Arachidonic Acid-derived Metabolites Research Articles

Linoleic acid blunts early osteoblast differentiation and impairs oxidative phosphorylation in vitro

BackgroundLinoleic acid (LNA), an essential polyunsaturated fatty acid (PUFA), plays a crucial role in cellular functions. However, excessive intake of LNA, characteristic of Western diets, can have detrimental effects on cells and organs. Human observational studies have shown an inverse relationship between plasma LNA concentrations and bone mineral density. The mechanism by which LNA impairs the skeleton is unclear, and there is a paucity of research on the effects of LNA on bone-forming osteoblasts. MethodsThe effect of LNA on osteoblast differentiation, cellular bioenergetics, and production of oxidized PUFA metabolites in vitro, was studied using primary mouse bone marrow stromal cells (BMSC) and MC3T3-E1 osteoblast precursors. ResultsLNA treatment decreased alkaline phosphatase activity, an early marker of osteoblast differentiation, but had no effect on committed osteoblasts or on mineralization by differentiated osteoblasts. LNA suppressed osteoblast commitment by blunting the expression of Runx2 and Osterix, key transcription factors involved in osteoblast differentiation, and other key osteoblast-related factors involved in bone formation. LNA treatment was associated with increased production of oxidized LNA- and arachidonic acid-derived metabolites and blunted oxidative phosphorylation, resulting in decreased ATP production. ConclusionOur results show that LNA inhibited early differentiation of osteoblasts and this inhibitory effect was associated with increased production of oxidized PUFA metabolites that likely impaired energy production via oxidative phosphorylation.

Different Structures-Similar Effect: Do Substituted 5-(4-Methoxyphenyl)-1H-indoles and 5-(4-Methoxyphenyl)-1H-imidazoles Represent a Common Pharmacophore for Substrate Selective Inhibition of Linoleate Oxygenase Activity of ALOX15?

Mammalian 15-lipoxygenases (ALOX15) are lipid peroxidizing enzymes that exhibit variable functionality in different cancer and inflammation models. The pathophysiological role of linoleic acid- and arachidonic acid-derived ALOX15 metabolites rendered this enzyme a target for pharmacological research. Several indole and imidazole derivatives inhibit the catalytic activity of rabbit ALOX15 in a substrate-specific manner, but the molecular basis for this allosteric inhibition remains unclear. Here, we attempt to define a common pharmacophore, which is critical for this allosteric inhibition. We found that substituted imidazoles induce weaker inhibitory effects when compared with the indole derivatives. In silico docking studies and molecular dynamics simulations using a dimeric allosteric enzyme model, in which the inhibitor occupies the substrate-binding pocket of one monomer, whereas the substrate fatty acid is bound at the catalytic center of another monomer within the ALOX15 dimer, indicated that chemical modification of the core pharmacophore alters the enzyme-inhibitor interactions, inducing a reduced inhibitory potency. In our dimeric ALOX15 model, the structural differences induced by inhibitor binding are translated to the hydrophobic dimerization cluster and affect the structures of enzyme-substrate complexes. These data are of particular importance since substrate-specific inhibition may contribute to elucidation of the putative roles of ALOX15 metabolites derived from different polyunsaturated fatty acids in mammalian pathophysiology.

Accumulation of polyunsaturated fatty acid-derived metabolites in the sarcopenic muscle of aging mice.

Although it is known that advanced age alters skeletal muscle lipid metabolism, the role(s) of polyunsaturated fatty acid-derived metabolites (mostly eicosanoids and docosanoids) in sarcopenia are not clear. We therefore examined the changes in the metabolites of arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid in the sarcopenic muscle of aged mice. We used 6- and 24-month-old male C57BL/6J mice as healthy and sarcopenic muscle models, respectively. Skeletal muscles were removed from the lower limb and subjected to a liquid chromatography-tandem mass spectrometry analysis. The liquid chromatography-tandem mass spectrometry analysis detected distinct changes of metabolites in the muscles of the aged mice. Of the 63 metabolites identified, nine were significantly higher in the sarcopenic muscle of aged mice compared with the healthy muscle of young mice. In particular, prostaglandin E2 , prostaglandin F2a , thromboxane B2 , 5-hydroxyeicosatetraenoic acid, and 15-oxo-eicosatetraenoic acid (arachidonic acid-derived metabolites), 12-hydroxy-eicosapentaenoic acid and 14,15-epoxy-eicosatetraenoic acid (eicosapentaenoic acid-derived metabolites) and 10-hydroxydocosa-hexaenoic acid and 14-hydroxyoctadeca-pentaenoic acid (docosahexaenoic acid-derived metabolites) were significantly higher in aged tissue compared with young tissue (all P< 0.05). We observed the accumulation of metabolites in the sarcopenic muscle of aged mice. Our results may provide new insights into the pathogenesis and progression of aging- or disease-related sarcopenia. Geriatr Gerontol Int 2023; 23: 297-303.

