Cyclooxygenase Metabolites Of Arachidonic Acid Research Articles

Role of prostaglandins in tumor microenvironment.

Tumor tissue is composed of tumor cells and surrounding non-tumor endothelial and immune cells, collectively known as the tumor microenvironment. Tumor cells manipulate tumor microenvironment to obtain sufficient oxygen and nutrient supply, and evade anti-tumor immunosurveillance. Various types of signaling molecules, including cytokines, chemokines, growth factors, and lipid mediators, are secreted, which co-operate to make up the complex tumor microenvironment. Prostaglandins, cyclooxygenase metabolites of arachidonic acid, are abundantly produced in tumor tissues. Ever since treatment with nonsteroidal anti-inflammatory drugs showed anti-tumor effect in mouse models and human patients by inhibiting whole prostaglandin production, investigators have focused on the importance of prostaglandins in tumor malignancies. However, most studies that followed focused on the role of an eminent prostaglandin, prostaglandin E2, in tumor onset, growth, and metastasis. It remained unclear how other prostaglandin species affected tumor malignancies. Recently, we identified prostaglandin D2, a well-known sleep-inducing prostaglandin, as a factor with strong anti-angiogenic and anti-tumor properties, in genetically modified mice. In this review, we summarize recent studies focusing on the importance of prostaglandins and their metabolites in the tumor microenvironment.

Ultrasound stimulates formation and release of vasoactive compounds in brain endothelial cells.

Stroke outcome is improved by therapeutic ultrasound. This benefit is presumed to be principally from ultrasound-mediated thrombolysis. We hypothesized that the therapeutic benefit of ultrasound in stroke may, in part, be mediated by the release of beneficial vasoactive substances. Accordingly, we investigated the effect of ultrasound on levels of cytochrome P-450, lipoxygenase, and cyclooxygenase metabolites of arachidonic acid as well as adenosine release and endothelial nitric oxide synthase (eNOS) phosphorylation in primary brain endothelial cells in vitro. Brain endothelial cells were exposed to 1.05-MHz ultrasound at peak rarefactional acoustic pressure amplitudes of 0.35, 0.55, 0.90, and 1.30 MPa. Epoxyeicosatrienoic acids (EETs), hydroxyeicosatetraenoic acids (HETEs), PGE2, adenosine, nitrate/nitrite, and eNOS phosphorylation were measured after ultrasound exposure. Levels of 8,9-EET, 11,12-EET, and 14,15-EET increased by 230 ± 28%, 240 ± 30%, and 246 ± 31% (P < 0.05), respectively, whereas 5-HETE and 15-HETE levels were reduced to 24 ± 14% and 10 ± 3% (P < 0.05), respectively, compared with cells not exposed to ultrasound. PGE2 levels were reduced to 56 ± 14% of control. Adenosine increased more than sixfold after ultrasound exposure compared with unstimulated cells (1.36 ± 0.22 vs. 0.37 ± 0.10 ng/ml, P < 0.05), nitrate/nitrite was below levels of quantification, and eNOS phosphorylation was not altered significantly. Our results suggest that ultrasound may enhance tissue perfusion during stroke by augmenting the generation of vasodilator compounds and inhibiting that of vasoconstrictors. Such regulation supports a beneficial role for therapeutic ultrasound in stroke independent of its effect on the occlusive thrombus.

Impact of IL-33 on PGD2 Generation by Activated Human Cord Blood-Derived Mast Cell: Lack of Effect on Tryptase Release

Mast cells are important not only in allergic reactions, but also in inflammation and are involved in a variety of responses including the immediate release of potent inflammatory mediators after activation by cross-linking of FcεRI molecules. Prostaglandin D2 (PGD2) is a major cyclooxygenase metabolite of arachidonic acid produced by mast cells and it is released following allergen challenge in allergic diseases. IL-33 is an iflammatory cytokine which is critically involved in the regulation of in vitro and in vivo cyclooxygenase production, providing a potential therapeutic target for inflammatory disorders. In this study, using human derived umbelical cord blood mast cells, we show that IL-33 (50 ng/ml), and calcium ionophore A 23187 (0.5 μg/ml), compound 48/80 (10−5 M) or anti-IgE (10 μg/ml), enhaced the production of PGD2 and this effect was inhibited by indomethacin. However, IL-33 was unable to induce tryptase release in these cells. These effects confirm the inflammatory property of IL-33 by stimulating PGD2 but not tryptase in human mast cells. The inhibitory effect of this new cytokine may have a potential therapeutic response in allergic and inflammatory diseases.

