Arachidonic Acid Derivatives Research Articles

The discovery of the leukotrienes.

In a study of the transformation of polyunsaturated fatty acids in rabbit polymorphonuclear leukocytes in 1976 it was discovered that arachidonic acid is oxygenated at C-5 (1). Subsequently a number of derivatives, including leukotriene B 4 (LTB 4 ), were identified (2-4). Extension of these studies led in 1979 to the discovery of the pivot epoxide intermediate, LTA 4 (5), and the elucidation of the structure of SRS-A (slow-reacting substance of anaphylaxis) as a group of cysteinyl-leukotrienes, namely LTC 4 , LTD 4 , and LTE 4 (6-10). Earlier work had shown that the prostaglandins are formed by oxygenation and further transformation of arachidonic acid and other polyunsaturated fatty acids (11). The first intermediate in the formation of prostaglandins, the endoperoxide PGG 2 , was isolated and identified in 1974 (12). At the same time we introduced the name cyclooxygenase for the enzyme catalyzing the transformation of arachidonic acid into PGG 2 (12). However, the endoperoxides, PGG 2 and PGH 2 , had some biological effects that could not be explained by their conversion to the known prostaglandins (12). This finding eventually led to the discovery of thromboxane A 2 , an unstable platelet-aggregating and vasoconstrictor substance (13). Subsequent work showed that the endoperoxide can also be converted into a derivative, prostacyclin (PGI 2 ), with opposite biological effects (14). Prostaglandins E 2 and I 2 have strong proinflammatory effects. Aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs) inhibit the enzyme (cyclooxygenase) responsible for conversion of arachidonic acid into prostaglandins and thromboxanes (15). An induced form of the cyclooxygenase (COX-2) seems to play an important role in inflammation, thus opening the possibility of developing antiinflammatory NSAIDs that lack the side effects of the previous generation of such drugs (16, 17). In 1975 antiinflammatory steroids were proposed to inhibit prostaglandin formation by blocking of the release of the precursor acid from phospholipid stores (18). Since steroids and NSAIDs have significantly different antiinflammatory effects it seemed conceivable that some of these differences might be explained by the formation of proinflammatory derivatives of arachidonic acid by cyclooxygenase-independent reactions. Our studies of the metabolism of arachidonic acid in leukocytes unraveled a new metabolic pathway and led to the discovery of the leukotrienes (8). These compounds play a role in allergy and asthma and have pronounced proinflammatory effects (19, 20).

Subcellular localization of prostaglandin H synthase-2 in a human amnion cell line: implications for nuclear localized prostaglandin signaling pathways

We have determined that prostaglandin H synthase-2 localises strongly to the nuclear membrane as well as being found in the endoplasmic reticulum in human amnion-derived WISH cells which have been stimulated with interleukin 1β and phorbol ester. This is consistent with findings in cells of non-reproductive origin. There is strong evidence that prostaglandin J2derivatives, which in other tissues exhibit tumour suppressing, antiproliferative and/or differentiation promoting activities, act through binding of intracellular receptors which then enter the nucleus. In addition, some arachidonic acid derivatives are clearly generated by enzymes at the nuclear envelope and localise to sites in nuclei or bind sites in nuclei. The WISH cell line will make an excellent system for studying these perinuclear intracellular prostanoid signaling mechanisms.

Interaction of 12-lipoxygenase with cellular proteins

Eicosapentaenoic acid (EPA) has been reported to have a potent anti-aggregatory activity and to be efficiently metabolized by 12-lipoxygenase, not by cyclooxygenase in platelets. In vitro effect of 12-lipoxygenase metabolites of EPA on platelet function was studied and compared with those of arachidonic acid (AA). The 12-lipoxygenase metabolites of AA and EPA; 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 12-hydroperoxyeicosapentaenoic acid (12-HPEPE), and their hydroxy derivatives, 12-hydroxyeicosatetraenoic acid (12-HETE) and 12-hydroxyeicosapentaenoic acid (12-HEPE) were prepared enzymatically using human platelet lysate. These compounds were purified by high performance liquid chromatography and identified by gas chromatography-mass spectrometry. 12-HPETE and 12-HPEPE inhibited dose-dependently washed human platelet aggregation and serotonin (5-HT) release induced by AA and collagen. The potency of 12-HPEPE was almost equal to that of 12-HPETE. Their hydroxy derivatives, 12-HETE and 12-HEPE were less potent. 12-hydroperoxy derivatives of AA and EPA were the most potent in inhibiting platelet aggregation and 5-HT release among 5-, 12- and 15-hydroperoxy isomers of AA and EPA. The inhibitory effects of 12-HPETE and 12-HPEPE on platelet aggregation were additive.

