8-11-14-eicosatrienoic-acid and 8-hydroxy-11-12-epoxyeicosa-5-9-14-trienoic-acid

This review summarizes recent developments in the role of soluble mediators of inflammation, particularly arachidonic acid metabolites, in inflammatory bowel disease (IBD).. The role of prostaglandin E2 in immune regulation has been better defined. Prostaglandin E2 promotes not only immune tolerance and epithelial homeostasis but also the proinflammatory Th17 pathway. Prostaglandin D2 has been established as promoting the resolution of inflammation in the gastrointestinal mucosa. The 12-lipoxygenase product hepoxilin A3 mediates the migration of neutrophils from the mucosa into the lumen.. Recent studies of soluble mediators, especially arachidonic acid metabolites, have defined their proinflammatory and anti-inflammatory roles in IBD.

Topics: 8,11,14-Eicosatrienoic Acid; Anti-Inflammatory Agents; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acid; Cell Movement; Dinoprostone; Humans; Inflammation; Inflammatory Bowel Diseases; Intestinal Mucosa; Neutrophils; Prostaglandin D2; Signal Transduction; Th17 Cells

The transmigration of polymorphonuclear leukocytes (PMNs; neutrophils) into the intestinal lumen is a classical phenomenon associated with a wide variety of disease states, including those of both pathogenic and autoimmune/idiopathic origin. While PMNs are highly effective at killing invading pathogens by releasing microbiocidal products, excessive or unnecessary release of these substances can cause substantial damage to the intestinal epithelium. Therefore, it is necessary to understand the underlying mechanisms that lure neutrophils into the lumen allowing them to perform their desired functions, so that researchers may begin to identify which processes may be potential targets for inhibiting the transmigration of PMNs during noninfectious states.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Drug Delivery Systems; Enterocolitis; Humans; Immunity, Cellular; Infections; Intestinal Mucosa; Models, Biological; Neutrophil Infiltration; Signal Transduction

Bacterial infections at epithelial surfaces, such as those that line the gut and the lung, stimulate the migration of neutrophils through the co-ordinated actions of chemoattractants secreted from pathogen-stimulated epithelial cells. One such factor involved in attracting polymorphonuclear leukocytes across the epithelium and into the lumen has until recently remained elusive. In 2004, we identified the eicosanoid, hepoxilin A(3), to be selectively secreted from the apical surface of human intestinal or lung epithelial cells stimulated with Salmonella enterica serotype Typhimurium or Pseudomonas aeruginosa, respectively. In this role, the function of hepoxilin A(3) is to guide neutrophils, via the establishment of a gradient, across the epithelial tight junction complex. Interestingly, interruption of the synthetic pathway of hepoxilin A(3) blocks the apical release of hepoxilin A(3)in vitro and the transmigration of neutrophils induced by S. typhimurium both in in vitro and in vivo models of inflammation. Such results have led to the discovery of a completely novel pathway that is not only critical for responses to bacterial pathogens but also has broad implications for inflammatory responses affecting mucosal surfaces in general. Thus, the objective of this review was to highlight the recent findings that implicate hepoxilin A(3) as a key regulator of mucosal inflammation.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acids; Chemotaxis; Humans; Inflammation; Inflammation Mediators; Neutrophils

Hepoxilins constitute a group of 12S-hydroperoxyeicosatetraenoic acid (12S-HpETE)-derived epoxy-hydroxy fatty acids that have been detected in various cell types and tissues. Although hepoxilin A3 (HXA3) exhibits a myriad of biological activities, its biosynthetic mechanism was not investigated in detail. Here we review the isolation, cloning, and characterization of a leukocyte-type 12S-lipoxygenase (12S-LOX) from rat insulinoma cells RINm5F, which exhibits an intrinsic hepoxilin A3 synthase activity. Confirmation for this observation was achieved by coimmunoprecipitation of HXA3 synthase activity with an anti-leukocyte 12S-LOX antibody, preparation of recombinant rat 12S-LOX enzyme from RINm5F cells, and assay of HXA3 synthase activity therein. Site-directed mutagenesis studies performed on rat 12S-LOX showed that 12-lipoxygenating enzyme species exhibit a strong HXA3 synthase activity that is impaired when the positional specificity of arachidonic acid is altered in favor of 15-lipoxygenation. Inasmuch as cellular glutathione peroxidases (cGPx and PHGPx) and HXA3 synthase compete for the same substrate 12S-HpETE, it can be proposed that the overall activity of glutathione peroxidases, representing the overall peroxide tone, finely tunes the rate of HXA3 formation.

Topics: 8,11,14-Eicosatrienoic Acid; Amino Acid Sequence; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Cell Line, Tumor; Intramolecular Oxidoreductases; Lipoxygenase Inhibitors; Molecular Sequence Data; Neoplasm Proteins; Rats; Sequence Alignment

This article reviews published evidence describing the enzymatic and nonenzymatic formation and the routes of metabolism of the hepoxilins. Also treated are the major approaches used for the chemical synthesis of these compounds and for some of their analogs.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Drug Stability; Leukotrienes; Molecular Structure

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Hemin; Humans; Leukotrienes; Lung; Molecular Conformation; Molecular Structure; Pineal Gland

This review is intended to summarize the biological actions of the hepoxilins reported to date. These actions appear to have, as their basis, changes in intracellular concentrations of ions including calcium and potassium ions as well as changes in second messenger systems. Recent evidence suggests that the biological actions of the hepoxilins may be receptor-mediated as indicated from data showing the existence of hepoxilin-specific binding proteins in the human neutrophil. Such evidence also implicates the association of G-proteins both in hepoxilin-binding as well as in hepoxilin action. The potential use of stable analogs of the hepoxilins is discussed as well as the directions in which this area is heading.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Cations; Humans; Second Messenger Systems

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Calcium; Chemotaxis, Leukocyte; Humans; Insulin; Insulin Secretion; Neutrophils

1. This article reviews the formation, metabolism and pharmacological actions of the hepoxilins. These are biologically active hydroxy epoxide derivatives of arachidonic acid formed through the 12-lipoxygenase pathway. 2. This review summarizes literature data available at the time of writing of this article.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Humans

In this chapter, we will review recent findings which implicate the hepoxilins as modulators of second messenger systems in the human neutrophil. We have shown that the hepoxilins affect calcium homeostasis in the cell and that they stimulate the release of arachidonic acid and diradylglycerol but not inositol phosphate indicating a mode of action for these 12-lipoxygenase metabolites that is independent of phospholipase C activation. In fact lipid analyses indicate that the phospholipid affected by the hepoxilins is phosphatidyl choline, and that this phospholipid is hydrolyzed by a phospholipase D. These findings indicate that the hepoxilins, which are formed by the platelet as well as the neutrophil, may affect neutrophil activation through a potential cell-cell interaction in the circulation or at pathologic sites to initiate or potentiate the inflammatory process.

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonic Acids; Fatty Acids, Unsaturated; Humans; Models, Biological; Neutrophils; Propranolol; Second Messenger Systems

A feature shared by many inflammatory lung diseases is excessive neutrophilic infiltration. Neutrophil homing to airspaces involve multiple factors produced by several distinct cell types. Hepoxilin A(3) is a neutrophil chemoattractant produced by pathogen-infected epithelial cells that is hypothesized to facilitate neutrophil breach of mucosal barriers. Using a Transwell model of lung epithelial barriers infected with Pseudomonas aeruginosa, we explored the role of hepoxilin A(3) in neutrophil transepithelial migration. Pharmacological inhibitors of the enzymatic pathways necessary to generate hepoxilin A(3), including phospholipase A(2) and 12-lipoxygenase, potently interfere with P. aeruginosa-induced neutrophil transepithelial migration. Both transformed and primary human lung epithelial cells infected with P. aeruginosa generate hepoxilin A(3) precursor arachidonic acid. All four known lipoxygenase enzymes capable of synthesizing hepoxilin A(3) are expressed in lung epithelial cell lines, primary small airway epithelial cells, and human bronchial epithelial cells. Lung epithelial cells produce increased hepoxilin A(3) and lipid-derived neutrophil chemotactic activity in response to P. aeruginosa infection. Lipid-derived chemotactic activity is soluble epoxide hydrolase sensitive, consistent with hepoxilin A(3) serving a chemotactic role. Stable inhibitory structural analogs of hepoxilin A(3) are capable of impeding P. aeruginosa-induced neutrophil transepithelial migration. Finally, intranasal infection of mice with P. aeruginosa promotes enhanced cellular infiltrate into the airspace, as well as increased concentration of the 12-lipoxygenase metabolites hepoxilin A(3) and 12-hydroxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid. Data generated from multiple models in this study provide further evidence that hepoxilin A(3) is produced in response to lung pathogenic bacteria and functions to drive neutrophils across epithelial barriers.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonate 12-Lipoxygenase; Blood-Air Barrier; Epithelial Cells; Female; Humans; Male; Mice; Neutrophils; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mucosa; Transendothelial and Transepithelial Migration

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonate 12-Lipoxygenase; Bacillus subtilis; Bacterial Proteins; Bacterial Toxins; Cell Line; Cell Membrane; Clostridium septicum; Female; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophil Infiltration; Neutrophils; Pneumococcal Infections; Streptococcus pneumoniae; Streptolysins; Transendothelial and Transepithelial Migration; Virulence Factors

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonate 12-Lipoxygenase; Cell Movement; Epithelial Cells; Humans; Neutrophils

Zika virus (ZIKV) has emerged as one of the most medically relevant viral infections of the past decades; the devastating effects of this virus over the developing brain are a major matter of concern during pregnancy. Although the connection with congenital malformations are well documented, the mechanisms by which ZIKV reach the central nervous system (CNS) and the causes of impaired cortical growth in affected fetuses need to be better addressed. We performed a non-invasive, metabolomics-based screening of saliva from infants with congenital Zika syndrome (CZS), born from mothers that were infected with ZIKV during pregnancy. We were able to identify three biomarkers that suggest that this population suffered from an important inflammatory process; with the detection of mediators associated with glial activation, we propose that microcephaly is a product of immune response to the virus, as well as excitotoxicity mechanisms, which remain ongoing even after birth.

Topics: 8,11,14-Eicosatrienoic Acid; Biomarkers; Female; Fetal Development; Fetus; Humans; Infant; Infant, Newborn; Inflammation; Longitudinal Studies; Male; Metabolomics; Microcephaly; Mothers; Parturition; Pregnancy; Pregnancy Complications, Infectious; Saliva; Virus Diseases; Zika Virus; Zika Virus Infection

Hepoxilins (HXs) and trioxilins (TrXs) are involved in physiological processes such as inflammation, insulin secretion and pain perception in human. They are metabolites of polyunsaturated fatty acids (PUFAs), including arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid, formed by 12-lipoxygenase (LOX) and epoxide hydrolase (EH) expressed by mammalian cells. Here, we identify ten types of HXs and TrXs, produced by the prokaryote Myxococcus xanthus, of which six types are new, namely, HXB

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonate 12-Lipoxygenase; Arachidonate Lipoxygenases; Bacterial Proteins; Biotransformation; Epoxide Hydrolases; Fatty Acids, Unsaturated; Metabolic Networks and Pathways; Molecular Structure; Myxococcus xanthus

Although nonsteroidal anti-inflammatory drugs are the first line of therapeutics for the treatment of mild to moderate somatic pain, they are not generally considered to be effective for neuropathic pain. In the current study, direct activation of spinal Toll-like 4 receptors (TLR4) by the intrathecal (IT) administration of KDO2 lipid A (KLA), the active component of lipopolysaccharide, elicits a robust tactile allodynia that is unresponsive to cyclooxygenase inhibition, despite elevated expression of cyclooxygenase metabolites in the spinal cord. Intrathecal KLA increases 12-lipoxygenase-mediated hepoxilin production in the lumbar spinal cord, concurrent with expression of the tactile allodynia. The TLR4-induced hepoxilin production was also observed in primary spinal microglia, but not in astrocytes, and was accompanied by increased microglial expression of the 12/15-lipoxygenase enzyme 15-LOX-1. Intrathecal KLA-induced tactile allodynia was completely prevented by spinal pretreatment with the 12/15-lipoxygenase inhibitor CDC or a selective antibody targeting rat 15-LOX-1. Similarly, pretreatment with the selective inhibitors ML127 or ML351 both reduced activity of the rat homolog of 15-LOX-1 heterologously expressed in HEK-293T cells and completely abrogated nonsteroidal anti-inflammatory drug-unresponsive allodynia in vivo after IT KLA. Finally, spinal 12/15-lipoxygenase inhibition by nordihydroguaiaretic acid (NDGA) both prevents phase II formalin flinching and reverses formalin-induced persistent tactile allodynia. Taken together, these findings suggest that spinal TLR4-mediated hyperpathic states are mediated at least in part through activation of microglial 15-LOX-1.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Animals, Newborn; Anti-Inflammatory Agents, Non-Steroidal; Cells, Cultured; Chromatography, Liquid; Enzyme Inhibitors; Hyperalgesia; Lipopolysaccharides; Lipoxygenases; Male; Mass Spectrometry; Neuroglia; Physical Stimulation; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord; Toll-Like Receptor 4; Transfection

Neutrophil breach of the mucosal surface is a common pathological consequence of infection. We present an advanced co-culture model to explore neutrophil transepithelial migration utilizing airway mucosal barriers differentiated from primary human airway basal cells and examined by advanced imaging. Human airway basal cells were differentiated and cultured at air-liquid interface (ALI) on the underside of 3 µm pore-sized transwells, compatible with the study of transmigrating neutrophils. Inverted ALIs exhibit beating cilia and mucus production, consistent with conventional ALIs, as visualized by micro-optical coherence tomography (µOCT). µOCT is a recently developed imaging modality with the capacity for real time two- and three-dimensional analysis of cellular events in marked detail, including neutrophil transmigratory dynamics. Further, the newly devised and imaged primary co-culture model recapitulates key molecular mechanisms that underlie bacteria-induced neutrophil transepithelial migration previously characterized using cell line-based models. Neutrophils respond to imposed chemotactic gradients, and migrate in response to Pseudomonas aeruginosa infection of primary ALI barriers through a hepoxilin A3-directed mechanism. This primary cell-based co-culture system combined with µOCT imaging offers significant opportunity to probe, in great detail, micro-anatomical and mechanistic features of bacteria-induced neutrophil transepithelial migration and other important immunological and physiological processes at the mucosal surface.

