha. BK channel activation and vasorelaxation by iloprost were similar in lean and ZDF rats. Immunoblots showed a 73% reduction in PGI2 synthase (PGIS) expression in ZDF vessels compared with lean vessels, and there was no change in cyclooxygenase (COX) and BK channel expressions. Real-time PCR studies showed that mRNA levels of PGIS, COX-1, and COX-2 were similar between lean and ZDF vessels. We conclude that PGI2 is the major AA metabolite in lean coronaries, and AA-mediated BK channel activation is impaired in ZDF coronaries due to reduced PGIS activity.

Role of prostaglandin E receptor EP1 subtype in the development of renal injury in genetically hypertensive rats.

One of the major causes of end-stage renal diseases is hypertensive renal disease, in which enhanced renal prostaglandin (PG) E2 production has been shown. PGE2, a major arachidonic acid metabolite produced in the kidney, acts on 4 receptor subtypes, EP1 through EP4, but the pathophysiological importance of the PGE2/EP subtypes in the development of hypertensive renal injury remains to be elucidated. In this study, we investigated whether an orally active EP1-selective antagonist (EP1A) prevents the progression of renal damage in stroke-prone spontaneously hypertensive rats (SHRSP), a model of human malignant hypertension. Ten-week-old SHRSP, with established hypertension but with minimal renal damage, were given EP1A or vehicle for 5 weeks. After the treatment period, vehicle-treated SHRSP showed prominent proliferative lesions in arterioles, characterized by decreased alpha-smooth muscle actin expression in multilayered vascular smooth muscle cells. Upregulation of transforming growth factor-beta expression and tubulointerstitial fibrosis were also observed in vehicle-treated SHRSP. All these changes were dramatically attenuated in EP1A-treated SHRSP. Moreover, EP1A treatment significantly inhibited both increase in urinary protein excretion and decrease in creatinine clearance but had little effect on systemic blood pressure. These findings indicate that the PGE2/EP1 signaling pathway plays a crucial role in the development of renal injury in SHRSP. This study opens a novel therapeutic potential of selective blockade of EP1 for the treatment of hypertensive renal disease.

The DP receptor, allergic inflammation and asthma

Prostaglandin (PG) D 2 is the major cyclooxygenase metabolite of arachidonic acid produced by mast cells in response to allergen in diseases, such as asthma, atopic dermatitis, allergic rhinitis and allergic conjunctivitis. However, whether PGD 2 regulates allergic process per se, and, if so, whether it facilitates or down-regulates the disease process has remained unknown. PGD 2 exerts its actions by binding to two types of specific cell surface receptor. One is DP (the PGD receptor) and the other is chemoattractant receptor-homologous molecule expressed on Th2. Between the two, the DP receptor has been better characterized since its cDNA cloning in 1994, and novel class of DP antagonists have been and are being developed. Furthermore, mice deficient in DP were generated and have been subjected to several models of allergic diseases to reveal the role of PGD 2 in allergy. In this article, we summarize these findings and provide an overview of the current status of the DP receptor research to discuss the therapeutic potential of modulating the PGD 2-DP pathway in allergic diseases.

CytochromeP-450 4A isoform expression and 20-HETE synthesis in renal preglomerular arteries

20-Hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor and mediator of the myogenic response, is a major arachidonic acid metabolite in the microvasculature of the rat kidney formed primarily by the cytochrome P-450 (CYP) 4A isoforms, CYP4A1, CYP4A2, and CYP4A3. We examined CYP4A isoform expression and 20-HETE synthesis in microdissected interlobar, arcuate, and interlobular arteries; mRNA for all CYP4A isoforms was identified by RT-PCR. Western blot analysis indicated that the levels of CYP4A2/4A3-immunoreactive protein increased with decreased arterial diameter, whereas those of CYP4A1-immunoreactive protein remained unchanged. 20-HETE synthesis was the highest in the interlobular arteries (17 +/- 1.62 nmol. mg(-1). h(-1)) and, like CYP4A2/4A3-immunoreactive protein, decreased with increasing vessel diameter (4.5 +/- 1.21, 2.65 +/- 0.58, and 0.81 +/- 0.14 nmol. mg(-1). h(-1) in the arcuate, interlobar, and segmental arteries, respectively). 20-HETE synthesis in the renal artery and the abdominal aorta was undetectable. The observed decreased immunoreactivity of NADPH-cytochrome P-450 (c) oxidoreductase with increased arterial diameter provided a possible explanation for the decreased capacity to generate 20-HETE in the large arteries. The increase in CYP4A isoform expression and 20-HETE synthesis with decreasing diameter along the preglomerular arteries and the potent biological activity of 20-HETE underscore the significance of 20-HETE as a modulator of renal hemodynamics.