20-HETE agonists and antagonists in the renal circulation.

The present study examined the effects of a series of 20-hydroxyeicosatetraenoic acid (20-HETE) derivatives on the diameter of renal arterioles to determine the structural requirements of the vasoconstrictor response to 20-HETE. The vascular responses to 5-, 8-, 12-, 15-, 19-, 20-, 21-HETEs, arachidonic acid (AA), and saturated, partially saturated, dimethyl, carboxyl, and 19-carbon derivatives of 20-HETE (10(-8) to 10(-6) M) were assessed in rat renal interlobular arteries (65-125 micrometer). 20-HETE, 21-HETE, dimethyl-20-HETE, and a partially saturated derivative of 20-HETE, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid, reduced vessel diameter by 19 +/- 3, 17 +/- 3, 16 +/- 2, and 28 +/- 2%, respectively. In contrast, 5-, 8-, 12-, 15-, and 19-HETE, AA, saturated, partially saturated, carboxyl, and the 19-carbon derivatives of 20-HETE had no effect on vessel diameter. Pretreatment with 5-, 15-, and 19-HETE, the 19-carbon derivative or 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (1 microM) completely blocked the vasoconstrictor response to 20-HETE in renal arterioles. Pretreatment with AA, carboxyl, saturated 19-carbon, and saturated 20-HETE derivatives (1 microM) partially blocked the response, whereas 8- and 12-HETE (1 microM) had no effect on the vasoconstrictor response to 20-HETE. These findings suggest that 20-HETE agonists and antagonists require a carboxyl or an ionizable group on carbon 1 and a double bond near the 14 or 15 carbon. 20-HETE agonists also require a functional group capable of hydrogen bonding on carbon 20 or 21, whereas antagonists lack this reactive group.

Indirect enhancement of neutrophil activity and adhesion to cultured human umbilical vein endothelial cells by isoprostanes (iPF 2α-III and iPE 2-III)

Isoprostanes are metabolites of arachidonic acid found in blood under various conditions of oxidative stress. Because arachidonic acid derivatives are major mediators of inflammation, we investigated the potential inflammatory effects of iPF 2α-III (previously 8-isoPGF 2α) and iPE 2-III (8-isoPGE 2) on human polymorphonuclear granulocytes (PMN), as well as on human umbilical vein endothelial cells (HUVECs). The early activation marker CD11b on PMN and the adhesion molecules ICAM-1, E-selectin, and P-selectin on HUVECs were quantified by flow cytometry. Levels of the cytokines interleukin (IL)-6 and IL-8 were measured in the culture supernatant by enzyme-linked immunosorbent assay. Furthermore, adhesion of PMN to HUVECs was assessed. Neither isoprostane showed any direct stimulatory effects on PMN or HUVECs at concentrations of 0.1 or 1 μM: there was no acute elevation in expression of CD11b or P-selectin and no change of ICAM-1 or E-selectin after 4 or 24 h of incubation, respectively. The levels of interleukin IL-6 and IL-8 were also unaltered. However, PMN adhesion was significantly enhanced both after 4 and 24 h of incubation of HUVECs with iPF 2α-III, and CD11b expression on PMN was elevated by contact of these cells with the supernatant of pre-exposed HUVECs. Neither of these actions were inhibited by an endothelin receptor antagonist (bosentan) or a combined thromboxane A2/isoprostane-receptor antagonist (SQ29548). Thus, although not having a direct pro-inflammatory potential, isoprostanes might indirectly accentuate PMN stimulation. This seems to occur via a receptor-independent mechanism, perhaps the production of an active metabolite of isoprostanes by endothelial cells.