Topics: 8,11,14-Eicosatrienoic Acid; Cell Culture Techniques; Cell Line; Cell Movement; Cell Polarity; Chemotaxis, Leukocyte; Coculture Techniques; Epithelial Cells; Fluorescent Antibody Technique; Humans; Inflammation; Neutrophil Infiltration; Neutrophils; Respiratory Mucosa

Eicosanoids are a group of bioactive lipids that are shown to be important mediators of neutrophilic inflammation; selective targeting of their function confers therapeutic benefit in a number of diseases. Neutrophilic airway diseases, including cystic fibrosis, are characterized by excessive neutrophil infiltration into the airspace. Understanding the role of eicosanoids in this process may reveal novel therapeutic targets. The eicosanoid hepoxilin A3 is a pathogen-elicited epithelial-produced neutrophil chemoattractant that directs transepithelial migration in response to infection. Following hepoxilin A3-driven transepithelial migration, neutrophil chemotaxis is amplified through neutrophil production of a second eicosanoid, leukotriene B4 (LTB4). The rate-limiting step of eicosanoid generation is the liberation of arachidonic acid by phospholipase A2, and the cytosolic phospholipase A2 (cPLA2)α isoform has been specifically shown to direct LTB4 synthesis in certain contexts. Whether cPLA2α is directly responsible for neutrophil synthesis of LTB4 in the context of

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Antigens, Human Platelet; Cell Communication; Cell Line; Chemotaxis; Coculture Techniques; Cystic Fibrosis; Cytosol; Humans; Leukotriene B4; Mice; Neutrophils; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mucosa; Tomography, Optical Coherence; Transendothelial and Transepithelial Migration

12/15-lipoxygenase (12/15-LO) metabolizes arachidonic acid (AA) into several vasoactive eicosanoids. In mouse arteries, we previously characterized the enzyme's 15-LO metabolites 12(S)-hydroxyeicosatetraenoic acid (HETE), 15-HETE, hydroxyepoxyeicosatrienoic acids (HEETAs) and 11,12,15-trihydroxyeicosatrienoic acids (11,12,15-THETAs) as endothelium-derived relaxing factors. However, the observed 12-LO metabolites remained uncharacterized. The purpose of this study was to determine the structure and biological functions of eicosanoids generated by the enzyme's 12-LO activity.. Metabolites extracted from aortas of C57BL/6 male mice were separated using a series of reverse and normal phase chromatographic steps and identified as hepoxilin A. All identified vascular 12-LO metabolites were biologically active. In mouse mesenteric arteries, trioxilin A. Trioxilin A

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Aorta; HEK293 Cells; Humans; Male; Mesenteric Arteries; Mice; Mice, Inbred C57BL; Receptors, Thromboxane; Vasodilation; Vasodilator Agents

Streptococcus pneumoniae infections are an important cause of morbidity and mortality in older patients. Uncontrolled neutrophil-driven pulmonary inflammation exacerbates this disease. To test whether the α-tocopherol (α-Toc) form of vitamin E, a regulator of immunity, can modulate neutrophil responses as a preventive strategy to mitigate the age-associated decline in resistance to S. pneumoniae, young (4 mo) and old (22-24 mo) C57BL/6 mice were fed a diet containing 30-PPM (control) or 500-PPM (supplemented) α-Toc for 4 wk and intratracheally infected with S. pneumoniae. Aged mice fed a control diet were exquisitely more susceptible to S. pneumoniae than young mice. At 2 d postinfection, aged mice suffered 1000-fold higher pulmonary bacterial burden, 2.2-fold higher levels of neutrophil recruitment to the lung, and a 2.25-fold higher rate of lethal septicemia. Strikingly, α-Toc supplementation of aged mice resulted in a 1000-fold lower bacterial lung burden and full control of infection. This α-Toc-induced resistance to pneumococcal challenge was associated with a 2-fold fewer pulmonary neutrophils, a level comparable to S. pneumoniae-challenged, conventionally fed young mice. α-Toc directly inhibited neutrophil egress across epithelial cell monolayers in vitro in response to pneumococci or hepoxilin-A3, an eicosanoid required for pneumococcus-elicited neutrophil trans-epithelial migration. α-Toc altered expression of multiple epithelial and neutrophil adhesion molecules involved in migration, including CD55, CD47, CD18/CD11b, and ICAM-1. These findings suggest that α-Toc enhances resistance of aged mice to bacterial pneumonia by modulating the innate immune response, a finding that has potential clinical significance in combating infection in aged individuals through nutritional intervention.

Topics: 8,11,14-Eicosatrienoic Acid; Age Factors; alpha-Tocopherol; Animals; Cell Adhesion Molecules; Dietary Supplements; Disease Models, Animal; Disease Resistance; Disease Susceptibility; Epithelium; Gene Expression; Male; Mice; Neutrophil Infiltration; Neutrophils; Pneumonia, Pneumococcal; Streptococcus pneumoniae; Transendothelial and Transepithelial Migration

Neutrophilic infiltration is a leading contributor to pathology in a number of pulmonary disease states, including cystic fibrosis. Hepoxilin A3 (HXA3) is a chemotactic eicosanoid shown to mediate the transepithelial passage of neutrophils in response to infection in several model systems and at multiple mucosal surfaces. Another well-known eicosanoid mediating general neutrophil chemotaxis is leukotriene B4 (LTB4). We sought to distinguish the roles of each eicosanoid in the context of infection of lung epithelial monolayers by Pseudomonas aeruginosa. Using human and mouse in vitro transwell model systems, we used a combination of biosynthetic inhibitors, receptor antagonists, as well as mutant sources of neutrophils to assess the contribution of each chemoattractant in driving neutrophil transepithelial migration. We found that following chemotaxis to epithelial-derived HXA3 signals, neutrophil-derived LTB4 is required to amplify the magnitude of neutrophil migration. LTB4 signaling is not required for migration to HXA3 signals, but LTB4 generation by migrated neutrophils plays a significant role in augmenting the initial HXA3-mediated migration. We conclude that HXA3 and LTB4 serve independent roles to collectively coordinate an effective neutrophilic transepithelial migratory response.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Bacteria; Bacterial Infections; Calcium Signaling; Cell Line; Chemotaxis, Leukocyte; Disease Models, Animal; Female; Gene Knockdown Techniques; Humans; Leukotriene B4; Mice; Neutrophil Infiltration; Neutrophils; Pseudomonas aeruginosa; Receptors, Leukotriene B4; Transendothelial and Transepithelial Migration

Pulmonary exacerbations in cystic fibrosis airways are accompanied by inflammation, neutrophilia, and mucous thickening. Cystic fibrosis sputum contains a large amount of uncleared DNA contributed by neutrophil extracellular trap (NET) formation from neutrophils. The exact mechanisms of the induction of NETosis in cystic fibrosis airways remain unclear, especially in uninfected lungs of patients with early cystic fibrosis lung disease. Here we show that Hepoxilin A3, a proinflammatory eicosanoid, and the synthetic analog of Hepoxilin B3, PBT-3, directly induce NETosis in human neutrophils. Furthermore, we show that Hepoxilin A3-mediated NETosis is NADPH-oxidase-dependent at lower doses of Hepoxilin A3, while it is NADPH-oxidase-independent at higher doses. Together, these results demonstrate that Hepoxilin A3 is a previously unrecognized inducer of NETosis in cystic fibrosis lungs and may represent a new therapeutic target for treating cystic fibrosis and other inflammatory lung diseases.

Topics: 8,11,14-Eicosatrienoic Acid; Cells, Cultured; Cystic Fibrosis; Extracellular Traps; Humans; Neutrophils

Pathogen induced migration of neutrophils across mucosal epithelial barriers requires epithelial production of the chemotactic lipid mediator, hepoxilin A3 (HXA3). HXA3 is an eicosanoid derived from arachidonic acid. Although eosinophils are also capable of penetrating mucosal surfaces, eosinophilic infiltration occurs mainly during allergic processes whereas neutrophils dominate mucosal infection. Both neutrophils and eosinophils can respond to chemotactic gradients of certain eicosanoids, however, it is not known whether eosinophils respond to pathogen induced lipid mediators such as HXA3. In this study, neutrophils and eosinophils were isolated from human blood and placed on the basolateral side of polarized epithelial monolayers grown on permeable Transwell filters and challenged by various chemotactic gradients of distinct lipid mediators. We observed that both cell populations migrated across epithelial monolayers in response to a leukotriene B4 (LTB4) gradient, whereas only eosinophils migrated toward a prostaglandin D2 (PGD2) gradient. Interestingly, while pathogen induced neutrophil trans-epithelial migration was substantial, pathogen induced eosinophil trans-epithelial migration was not observed. Further, gradients of chemotactic lipids derived from pathogen infected epithelial cells known to be enriched for HXA3 as well as purified HXA3 drove significant numbers of neutrophils across epithelial barriers, whereas eosinophils failed to respond to these gradients. These data suggest that although the eicosanoid HXA3 serves as an important neutrophil chemo-attractant at mucosal surfaces during pathogenic infection, HXA3 does not appear to exhibit chemotactic activity toward eosinophils.

Topics: 8,11,14-Eicosatrienoic Acid; Cell Line; Chemokine CCL26; Chemokines, CC; Chemotaxis, Leukocyte; Eosinophils; Epithelial Cells; Humans; Interleukin-8; Leukotriene B4; Neutrophil Infiltration; Peroxidase; Pseudomonas aeruginosa; Respiratory Mucosa; Transendothelial and Transepithelial Migration

Acute pulmonary infection by Streptococcus pneumoniae is characterized by high bacterial numbers in the lung, a robust alveolar influx of polymorphonuclear cells (PMNs), and a risk of systemic spread of the bacterium. We investigated host mediators of S. pneumoniae-induced PMN migration and the role of inflammation in septicemia following pneumococcal lung infection. Hepoxilin A3 (HXA3) is a PMN chemoattractant and a metabolite of the 12-lipoxygenase (12-LOX) pathway. We observed that S. pneumoniae infection induced the production of 12-LOX in cultured pulmonary epithelium and in the lungs of infected mice. Inhibition of the 12-LOX pathway prevented pathogen-induced PMN transepithelial migration in vitro and dramatically reduced lung inflammation upon high-dose pulmonary challenge with S. pneumoniae in vivo, thus implicating HXA3 in pneumococcus-induced pulmonary inflammation. PMN basolateral-to-apical transmigration in vitro significantly increased apical-to-basolateral transepithelial migration of bacteria. Mice suppressed in the expression of 12-LOX exhibited little or no bacteremia and survived an otherwise lethal pulmonary challenge. Our data suggest that pneumococcal pulmonary inflammation is required for high-level bacteremia and systemic infection, partly by disrupting lung epithelium through 12-LOX-dependent HXA3 production and subsequent PMN transepithelial migration.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonate 12-Lipoxygenase; Bacillus subtilis; Bacteremia; Cell Line, Tumor; Cell Movement; Chemotactic Factors; Humans; Inflammation; Lung; Lung Diseases; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophils; Pneumococcal Infections; Streptococcus pneumoniae; Transendothelial and Transepithelial Migration