Novel eicosanoid activators of PPAR gamma formed by RAW 264.7 macrophage cultures.

Prostaglandin D2 (PGD2) is a major arachidonic acid metabolite in mast cells, megakaryoblastic CMK cells and antigen presenting cells, for example various types of macrophages. It induces pulmonary vasoconstriction and bronchoconstriction. Increased formation of PGD2 may be responsible for the symptoms of mastocytosis, it may contribute to the development of allergic symptoms and there is evidence that PGD2 regulates sleep. In the presence of serum albumin, PGD2 is converted to several products (1,2) which exhibit antiproliferative properties. These prostaglandin J2 compounds have been proposed to bind to intracellular receptors and translocate to the cell nucleus (3-6). In 1995, one of the antiproliferative prostaglandins, 15-deoxy-Al2“4 —PGJ2 was suggested to be a natural ligand for peroxisome proliferator-activated receptor-y (PPAR y)and to play a role in the terminal differentiation of fat cells (7,8). Recent studies show that PGD2 inhibits the expression of the genes encoding inducible nitric oxide synthase, gelatinase B and LDL scavenger receptor RAW 264.7 macrophages (9). PGD2 itself is a poor PPARy ligand. Therefore, we assumed that the activity observed in (9) was mediated by PGD2 metabolites. The purpose of this investigation was to identify PGD2 metabolites which activate PPARy in macrophages.KeywordsMacrophage CultureMethoxyamine HydrochlorideUnlabeled Fatty AcidEicosanoid ActivatorCouple Reticulocyte Lysate SystemThese 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.

New polymorphisms of haematopoietic prostaglandin D synthase and human prostanoid DP receptor genes.

Prostaglandin D2 (PGD2), a major cyclo-oxygenase metabolite of arachidonic acid in mast cells, induces bronchoconstriction in the human lung. It has been reported that mice lacking PGD receptor fail to develop the bronchial hyper-responsiveness upon ovalbumin challenge, suggesting that PGD2 functions as a mediator of allergic asthma. To determine if there are any mutations associated with the development of asthma in the haematopoietic prostaglandin D synthase (H-PGDS) gene and the human prostanoid DP receptor (PTGDR) gene. We screened the 5'flanking and coding regions of the H-PGDS gene and the PTGDR gene by direct sequence. We identified one variant in intron 2 (IVS2 + 11 A > C) and one variant in intron 3 (IVS3 + 13T > C) of the H-PGDS gene, and two variants in the 5'flanking region of the PTGDR gene (-197T > C and -2C > T). The IVS3 + 13T > C and -197T > C variants were rare, appearing only once in 48 subjects. transmission disequilibrium test (TDT) analysis of 144 asthmatic families revealed that the IVS2 + 11 A allele of the H-PGDS gene was significantly transmitted preferentially to asthma-affected children (P = 0.0056), but no association was observed between -2C/T polymorphism of the PTGDR gene and asthma (P > 0.05). Our results suggest that the IVS2 + 11A/C allele may be involved in the development of asthma in the Japanese population.

Activation-dependent internalization of the human prostacyclin receptor.

Prostacyclin (PGI2), the major cyclooxygenase-derived arachidonic acid metabolite formed in macrovascular endothelial cells’, is a potent vasodilator, an inhibitor of platelet aggregation2 and smooth muscle proliferation3 in vitro and an antithrombotic in mice4. Given the absence of PGI2 antagonists its role in the prevention of vascular disease in vivo has remained speculative.