Effects of ATP on phosphoinositide hydrolysis and prostaglandin E2 generation in rabbit astrocytes.

Extracellular ATP secreted from stimulated nerves plays a role in neurotransmission. This study examined the effects of extracellular ATP on phospholipase A2 and C signalling pathways in rabbit astrocytes. ATP caused prostaglandin E2 (PGE2) generation and phosphoinositide hydrolysis in a time- and concentration-dependent manner. A P2y purinoceptor-selective agonist, 2-methylthio-ATP also caused phosphoinositide hydrolysis, but not PGE2 generation. A P2x purinoceptor-selective agonist, alpha, beta-methylene-ATP did not cause either phosphoinositide hydrolysis or PGE2 generation. Although pertussis toxin had no effect on 2-methylthio-ATP-induced phosphoinositide hydrolysis, it markedly decreased ATP-induced PGE2 generation, with significant inhibition of phosphoinositide hydrolysis. Dexamethasone and indomethacin which potently inhibited ATP-induced PGE2 generation, caused partial inhibition of phosphoinositide hydrolysis, suggesting that pertussis toxin-sensitive component of ATP-induced phospholipase C activation is mediated by cyclo-oxygenase metabolites of arachidonic acid. These results suggest that a stimulation of P2y receptor results in phospholipase C activation in a pertussis toxin-insensitive manner, and that a P2 receptor other than the P2y or P2x subtypes is involved in ATP-induced phospholipase A2 activation via a pertussis toxin-sensitive G protein.

Prostanoid Receptors as Possible Targets for Anti-Allergic Drugs: Recent Advances in Prostanoids on Allergy and Immunology

Prostanoids, consisting of prostaglandins and thromboxane, are cyclooxygenase metabolites of arachidonic acid released in various pathophysiological conditions which exert a range of actions mediated through their respective receptors expressed on target cells. Although it has been difficult to analyze the physiological role of prostanoids, recent developments in both the disruption of the respective gene and receptor selective compounds have enabled us to investigate the physiological roles for each receptor. It has been demonstrated that each prostanoid receptor has multiple functions, and that their expression is regulated in a context-dependent manner that sometimes results in opposite, excitatory and inhibitory, outcomes. The balance of prostanoid production and receptor expression has been revealed to be important for homeostasis of the human body. Here, we review new findings on the roles of prostanoids in allergic and immune diseases, focusing on contact dermatitis, atopic dermatitis, asthma, rheumatoid arthritis, and encephalomyelitis, and also discuss the clinical potentials of receptor-selective drugs.

Mast Cells and Arachidonic Acid Cascade in Inflammation

Prostaglandin D2 PGD2 is a major cyclooxygenase metabolite of arachidonic acid produced by mast cells and it is released following allergen challenge in diseases, such as allergic diseases. PGD2 may act as a neuromodulator and as an allergic and inflammatory mediator. In allergic diseases, activated mast cell synthesizes prostaglandin D2 (first cyclo-oxygenate mediator) which has bronchoconstrictive and vasodilating effects and attracts several leukocytes. It has been found that activated mast cells, challenged with physiological and non- physiological secretagogues, release elevated histamine and tryptase and chymase, leukotrienes B4, C4 and D4, 5-hydroxyeicosatetraenoic acid, PGD2, Platelet Activating Factor (PAF), heparin, and high-molecular-weight neutrophil chemotactic factor and cytokines/chemokines. PGD2 exerts its biological activity through the DP and CRTH2 receptors and their cDNA cloning which were characterized 15 years ago. In this report, we revisited the biological effects of arachidonic acid compounds released by activated mast cells in allergic and inflammatory states.

A bioluminescent enzyme immunoassay for prostaglandin E2 using Cypridina luciferase

Prostaglandin E(2) is one of the major cyclooxygenase metabolites of arachidonic acid. We developed a competitive immunosorbent assay for prostaglandin E(2) utilizing a bioluminescent enzyme Cypridina luciferase. The prostaglandin E(2) amount could be quantified over the concentration ranging from 7.8 to 500 pg/mL. The amount of unlabeled prostaglandin E(2) required to displace 50% of the maximal binding of Cypridina luciferase-labeled prostaglandin E(2) (B/B(0)) was approximately 35 pg/mL. The results show a great potential of Cypridina luciferase as a new labeling enzyme for enzyme-linked immunosorbent assay.