Endocrine and other responses to acute administration of cannabinoid compounds to non-stressed male calves

There is an abundance of cannabinoid (CB) receptors for derivatives of cannabis plants in the brain and throughout the body, and several naturally occurring arachidonic acid derivatives can activate these receptors. The specific objective of this study was to activate these CB receptors in castrated male calves through administration of several CB agonists and to measure immediate changes in concentrations of several serum hormones, respiration rate, and sensitivity to pain. The rationale for the study was that exogenous activation of CB receptors might reveal whether the endogenous CB system (consisting of receptors and endogenous ligands) plays a role in the stress response of animals and specifically whether the activated CB system might be part of a coping mechanism to combat stress. Intravenous administration of three CB agonists (anandamide, methanandamide and WIN 55212-2) to nine castrated male calves under nonstress conditions provoked immediate increases of serum cortisol and respiration rate as well as rapidly caused hypoalgesia to cutaneous pain and thermal stimuli. Although anandamide and methanandamide did not affect serum prolactin, administration of another CB agonist (WIN 55212-2) did increase serum prolactin abruptly. None of the CB agonists affected serum growth hormone. In summary, many of the changes following administration of CB agonists were similar to a stress response in this species, but there were some agonist-specific differences, notably regarding prolactin secretion, as well as differences between calves and observations made in other species. Although CB receptors in calves may be activated by endogenous ligands during exposure to some stressors, the present results are also consistent with this CB system being part of a coping mechanism that helps animals deal with imposed stressors.

Stage-specific excitation of cannabinoid receptor exhibits differential effects on mouse embryonic development.

Anandamide (N-arachidonoylethanolamine), an arachidonic acid derivative, is an endogenous ligand for both the brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors. We have previously demonstrated that preimplantation mouse embryos express mRNA for these receptors and that the periimplantation uterus contains the highest level of anandamide yet discovered in a mammalian tissue. We further demonstrated that 2-cell mouse embryos exposed to low levels of anandamide (7 nM) or other known cannabinoid agonists in culture exhibit markedly compromised embryonic development to blastocysts and that this effect is mediated by CB1-R. In contrast, the present study demonstrates that blastocysts exposed in culture to the same low levels of cannabinoid agonists exhibited accelerated trophoblast differentiation with respect to fibronectin-binding activity and trophoblast outgrowth. Again, these effects resulted from activation of embryonic CB1-R. There was a differential concentration-dependent effect of cannabinoids on the trophoblast, with an observed inhibition of differentiation at higher doses. These results provide evidence for the first time that cannabinoid effects are differentially executed depending on the embryonic stage and cannabinoid levels in the environment. Since uterine anandamide levels are lowest at the sites of implantation and highest at the interimplantation sites, the new findings imply that site-specific levels of anandamide and/or other endogenous ligands in the uterus may regulate implantation spatially by promoting trophoblast differentiation at the sites of blastocyst implantation.

Investigation of structural analogs of prostaglandin amides for binding to and activation of CB1 and CB2 cannabinoid receptors in rat brain and human tonsils.

Arachidonyl ethanolamide (anandamide, AEA) is an endogenously produced lipid amide which binds to and activates the CB1 and CB2 cannabinoid receptors. Further, administration of arachidonyl ethanolamide to animals produces many of the same physiological effects as classical and non-classical cannabinoids1,2. The CB1 cannabinoid receptors are found in the brain and central nervous system; the CB2 cannabinoid receptors are found in immune cells and tissue. Because arachidonyl ethanolamide is an arachidonic acid derivative, it is possible that it may be metabolized to a prostaglandin derivative. The carboxylic acid moiety is not necessary for binding to cyclooyxgenase, the enzyme which begins the arachidonic acid cascade3,4, suggesting that an ethanol amide derivative might be possible as a substrate for this enzyme. This lends potential physiological relevance to a prostaglandin amide.

Vitamin E: blocking the beginnings of atherosclerosis?

Apolipoprotein E-deficient (apoE–/–) mice are a useful model of human atherosclerosis as they develop vascular lesions with many of the histopathological features of the human disease. In this paper1xVitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis in ApoE-deficient mice. Pratico, D. et al. Nat. Med. 1998; 4: 1189–1192Crossref | PubMed | Scopus (429)See all References1, apoE–/– mice have been used to demonstrate the functional importance of oxidative stress in the progression of atherosclerosis.The authors focus their attention on isoprostanes (iPs), which are oxidized derivatives of arachidonic acid. iPs were previously shown to accumulate in human atherosclerotic plaques and thus can serve as a reliable indicator of oxidative stress. In apoE–/– mice, the iP level in urine and plasma gradually increases with age and is always higher than in control mice. This increase in iP levels is apparent even at eight weeks of age, when no anatomical atherosclerotic lesions can be detected. This is consistent with the hypothesis that lipid oxidation precedes the development of overt atherosclerosis. Addition of vitamin E to the diet of the apoE–/– mice significantly decreases iP levels in urine and plasma. Remarkably, the progression of aortic atherosclerosis also visibly slows down in the vitamin E-fed apoE–/– mice, despite persistently high levels of cholesterol.The conclusion of this paper is clear: lipid oxidation plays an important role in the progression of atherosclerosis. Furthermore, if apoE–/– mice are a faithful approximation to patients with atherosclerosis, consumption of vitamin E could be a great weapon to fight the disease.