Peripheral inflammation initiates changes in spinal nociceptive processing leading to hyperalgesia. Previously, we demonstrated that among 102 lipid species detected by LC-MS/MS analysis in rat spinal cord, the most notable increases that occur after intraplantar carrageenan are metabolites of 12-lipoxygenases (12-LOX), particularly hepoxilins (HXA(3) and HXB(3)). Thus, we examined involvement of spinal LOX enzymes in inflammatory hyperalgesia. In the current work, we found that intrathecal (IT) delivery of the LOX inhibitor nordihydroguaiaretic acid prevented the carrageenan-evoked increase in spinal HXB(3) at doses that attenuated the associated hyperalgesia. Furthermore, IT delivery of inhibitors targeting 12-LOX (CDC, Baicalein), but not 5-LOX (Zileuton) dose-dependently attenuated tactile allodynia. Similarly, IT delivery of 12-LOX metabolites of arachidonic acid 12(S)-HpETE, 12(S)-HETE, HXA(3), or HXB(3) evoked profound, persistent tactile allodynia, but 12(S)-HpETE and HXA(3) produced relatively modest, transient heat hyperalgesia. The pronociceptive effect of HXA(3) correlated with enhanced release of Substance P from primary sensory afferents. Importantly, HXA(3) triggered sustained mobilization of calcium in cells stably overexpressing TRPV1 or TRPA1 receptors and in acutely dissociated rodent sensory neurons. Constitutive deletion or antagonists of TRPV1 (AMG9810) or TRPA1 (HC030031) attenuated this action. Furthermore, pretreatment with antihyperalgesic doses of AMG9810 or HC030031 reduced spinal HXA(3)-evoked allodynia. These data indicate that spinal HXA(3) is increased by peripheral inflammation and promotes initiation of facilitated nociceptive processing through direct activation of TRPV1 and TRPA1 at central terminals.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonate 12-Lipoxygenase; Hyperalgesia; Inflammation; Mice; Spinal Cord; Transient Receptor Potential Channels; TRPA1 Cation Channel; TRPV Cation Channels

Airway neutrophil infiltration is a pathological hallmark observed in multiple lung diseases including pneumonia and cystic fibrosis. Bacterial pathogens such as Pseudomonas aeruginosa instigate neutrophil recruitment to the air space. Excessive accumulation of neutrophils in the lung often contributes to tissue destruction. Previous studies have unveiled hepoxilin A(3) as the key molecular signal driving neutrophils across epithelial barriers. The eicosanoid hepoxilin A(3) is a potent neutrophil chemoattractant produced by epithelial cells in response to infection with P. aeruginosa. The enzyme phospholipase A(2) liberates arachidonic acid from membrane phospholipids, the rate-limiting step in the synthesis of all eicosanoids, including hepoxilin A(3). Once generated, aracidonic acid is acted upon by multiple cyclooxygenases and lipoxygenases producing an array of functionally diverse eicosanoids. Although there are numerous phospholipase A(2) isoforms capable of generating arachidonic acid, the isoform most often associated with eicosanoid generation is cytoplasmic phospholipase A(2)α. In the current study, we observed that the cytoplasmic phospholipase A(2)α isoform is required for mediating P. aeruginosa-induced production of certain eicosanoids such as prostaglandin E(2). However, we found that neutrophil transepithelial migration induced by P. aeruginosa does not require cytoplasmic phospholipase A(2)α. Furthermore, P. aeruginosa-induced hepoxilin A(3) production persists despite cytoplasmic phospholipase A(2)α suppression and generation of the 12-lipoxygenase metabolite 12-HETE is actually enhanced in this context. These results suggest that alterative phospholipase A(2) isoforms are utilized to synthesize 12-lipoxygenase metabolites. The therapeutic implications of these findings are significant when considering anti-inflammatory therapies based on targeting eicosanoid synthesis pathways.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Arachidonic Acid; Base Sequence; Cell Line; Cytoplasm; Dinoprostone; Eicosanoids; Epithelial Cells; Group IV Phospholipases A2; Humans; Lung; Neutrophil Infiltration; Phospholipases A2; Pseudomonas aeruginosa; Pseudomonas Infections; RNA, Small Interfering; Transendothelial and Transepithelial Migration

Hepoxilins are lipid signaling molecules derived from arachidonic acid through the 12-lipoxygenase pathway. These trans-epoxy hydroxy eicosanoids play a role in a variety of physiological processes, including inflammation, neurotransmission, and formation of skin barrier function. Mammalian hepoxilin hydrolase, partly purified from rat liver, has earlier been reported to degrade hepoxilins to trioxilins. Here, we report that hepoxilin hydrolysis in liver is mainly catalyzed by soluble epoxide hydrolase (sEH): i) purified mammalian sEH hydrolyses hepoxilin A₃ and B₃ with a V(max) of 0.4-2.5 μmol/mg/min; ii) the highly selective sEH inhibitors N-adamantyl-N'-cyclohexyl urea and 12-(3-adamantan-1-yl-ureido) dodecanoic acid greatly reduced hepoxilin hydrolysis in mouse liver preparations; iii) hepoxilin hydrolase activity was abolished in liver preparations from sEH(-/-) mice; and iv) liver homogenates of sEH(-/-) mice show elevated basal levels of hepoxilins but lowered levels of trioxilins compared with wild-type animals. We conclude that sEH is identical to previously reported hepoxilin hydrolase. This is of particular physiological relevance because sEH is emerging as a novel drug target due to its major role in the hydrolysis of important lipid signaling molecules such as epoxyeicosatrienoic acids. sEH inhibitors might have undesired side effects on hepoxilin signaling.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Blotting, Western; Chromatography, Liquid; Electrophoresis, Polyacrylamide Gel; Epoxide Hydrolases; Humans; Liver; Mice; Mice, Inbred C57BL; Models, Biological; Rats; Tandem Mass Spectrometry

Pancreatic β-cells have a deficit of scavenging enzymes such as catalase (Cat) and glutathione peroxidase (GPx) and therefore are susceptible to oxidative stress and apoptosis. Our previous work showed that, in the absence of cytosolic GPx in insulinoma RINm5F cells, an intrinsic activity of 12 lipoxygenase (12(S)-LOX) converts 12S-hydroperoxyeicosatetraenoic acid (12(S)-HpETE) to the bioactive epoxide hepoxilin A(3) (HXA(3)). The aim of the present study was to investigate the effect of HXA(3) on apoptosis as compared to its precursor 12(S)-HpETE and shed light upon the underlying pathways. In contrast to 12(S)-HpETE, which induced apoptosis via the extrinsic pathway, we found HXA(3) not only to prevent it but also to promote cell proliferation. In particular, HXA(3) suppressed the pro-apoptotic BAX and upregulated the anti-apoptotic Bcl-2. Moreover, HXA(3) induced the anti-apoptotic 12(S)-LOX by recruiting heat shock protein 90 (HSP90), another anti-apoptotic protein. Finally, a co-chaperone protein of HSP90, protein phosphatase 5 (PP5), was upregulated by HXA(3), which counteracted oxidative stress-induced apoptosis by dephosphorylating and thus inactivating apoptosis signal-regulating kinase 1 (ASK1). Taken together, these findings suggest that HXA(3) protects insulinoma cells from oxidative stress and, via multiple signaling pathways, prevents them from undergoing apoptosis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Apoptosis; Arachidonate 12-Lipoxygenase; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Survival; DNA Fragmentation; Dose-Response Relationship, Drug; Flow Cytometry; Gene Expression Regulation, Neoplastic; Immunoblotting; Insulin-Secreting Cells; MAP Kinase Kinase Kinase 5; Nuclear Proteins; Phosphoprotein Phosphatases; Proto-Oncogene Proteins c-bcl-2; Rats; Reverse Transcriptase Polymerase Chain Reaction

Neutrophil transmigration across mucosal surfaces contributes to dysfunction of epithelial barrier properties, a characteristic underlying many mucosal inflammatory diseases. Thus, insight into the directional movement of neutrophils across epithelial barriers will provide important information relating to the mechanisms of such inflammatory disorders. The eicosanoid hepoxilin A(3), an endogenous product of 12-lipoxygenase activity, is secreted from the apical surface of the epithelial barrier and establishes a chemotactic gradient to guide neutrophils from the submucosa across epithelia to the luminal site of an inflammatory stimulus, the final step in neutrophil recruitment. Currently, little is known regarding how hepoxilin A(3) is secreted from the intestinal epithelium during an inflammatory insult. In this study, we reveal that hepoxilin A(3) is a substrate for the apical efflux ATP-binding protein transporter multidrug resistance-associated protein 2 (MRP2). Moreover, using multiple in vitro and in vivo models, we show that induction of intestinal inflammation profoundly up-regulates apical expression of MRP2, and that interfering with hepoxilin A(3) synthesis and/or inhibition of MRP2 function results in a marked reduction in inflammation and severity of disease. Lastly, examination of inflamed intestinal epithelia in human biopsies revealed up-regulation of MRP2. Thus, blocking hepoxilin A(3) synthesis and/or inhibiting MRP2 may lead to the development of new therapeutic strategies for the treatment of epithelial-associated inflammatory conditions.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonate 12-Lipoxygenase; Gene Expression Regulation; Humans; Inflammation; Intestinal Diseases; Intestinal Mucosa; Mice; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Neutrophil Infiltration; Neutrophils

A common feature underlying active states of inflammation is the migration of neutrophils (PMNs) from the circulation and across a number of tissue barriers in response to chemoattractant stimuli. Although our group has recently established a discreet role for the PMN chemoattractant, hepoxilin A3 (HXA3) in the process of PMN recruitment, very little is known regarding the interaction of HXA3 with PMNs. To characterize further the event of HXA3-induced PMN transepithelial migration, we sought to determine the adhesion molecules required for migration across different epithelial surfaces (T84 intestinal and A549 airway cells) relative to two well-studied PMN chemoattractants, formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 (LTB4). Our findings reveal that the adhesion interaction profile of PMN transepithelial migration in response to HXA3 differs from the adhesion interaction profile exhibited by the structurally related eicosanoid LTB4. Furthermore, unique to PMN transepithelial migration induced by gradients of HXA3 was the critical dependency of all four major surface adhesion molecules examined (i.e. CD18, CD47, CD44 and CD55). Our results suggest that the particular chemoattractant gradient imposed, as well as the type of epithelial cell monolayer, each plays a role in determining the adhesion molecules involved in transepithelial migration. Given the complexities of these interactions, our findings are important to consider with respect to adhesion molecules that may be targeted for potential drug development.

Topics: 8,11,14-Eicosatrienoic Acid; Cell Adhesion Molecules; Chemotactic Factors; Chemotaxis, Leukocyte; Dose-Response Relationship, Drug; Epithelial Cells; Humans; Leukotriene B4; Neutrophils; Tumor Cells, Cultured

Salmonella spp. and Shigella spp. are responsible for millions of cases of enteric disease each year worldwide. While these pathogens have evolved distinct strategies for interacting with the human intestinal epithelium, they both induce significant proinflammatory responses that result in massive transepithelial migration of neutrophils across the intestinal mucosa. It has previously been shown with Salmonella enterica serotype Typhimurium that the process of neutrophil transmigration is mediated in part by the secretion of hepoxilin A(3) (HXA(3); 8-hydroxy-11,12-epoxy-eicosatetraenoic acid), a potent neutrophil chemoattractant, from the apical surface of infected model intestinal epithelium. This study confirms that HXA(3) is also secreted in response to infection by Shigella flexneri, that it is produced by a pathway involving 12/15-lipoxygenase (12/15-LOX), and that S. enterica serovar Typhimurium and S. flexneri share certain elements in the mechanism(s) that underlies the otherwise separate signal transduction pathways that are engaged to induce polymorphonuclear leukocyte (PMN) transepithelial migration (protein kinase C and extracellular signal-regulated kinases 1 and 2, respectively). PMN transepithelial migration in response to infection with S. flexneri was dependent on 12/15-LOX activity, the enzyme responsible for the initial metabolism of arachidonic acid to HXA(3). Probing further into this pathway, we also found that S. enterica serovar Typhimurium and S. flexneri activate different subtypes of phospholipase A(2), a critical enzyme involved in the liberation of arachidonic acid from cellular membranes. Thus, although S. enterica serovar Typhimurium and S. flexneri utilize different mechanisms for triggering the induction of PMN transepithelial migration, we found that their reliance on 12/15-LOX is conserved, suggesting that enteric pathogens may ultimately stimulate similar pathways for the synthesis and release of HXA(3).