Inhibition of Cytosolic Phospholipase A2 in Human Neutrophils by Oxatomide

Background: Lipid mediators play a pivotal role in the pathogenesis of allergic and inflammatory reactions. These molecules include metabolites of arachidonic acid (AA) and the group of platelet-activating factor (PAF)-related phospholipids. The initial step in the synthetic pathway of both classes of lipid mediators is catalyzed by members of the phospholipase A₂ (PLA₂) family. Oxatomide is a histamine H₁ receptor antagonist currently used in the treatment of allergic disorders. Preliminary evidence indicates that oxatomide may exert anti-inflammatory activities unrelated to H₁ receptor antagonism. Methods: We investigated the effect of oxatomide on lipid mediator production by human neutrophils. Results: Preincubation (15 min, 37°C) of neutrophils with oxatomide (10–100 µM) concentration-dependently inhibited (10–70%) the release of AA induced by the Ca²⁺ ionophore A23187 (0.5 µM). Oxatomide also comparably inhibited the release of the four major metabolites of AA induced by A23187 (LTB₄, 20-OH-LTB₄, 20-COOH-LTB₄ and 5-HETE). In addition, oxatomide reduced by 60% the production of PAF induced by A23187. The simultaneous inhibition of the production of AA metabolites and PAF suggested that oxatomide could influence the activity of cytosolic PLA₂ (cPLA₂). To test this hypothesis, we evaluated the functional activity of cPLA₂ in neutrophils preincubated with oxatomide. This preincubation inhibited (72 ± 24%) the increase in cPLA₂ activity induced by A23187. Conclusions: These results indicate that oxatomide reduces the biosynthesis of lipid mediators in human neutrophils by inhibiting cPLA₂. This inhibitory effect of oxatomide may contribute to the anti-inflammatory activity of this drug in allergic diseases.

Protein Kinases C Translocation Responses to Low Concentrations of Arachidonic Acid

Arachidonic acid (AA) directly activates protein kinases C (PKC) and may thereby serve as a regulatory signal during cell stimulation. The effect, however, requires a > or =20 microm concentration of the fatty acid. We find that human polymorphonuclear neutrophils (PMN) equilibrated with a ligand for the diacylglycerol receptor on PKC, [(3)H]phorbol dibutyrate (PDB), increased binding of [(3)H]PDB within 15 s of exposure to > or =10-30 nm AA. Other unsaturated fatty acids, but not a saturated fatty acid, likewise stimulated PDB binding. These responses, similar to those caused by chemotactic factors, resulted from a rise in the number of diacylglycerol receptors that were plasma membrane-associated and therefore accessible to PDB. Unlike chemotactic factors, however, AA was fully active on cells overloaded with Ca(2+) chelators. The major metabolites of AA made by PMN, leukotriene B(4) and 5-hydroxyicosatetraenoate, did not mimic AA, and an AA antimetabolite did not block responses to AA. AA also induced PMN to translocate cytosolic PKCalpha, beta(II), and delta to membranes. This response paralleled PDB binding with respect to dose requirements, time, Ca(2+)-independence, resistance to an AA antimetabolite, and induction by another unsaturated fatty acid but not by a saturated fatty acid. Finally, HEK 293 cells transfected with vectors encoding PKCbeta(I) or PKCdelta fused to the reporter enhanced green fluorescent protein (EGFP) were studied. AA caused EGFP-PKCbeta translocation from cytosol to plasma membrane at > or =0.5 microm, and EGFP-PKCdelta translocation from cytosol to nuclear and, to a lesser extent, plasma membrane at as little as 30 nm. We conclude that AA induces PKC translocations to specific membrane targets at concentrations 2-4 orders of magnitude below those activating the enzymes. These responses, at least as they occur in PMN, do not require changes in cell Ca(2+) or oxygenation of the fatty acid. AA seems more suited for signaling the movement than activation of PKC.

Effects of 20-HETE and 19(S)-HETE on rabbit proximal straight tubule volume transport.

The kidney has the highest abundance of cytochrome P-450 of all extrahepatic organs. Within the kidney, the highest concentration of cytochrome P-450 is found in the proximal tubule. Whether 20- or 19(S)-hydroxyeicosatetraenoic acid (HETE), the major P-450 metabolites of arachidonic acid in the proximal tubule, affect transport in this segment has not been previously investigated. We examined the direct effects of 20- and 19(S)-HETE on volume absorption (J(v)) in the rabbit proximal straight tubule (PST). Production of 20-HETE by rabbit PST was demonstrated by incubating microdissected tubules with [(3)H]arachidonic acid and separating the lipid extract by HPLC. There was significant conversion of [(3)H]arachidonic acid to 20-HETE in control tubules that was inhibited by 10(-5) M N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). Addition of exogenous 20-HETE had no effect on PST volume transport. However, inhibition of endogenous production of 20-HETE using DDMS stimulated transport. In the presence of DDMS, 20-HETE inhibited PST J(v). 19(S)-HETE in the bathing solution stimulated PST J(v) alone and in the presence of DDMS. Thus omega- and omega-1-hydroxylase products of arachidonic acid have direct effects on PST transport. Endogenous production of 20-HETE may play a role in tonic suppression of transport and may therefore be an endogenous regulator of transport in the proximal tubule.