Chronic nitric oxide inhibition model six years on.

The discovery in 1987 that endothelium-derived nitric oxide (NO) mediates the vasodilatory effect of certain endothelium-dependent agonists1 2 inaugurated the current huge field of NO biology. It is now recognized that NO plays essential roles in many diverse physiological processes and in some pathophysiologic events. Development of these concepts has been based largely on evidence obtained by limiting NO biosynthesis. This review is centered on the cardiovascular and particularly the renal functional and structural consequences of chronic pharmacologic NO inhibition by l-arginine analogues. We devoted special attention to the mechanisms of hypertension and organ injury that occur under these circumstances, while appreciating the inherent limitations surrounding interpretation of this data. NO is made by the enzymatic action of several widely distributed NO synthases (NOS). In the presence of the substrates l-arginine and oxygen, as well as a number of essential cofactors, NO is produced in response to appropriate stimuli. The constitutively expressed NOS play a major role in the physiological control of vascular tone and kidney function.3 4 Vascular endothelial NOS (eNOS) and brain-type NOS (bNOS) are widely distributed throughout the kidney5 as well as the cardiovascular system and in strategic locations in the peripheral and central nervous system (CNS).6 7 Both eNOS and particularly bNOS are abundant in the kidney, glomeruli, and vasculature as well as in most segments of the tubule,3 5 and NOS activity in medulla is considerably greater than in cortex.8 NO generated within the kidney controls the glomerular filtration rate (GFR), total renal and medullary blood flow, pressure natriuresis, epithelial sodium transport, and production of various vasoactive factors including renin.3 4 5 eNOS is distributed throughout most parts of the arterial and venous circulation, although there is considerable heterogeneity in the extent to which NO controls …

Role of Human CYP4F2 in Hepatic Catabolism of the Proinflammatory Agent Leukotriene B4

Leukotriene B4 (LTB4), an arachidonic acid derivative, is a potent proinflammatory agent whose actions are terminated by catabolism via a microsomal ω-hydroxylation pathway. Although the liver serves as the principal site for LTB4 clearance from the systemic circulation, the attributes of hepatic LTB4 metabolism are ill defined in humans. Thus, we examined metabolism of LTB4 to its ω-hydroxylated metabolite 20-hydroxyleukotriene B4 (20-OH LTB4) by human liver microsomes and also purified the hepatic P450 enzyme underlying this reaction. Liver microsomes from 10 different subjects converted LTB4 to 20-OH LTB4 at similar rates (1.06 ± 0.3 nmol/min/nmol P450; 0.25 ± 0.1 nmol/min/mg protein). Analysis of the microsomal LTB4 20-hydroxylation reaction revealed kinetic parameters (apparentKmof 74.8 μM with aVMAXof 2.42 nmol/min/nmol P450) consistent with catalysis by a single P450 enzyme. Conventional chromatography combined with immunochemical screening with rat CYP4A1 antibodies was then used to isolate a P450 enzyme from human liver microsomes with a molecular weight of 57,000 and an NH2-terminal amino acid sequence 94% homologous (12Trp →12Gly) over the first 17 residues with the humanCYP4F2cDNA-derived sequence. Upon reconstitution with P450 reductase and phospholipid, CYP4F2 converted LTB4 to 20-OH LTB4 at a turnover rate of 392 pmol/min/nmol P450, whereas the other human liver P450s tested, including CYP4A11, exhibited neglible LTB4 ω-hydroxylase activity. Polyclonal antibodies to CYP4F2 were found to markedly inhibit (91.9 ± 5%;n= 5) LTB4 20-hydroxylation by human liver microsomes. Microsomal 20-OH LTB4 formation was also inhibited 30% by arachidonic acid, a known CYP4F2 substrate, and 50% by prostaglandin A1but was unaffected by lauric acid, palmitic acid, and PGF2α. Finally, a strong correlation (r= 0.86;P< 0.002;n= 10) was observed between CYP4F2 content and LTB4 20-hydroxylase activity in the human liver samples. Our results indicate that CYP4F2 is the principle LTB4 ω-hydroxylating enzyme expressed in human liver and, as such, may play an important role in regulating circulating as well as hepatic levels of this powerful proinflammatory eicosanoid.