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Cell Line; Cell Migration Assays, Leukocyte; Cell Movement; Humans; Intestinal Mucosa; Isoenzymes; Neutrophils; Phospholipases A2; Salmonella typhimurium; Shigella flexneri

Arachidonic acid can be transformed into a specific epoxyalcohol product via the sequential action of two epidermal lipoxygenases, 12R-LOX and eLOX3. Functional impairment of either lipoxygenase gene (ALOX12B or ALOXE3) results in ichthyosis, suggesting a role for the common epoxyalcohol product or its metabolites in the differentiation of normal human skin. Here we tested the ability of products derived from the epidermal LOX pathway to activate the peroxisome proliferator-activated receptors PPARalpha, gamma, and delta, which have been implicated in epidermal differentiation. Using a dual luciferase reporter assay in PC3 cells, the 12R-LOX/eLOX3-derived epoxyalcohol, 8R-hydroxy-11R,12R-epoxyeicosa-5Z,9E,14Z-trienoic acid, activated PPARalpha with similar in potency to the known natural ligand, 8S-hydroxyeicosatetraenoic acid (8S-HETE) (both at 10 microM concentration). In contrast, the PPARgamma and PPARdelta receptor isoforms were not activated by the epoxyalcohol. Activation of PPARalpha was also observed using the trihydroxy hydrolysis products (trioxilins) of the unstable epoxyalcohol. Of the four trioxilins isolated and characterized, the highest activation was observed with the isomer that is also formed by enzymatic hydrolysis of the epoxyalcohol. Formation of a ligand for the nuclear receptor PPARalpha may be one possibility by which 12R-LOX and eLOX3 contribute to epidermal differentiation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Arachidonate Lipoxygenases; Cell Differentiation; Cell Line; Epidermal Cells; Epidermis; Genes, Reporter; Humans; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Luciferases; PPAR alpha; PPAR delta; PPAR gamma; Receptors, Cytoplasmic and Nuclear; Signal Transduction

Inflammation resulting from bacterial infection of the respiratory mucosal surface during pneumonia and cystic fibrosis contributes to pathology. A major consequence of the inflammatory response is recruitment of polymorphonuclear cells (PMNs) to the infected site. To reach the airway, PMNs must travel through several cellular and extracellular barriers, via the actions of multiple cytokines, chemokines, and adhesion molecules. Using a model of polarized lung epithelial cells (A549 or Calu-3) grown on Transwell filters and human PMNs, we have shown that Pseudomonas aeruginosa induces PMN migration across lung epithelial barriers. The process is mediated by epithelial production of the eicosanoid hepoxilin A(3) (HXA(3)) in response to P. aeruginosa infection. HXA(3) is a PMN chemoattractant metabolized from arachidonic acid (AA). Given that release of AA is believed to be the rate-limiting step in generating eicosanoids, we investigated whether P. aeruginosa infection of lung epithelial cells resulted in an increase in free AA. P. aeruginosa infection of A549 or Calu-3 monolayers resulted in a significant increase in [(3)H]AA released from prelabeled lung epithelial cells. This was partially inhibited by PLA(2) inhibitors ONO-RS-082 and ACA as well as an inhibitor of diacylglycerol lipase. Both PLA(2) inhibitors dramatically reduced P. aeruginosa-induced PMN transmigration, whereas the diacylglycerol lipase inhibitor had no effect. In addition, we observed that P. aeruginosa infection caused an increase in the phosphorylation of cytosolic PLA(2) (cPLA(2)), suggesting a mechanism whereby P. aeruginosa activates cPLA(2) generating free AA that may be converted to HXA(3), which is required for mediating PMN transmigration.

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonic Acid; Cell Line; Cell Polarity; Chemotactic Factors; Cytosol; Enzyme Inhibitors; Epithelial Cells; Group IV Phospholipases A2; Humans; Lung; Male; Middle Aged; Neutrophil Infiltration; Phospholipases A; Phospholipases A2; Phosphorylation; Pseudomonas Infections

We have chemically synthesized several stable analogs of the naturally occurring hepoxilins, 12-LO products derived from arachidonic acid, which we found to have promising actions in a variety of test systems of disease. The analogs, PBTs, afford chemical and biological stability to the hepoxilin molecule. This article reviews some of our latest observations with the PBTs in the areas of inflammation (inhibition of the bleomycin-evoked lung fibrosis in mice in vivo), platelet aggregation (antagonism of the thromboxane receptor in human platelets in vitro) and thrombosis (inhibitors in vivo), and cancer (apoptosis of the human leukemia cell line, K562 in vitro and in vivo). The demonstration that the PBTs are active in vivo suggests that they can serve as a platform for their further development as novel therapeutics in disease.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Apoptosis; Bleomycin; Blood Platelets; Disease Models, Animal; Fibrinolytic Agents; Humans; K562 Cells; Leukemia, Experimental; Lung; Mice; Platelet Aggregation; Pulmonary Fibrosis

The mechanism by which neutrophils [polymorphonuclear leukocyte (PMNs)] are stimulated to move across epithelial barriers at mucosal surfaces has been basically unknown in biology. IL-8 has been shown to stimulate PMNs to leave the bloodstream at a local site of mucosal inflammation, but the chemical gradient used by PMNs to move between adjacent epithelial cells and traverse the tight junction at the apical neck of these mucosal barriers has eluded identification. Our studies not only identify this factor, previously termed pathogen-elicited epithelial chemoattractant, as the eicosanoid hepoxilin A(3) (hepA(3)) but also demonstrate that it is a key factor promoting the final step in PMN recruitment to sites of mucosal inflammation. We show that hepA(3) is synthesized by epithelial cells and secreted from their apical surface in response to conditions that stimulate inflammatory events. Our data further establish that hepA(3) acts to draw PMNs, via the establishment of a gradient across the epithelial tight junction complex. The functional significance of hepA(3) to target PMNs to the lumen of the gut at sites of inflammation was demonstrated by the finding that disruption of the 12-lipoxygenase pathway (required for hepA(3) production) could dramatically reduce PMN-mediated tissue trauma, demonstrating that hepA(3) is a key regulator of mucosal inflammation.

Topics: 8,11,14-Eicosatrienoic Acid; Humans; Inflammation; Intestinal Mucosa; Neutrophils; Salmonella typhimurium

Lung inflammation resulting from bacterial infection of the respiratory mucosal surface in diseases such as cystic fibrosis and pneumonia contributes significantly to the pathology. A major consequence of the inflammatory response is the recruitment and accumulation of polymorphonuclear cells (PMNs) at the infection site. It is currently unclear what bacterial factors trigger this response and exactly how PMNs are directed across the epithelial barrier to the airway lumen. An in vitro model consisting of human PMNs and alveolar epithelial cells (A549) grown on inverted Transwell filters was used to determine whether bacteria are capable of inducing PMN migration across these epithelial barriers. A variety of lung pathogenic bacteria, including Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa are indeed capable of inducing PMN migration across A549 monolayers. This phenomenon is not mediated by LPS, but requires live bacteria infecting the apical surface. Bacterial interaction with the apical surface of A549 monolayers results in activation of epithelial responses, including the phosphorylation of ERK1/2 and secretion of the PMN chemokine IL-8. However, secretion of IL-8 in response to bacterial infection is neither necessary nor sufficient to mediate PMN transepithelial migration. Instead, PMN transepithelial migration is mediated by the eicosanoid hepoxilin A3, which is a PMN chemoattractant secreted by A549 cells in response to bacterial infection in a protein kinase C-dependent manner. These data suggest that bacterial-induced hepoxilin A3 secretion may represent a previously unrecognized inflammatory mechanism occurring within the lung epithelium during bacterial infections.

Topics: 8,11,14-Eicosatrienoic Acid; Cell Line, Tumor; Cell Migration Inhibition; Cell Movement; Cells, Cultured; Enzyme Inhibitors; Humans; Interleukin-8; Male; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neutrophils; Phosphorylation; Protein Kinase C; Pseudomonas aeruginosa; Pulmonary Alveoli; Respiratory Mucosa

The barnacle life cycle has two key stages at which eicosanoids are believed to be involved in cellular communication pathways, namely the hatching of nauplii and the settlement of cypris larvae. Barnacle egg-hatching activity has previously been reported to reside in a variety of eicosanoids, including 8-hydroxyeicosapentaenoic acid and a number of tri-hydroxylated polyunsaturated fatty acid derivatives, the trioxilins. The production of the eicosapentaenoic acid metabolite trioxilin A4 (8,11,12-trihydroxy-5,9,14,17-eicosatetraenoic acid) by the barnacles Balanus amphitrite and Elminius modestus was confirmed using a combination of high-performance liquid chromatography and gas chromatography, both linked to mass spectrometry. In addition, both species also generated trioxilin A3 (8,11,12-trihydroxy-5,9,14-eicosatrienoic acid; an arachidonic acid-derived product), 8,11,12-trihydroxy-9,14,17-eicosatrienoic acid (a omega3 analogue of trioxilin A3; derived from omega3 arachidonic acid) and 10,13,14-trihydroxy-4,7,11,16,19-docosapentaenoic acid (a docosahexaenoic acid-derived product). In contrast to earlier reports, trioxilin A3 had no E. modestus egg-hatching activity at any of the concentrations tested (10(-9)-10(-6) mol l(-1)). The unstable epoxide precursor hepoxilin A3, however, caused significant levels of hatching at 10(-6) mol l(-1). Furthermore, the stable hepoxilin B3 analogue PBT-3 stimulated hatching at 10(-7) mol l(-1). Neither trioxilin A3, hepoxilin A3 or PBT-3 at 0.25-30 micromol l(-1) served as settlement cues for B. amphitrite cypris larvae.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Larva; North Carolina; Ovum; Reproduction; Signal Transduction; Thoracica; United Kingdom

In this study we set out to investigate whether the inflammatory agents, bradykinin (BK) and platelet activating factor (PAF), affect the lipoxygenase pathway in rat skin in vivo and whether the main products so formed may be involved in the inflammatory actions of these agents. In vitro preparations of epidermis were also investigated to determine whether lipoxygenases are stimulated by these agents. We also investigated the actions of arachidonic acid and 12(S)-HPETE as substrates for the lipoxygenases. Our results indicated that 12-lipoxygenase is actively and selectively stimulated in a dose-dependent way in both preparations by the administration of BK and PAF; the main product, 12-HETE, was shown by chiral analysis to be exclusively of the S-configuration, indicating that 12(S)-lipoxygenase was present in the rat skin and was stimulated by these inflammatory agents. Hepoxilins were also formed but to a lesser extent in both in vivo and in vitro preparations. In separate experiments, 12(S)-HETE administered intradermally on its own (40 ng/site), increased vascular permeability as also seen with bradykinin (100 ng/site) and PAF (10 ng/site). However, unlike previously observed with hepoxilin A3 administration, 12(S)-HETE did not stimulate the action of BK on vascular permeability, suggesting that the two compounds may have different mechanisms of action to enhance inflammation. These observations suggest that the vascular permeability and plasma extravasation observed with both inflammatory agents (BK and PAF) may be mediated at least in part through the activation of 12(S)-lipoxygenase, resulting in enhanced formation of 12(S)-HETE which causes acute inflammation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Arachidonate 12-Lipoxygenase; Bradykinin; Capillary Permeability; Enzyme Activation; Inflammation; Inflammation Mediators; Male; Platelet Activating Factor; Rats; Rats, Wistar; Skin

We have previously shown that the methyl ester of hepoxilin A3 causes a receptor-induced rise in intracellular calcium through the release from intracellular stores in suspended human neutrophils. The corresponding free acid was devoid of activity. We now report that the action of the free acid form of hepoxilin A3 is dependent on the type of vehicle used, i.e. it is active in releasing calcium when used in an ethanol vehicle but not in DMSO. The methyl ester is equally active in either vehicle. The pattern of calcium release between the free acid and the methyl ester is qualitatively different. Both compounds show a biphasic pattern, i.e. an initial rapid phase followed by a slow decline in calcium levels but never reaching pre-hepoxilin A3 baseline levels. The methyl ester appears slightly more potent in the initial phase of calcium release than the free acid (methyl = 188+/-14 S.D., free acid = 135+/-11 S.D. nM, P < 0.0005). Both compounds appear to reach the same calcium levels at the plateau of the second prolonged phase (methyl = 88+/-8 S.D., free acid = 107+/-15 S.D. nM, not significant). Lanthanum chloride (an inhibitor of calcium influx) interfered with the second phase of the curve causing calcium levels to return to normal pre-hepoxilin levels for both compounds. Addition of lanthanum chloride prior to the hepoxilin addition or carrying out the experiments in calcium-free medium, eliminated the second phase completely, with the calcium peak returning rapidly to normal baseline levels, suggesting that the second phase is due to calcium influx. Again the methyl ester is more active than the free acid (methyl, 189+/-12; free acid, 145+/-6 S.D. nM, P<0.005). Additional experiments with tritium-labelled methyl ester of hepoxilin A3 demonstrated that the compound is hydrolyzed into the free acid intracellularly. These experiments demonstrate that DMSO interacts with hepoxilin free acid, interfering with its entry into the cell while ethanol does not. Once inside the cell, hepoxilin interacts with its own receptor to release calcium rapidly from stores, but it also causes a more prolonged influx of calcium from the extracellular milieu.

Topics: 8,11,14-Eicosatrienoic Acid; Calcium; Cells, Cultured; Esterification; Humans; Intracellular Fluid; Neutrophils; Signal Transduction

We recently found that normal human epidermis produces relatively high amounts of hepoxilins and trioxilins in vitro. Therefore, the aim of this study was to demonstrate the presence of these compounds in psoriatic lesions. Extracts from scales of patients with chronic stable plaque psoriasis were analyzed by a combination of high performance liquid chromatography and gas chromatography-mass spectrometry techniques. We found that the levels of hepoxilin B3 were more than 16-fold higher in psoriatic scales than in normal epidermis (3.2+/-2.3 and < 0.2 ng per mg, respectively), whereas hepoxilin A3 was not detected in any sample. Trioxilins were semiquantitated and referred to 12-hydroxyeicosatetraenoic acid, ratios of trioxilins A3 and B3 12-hydroxyeicosatetraenoic acid in psoriatic lesions were 0.65+/-0.23 and 0.32+/-0.28, respectively, and they were not detected in normal epidermis. The presence of a great amount of trioxilin A3 strongly suggests that hepoxilin A3 was present in psoriatic lesions and it was totally degraded to trioxilin A3 during the analysis procedure. Our results demonstrate that hepoxilins and trioxilins are produced by human skin in vivo and that the levels of these compounds are increased in psoriasis. The reported biologic activities of hepoxilins indicate that they could amplify and maintain the inflammatory response. Our results reinforce the idea that these compounds could play a role as mediators in the inflammatory response in skin, particularly in psoriasis.