Induction of cyclooxygenase expression and enhancement of malignant cell transformation by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The potential role of arachidonic acid metabolism in the enhancement (promotion) of malignant transformation of C3H/M2 mouse fibroblasts by the tumor promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated using inhibitors of cyclooxygenase and lipoxygenase activities. The promoting effects of TCDD (1.5 pM) and of the reference tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA; 0.4 mM) on carcinogen (N-methyl-N'-nitro-N-nitrosoguanidine or 3-methylcholanthrene)-pre-treated fibroblasts was abolished by cotreatment with indomethacin, hydrocortisone, caffeic acid or nordihydroguaiaretic acid. A differential inhibition was found with N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide, a selective inhibitor of the cyclooxygenase isoenzyme COX-2: the promoting effect of TPA, but not that of TCDD, was abolished. Therefore, the role of the cyclooxygenase isoenzymes COX-1 and COX-2 during chronic exposure to TCDD was studied in more detail. Long-term treatment with TCDD (4-7 weeks) induced the expression of COX-1 and COX-2 mRNA in C3H/M2 fibroblasts (up to 2-fold). The enhanced expression of COX-2 protein in TCDD-treated fibroblasts was confirmed by western blot analysis. Concomitantly, the accumulation of the prostaglandins (PGs) PGE(2) and 6-keto-PGF(1alpha), which were identified as major metabolites of arachidonic acid in C3H/M2 cell cultures, was enhanced ( approximately 2-fold) following long-term treatment with TCDD (0.15 and 1.5 pM). The results suggest that the stimulation of arachidonic acid metabolism caused by a sustained cyclooxygenase induction is a critical event in the promoting action of TCDD in mouse fibroblasts in vitro. However, in contrast to TPA, the TCDD-mediated enhancement of malignant cell transformation may not specifically depend on the induction of COX-2 but, additionally, the induction of COX-1 activity may be necessary.

Activation-dependent internalization of the human prostacyclin receptor

Prostacyclin (PGI2), the major cyclooxygenase-derived arachidonic acid metabolite formed in macrovascular endothelial cells’, is a potent vasodilator, an inhibitor of platelet aggregation2 and smooth muscle proliferation3 in vitro and an antithrombotic in mice4. Given the absence of PGI2 antagonists its role in the prevention of vascular disease in vivo has remained speculative.

Lipoxygenase Products Increase Monocyte Adhesion to Human Aortic Endothelial Cells

The development of atherosclerosis is accelerated in individuals with type 2 diabetes. Adhesion of monocytes to the vascular endothelium is a key initial step in atherogenesis. We have previously shown that monocyte adhesion to human aortic endothelial cells (HAECs) cultured long-term in high-glucose medium (25 mmol/L, 2 passages) is increased compared with cells grown in normal glucose (5 mmol/L). One potential mechanism for increased monocyte adhesion to HAECs under hyperglycemic conditions is via the 12-lipoxygenase (12-LO) pathway. In this study, we demonstrated in HAECs that the major LO metabolite of arachidonic acid was the 12-LO product, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], which was increased severalfold in HAECs cultured under high-glucose conditions. Furthermore, treatment of HAECs with 12(S)-HETE induced monocyte, but not neutrophil, adhesion an average of 3-fold (range of 1.5- to 5-fold) compared with untreated cells (75+/-5 versus 26+/-1 monocytes per field, respectively, P<0.001). Expression of the adhesion molecules vascular cell adhesion molecule-1, E-selectin, and intercellular adhesion molecule-1 was not significantly increased. However, both glucose and 12(S)-HETE induced a 60% increase in HAEC surface expression of connecting segment-1 (ie, CS-1) fibronectin, a ligand for very late-acting antigen-4 (VLA-4). The antibodies used to block monocyte integrin VLA-4 and leukocyte function-related antigen-1, a monocytic counterreceptor for intercellular adhesion molecule-1, inhibited the ability of both 12-LO products and high glucose to induce monocyte adhesion. These results definitively demonstrate for the first time in HAECs that the 12-LO pathway can induce monocyte-endothelial cell interaction and that the effects of glucose may be mediated, at least in part, through this pathway. Thus, these results suggest that the 12-LO pathway may play a role in the increased susceptibility of diabetics to atherosclerosis.