A genetic approach for studying the role of thromboxane A2 in the kidney

Thromboxane A2 (TxA2) has been implicated in a number of processes in normal kidney physiology and as a mechanism for injury in renal disease. TxA2 is a biologically active derivative of arachidonic acid and has potent vasoconstrictive and platelet-activation functions. Its actions are mediated by binding to specific G-protein-associated receptors designated TP receptors. There are at least two isoforms of the human TP receptor, and pharmacological evidence suggests TP receptor heterogeneity in other species. TP receptors are located in the renal cortex, and there may also be TxA2 binding sites in the medulla. TP receptors are involved in some normal functions of the kidney, and there is considerable evidence that TP receptors may mediate renal damage in disease states. To assess directly the role of the TxA2 in normal kidney function and in murine models of human disease, we have used gene targeting to eliminate expression of the TP receptor in mice.

Low-molecular-weight hormonal factors that affect head formation in Hydra.

Support or inhibition of DAG-induced head formation: Hydra magnipapillata can be caused to form ectopic head structures by periodictreatmentwith PKC activators such as diacylglycerol (DAG). This ectopic head formation is supported by an extract from the ovine pineal gland that contains low-molecular-weight compounds. The frequency of ectopic head formation is also increased when DAG is paired with one of several lipophilic hormonal factors: (1) pinoline, a putative pineal hormone derived from tryptophan, (2) 12-S-HETE, a paracrine derivative of arachidonic acid, or (3) 1alpha, 25 dihydroxyvitamine D3, a hormonal factor also known as calcitriol. Dose-response curves were non-monotonic passing a maximum at low dosages. By contrast, DAG lost its capacity to induce ectopic head structures when it was paired with the provitamin D3, cholecalciferol. Patterning the head: one eicosanoid was found which influences patterning without being combined with DAG: 12-R-HETE caused growth-based elongation of the tentacles and an increase in the number of tentacles without affecting the longitudinal body pattern. Similar effects are brought about by substances that interfere with tyrosine phosphorylation, most potently bythe phosphotyrosine phosphatase inhibitor bisperoxo-(1,10-phenanthroline)-oxovanadate (V). The results speak for the existence of head-inducing hormonal factors, underline the significance of content protein kinases to pattern formation and point to a negative influence of the vitamin D3 content of the food on the capacity of the animals to develop head structures.

Phospholipase A2-mediated activation of mitogen-activated protein kinase by angiotensin II.

In renal proximal tubule epithelial cells, a membrane-associated phospholipase A2 (PLA2) is a major signaling pathway linked to angiotensin II (Ang II) type 2 receptor (AT2). The current studies were designed to test the hypothesis that membrane-associated PLA2-induced release of arachidonic acid (AA) and/or its metabolites may serve as an upstream mediator of Ang II-induced mitogen-activated protein kinase (MAPK) activation. Ang II stimulated transient dose-dependent phosphorylation of MAPK with a maximum at 1 microM (10 min). Inhibition of PLA2 by mepacrine diminished both AA release and MAPK phosphorylation, induced by Ang II. Furthermore, AA itself induced time- and dose-dependent phosphorylation of MAPK, supporting the importance of PLA2 as a mediator of Ang II signaling. The effects of both Ang II and AA on MAPK phosphorylation were protein kinase C independent and abolished by the inhibitor of cytochrome P450 isoenzyme, ketoconazole. Moreover, 5,6-epoxyeicosatrienoic acid and 14,15-epoxyeicosatrienoic acid, the cytochrome P450-dependent metabolites of AA, significantly stimulated MAPK activity in renal proximal tubule epithelial cells. These observations document a mechanism of Ang II-induced MAPK phosphorylation, mediated by PLA2-dependent release of AA and cytochrome P450-dependent production of epoxy derivatives of AA.