Topics: 8,11,14-Eicosatrienoic Acid; Chromatography, High Pressure Liquid; Epidermis; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyeicosatetraenoic Acids; Psoriasis; Reference Values

Hepoxilin A3 has previously been shown to cause a rapid dose-dependent rise in intracellular calcium in intact human neutrophils in suspension. Two components have been observed, an initial rapid phase of intracellular calcium rise, followed by a slow decline to plateau levels that remain above the original baseline calcium levels. These changes have been suggested to involve the release of calcium from intracellular stores in the ER (initial rapid phase), while the slower rate of decline (plateau phase) was presumed to be due to calcium influx as it was abolished in zero calcium extracellular medium. The present study used confocal microscopy to examine the response to hepoxilin A3 at the subcellular level. Our results show that calcium dynamics in response to hepoxilin A3 varies in different subcellular compartments within the cell and that hepoxilin A3 evoked a persistent accumulation of calcium in organelles. The hepoxilin-evoked calcium sequestration was eliminated by prior exposure to CCCP, a mitochondrial uncoupler. CCCP also eliminated the plateau phase of the calcium response in cell suspension, suggesting that this phase was due to mitochondrial accumulation of calcium rather than calcium influx. Experiments with DiI-loaded cells, a membrane marker, showed that the nuclear calcium was not elevated by hepoxilin addition to the cells. These results demonstrate that hepoxilins evoke the release of calcium from the ER which is taken up by the mitochondria where it is tightly sequestered. These results offer an explanation of observations previously made with cell suspensions in which hepoxilin A3 was shown to inhibit the calcium mobilizing effects of chemotactic agents.

Topics: 8,11,14-Eicosatrienoic Acid; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cells, Cultured; Humans; Microscopy, Confocal; Mitochondria; Neutrophils; Uncoupling Agents

We describe here the effects of two 12-lipoxygenase products, 12-HETE (12-hydroxyeicosa (5Z,8Z,10E,14Z) tetraenoic acid) and 12-HPETE (12-hydroperoxyeicosa (5Z,8Z,10E,14Z) tetraenoic acid), on the release of intracellular calcium in intact human neutrophils using the INDO-1 AM fluorescent dye technique. Both products dose dependently stimulate intracellular release, with 12-HETE being more powerful than 12-HPETE. The threshold concentration for 12-HETE was 5 ng/ml (1.5 x 10-8 M), while that for 12-HPETE was 10 ng/ml. The (12S) regioisomer was slightly more active than the (12R) isomer. The laser potency of 12-HPETE may be due to its conversion into the less active hepoxilins as incubation of neutrophils with (12S/R)-HPETE in a nonradioactive assay, using fluorescent ADAM esters of the products, generated mostly hepoxilin A3 (8-hydroxy-(11S,12S) epoxyeicosa (5Z,9E,14Z)trienoic acid), indicative of an enzymatic process. In contrast, boiled neutrophil preparations converted 12-HPETE primarily into hepoxilin B3 which previously showed to be derived nonenzymatically. This data demonstrates that 12-HETE, known to be generated in significant amounts by platelets, can act transcellularly to modify intracellular concentrations of calcium in neutrophils. This may in turn affect the responsiveness of these cells to other chemotactic factors.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Arachidonate 12-Lipoxygenase; Arachidonic Acid; Blood Platelets; Calcium; Chelating Agents; Fluorescent Dyes; Humans; Indoles; Leukotrienes; Neutrophils

Hepoxilins (Hx) are biologically active metabolites of arachidonic acid (AA) formed regioselectively from 12(S)-HPETE by 'hepoxilin synthase'. Hx modulate synaptic neurotransmission in hippocampal CA1 neurons, and inhibit norepinephrine release in hippocampal slices. During the course of our studies we investigated whether docosahexaenoic acid (DHA) was a substrate for hepoxilin formation. We used two tissues, the pineal gland and hippocampal slices. Tissues were incubated alone or with AA (20 microg/ml) or DHA (20 microg/ml). After 60 min at 37 degrees C, samples were acid-extracted to convert Hx into their stable trioxilin (TrX) form and analyzed as the Me-TMSi derivatives by EI-GC/MS to determine the structures of the DHA metabolites, and as PFB-TMSi derivatives by GC/MS in the NICI mode using SIM to simultaneously quantify TrX products of the 3-series (derived from AA) monitored at m/z 569, while those of the 5-series (derived from DHA) were monitored at m/z 593. Results show good conversion of both substrate fatty acids by the rat pineal gland and hippocampal slices, into the 3-series (21.3 +/- 5.8 and 12.5 +/- 2.2 ng/microg protein, respectively) and 5-series TrX (12.3 +/- 2.7 and 2.9 +/- 0.4 ng/microg protein, respectively). Surprisingly though, experiments with DHA, in both tissues, also showed formation of TrX derived from endogenous AA (3-series) (10.4 +/- 8.3 and 3.1 +/- 2.1 ng/microg protein, respectively). These experiments demonstrate previously unreported actions of DHA causing the accumulation of AA, which is converted into hepoxilins. In order to prove that AA is accumulated during DHA stimulation of the tissue, we carried out separate experiments with hippocampal slices in which the neutral lipids and phospholipids were labeled with [14C]AA. DHA caused a time-dependent appearance of free [14C]AA which was released mostly from the TG pool. Measurement of the AA/DHA ratio in the TG pool by GC/MS further indicated that DHA is incorporated into the TG at the expense of AA. These results demonstrate that DHA competes with AA for acylation into the metabolically active TG fraction, and both fatty acids are converted into hepoxilins of the corresponding series.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Chromatography, Thin Layer; Docosahexaenoic Acids; Gas Chromatography-Mass Spectrometry; Hippocampus; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Male; Molecular Structure; Pineal Gland; Rats; Rats, Wistar

Topics: 8,11,14-Eicosatrienoic Acid; Acute Disease; Chronic Disease; Eicosanoids; Endothelium, Vascular; Hemorheology; Humans; Myocardial Infarction; Myocardial Ischemia; Platelet Activation; Stress, Mechanical

Hepoxilin A3-methyl ester is taken up by intact human neutrophils where it is first hydrolyzed into the free acid which is subsequently converted into a single major metabolite. The structure of this metabolite was determined through mass spectral analysis of several derivatives, and through identity with an authentic compound prepared by chemical synthesis. The metabolite was identified as omega-hydroxy-hepoxilin A3 showing that the epoxide functionality of the parent hepoxilin is not opened during incubation with human neutrophils. All attempts to investigate hepoxilin metabolism in broken cells, despite the presence of protease inhibitors (Aproteinin, PMSF, DFP) and supplementation with NADPH were unsuccessful. Metabolism of hepoxilin A3 required the intact cell, while parallel experiments with LTB4 as substrate demonstrated that this eicosanoid was metabolized into its omega-hydroxy metabolite regardless of whether intact or broken cell preparations were used provided that NADPH was present in the latter. Hepoxilin metabolism in intact cells was inhibited dose-dependently by CCCP (0.01-100 microM), a mitochondrial uncoupler, whereas LTB4 metabolism was unaffected by CCCP. This data suggests that metabolism of hepoxilin A3 occurs in intact human neutrophils through omega-oxidation, is likely located in the mitochondrial compartment of the cell (inhibition by CCCP) and is carried out by an activity that is independent of the well characterized, relatively stable microsomal LTB4 omega-hydroxylase.

Topics: 8,11,14-Eicosatrienoic Acid; Adenosine Triphosphate; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Chromatography, Thin Layer; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 4; Gas Chromatography-Mass Spectrometry; Humans; Hydrolysis; Leukotriene B4; Mixed Function Oxygenases; NAD; NADP; Neutrophils; Oxidation-Reduction

Topics: 8,11,14-Eicosatrienoic Acid; Calcium; Humans; In Vitro Techniques; Kinetics; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Platelet Activating Factor

Hepoxilins have been shown to release calcium from intracellular stores in human neutrophils [Dho, Grinstein, Corey, Su and Pace-Asciak (1990) Biochem. J. 266, 63-68; Laneuville, Reynaud, Grinstein, Nigam and Pace-Asciak (1993) Biochem. J. 295, 393-397]. In this paper we report that tritium-labelled hepoxilin A3 (8S) binds to broken neutrophil membranes in a time-, substrate- and temperature-dependent fashion. Specific binding was displaced with unlabelled hepoxilin A3. Specific binding was greatest at 37 degrees C. Competitive binding was best observed with unlabelled hepoxilin A3 (8S); the glutathione conjugate, HxA3-C (8S or 8R), or 12(S)-hydroxyeicosatetraenoic acid was less active. Similarly inactive in displacing the bound radiolabelled hepoxilin A3 was leukotriene B4 as well as a variety of prostaglandins and thromboxane B2. Formylmethionyl-leucylphenylalanine was similarly inactive in competing for the hepoxilin binding sites. Specific binding was inhibited by pretreatment of the broken membranes during 30 min at 37 degrees C with proteinase K, while specific binding of the intact cells was unaffected. Scatchard analysis of binding data revealed a single population of binding sites with apparent KD and Bmax. of 79.3 +/- 9.1 nM and 8.86 +/- 1.4 pmol/ml per 2 x 10(6) cells (+/- S.E.M.) respectively reflecting approx. 2.67 x 10(6) sites/cell. These results demonstrate for the first time that neutrophils contain specific binding sites to hepoxilin A3.

Topics: 8,11,14-Eicosatrienoic Acid; Binding Sites; Endopeptidase K; Humans; Hydrolysis; Kinetics; Neutrophils; Serine Endopeptidases; Tritium

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Blood Proteins; Bradykinin; Capillary Permeability; Drug Synergism; Injections, Intradermal; Male; Platelet Activating Factor; Rats; Rats, Wistar; Regional Blood Flow; Skin

Topics: 8,11,14-Eicosatrienoic Acid; Binding Sites; Calcium; GTP-Binding Proteins; Humans; Intracellular Fluid; Neutrophils

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Chromatography, High Pressure Liquid; Leukotrienes; Pineal Gland; Rats

Facile methods for the detection of intact hepoxilins, monohydroxy-epoxide derivatives of arachidonic acid formed through the 12-lipoxygenase pathway, are unavailable because (i) an absence in these compounds of an appropriate chromophore for sensitive detection by uv exists, (ii) these compounds are sensitive to the acidic workup leading to varying degrees of decomposition, and (iii) they decompose to the derivatization procedures required for their analysis by gas chromatography mass spectrometry. Herein we apply a method which introduces a fluorescent ester chromophore to the carboxylic group of the hepoxilins under conditions which do not require acidification leading to stabilization of the derivative which is extracted into an organic solvent in situ. This procedure quantitatively derivatizes hepoxilins in a biological sample, permitting the detection of hepoxilins after a TLC purification with a limit of 50 pg/sample. This method permits the profiling of 12-HETE, hepoxilins A3 and B3, as well as the corresponding epoxide hydrolase products, trioxilins A3 and B3, in a biological sample by reverse-phase HPLC with fluorescent detection. We also report on the fluorescent and mass spectral properties of these derivatives using a liquid chromatography mass spectrometry LCMS interface with thermospray ionization.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Anthracenes; Arachidonic Acid; Chromatography, High Pressure Liquid; Eicosanoids; Esters; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Pineal Gland; Rats; Spectrometry, Fluorescence

Hepoxilins have previously been shown to release intracellular calcium in human neutrophils. We show herein that tritium-labeled hepoxilin A3 of high specific radioactivity binds to human neutrophils, and this binding is reversed by the addition of unlabeled compound, demonstrating that specific binding for these compounds exists in these cells. Specific binding of both the methyl ester derivative as well as the free acid form of the hepoxilin takes place in broken membrane fragments. In contrast only the methyl ester derivative binds specifically to the intact cells. We also show that intact neutrophils form hepoxilin A3 when incubated in the presence of the hepoxilin precursor, 12(S)-HPETE. These data demonstrate that hepoxilin synthesis can occur in the neutrophil and that hepoxilin binding sites, which appear to be located intracellularly, exist in these cells.

Topics: 8,11,14-Eicosatrienoic Acid; Analysis of Variance; Binding Sites; Cell Membrane; Humans; In Vitro Techniques; Kinetics; Leukotrienes; Neutrophils; Reference Values; Tritium

Fluorescent anthryl (ADAM) derivatives of hepoxilins have been shown to possess good chromatographic properties affording good sensitivity for the high-performance liquid chromatographic analysis and detection of these compounds and related eicosanoids (12-hydroxyeicosatetraenoic acid) in biological samples. We report herein the separation of all possible stereoisomers of hepoxilins A3 and B3 as their methyl esters as well as their ADAM ester and acetate derivatives on a cellulose trisdimethyphenylcarbamate chiral stationary phase (Chiracel OD) in the normal-phase mode. This methodology is important to address the mechanistic route of biosynthesis of these products.