The Potential of Selected Prostanoid Receptors as Targets in a New Therapeutic Strategy for Allergy and Immune Diseases

Allergy and immune diseases including asthma, atopic dermatitis, rhinitis, and autoimmune diseases have been treated mainly with steroid based therapy that has multiple side effects. Even then, these allergic diseases are sometimes still not easy to control. Therefore, new effective treatments with fewer side effects are expected to be developed. Prostanoids consisting of prostaglandins and thromboxane are cyclooxygenase metabolites of arachidonic acid. They exert a range of actions mediated through their respective receptors expressed in target cells. In the skin, a wide variety of prostanoids and their receptors are highly expressed under pathophysiological conditions. However, their roles have not been well clarified. Recent developments in molecular biology have enabled us to investigate the physiological roles for each receptor via the disruption of the respective gene in combination with receptor selective compounds. It has been demonstrated that each prostanoid receptor has multiple functions whose expression is regulated in a context dependent manner, sometimes resulting in opposite, excitatory and inhibitory, outcomes. The balance of prostanoid production and receptor expression is important for homeostasis of the human body. Here, we review new findings on the roles of prostanoids in allergy and immune diseases, focusing on skin disorders as a representative, and discuss the clinical potentials of receptor-selective drugs.

Role of prostaglandin E2 as a protective mediator for airway inflammation

SummaryProstaglandin (PG) E2 is a cyclooxygenase metabolite of arachidonic acid that has anti‐inflammatory properties including suppression of lymphocyte proliferation, inhibition of cytokine/chemokine production, and dilation of human bronchi. Recently, four types of PGE2 receptors (EP1, EP2, EP3, and EP4) were identified, and their roles have been extensively examined. We found mRNA expression of all four PGE2 receptors in human airway epithelial cells, and EP3 and EP4 agonists significantly inhibited mucus secretion and inflammatory cell infiltration induced by lipopolysaccharide (LPS) or by allergic inflammation in rat nasal epithelium. These EP agonists significantly inhibited LPS‐induced secretion of IL‐8 and mucus in cultured airway epithelial cells. These results indicate that PGE2 acts as an anti‐inflammatory mediator via EP receptors of airways. EP agonists may serve as a new therapeutic modality for the treatment of airway inflammation.

Involvement of cAMP-response element binding protein-1 in arachidonic acid-induced vascular smooth muscle cell motility

In addition to their role in many vital cellular functions, arachidonic acid (AA) and its eicosanoid metabolites are involved in the pathogenesis of several diseases, including atherosclerosis and cancer. To understand the potential mechanisms by which these lipid molecules could influence the disease processes, particularly cardiovascular diseases, we studied AA's effects on vascular smooth muscle cell (VSMC) motility and the role of cAMP-response element binding protein-1 (CREB-1) in this process. AA exerted differential effects on VSMC motility; at lower doses, it stimulated motility, whereas at higher doses, it was inhibitory. AA-induced VSMC motility requires its conversion via the lipoxygenase (LOX) and cyclooxygenase (COX) pathways. AA stimulated the phosphorylation of extracellular signal-regulated kinases (ERKs), Jun N-terminal kinases (JNKs), and p38 mitogen-activated protein kinase (p38MAPK) in a time-dependent manner, and blockade of these serine/threonine kinases significantly attenuated AA-induced VSMC motility. In addition, AA stimulated CREB-1 phosphorylation and activity in a manner that was also dependent on its metabolic conversion via the LOX and COX pathways and the activation of ERKs and p38MAPK but not JNKs. Furthermore, suppression of CREB-1 activation inhibited AA-induced VSMC motility. 15(S)-Hydroxyeicosatetraenoic acid and prostaglandin F2alpha, the 15-LOX and COX metabolites of AA, respectively, that are produced by VSMC at lower doses, were also found to stimulate motility in these cells. Together, these results suggest that AA induces VSMC motility by complex mechanisms involving its metabolism via the LOX and COX pathways as well as the ERK- and p38MAPK-dependent and JNK-independent activation of CREB-1.