Study on the functionality and molecular properties of the AT4 receptor.

Whereas the role of angiotensin II (Ang II) has been clarified in numerous tissues and cell types, the physiological relevance of its C-terminal (3–8) degradation fragment, angiotensin IV (Ang IV), is unclear. Previously, we characterized a specific binding site for Ang IV in the bovine adrenal cortex and on bovine aortic endothelial cells (BAEC). In the present study, we tried to assess the functionality and mechanism of action of this receptor for Ang IV (AT4 receptor). Our results revealed that none of the classical second messengers (i.e., cAMP, Ca2+, inositol phosphates, nitric oxide or arachidonic acid derivatives) was modified significantly during acute (less than 1 h) stimulation of cells with Ang IV. Under normal culture conditions, BAEC efficiently internalized 125I-Ang IV. After a 2 h incubation at 37°C, acid-resistant binding corresponded to about 50% of total cell-associated radioactivity. This rapid internalization process suggests that the AT4 receptor is a functional protein. With a photoaffinity labeling approach, we revealed some properties of the AT4 receptor that are consistent with those of a growth factor or cytokine receptor.

Studies on the effects of anandamide in rat hepatic artery.

1. The effects of anandamide on K+ currents and membrane potential have been examined in freshly-isolated smooth muscle cells from rat hepatic artery and the results compared with the effects of this arachidonic acid derivative on tension and membrane potential changes in segments of whole artery. 2. In the presence of 0.3 mM L-NOARG and 10 microM indomethacin, anandamide (0.1-100 microM) and endothelium-derived hyperpolarizing factor (EDHF; liberated by acetylcholine, 0.01-10 microM) each relaxed endothelium-intact segments of hepatic artery precontracted with phenylephrine. These effects of anandamide, but not those of EDHF, were antagonized by the cannabinoid receptor antagonist, SR141716A (3 microM). 3. The relaxant effects of anandamide were unaffected by a toxin combination (apamin plus charybdotoxin, each 0.3 microM) which abolishes EDHF relaxations and were essentially unchanged in endothelium-denuded arteries. The relaxant effects of anandamide in endothelium-intact arteries were significantly reduced in a physiological salt solution containing 30 mM KCl and abolished when the K+ concentration was raised to 60 mM. 4. Anandamide (10 microM), acetylcholine (1 microM, via release of EDHF) and levcromakalim (10 microM) each markedly hyperpolarized the membrane potential of the smooth muscle cells of endothelium-intact arteries. However, when the endothelium was removed, the hyperpolarizing effects of both anandamide (10 microM) and acetylcholine were essentially abolished whereas those of levcromakalim (10 microM) were unaffected. 5. Under voltage-clamp conditions, anandamide (10 microM) abolished spontaneous transient outward currents (STOCs) in freshly-isolated single hepatic artery cells held at 0 mV but had no effect on the holding current at this potential. In current-clamp mode, the spontaneous hyperpolarizing potentials which corresponded to the STOCs were abolished with no significant change in basal membrane potential. 6. Anandamide (10 microM) abolished the iberiotoxin-sensitive K+ current (IBK(Ca)) produced by caffeine and the corresponding hyperpolarizations generated by this xanthine derivative in current-clamp mode. In contrast, anandamide had no effect on IBK(Ca) generated on exposure to NS1619 (30 microM). 7. It was concluded that anandamide is not EDHF in the rat hepatic artery. Anandamide-induced hyperpolarization is exerted indirectly and requires the presence of the endothelium. Anandamide also acts on the smooth muscle cells to inhibit processes which require functional intracellular calcium stores. This direct action seems more important than membrane hyperpolarization in relaxing phenylephrine-contracted vessels.

Enantiomeric separation of various lipoxygenase derived monohydroxy polyunsaturated fatty acid methyl esters by high-performance liquid chromatography

Lipoxygenase derived monohydroxy polyunsaturated fatty acid methyl esters were separated by chiral high-performance liquid chromatography (HPLC) with a Chiralcel OD-H column in the normal-phase mode. Major lipoxygenase derivatives of linoleic, α-linolenic and arachidonic acids are well resolved by this column, provided they have been individually purified. Our method allows an easy and rapid determination of lipoxygenases enantioselectivity. In all cases tested the R enantiomer is eluted first.