Topics: 8,11,14-Eicosatrienoic Acid; Acetates; Anthracenes; Chromatography, High Pressure Liquid; Esters; Fluorescent Dyes; Stereoisomerism

Metabolites of arachidonic acid are known to be formed in the mammalian central nervous system. When intact hippocampal slices were incubated in artificial cerebrospinal fluid, 12-hydroxyeicosatetraenoic acid and two isomers of hepoxilin A3 (8R and 8S) were released as measured by gas chromatography-mass spectrometry. These compounds were released in greater amounts in the presence of noradrenaline or when arachidonic acid was added to the slices. The neuronal actions of chemically derived preparations of 8R and 8S hepoxilins and the glutathione conjugate, hepoxilin A3-C, were examined using intracellular and whole-cell electrophysiological recordings in hippocampal CA1 neurons in vitro. All compounds had the excitatory effects of lowering spike threshold and decreasing spike frequency adaptation, and the inhibitory actions of membrane hyperpolarization, enhanced postspike train afterhyperpolarizations and increased inhibitory postsynaptic potentials or currents. A synthetic analog of hepoxilin A3-C, in which the glutathione moiety is placed at carbon position 9 instead of carbon position 11 as in hepoxilin A3-C, was inactive. The actions of the hepoxilins showed a sharp dose-response relationship, with minimal threshold or no effect at 3 nM (n = 21) and maximal effects at 10 nM (n = 33). There were no significant differences between the responses to either the 8R or 8S isomers, or between hepoxilin A3 and hepoxilin A3-C. These data suggest that hepoxilins formed by the brain have significant neuromodulatory actions.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; 8,11,14-Eicosatrienoic Acid; Animals; Electrophysiology; Gas Chromatography-Mass Spectrometry; Hippocampus; Kainic Acid; Male; Quinoxalines; Rats; Rats, Wistar; Receptors, GABA-B

The effect of short duration of shear-stress (350 dyne/cm2, 20 ms) on platelet-aggregation has been assessed. This treatment inhibits thrombin-induced but not ADP- or collagen-induced aggregation. The inhibitory effect is mediated by endogenous hepoxilin A3. This conclusion is based on the following observations: (a) The shear-stress effect is abolished by lipoxygenase inhibitors. (b) Hepoxilin A3 mimics the shear-stress effect.

Topics: 8,11,14-Eicosatrienoic Acid; Blood Platelets; Dose-Response Relationship, Drug; Humans; Lipoxygenase Inhibitors; Platelet Aggregation; Platelet Aggregation Inhibitors; Thrombin; Time Factors

The present study was undertaken to investigate the possible formation of hepoxilin A3 in the rat pineal gland and to study the potential physiological role for this compound in this tissue. Incubation of homogenates of rat pineal glands with arachidonic acid (66 microM) led to the appearance of hepoxilin A3 (HxA3) analyzed as its stable trihydroxy derivative, trioxilin A3 by gas chromatography in both the electron impact and negative ion chemical ionization modes. Endogenous formation of HxA3 is estimated to be 1.43 +/- 0.66 ng/micrograms of protein. This amount is not modified when the tissue is boiled (2.07 +/- 0.66 ng/micrograms of protein). However, the formation of this compound was stimulated to 21.26 +/- 5.82 ng/micrograms of protein when exogenous arachidonic acid was added to the homogenate. Addition of the dual cyclooxygenase/lipoxygenase inhibitor BW 755C (10 micrograms) resulted in a partial blockade of hepoxilin formation. Using [1-14C]HxA3, we demonstrated that the pineal gland contained hepoxilin epoxide hydrolase, which hydrolyzed HxA3 into trioxilin A3. This hydrolysis was inhibited by 1 mumol/L of 3,3,3-trichloropropene-1,2-oxide. In a separate study, HxA3 in the presence of 3,3,3-trichloropropene-1,2-oxide to block the hydrolysis of HxA3 decreased the production of cyclic AMP in cultured organ rat pineals after stimulation with 5'-N-ethylcarboxamidoadenosine, an A1/A2 adenosine receptor agonist. This effect is stereospecific because the (8S)-enantiomer is more active in decreasing cyclic AMP production (-88.7%) than the (8R)-enantiomer. This is the first demonstration of the presence, metabolism, and action of HxA3 in the rat pineal gland.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; 8,11,14-Eicosatrienoic Acid; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Arachidonic Acid; Cells, Cultured; Cyclic AMP; Gas Chromatography-Mass Spectrometry; Male; Pineal Gland; Rats; Rats, Sprague-Dawley; Rats, Wistar; Tritium

Platelets revert hypotonic-induced swelling by the process of regulatory volume decrease (RVD). We have recently shown that this process is under the control of endogenous hepoxilin A3. In this work, we investigated the mechanical-biochemical transduction that leads to hepoxilin A3 formation. We demonstrate that this process is mediated by pertussis-toxin-sensitive G protein, which activates Ca(2+)-insensitive phospholipase A2, and the sequential release of arachidonic acid. This conclusion is supported by the following observations: (i) RVD response is blocked selectively by the phospholipase A2 inhibitors manoalide and bromophenacyl-bromide (0.2 and 5 microM, respectively) but not by phospholipase C inhibitors. The addition of arachidonic acid overcame this inhibition; (ii) extracellular Ca2+ depletion by EGTA (up to 10 mM) does not affect RVD; (iii) intracellular Ca2+ depletion by BAPTA-AM (100 microM) inhibits RVD but not hepoxilin A3 formation, as tested by the RVD reconstitution assay; (iv) RVD is inhibited by the G-protein inhibitors, GDP beta S (1 microM) and pertussis toxin (1 ng/ml). This inhibition is overcome by addition of arachidonic acid or hypotonic cell-free eluate that contains hepoxilin A3; (v) NaF, 1 mM, induces hepoxilin A3 formation, tested by the RVD reconstitution assay; and (vii) GDP beta S inhibits hepoxilin A3 formation associated with flow. Therefore, it seems that G proteins are involved in the initial step of the mechanical-biochemical transduction leading to hepoxilin A3 formation in human platelets.

Topics: 8,11,14-Eicosatrienoic Acid; Adult; Blood Platelets; Calcium; Cell Size; GTP-Binding Proteins; Guanosine Diphosphate; Humans; Hypotonic Solutions; In Vitro Techniques; Mechanoreceptors; Models, Biological; Osmotic Pressure; Pertussis Toxin; Phospholipases A; Phospholipases A2; Protein Kinase Inhibitors; Sodium Fluoride; Stress, Mechanical; Thionucleotides; Virulence Factors, Bordetella

The effects of two acyclic derivatives of arachidonic acid which are formed under the action of 5- and 12-lipoxygenases 5(S)-hydroxy-(6,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE) and (8R/S)-hydroxy-(11S,12S)-epoxy-5Z,9E,14Z-eicosatrienoi c acid (hepoxilin A3) on extinction of inward current evoked by local acetylcholine (ACh-current) application on soma of Helix lucorum RPa3 and LPa3 neurons were studied by the double-electrode voltage clamp technique. It was shown an increase in ACh-current extinction by 5-HETE. Hepoxilin A3 did not influence cholinoreceptor plasticity. The present results confirm earlier assumptions concerning the regulation of cholinoreceptor plasticity by acyclic eicosanoids which were formed from arachidonic acid under the influence of 5-lipoxygenase and the lack of 12-lipoxygenase metabolites in this regulation.

Topics: 8,11,14-Eicosatrienoic Acid; Acetylcholine; Animals; Helix, Snails; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Membrane Potentials; Microelectrodes; Neuronal Plasticity; Neurons; Receptors, Cholinergic

We have previously shown that the hepoxilins are capable of increasing the intracellular free concentration of calcium ([Ca2+]i) in human neutrophils through a pertussis toxin-sensitive, extracellular calcium-independent pathway involving the mobilization of calcium from internal stores. A subsequent hepoxilin-induced and extracellular calcium-dependent influx of calcium is observed. In an effort to investigate further the role of these compounds in the human neutrophil, we investigated their potential effects on the action of known agonists such as formyl-methionine-leucine-phenylalanine (fMLP), platelet-activating factor (PAF) and leukotriene B4 (LTB4) on the mobilization of calcium. Hepoxilis dose-dependently inhibited the increases in [Ca2+]i induced by fMLP, PAF and LTB4. The hepoxilin concentration required for inhibition was around 100 ng/ml (3 x 10(-7) M). This concentration of hepoxilin did not cause any measurable change in [Ca2+]i. The extent of inhibition of the agonist-evoked rise in [Ca2+]i by hepoxilins was proportional to the increase in the calcium response evoked by hepoxilin beyond its threshold concentration. Additional experiments were carried out to investigate the mechanism for the hepoxilin effect. Using calcium-free medium and in the presence of sufficient amounts of thapsigargin (200 ng/ml) to maximally block the calcium pump (thereby achieving a constant rate of calcium leakage from stores), hepoxilin A3 increased further this rate of calcium leakage, indicating that hepoxilin acts by rapidly draining calcium from stores. Its potential (additional) thapsigargin-like action in blocking the pump, however, cannot be ruled out by these experiments. These observations suggest that the hepoxilins may serve an important negative regulatory function in the agonist-induced mobilization of calcium in these cells by depleting calcium stores.

Topics: 8,11,14-Eicosatrienoic Acid; Calcium; Calcium-Transporting ATPases; Humans; In Vitro Techniques; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Platelet Activating Factor; Stereoisomerism; Terpenes; Thapsigargin

Available data obtained so far has indicated that hepoxilin formation from 12-HPETE is catalyzed by hemin and hemoglobin and is not affected through heating of these ferri-heme compounds suggestive of nonenzymatic processes. The present paper demonstrates for the first time that 12-HPETE is transformed into the hepoxilins A3 and B3 by intact cells (skin subcutis layer) and slices of several tissues (brain hippocampus and pineal gland) and that this transformation is inhibited by tissue boiling, indicating an enzymatic catalysis. The tissues employed are pharmacologically responsive to hepoxilins and hence the present data offer biochemical support of a potential biological role for the hepoxilins in these tissues.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Hippocampus; In Vitro Techniques; Leukotrienes; Male; Pineal Gland; Rats; Rats, Wistar; Skin

When human blood platelets are exposed to hypotonic medium they swell first but, shortly thereafter, revert toward their original volume in a process termed regulatory volume decrease (RVD). RVD is the result of an enhanced efflux of K+ and Cl- ions and associated water. Platelet RVD is controlled by a short-lived lipoxygenase-derived product (LP). By using a combination of high-performance liquid chromatography, gas chromatography-mass spectrometry, and RVD reconstitution bioassay, we show that LP is identical with hepoxilin A3. In addition we demonstrate that authentic hepoxilin A3 possesses the same biological properties on RVD reconstitution as LP and that the activity of both compounds is amplified through epoxide hydrolase inhibition with 3,3,3-trichloropropene-1,2-oxide. Therefore, we report here that volume expansion causes the formation and release of hepoxilin A3 from intact human platelets and that this hepoxilin plays a major role in volume regulation.

Topics: 8,11,14-Eicosatrienoic Acid; Adult; Blood Platelets; Chromatography, High Pressure Liquid; Humans; Hypotonic Solutions; In Vitro Techniques; Kinetics; Lipoxygenase; Masoprocol; Time Factors

1. Hepoxilin A3 (8R and 8S isomers) (HxA3), hepoxilin A3-C (8R and 8S isomers) (HxA3-C) and trioxilin A3 (8S isomer) (TrXA3, the stable derivative of HxA3) were tested for their effects on helicoidal strips of guinea-pig isolated tracheae. 2. None of the compounds (10(-9)-10(-6) M) tested had a direct effect on resting tension of trachea. 3. HxA3 (8S) and HxA3-C (8R) (10(-8) M) produced a significant leftward shift of the log concentration-response curves to neurokinin A (NKA) (EC50 (nM), control = 29.0 +/- 2.8, HxA3 (8S) = 21.7 +/- 3.7, HxA3-C (8R) = 13.8 +/- 3.8, n = 6 for each). Also the maximal response to NKA was significantly increased when the tissues were exposed to these hepoxilins (% of the maximal response to NKA, control = 100, HxA3 (8S) = 114.5 +/- 5.3, HxA3-C (8R) = 139.0 +/- 6.2, n = 6 for each). The threshold concentrations for both hepoxilins was 10(-8) M and their effects were dose-related. 4. Stereochemical specificity was observed. The 8S-isomer of HxA3 was active in potentiating the NKA-induced contraction of the trachea while the 8R isomer was inactive. In contrast, the 8R isomer of HxA3-C was active while the 8S isomer was inactive. The trihydroxy metabolite of the active isomer of HxA3 (8S), i.e. TrXA3 (8S) (10(-6) M), was inactive in potentiating the NKA-induced contraction of the trachea. 5. It is concluded that hepoxilins sensitize the guinea-pig isolated trachea to the potent bronchoconstrictor, NKA.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Bronchoconstriction; Drug Synergism; Guinea Pigs; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Neurokinin A; Stereoisomerism; Trachea

