prostaglandin-h2 and 15-hydroxy-5-8-11-13-eicosatetraenoic-acid

The endocannabinoid, 2-arachidonylglycerol (2-AG), is an endogenous ligand for the central (CB1) and peripheral (CB2) cannabinoid receptors and has been shown to be efficiently and selectively oxygenated by cyclooxygenase (COX)-2. We have investigated 2-AG/COX-2 interactions through site-directed mutagenesis. An evaluation of more than 20 site-directed mutants of murine COX-2 has allowed for the development of a model of 2-AG binding within the COX-2 active site. Most strikingly, these studies have identified Arg-513 as a critical determinant in the ability of COX-2 to efficiently generate prostaglandin H(2) glycerol ester, explaining, in part, the observed isoform selectivity for this substrate. Mutational analysis of Leu-531, an amino acid located directly across from Arg-513 in the COX-2 active site, suggests that 2-AG is shifted in the active site away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Despite this difference, aspirin-treated COX-2 oxygenates 2-AG to afford 15-hydroxyeicosatetraenoic acid glycerol ester in a reaction analogous to the C-15 oxygenation of arachidonic acid observed with acetylated COX-2. Finally, the differences in substrate binding do not alter the stereospecificity of the cyclooxygenase reaction; 2-AG-derived and arachidonic acid-derived products share identical stereochemistry.

Topics: Amino Acid Sequence; Amino Acids; Animals; Arachidonic Acid; Arachidonic Acids; Arginine; Binding Sites; Cannabinoid Receptor Modulators; Cannabinoids; Cyclooxygenase 1; Cyclooxygenase 2; DNA Mutational Analysis; Endocannabinoids; Esters; Glycerides; Glycerol; Hydroxyeicosatetraenoic Acids; Isoenzymes; Leucine; Mass Spectrometry; Membrane Proteins; Mice; Models, Chemical; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Oxygen; Prostaglandin H2; Prostaglandin-Endoperoxide Synthases; Prostaglandins H; Protein Binding; Protein Isoforms; Time Factors

Capsaicin, a pungent ingredient of hot peppers, causes excitation of small sensory neurons, and thereby produces severe pain. A nonselective cation channel activated by capsaicin has been identified in sensory neurons and a cDNA encoding the channel has been cloned recently. However, an endogenous activator of the receptor has not yet been found. In this study, we show that several products of lipoxygenases directly activate the capsaicin-activated channel in isolated membrane patches of sensory neurons. Among them, 12- and 15-(S)-hydroperoxyeicosatetraenoic acids, 5- and 15-(S)-hydroxyeicosatetraenoic acids, and leukotriene B(4) possessed the highest potency. The eicosanoids also activated the cloned capsaicin receptor (VR1) expressed in HEK cells. Prostaglandins and unsaturated fatty acids failed to activate the channel. These results suggest a novel signaling mechanism underlying the pain sensory transduction.

Topics: Animals; Capsaicin; Cell Line; Cells, Cultured; Dinoprostone; Eicosanoids; Ganglia, Spinal; Humans; Hydroxyeicosatetraenoic Acids; Inflammation; Ion Channel Gating; Leukotriene B4; Leukotrienes; Ligands; Lipid Peroxides; Lipoxygenase; Molecular Structure; Neurons, Afferent; Prostaglandin D2; Prostaglandin H2; Prostaglandins H; Rats; Receptors, Drug; Structure-Activity Relationship

We examined the effects of diabetes on eicosanoid metabolism and endothelium-dependent relaxation in isolated aorta from alloxan-induced diabetic rabbits and that from normal rabbits incubated in increased concentrations (44 mM) of glucose in vitro for 6 h. Immunoreactive 15-hydroxyeicosatetraenoic acid (HETE) was assayed in the incubation media of isolated aortic segments. Basal and acetylcholine (ACh)-stimulated release of 15-HETE was significantly greater in aorta of diabetic animals as compared with those of normal rabbits. Incubation of aortic segments from normal rabbits in increased concentrations of glucose caused a significant increase in basal and ACh-stimulated release of 15-HETE; and the release was significantly greater in aortic segments with endothelium than in segments without endothelium. Basal and ACh-stimulated release of 15-HETE was inhibited by indomethacin, a cyclooxygenase inhibitor. 15-HETE caused contractions of aortic rings that were inhibited by the prostaglandin H2 (PGH2) thromboxane A2 (TXA2) receptor blocker SQ-29548, but not by the TXA2 synthase inhibitor carbethoxyhexyl imidazole or indomethacin. Treatment of aortic rings with subthreshold concentrations of 15-HETE impaired ACh-induced relaxation; this was prevented by treatment with SQ-29548. Thus, abnormal release of endothelium-derived 15-HETE may play a role in endothelial cell dysfunction and increased vasoconstriction in diabetes by a mechanism that involves interaction with PGH2/TXA2 receptors.

Topics: Acetylcholine; Animals; Aorta, Abdominal; Bridged Bicyclo Compounds, Heterocyclic; Cytochrome P-450 Enzyme Inhibitors; Diabetes Mellitus, Experimental; Disease Models, Animal; Endothelium, Vascular; Fatty Acids, Unsaturated; Glucose; Hydrazines; Hydroxyeicosatetraenoic Acids; Imidazoles; Indomethacin; Male; Muscle Contraction; Muscle, Smooth, Vascular; Proadifen; Prostaglandin H2; Prostaglandins H; Rabbits; Radioimmunoassay; Superoxide Dismutase; Thromboxane A2

A preparative HPLC purification scheme for the isolation of prostaglandin endoperoxides prepared by short-time incubation of [1-14C]-labelled arachidonic acid (AA) with sheep seminal vesicle microsomes was developed. Milligram quantities of prostaglandin G2 (PGG2) and prostaglandin H2 (PGH2) were obtained in greater than or equal to 95% purity within shortest time. Furthermore, careful application of this HPLC technique led to the isolation of two minor [1-14C]-labelled fractions which according to their spectral and chromatographic characteristics, were identical with 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). Another HETE substituted at either C11 or C12 was also present. The formation of these products was mediated by cyclooxygenase as evidenced by aspirin (100 microM) and indomethacin (10 microM) inhibition. Sulfhydryl-blocking agents such as p-hydroxymercuribenzoate (1 mM) and/or the 12-lipoxygenase inhibitor esculetin (100 microM) were without effect. In addition to these AA metabolites four other fractions contained arachidonate-derived endoperoxides with antiaggregatory properties, all of which released malondialdehyde upon incubation with thromboxane A2 synthase. No thromboxane formation was observed although turnover numbers were comparable to those of PGG2 and PGH2. The formation of these endoperoxides did not occur via enzymatic or non-enzymatic degradation of PGG2 or PGH2. The exact chemical nature of these endoperoxides remains to be established.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxides; Male; Microsomes; Platelet Aggregation; Prostaglandin Endoperoxides; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandin-Endoperoxide Synthases; Prostaglandins G; Prostaglandins H; Seminal Vesicles; Sheep