Consultation with the Specialist: Dysmenorrhea

1. Paula J. Adams Hillard, MD* 1. *Professor, Department of Obstetrics & Gynecology and of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio After completing this article, readers should be able to: 1. Recognize the prevalence of dysmenorrhea in adolescents and its common results. 2. Describe a menstrual history that is most consistent with primary dysmenorrhea. 3. List the mediators of uterine pain and contractions believed to be involved in the pathophysiology of primary dysmenorrhea. 4. Recommend the appropriate evaluation and management of primary dysmenorrhea, including the appropriate choice and dosing of both over-the-counter and prescription medications. 5. Recognize patients who should be referred for evaluation of possible causes of secondary dysmenorrhea. A 15½-year-old girl came to the office with complaints of the recent onset of painful menstrual periods. She experienced her first menstrual period at the age of 13 years and except for an occasional mildly uncomfortable menstrual period, had not experienced significant pain until recently. She reported regular monthly menses, although she did not write down the dates. She described additional symptoms that also had not been present previously, including headache, loose stools, and breast tenderness. She has tried several over-the-counter medications, including acetaminophen, without significant relief. She has missed 3 days of school in the last 6 weeks because of these complaints. She is otherwise healthy, having no major illnesses, no history of surgeries, and no other genitourinary complaints. There is no family history of endometriosis; severe dysmenorrhea; menorrhagia; irregular periods; infertility; uterine fibroids; or breast, ovarian, colon, or uterine cancer. Her mother recalled that she had been prescribed oral contraceptives at the age of 16 years to alleviate her severe menstrual cramps. When spoken with privately, having been assured confidentiality, the girl denied sexual activity or abuse. She was doing well in school, making As and Bs, and felt that the relationship with her parents and one sister were good. She reported that she experienced crampy midline lower abdominal pain that began with the …

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.

INVOLVEMENT OF ENDOTHELIAL CYCLO‐OXYGENASE METABOLITES IN NORADRENALINE‐INDUCED CONTRACTION OF RAT CORONARY ARTERY

1. Noradrenaline (NA; 0.3 micromol/L) caused a contraction of the rat coronary artery that markedly increased in the presence of the nitric oxide synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME; 100 micromol/L) and arachidonic acid (1 micromol/L; P < 0.05). 2. The present experiments attempted to elucidate the endothelium dependency of the contraction and to pharmacologically characterize the factors involved in the contraction induced by NA (0.3 micromol/L) in the presence of L-NAME and arachidonic acid in ring preparations of the rat coronary artery. 3. The NA (0.3 micromol/L)-induced contraction was attenuated by a chemical remover of the endothelium (saponin at concentrations of 0.1 and 0.4 mg/mL) in a concentration-dependent manner (P < 0.05). 4. The cyclo-oxygenase (COX)-1 inhibitor flurbiprofen (0.01-1 micromol/L) and the COX-2 inhibitor nimesulide (0.01-1 micromol/L) attenuated the NA-induced contraction in a concentration-dependent manner and the inhibitory effect of flurbiprofen was significantly more potent than that of nimesulide (P < 0.05). The 5-lipoxigenase inhibitor ZM-230487 (1 micromol/L) did not affect the NA-induced contraction. 5. The thromboxane A2 (TXA2) synthetase inhibitor OKY-046 (30 micromol/L) and the TXA2 antagonist S-1452 (0.1-10 micromol/L) did not attenuate the NA-induced contraction. 6. These results indicate that the contraction induced by NA in the rat coronary artery in the presence of L-NAME and arachidonic acid is endothelium dependent and is due to endothelial COX metabolites of arachidonic acid.