1. The vascular activity of two stereoisomers of hepoxilin A3 (HxA3) (8R and 8S) and of its glutathione conjugate, hepoxilin A3-C (HxA3-C) (8R and 8S), was investigated on rat helicoidal strips of thoracic aorta and longitudinal strips of portal vein. 2. Neither of the hepoxilins tested had a direct effect on the tone of the aortic strip or on the spontaneous contractions of the portal vein. However, the noradrenaline (NA)-induced response of these vessels, as expressed by the dose required for half maximal contraction, (EC50) was greater in HxA3 (8S)- and HxA3-C (8R)-treated aorta. Increased frequency and strength of spontaneous contractions of the portal vein were detected at lower concentrations of NA in the presence of hepoxilins. 3. The threshold dose for both hepoxilins was 10(-8) M and their effect was not dose-related beyond 10(-8) M. The effect of hepoxilin appeared after a 45 min incubation period and could be observed even if the compounds were washed out after 15 min. 4. Stereochemical specificity was observed. The 8S isomer of HxA3 was active in potentiating the NA-induced contraction of these vessels while the 8R isomer was inactive. In contrast, the 8R isomer of HxA3-C was active while the 8S isomer was inactive. In both tissues, HxA3 (8S) was more potent than its glutathione conjugate, HxA3-C (8R). 5. In calcium-free buffer or in the presence of a calcium channel blocker (nifedipine 1 microM), no potentiation of NA-induced contraction by hepoxilins could be observed, suggesting the involvement of extracellular calcium in the actions of hepoxilins.6. These experiments suggest that hepoxilins may be involved in the modulation of vascular tone and contractility.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Aorta, Thoracic; Calcium; Drug Synergism; Glutathione; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth, Vascular; Nifedipine; Norepinephrine; Portal Vein; Rats; Rats, Inbred Strains; Stereoisomerism

In this paper we describe the release of hepoxilin A3 (HxA3) by intact pieces of the rat thoracic aorta and its stimulation by exogenous arachidonic acid but not by the calcium ionophore A23187. Homogenates of the rat aorta metabolize HxA3 via two competing pathways; one involves hepoxilin epoxide hydrolase to form the trihydroxy metabolite, trioxilin A3 (TrXA3), and a second pathway involves conjugation of HxA3 with glutathione via glutathione S-transferase to form a glutathione conjugate, which we refer to as hepoxilin A3-C (HxA3-C), a name based upon the accepted nomenclature for the glutathione conjugate leukotriene C. The formation of HxA3-C was dependent on the presence of reduced glutathione in the incubation medium. HxA3-C formation was greatly enhanced in the presence of TCPO, an epoxide hydrolase inhibitor which blocks utilization of the substrate via hepoxilin epoxide hydrolase. Comparison of HxA3-C formation by several arteries and veins indicated that glutathione conjugation was more evident in veins than arteries. The aorta from spontaneously hypertensive rats was essentially similar in HxA3-C formation to aorta from local normotensive Wistar rats although the aorta from the normotensive Wistar Kyoto rats was much more active than aorta from either of the two other rat types. The biological activity of HxA3 and HxA3-C was investigated on isolated helicoidal strips of the rat aorta. While both compounds were inactive on their own, HxA3 and to a lesser extent HxA3-C potentiated the contractile response induced by norepinephrine. The present results provide evidence of the presence in rat aorta of a new pathway of arachidonic acid metabolism whose products may possess potential regulatory properties on vascular tissue.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Aorta, Thoracic; Arachidonic Acid; Arachidonic Acids; Calcimycin; Epoxide Hydrolases; gamma-Glutamyltransferase; Glutathione; Male; Muscle Contraction; Muscle, Smooth, Vascular; Rats; Rats, Inbred Strains; Vasoconstriction

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Brain; Glutathione; Hippocampus; Membrane Potentials; Rats

In this paper we show that hepoxilin A3 induces the expression of heat shock protein expression in human neutrophils at a concentration of 100 nM using Western blotting techniques employing the use of a commercial monoclonal antibody to HSP72. No regiospecificity was observed as the 8S enantiomer of HxA3 was as active as the 8R enantiomer of HxA3. Comparison of the effects of HxA3 with 12S-HETE and PGA1 indicated that HxA3 was as effective as 12S-HETE although PGA1 was essentially inactive at the same concentration used for these 12-lipoxygenase products.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Antibodies, Monoclonal; Blotting, Western; Cytosol; Heat-Shock Proteins; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kinetics; Neutrophils; Prostaglandins A

Studies of the marine mollusk Aplysia californica indicate that products of the 12-lipoxygenase pathway may be involved in neuronal intracellular signaling. The nervous tissue of Aplysia has a 12-lipoxygenase activity that converts both exogenous and endogenous arachidonic acid to an array of products, which include 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and its metabolites hepoxilin A3, hepoxilin B3, 12-ketoeicosatetraenoic acid, and 12-oxododecatrienoic acid. These eicosanoids were identified using a combination of high-performance liquid chromatography, ultraviolet spectrometry and gas chromatography-mass spectrometry. Generation of 12-lipoxygenase products was stimulated by application of the neurotransmitters, histamine and FMRF-amide, or by stimulation of identified neural cells. In electrophysiological studies of identified L14 and sensory neurons it was found that 12-HPETE and its metabolic products exert physiological actions that resemble those of histamine and FMRF-amide. These results suggest that products of 12-HPETE metabolism may act as second messengers in Aplysia neurons.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Aplysia; Arachidonic Acid; Arachidonic Acids; Fatty Acids; Histamine; Hydroxyeicosatetraenoic Acids; Nervous System; Neurons

We have recently shown that hepoxilins are formed by and act on human neutrophils leading to an increase in intracellular levels of calcium and activation of the release of arachidonic acid and diacylglycerol [6, 9]. Since neutrophil activation and accumulation is involved in the inflammatory process resulting in vascular permeability in the rat skin, we investigated the effects of hepoxilins on this process. Hepoxilins administered s.c. resulted in a concentration- and time-dependent leakage of dye to the extravascular compartment of skin from rats to which Evans Blue had been administered. These results were compared to experiments in which prostaglandin E2 was used. The threshold dose of hepoxilin that elicited an effect reached a level of significance over control within 5 min of administration at the 10 ng dose (139% +/- 7%, n = 6). Similar findings were obtained with prostaglandin E2, the level of significance was reached also at 5 min at the 10 ng dose (177% +/- 7% of control, n = 6). The maximum effect observed for both hepoxilin and prostaglandin E2 was 60 min although this did not differ significantly from 30 min except for the highest dose of PGE2 (100 ng). However, the extent of the effect observed for prostaglandin E2 was greater than that for hepoxilin after longer periods, i.e. at 60 min, the prostaglandin E2 effect being 238% +/- 10% of control, and hepoxilin A3 being 167% +/- 10% of control. These results demonstrate that hepoxilins may participate in inflammatory processes.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Capillary Permeability; Dinoprostone; Dose-Response Relationship, Drug; Kinetics; Male; Rats; Rats, Inbred Strains; Skin

The effects of hepoxilin A3 (HxA3), a 12-lipoxygenase metabolite of arachidonic acid, on cytosolic calcium ([Ca2+]i), intracellular pH (pHi), transmembrane potential and right-angle light scattering in human neutrophils were investigated. A rapid, transient elevation of [Ca2+]i was observed with HxA3 which was dependent on the concentration used. The effect of HxA3 on [Ca2+]i was blocked by pertussis toxin, suggesting involvement of receptors coupled to GTP-binding proteins. Experiments in Ca2(+)-free medium and using intracellular Ca2+ chelators indicated that HxA3 mobilized Ca2+ from intracellular stores. At similar concentrations, HxA3 altered pHi, producing an initial acidification followed by an alkalinization. The initial acidification was decreased in cells loaded with a Ca2+ chelator. In the presence of N-ethyl-N-(1-methylethyl)amino amiloride, an inhibitor of the Na+/H+ antiport, HxA3 induced a greater acidification but failed to elicit the recovery phase, suggesting that the latter is due to activation of the antiport. HxA3 also depolarized the membrane potential, although this effect was small. A decrease in right-angle light scattering, qualitatively similar to that observed with chemotactic peptides, was seen with HxA3, indicating that the 12-lipoxygenase metabolite can induce shape changes in neutrophils. At the concentrations used for the above effects, HxA3 was unable to generate a respiratory burst. These findings suggest that hepoxilins, which are formed by stimulated neutrophils, may have a role as messengers in neutrophil activation.

Topics: 8,11,14-Eicosatrienoic Acid; Calcium; Cytosol; Egtazic Acid; Fatty Acids, Unsaturated; Humans; Hydrogen-Ion Concentration; Light; Membrane Potentials; Neutrophils; Oxygen Consumption; Pertussis Toxin; Scattering, Radiation; Virulence Factors, Bordetella

We have previously shown that hepoxilin A3 increases the intracellular concentration of Ca+2 in human neutrophils. Herein we address the initial events of hepoxilin action on the neutrophil which precede the rise in intracellular calcium. We show that hepoxilin A3 at 10-1000 nM concentrations releases from [1-14C]-arachidonic acid labeled neutrophils diacylglycerol and unesterified arachidonic acid in a time and concentration dependent fashion. The release of arachidonic acid and diacyglycerol are receptor-mediated events which are blocked by pertussis toxin. This data shows that hepoxilin A3 stimulates phospholipases C and A2 in the cell which may be involved in the rise in cytosolic calcium. Thus, hepoxilins may represent a hitherto unrecognised class of cellular mediators.

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonic Acids; Calcium; Diglycerides; Humans; In Vitro Techniques; Kinetics; Models, Biological; Neutrophils; Pertussis Toxin; Phosphatidic Acids; Receptors, Cell Surface; Virulence Factors, Bordetella

1-14C-Labeled hepoxillin A3 is transformed by a purified preparation of glutathione S-transferase in the presence of glutathione into a glutathionyl conjugate in which the glutathione is covalently coupled to the carbon 11 position of hepoxilin A3. We have termed the glutathione conjugate hepoxilin A3-C in keeping with the established nomenclature for glutathione conjugates in the leukotriene series. Using [3H]glutathione as cosubstrate, the kinetics of the reaction were followed. Among various rat liver glutathione S-transferase isozymes, a homodimer of the Yb2 subunit showed the best activity, while isozymes containing the Ya and Yc subunits showed marginal activity with hepoxilin A3 as substrate.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Glutathione; Glutathione Transferase; Isoenzymes; Kinetics; Liver; Macromolecular Substances; Rats; Substrate Specificity

Hepoxilin A3 was previously shown to display neuromodulatory actions on rat hippocampal CA1 neurons, with hyperpolarization of the membrane potential, an increase in the amplitude and duration of the post-spike train after hyperpolarization and an increase in the inhibitory post synaptic potential. The present report describes new biochemical evidence of a presynaptic action of hepoxilin A3 in rat hippocampal slices prelabeled with [3H]-norepinephrine. Hepoxilin A3 on its own had a marginal effect on the release of label, but blocked release which was induced by 4-aminopyridine (4-AP). Prostaglandin E2 also behaved in a similar way. These results demonstrate that hepoxilins modulate neurotransmission in the mammalian CNS through both pre- and postsynaptic actions.

Topics: 4-Aminopyridine; 8,11,14-Eicosatrienoic Acid; Animals; Dinoprostone; Hippocampus; Male; Norepinephrine; Rats; Rats, Inbred Strains; Synapses

Incubation of (8R)- and (8S)-[1-14C]hepoxilin A3 [where hepoxilin A3 is 8-hydroxy-11,12-epoxyeicosa-(5Z,9E,14Z)-trienoic acid] and glutathione with homogenates of rat brain hippocampus resulted in a product that was identified as the (8R) and (8S) diastereomers of 11-glutathionyl hepoxilin A3 by reversed-phase high performance liquid chromatographic comparison with the authentic standard made by total synthesis. Identity was further confirmed by cleavage of the isolated product with gamma-glutamyltranspeptidase to yield the corresponding cysteinylglycinyl conjugate that was identical by reversed-phase high performance liquid chromatographic analysis with the enzymic cleavage product derived from the synthetic glutathionyl conjugate. The glutathionyl and cysteinylglycinyl conjugate are referred to as hepoxilin A3-C and hepoxilin A3-D, respectively, by analogy with the established leukotriene nomenclature. Formation of hepoxilin A3-C was greatly enhanced with a concomitant decrease in formation of the epoxide hydrolase product, trioxilin A3, when the epoxide hydrolase inhibitor trichloropropene oxide was added to the incubation mixture demonstrating the presence of a dual metabolic pathway in this tissue involving hepoxilin epoxide hydrolase and glutathione S-transferase processes. Hepoxilin A3-C was tested using intracellular electrophysiological techniques on hippocampal CA1 neurons and found to be active at concentrations as low as 16 nM in causing membrane hyperpolarization, enhanced amplitude and duration of the post-spike train afterhyperpolarization, a marked increase in the inhibitory postsynaptic potential, and a decrease in the spike threshold. These findings suggest that these products in the hepoxilin pathway of arachidonic acid metabolism formed by the rat brain may function as neuromodulators.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Carbon Radioisotopes; Epoxide Hydrolases; Evoked Potentials; Fatty Acids, Unsaturated; Glutathione; Hippocampus; Indicators and Reagents; Male; Rats; Rats, Inbred Strains; Trichloroepoxypropane

In Aplysia neural tissue, the release and metabolism of arachidonic acid are stimulated by histamine or by activation of the identified L32 nerve cell circuit of the abdominal ganglion. Previously we found that histamine and intracellular stimulation of L32 cells, which are putatively histaminergic neurons, cause the production of 12-hydroxy-5,8,10,14-icosatetraenoic acid (12-HETE), a product of the 12-lipoxygenase pathway formed through 12-hydroperoxy-5,8,10,14-icosatetraenoic acid (12-HPETE). 12-HPETE, but not 12(S)-HETE, mimics the dual-action response of L14 ink motor neurons to histamine and stimulation of L32. 12-HPETE can also be further metabolized to 8-hydroxy-11,12-epoxy-5,9,14-icosatrienoic acid (8-HEpETE) which was identified by HPLC, enzymatic hydrolysis, and GC/MS. Production of 8-HEpETE is specific, as its positional isomer 10-hydroxy-11,12-epoxy-5,8,14-icosatrienoic acid is not formed after physiologic stimulation. 8-HEpETE can elicit the late component (hyperpolarization) of the dual-action response in L14 cells, suggesting that it may be a second messenger in Aplysia.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Aplysia; Electric Stimulation; Fatty Acids, Unsaturated; Ganglia; Gas Chromatography-Mass Spectrometry; Isomerism; Lipoxygenase; Neurons; Second Messenger Systems

The effects of arachidonic acid and its lipoxygenase metabolites, the hepoxilins, were investigated in rat hippocampal CA1 neurons in vitro by intracellular electrophysiological recordings. Both arachidonic acid and the hepoxilins cause a hyperpolarization which is sometimes followed by a later depolarization, augment the postspike train long-lasting afterhyperpolarization (AHP) and increase orthodromic inhibitory postsynaptic potentials (IPSPs). These data show that this arachidonic acid metabolic pathway has significant actions on mammalian central neurons, and may represent an important mechanism of neuromodulation.