Heparin-Induced Thrombocytopenia

Heparin's anticoagulation effect makes it the principal component in the immediate treatment of all thrombotic diseases. After hemorrhage, the most important complication of heparin is thrombocytopenia, the decrease in the number of blood platelets. Ten cases of "heparin-induced thrombocytopenia" are described, and guidelines for diagnosing heparin-induced thrombocytopenia in patients who present for cardiac and vascular surgery are given. Methods of treating patients with heparin-induced thrombocytopenia, including use of arachidonic acid derivatives, antiplatelet agents, other anticoagulants, heparinoids, natural and synthetic thrombin inhibitors, hirudin, and defibrinogenation with ancrod, are discussed.

Effect of the lipidic lipidosterolic extract of Serenoa repens (Permixon ®) on the ionophore A23187-stimulated production of leukotriene B 4 (LTB 4) from human polymorphonuclear neutrophils

Although the lipidic extract of Serenoa repens (LESSr, Permixon ®, Sereprostat ®) is widely used in patients suffering from benign prostatic hypertrophy (BPH), its mechanism of action is not fully elucidated. It has been demonstrated that infiltration of the prostate by inflammatory cells is one of the aetiologic factors involved in the development of BPH. These inflammatory cell types, such as polymorphonuclear neutrophils (PMNs), produce chemotactic mediators and contribute to the development of the disease. Among the chemotactic factors generated by inflammatory cell types, the derivatives of arachidonic acid have been extensively studied. For instance, leukotriene (LT) B 4 is one of the most potent chemotactic factors for PMNs and also exhibits a wide range of biological activities. In order to investigate the potential action of LESSr on arachidonate metabolism, and particularly on the synthesis of LTB 4, the effect of this extract on the in vitro synthesis of LT by human PMNs stimulated with the calcium ionophore A23187 was investigated. LESSr significantly inhibits the production of 5-lipoxygenase metabolites (5-HETE, 20-COOH LTB 4, LTB 4 and 20-OH LTB 4) at concentrations as low as 5 μg/ml. Such an effect of LESSr was also observed in the presence of exogenous arachidonic acid (20 μg/ml) and when f-MLP was used as the agonist, suggesting that inhibition of LTB 4 production by the extract was unrelated to phospholipase A 2 blockade and independent of the stimulating agent. The capability of LESSr to antagonize 5-lipoxygenase metabolites production may contribute, at least partly, to the understanding of its therapeutic activity on the inflammatory component of BPH.

The Organization, Promoter Analysis, and Expression of the Human PPARγ Gene

PPARgamma is a member of the PPAR subfamily of nuclear receptors. In this work, the structure of the human PPARgamma cDNA and gene was determined, and its promoters and tissue-specific expression were functionally characterized. Similar to the mouse, two PPAR isoforms, PPARgamma1 and PPARgamma2, were detected in man. The relative expression of human PPARgamma was studied by a newly developed and sensitive reverse transcriptase-competitive polymerase chain reaction method, which allowed us to distinguish between PPARgamma1 and gamma2 mRNA. In all tissues analyzed, PPARgamma2 was much less abundant than PPARgamma1. Adipose tissue and large intestine have the highest levels of PPARgamma mRNA; kidney, liver, and small intestine have intermediate levels; whereas PPARgamma is barely detectable in muscle. This high level expression of PPARgamma in colon warrants further study in view of the well established role of fatty acid and arachidonic acid derivatives in colonic disease. Similarly as mouse PPARgammas, the human PPARgammas are activated by thiazolidinediones and prostaglandin J and bind with high affinity to a PPRE. The human PPARgamma gene has nine exons and extends over more than 100 kilobases of genomic DNA. Alternate transcription start sites and alternate splicing generate the PPARgamma1 and PPARgamma2 mRNAs, which differ at their 5'-ends. PPARgamma1 is encoded by eight exons, and PPARgamma2 is encoded by seven exons. The 5'-untranslated sequence of PPARgamma1 is comprised of exons A1 and A2, whereas that of PPARgamma2 plus the additional PPARgamma2-specific N-terminal amino acids are encoded by exon B, located between exons A2 and A1. The remaining six exons, termed 1 to 6, are common to the PPARgamma1 and gamma2. Knowledge of the gene structure will allow screening for PPARgamma mutations in humans with metabolic disorders, whereas knowledge of its expression pattern and factors regulating its expression could be of major importance in understanding its biology.