Eicosanoids in preeclampsia

Preeclampsia is characterized by an imbalance between two cyclooxygenase metabolites of arachidonic acid, thromboxane and prostacyclin, that favors thromboxane. Because of the biologic actions of these two eicosanoids, this imbalance might explain major clinical symptoms of preeclampsia, such as hypertension, platelet aggregation and reduced uteroplacental blood flow. In the maternal circulation, this imbalance is primarily manifested by decreased production of prostacyclin by endothelial cells. Platelet thromboxane synthesis is only increased in severe preeclampsia. In the placenta and in leukocytes, the imbalance is exacerbated by increased production of thromboxane coupled with decreased production of prostacyclin in both mild and severe preeclampsia. Longitudinal measurements of urinary metabolites of thromboxane and prostacyclin reveal that the thromboxane/prostacyclin imbalance predates the onset of clinical symptoms of preeclampsia. The imbalance between thromboxane and prostacyclin is most likely caused by oxidative stress, which is manifest in preeclampsia by increased lipid peroxidation and decreased antioxidant protection. Oxidative stress may drive this imbalance because lipid peroxides activate the cyclooxygenase enzyme to increase thromboxane synthesis, but at the same time they inhibit prostacyclin synthase to decrease prostacyclin synthesis. Low-dose aspirin therapy (50–150 mg/day) has been considered for the prevention of preeclampsia because it selectively inhibits thromboxane synthesis. Several studies reported dramatic decreases in the incidence of preeclampsia with low-dose aspirin therapy. However, two large multicenter studies reported only modest decreases, which dampened enthusiasm. The two large studies were “intent to treat” studies which included patients who were noncompliant and who discontinued the use of aspirin. In one of the studies for which compliance statistics were available only 53% of the aspirin group had a compliance rate greater than 75%, which raises a question as to whether the effectiveness of aspirin was being tested. Low-dose aspirin therapy should not yet be dismissed for the prevention of preeclampsia, but be reconsidered with emphasis on compliance using doses of aspirin in the range of 100–150 mg/day combined with antioxidants.

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.

20-HETE and furosemide-induced natriuresis in salt-sensitive essential hypertension.

Cyclooxygenase metabolites of arachidonic acid modulate the natriuretic effect of furosemide. It is not known whether 20-HETE, a monooxygenase metabolite of arachidonic acid that also inhibits sodium transport, participates in the action of furosemide. We measured urine sodium (UNaV) and 20-HETE during furosemide diuresis (40 mg three times over 12 hours) in 12 salt-sensitive (SS) and 11 salt-resistant (SR), salt-replete hypertensive subjects (126+/-24 mmol/24 hours positive sodium balance produced by 160-mmol-sodium diet and 2 L saline infusion). Individual systolic blood pressure decreases from the salt-replete to the salt-depleted state were the index of salt-sensitivity. SS had low plasma renin with blunted responses to changes in salt balance, inappropriate plasma aldosterone, and an increased aldosterone/renin ratio. UNaV by furosemide was less in SS (263+/-25 mmol/12 hours) than in SR (351+/-25 mmol/12 hours, P<0.02) patients. 20-HETE was not different between SS and SR patients before (1.92+/-0.38 versus 1.37+/-0.34 microg/h) or after furosemide (1.52+/-0.27 versus 2.01+/-0.40 microg/h), but furosemide changed 20-HETE excretion in opposite direction in SR (0.63+/-0.26) versus SS (-0.40+/-0.17, P<0.005) patients. In all patients together, %Delta20-HETE by furosemide correlated with %DeltaUNaV (r=0.56, P<0.01) and negatively with salt-sensitivity of blood pressure (r=-0.55, P<0.01). In SS, Delta20-HETE by furosemide correlated with Deltaaldosterone/renin ratio (r=0.60, P<0.05), whereas 20-HETE during furosemide had a negative correlation with body mass index (r=-0.73, P<0.01). Our data suggest that 20-HETE modulates the natriuretic response to furosemide, and impaired natriuresis of SS involves a mechanism that alters the 20-HETE response to furosemide and is linked to salt-sensitivity of blood pressure.

Role of arachidonic acid and protein kinase C during maturation-inducing hormone-dependent meiotic resumption and ovulation in ovarian follicles of Atlantic croaker.