Topics: 8,11,14-Eicosatrienoic Acid; Action Potentials; Animals; Arachidonic Acid; Arachidonic Acids; Fatty Acids, Unsaturated; Hippocampus; In Vitro Techniques; Male; Membrane Potentials; Rats; Rats, Inbred Strains

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Brain; Cerebral Cortex; Evoked Potentials; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kinetics; Male; Membrane Potentials; Rats; Rats, Inbred Strains

We describe herein the metabolism of hepoxilin A3 (HxA3) by glutathione S-transferase (GST) into a glutathione conjugate. The reaction was carried out with HxA3 (unlabelled and 14C-labelled) and glutathione (unlabelled and tritium labelled). When two isomers of HxA3 were reacted with GST, two products were formed. Only one product was formed when a single isomer of HxA3 was used. The isomeric product HxB3 was marginally active indicating considerable specificity in the reaction with GST. The products were characterized by retention of tritium from glutathione and by comparison of their migration on high performance liquid chromatography with authentic reference compounds. The products bear the structure, 11-glutathionyl HxA3.

Topics: 8,11,14-Eicosatrienoic Acid; Biotransformation; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Fatty Acids, Unsaturated; Glutathione Transferase; Isoenzymes; Radioisotope Dilution Technique

Hepoxilin epoxide hydrolase activity was demonstrated in rat liver cytosol using as substrate [1-14C] hepoxilin A3, a recently described hydroxy epoxide derivative of arachidonic acid. The enzyme was isolated and purified to apparent homogeneity using conventional chromatographic procedures resulting in 41-fold purification. The protein eluted during isoelectric focusing at a pI in the 5.3-5.4 range. The specific activity of the purified protein was 1.2 ng/microgram protein/20 min at 37 degrees C. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under denaturing conditions, a molecular mass value of 53 kDa was observed. Using native polyacrylamide gel electrophoresis, enzyme activity corresponded to the main protein band. The purified protein used hepoxilin A3 as preferred substrate converting it to trioxilin A3. The enzyme was marginally active toward other epoxides such as leukotriene A4 and styrene oxide. The Mr, pI, and substrate specificity of the hepoxilin epoxide hydrolase indicate that this enzyme is different from the recently reported leukotriene A4 hydrolase from human erythrocytes and rat and human neutrophils and constitutes a hitherto undescribed form of epoxide hydrolase with specificity toward hepoxilin A3. Tissue screening for enzyme activity revealed that this enzyme is ubiquitous in the rat.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Cytosol; Enzyme Stability; Epoxide Hydrolases; Fatty Acids, Unsaturated; Liver; Male; Rats; Rats, Inbred Strains; Substrate Specificity

The temporal in vivo expression of the eicosanoids (products of the cyclooxygenase pathway and one product of the 12-lipoxygenase pathway, hepoxilin A3) was investigated after bolus intravenous injection of arachidonic acid in the normal rat and in the genetic rat model of type I insulin-dependent diabetes, the diabetic BB rat. The temporal relationship between the expression of these products and plasma insulin concentrations was also investigated to determine whether any correlation existed between the rise in plasma insulin levels and any of the newly formed eicosanoids. Measurements of the eicosanoids present in whole blood were carried out using the deuterium isotope dilution technique involving separation of pentafluorobenzyl esters, O-methyl oximes, and trimethylsilyl ether derivatives by high-resolution gas chromatography and specific detection by negative ion chemical ionisation mass spectrometry in the selected ion mode. Injection of arachidonic acid resulted in large and statistically significant increases in the blood concentrations of all products within 1 min, with thromboxane B2 (the stable product of thromboxane A2) and trioxilin A3 (the stable product of hepoxilin A3) being the highest (4.5-12 ng/mL). The mean concentrations of thromboxane B2 and trioxilin A3 in blood appeared greater in the diabetic BB rat than in the normal rat, while the opposite was found for 6-keto PGF1 alpha (the stable product of prostacyclin). The apparent greater ratio of thromboxane B2 to 6-keto PGF1 alpha in the diabetic BB rat than in the normal rat supports a prothrombotic nature of platelets associated with diabetes.

Topics: 6-Ketoprostaglandin F1 alpha; 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Blood Glucose; Blood Pressure; Diabetes Mellitus, Experimental; Fatty Acids, Unsaturated; Gas Chromatography-Mass Spectrometry; Insulin; Kinetics; Male; Prostaglandins; Rats; Rats, Inbred BB; Rats, Inbred Strains; Thromboxane B2

Incubation of homogenates of the rat cerebral cortex with arachidonic acid led to the appearance of hepoxilin A3, analysed as its stable trihydroxy derivative, trioxilin A3, by high resolution gas chromatography/electron impact mass spectrometry. Using the stable deuterium isotope dilution technique, it is estimated that the cerebral cortex generates 5.0 +/- 0.2 ng/mg protein of hepoxilin A3. The formation of this product was stimulated by the addition of exogenous arachidonic acid (12.9 +/- 1.5 ng/mg protein) and blocked by boiling of the tissue. Addition of the dual cyclooxygenase/lipoxygenase inhibitor BW 755C at a concentration of 75 microM did not result in a blockade of hepoxilin formation. Three other regions were also tested for their ability to form hepoxilin A3 upon stimulation with exogenous arachidonic acid, i.e. median eminence, 11.7 +/- 1.6 ng/mg protein, pituitary, 12.3 +/- 0.7 ng/mg protein; pons, 26.6 +/- 0.2 ng/mg protein. In a separate study, 14C-labelled hepoxilin A3 was transformed into 14C-labelled trioxilin A3 by homogenates of the rat whole brain, demonstrating the presence of epoxide hydrolases in the CNS which utilise the hepoxilins as substrates. This is the first demonstration of the occurrence of the hepoxilin pathway in the central nervous system.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acids; Cerebral Cortex; Chemical Phenomena; Chemistry; Chromatography, Thin Layer; Fatty Acids, Unsaturated; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Male; Rats; Rats, Inbred Strains

Bolus intravenous injection of arachidonic acid (10 mg/kg) in the rat led to the appearance of hepoxilin A3 in the circulation. The product was assayed as the Me t-BDMSi derivative of its stable trihydroxy product trioxilin A3, by capillary gas chromatography-electron impact mass spectrometry using the stable deuterium isotope dilution technique. Hepoxilin A3, was undetected in blood samples taken prior to the injection of arachidonic acid, but rapidly appeared (4.62 +/- 1.3 ng/ml blood, n = 3) within 1 minute after injection of arachidonic acid. The plasma concentration of insulin increased by 36% over the same period after injection of arachidonic acid. These experiments demonstrate for the first time the formation of this new class of insulin secretagogues in vivo and their temporal correlation with plasma insulin concentrations in vivo.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Fatty Acids, Unsaturated; Insulin; Insulin Secretion; Rats

Evidence is presented to show that pancreatic islets of Langerhans are capable of producing hepoxilins A3 and B3 from endogenous substrates as well as 14C-labeled 12-HPETE. Both hepoxilins are active in stimulating the release of insulin from these cells in the presence of 10 mM glucose. These experiments suggest that the hepoxilins may participate as potential endogenous mediators of insulin release in islets of Langerhans.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Fatty Acids, Unsaturated; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Insulin; Islets of Langerhans; Rats; Rats, Inbred Strains

1-14C-labelled hepoxilin A3 (8-hydroxy-11,12-epoxyeicosa-5,9,14-trienoic acid) was generated from 1-14C-labelled arachidonic acid during incubation with a rat lung preparation lacking epoxide hydratase activity. The HPLC purified hepoxilin A3 gave only two isomeric 8,11,12-triols (termed trioxilins A3) upon incubation with a rat lung preparation containing epoxide hydratase activity. Based on this simple reaction an assay was developed using only 2000 cpm/tube of substrate and aliquots of a homogenate of platelet membranes from man. Products were assayed by thin-layer radiochromatography. Males were noted to have higher epoxide hydratase activity for this substrate than females.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Blood Platelets; Chemical Phenomena; Chemistry; Chromatography, Thin Layer; Epoxide Hydrolases; Fatty Acids, Unsaturated; Female; Humans; Lung; Rats

Pancreatic islets of Langerhans were perifused with Krebs-bicarbonate solution containing glucose (5 and 10 mM). The perifusate was spiked with tetradeuterated hepoxilin A3 and was extracted and analysed by gas chromatography-mass spectrometry using NICI detection. Evidence is presented showing the presence of hepoxilin A3 as the hydrolysis product trioxilin A3. These results demonstrate for the first time that this pathway is active in intact cells; this finding, taken together with our previous evidence that hepoxilins possess insulin secretagogue properties further supports our hypothesis that these products could play a role as endogenous mediators of insulin release.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acids; Gas Chromatography-Mass Spectrometry; Insulin; Islets of Langerhans; Rats; Rats, Inbred Strains

Isolated Krebs perifused rat pancreatic islets in the presence of 10 mM glucose convert 12S-HPETE into two hydroxy-epoxides, 8H-11,12-EPETE and 10H-11,12-EPETE for which we propose the name Hepoxilin A and B respectively. Hepoxilin A was investigated for its capacity to release insulin by this preparation. Insulin secretion by these cells, measured in the perifusate by radioimmunoassay, was dependent on the glucose concentration in the perifusing medium. Hepoxilin A dose dependently enhanced further the release of insulin during glucose (10 mM) stimulation (120 +/- 51% at 0.5 x 10(-6) M (n = 3) and 282 +/- 58% at 2.1 x 10(-6) M (n = 3) above control). These results suggest that Hepoxilin A (and possibly also Hepoxilin B) could be the active intermediate(s) involved in the potentiation of glucose dependent insulin secretion by both arachidonic acid and 12-HPETE.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acids; Carbon Radioisotopes; Dinoprostone; In Vitro Techniques; Insulin; Insulin Secretion; Islets of Langerhans; Kinetics; Leukotrienes; Prostaglandins E; Rats; Rats, Inbred Strains

The effect of hepoxilin A, a newly isolated hydroxyepoxide metabolite of arachidonic acid, on calcium transport across the visceral yolk sac membrane of the guinea pig was investigated in vitro in Ussing chambers. While 1-14C-labelled hepoxilin A itself was not transported across the membrane, it increased the rate of transport of calcium toward the side to which hepoxilin A was added. The degree of increase in calcium transport was similar whether hepoxilin A was added to the maternal side or to the fetal side of the membrane. The observed effect was dependent on the concentration of hepoxilin A over a narrow range (0.5-1.0 X 10(-6) M). It was also dependent on the time of incubation reaching maximal effect by 25 min. We have recently observed that hepoxilin A is formed from platelet-derived 12-hydroperoxyeicosatetraenoic acid (12-HPETE) through hemin and hemoglobin catalysis as well as during perifusion of 12-HPETE through isolated pancreatic islets. The present study suggests that hepoxilin A, if formed in vivo, could play a role in the mobilization of calcium.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Biological Transport; Calcium; Female; Guinea Pigs; In Vitro Techniques; Pregnancy; Yolk Sac

Arachidonic acid and 12-hydroperoxyeicosa-5,8, 10, 14-tetraenoic acid are converted by a 0-30% ammonium sulfate fraction (Fraction A) of the high speed supernatant of rat lung into two hydroxy epoxides (EH-1 and EH-2) which have been purified by high performance liquid chromatography. These hydroxy epoxides are converted quantitatively into two triols (10,11,12- from EH-1 and 8,11,12- from EH-2) by a 30-50% ammonium sulfate fraction (Fraction B) of the high speed supernatant. We propose the structures, 8-hydroxy-11,12-epoxyeicosa-5,9,14-trienoic acid (EH-2) and 10-hydroxy-11,12-epoxyeicosa-5,8,14-trienoic acids (EH-1) for these intermediates on the basis of mass spectral interpretation of several derivatives including the lithium aluminum hydride reduction product of both natural and 18Oxygenated derivatives.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Epoxy Compounds; Ethers, Cyclic; Fatty Acids, Unsaturated; Isomerism; Lung; Male; Mass Spectrometry; Rats; Rats, Inbred Strains; Tritium