The roles of arachidonic acid (AA) and protein kinase C (PKC) during in vitro maturation-inducing hormone (MIH)-dependent meiotic resumption (maturation) and ovulation were studied in ovarian follicles of Atlantic croaker (Micropogonias undulatus). The requirement for cyclooxygenase (COX) metabolites of AA was examined using a nonspecific COX inhibitor, indomethacin (IM), as well as two COX products, prostaglandin (PG) F(2alpha) and PGE(2), whereas the role of lipoxygenase (LOX) was investigated using a specific LOX inhibitor, nordihydroguaiaretic acid (NDGA). The involvement of PKC was examined using phorbol 12-myristate 13-acetate (PMA), a PKC activator, as well as GF109203X (GF), a specific inhibitor of PKC and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), nonspecific inhibitor of protein kinases. Genomic mechanisms were examined with the transcription-inhibitor actinomycin D (ActD) and the functionality of heterologous (oocyte-granulosa) gap junctions (GJ) with a dye transfer assay. The AA (100 microM) and PGF(2alpha) (5 microM) did not induce maturation, and NDGA (10 microM) did not affect MIH-dependent maturation. However, IM (100 microM) partially inhibited MIH-dependent maturation. Conversely, AA and both PGs induced, and IM and NDGA inhibited, MIH-dependent ovulation in matured follicles. The PMA (1 microg/ml) did not induce maturation but caused ovulation in matured follicles, whereas PKC inhibitors (GF, 5 microM; H7, 50 microM) did not affect MIH-dependent maturation but inhibited MIH- and PMA-dependent ovulation. The PMA-dependent ovulation was inhibited by IM but not by NDGA. In addition, ActD (5 microM) blocked MIH-dependent, but not PMA-dependent, ovulation, and PGF(2alpha) restored MIH-dependent ovulation in ActD-blocked follicles. The AA and PGs did not induce, and GF did not inhibit, MIH-dependent heterologous GJ uncoupling. In conclusion, AA and PKC mediate MIH-dependent ovulation but not meiotic resumption or heterologous GJ uncoupling in croaker follicles, but a permissive role of COX products of AA during maturation is possible. A novel model of MIH-dependent ovulation is proposed in which 1). LOX and COX metabolites of AA are both required for ovulation, but at upstream and downstream sites of the pathway, respectively, relative to PKC, and 2). PKC is downstream of genomic activation.

Liquid chromatographic–mass spectrometric determination of cyclooxygenase metabolites of arachidonic acid in cultured cells

A liquid chromatographic–electrospray ionization-mass spectrometric (LC-ESI-MS) technique was developed to simultaneously determine the cyclooxygenase metabolites of arachidonic acid (6-keto-PGF 1α, PGD 2, PGE 2, PGF 2α, and PGJ 2) produced by cultured cells. Samples were separated on a C 18 column with water–acetonitrile mobile phase, ionized by electrospray, and detected in the positive mode. Selected ion monitoring (SIM) of m/ z 353, 335, 335, 319, and 317 were used for quantifying 6-keto-PGF 1α, PGD 2, PGE 2, PGF 2α, and PGJ 2, respectively. Prostaglandins were detected at concentrations as low as 1 pg ( S/ N=3) on the column. The method was used to determine the production of PGs from bovine coronary artery endothelial cells (ECs) and human prostate cancer cells (PC-3) with different degree of invasiveness. Bradykinin (10 −6 M) stimulated a marked increase in the production of 6-keto-PGF 1α, PGE 2, and PGF 2α and a small increase of PGD 2 by ECs. 6-Keto-PGF 1α was the major metabolite in these cells. The production of PGE 2 was threefold higher and PGD 2 was twofold higher in PC-3-S (invasive) cells than in PC-3-U (non-invasive) cells.

Arachidonic acid stimulates internalisation of leptin by human placental choriocarcinoma (BeWo) cells

Arachidonic acid at 100 nM stimulated internalisation of 125I-leptin in human placental choriocarcinoma (BeWo) cells by 3-fold compared with controls. In contrast, eicosapentaenoic acid at similar concentration decreased internalisation of leptin by 2-fold. Use of ibuprofen and indomethacin (inhibitors of prostaglandin synthesis) inhibited the stimulatory effect of arachidonic acid. Prostaglandin E 2, a cyclooxygenase metabolite of arachidonic acid, stimulated internalisation of leptin by these cells. All these data demonstrate that stimulation of leptin internalisation by arachidonic acid in placental trophoblasts may be mediated via prostaglandin E 2.

Genetic analysis on roles of prostanoids in animal physiology and pathology; studies with receptor knockout mice

Prostaglandins (PGs) and thromboxane (TX) are the cyclooxygenase metabolites of arachidonic acid and are collectively termed prostanoids. The roles of prostanoids in our body have been examined by comparing the effects of aspirin-like drugs that inhibit cyclooxygenase, with those of prostanoids added exogenously. However, such studies do not clearly indicate which type of prostanoid is involved in a given process, and how critical the actions of prostanoids might be. To address these questions, others and we have generated mice deficient in each prostanoid receptor. Here we review the findings of these studies and discuss the roles of prostanoids in animal physiology and pathology.