Role of Arachidonic Acid-Derived Cyp450 Metabolites In Acute Kidney Injury

Acute Kidney Injury (AKI) remarkblely affect public health and need higher healthcare costs, which is life-threatening and characterized of acute decline with function. Arachidonic Acid (AA), the precursor for synthesis of various biologically active molecules and be involved in regulation of homeostasis and metabolism. Recent studies have shown that the AA cytochrome P450 (CYP450) pathway contribute to the development of AKI by EETs (the product of catalyzing epoxidation reactions), 20-HETE (the product of omega-hydroxylation reactions) and maybe potent key for the prevention and treatment of AKI. This review summarizes recent advances in AA metabolites in AKI.

Creating a mouse model resistant to induced ischemic stroke and cardiovascular damage

Vascular prostanoids, isomerized from an intermediate prostaglandin (PG), H2, produced by cyclooxygenase (COX), exert various effects on the vascular system, both protective and destructive. During endothelial dysfunction, vascular protector prostacyclin/prostaglandin I2 (PGI2) is decreased, while inflammatory PGE2 and thrombotic TXA2 are increased. Therefore, our research aim was to reverse the event by controlling PGH2 metabolism by generating an in vivo model via enzymatic engineering of COX-1 and prostacyclin synthase (PGIS). The COX-1 and PGIS genes were linked to a 10-residue amino acid linker to form a Single-chain Enzyme Complex (SCHEC), COX-1-10aa-PGIS. Transgenic (CP-Tg) mice in a FVB/N background were generated using the pronuclear microinjection method. We first confirmed mRNA and protein expression of COX-1-10aa-PGIS in various CP-Tg mouse tissues, as well as upregulation of circulating PGI2. We then examined the cardiovascular function of these mice. Our CP-Tg mice exhibited marked resistance to vascular assault through induced carotid arterial blockage, acute thrombotic stroke and arterial arrest, angiotensin-induced peripheral vasoconstriction, and hepatic lipid accumulation after receiving a high-fat diet. They also had a longer lifespan compared with wild-type mice. This study raises the possibility of fighting cardiovascular diseases by regulating cellular arachidonic acid-derived PGH2 metabolites using enzymatic engineering.

Compensatory Vasodilator Mechanisms in the Ophthalmic Artery of Endothelial Nitric Oxide Synthase Gene Knockout Mice

Nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) plays an important role in the maintenance of ocular vascular homeostasis. Therefore, perturbations in vascular NO synthesis have been implicated in the pathogenesis of several ocular diseases. We recently reported that eNOS contributes significantly to vasodilation of the mouse ophthalmic artery. Interestingly, dilatory responses were also retained in eNOS gene-deficient mice (eNOS−/−), indicating inherent endothelial adaptive mechanism(s) that act as back-up systems in chronic absence of eNOS to preserve vasorelaxation. Thus, this study endeavoured to identify the compensatory mechanism(s) in the ophthalmic artery of eNOS−/− mice employing isolated arterial segments and pharmacological inhibitors in vitro. Endothelium removal virtually abolished acetylcholine (ACh)-induced vasodilation, suggesting an obligatory involvement of the endothelium in cholinergic control of vascular tone. However, non-NOS and non-cyclooxygenase components compensate for eNOS deficiency via endothelium-derived hyperpolarizing factors (EDHFs). Notably, arachidonic acid-derived metabolites of the 12-lipoxygenase pathway were key mediators in activating the inwardly rectifying potassium channels to compensate for chronic lack of eNOS. Conclusively, endothelium-dependent cholinergic responses of the ophthalmic artery in the eNOS−/− mice are largely preserved and, this vascular bed has the ability to compensate for the loss of normal vasodilator responses solely via EDHFs.

N-3 Polyunsaturated Fatty Acids Decrease the Protein Expression of Soluble Epoxide Hydrolase via Oxidative Stress-Induced P38 Kinase in Rat Endothelial Cells.

N-3 polyunsaturated fatty acids (PUFAs) improve endothelial function. The arachidonic acid-derived metabolites (epoxyeicosatrienoic acids (EETs)) are part of the endothelial hyperpolarization factor and are vasodilators independent of nitric oxide. However, little is known regarding the regulation of EET concentration by docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in blood vessels. Sprague-Dawley rats were fed either a control or fish oil diet for 3 weeks. Compared with the control, the fish oil diet improved acetylcholine-induced vasodilation and reduced the protein expression of soluble epoxide hydrolase (sEH), a key EET metabolic enzyme, in aortic strips. Both DHA and EPA suppressed sEH protein expression in rat aorta endothelial cells (RAECs). Furthermore, the concentration of 4-hydroxy hexenal (4-HHE), a lipid peroxidation product of n-3 PUFAs, increased in n-3 PUFA-treated RAECs. In addition, 4-HHE treatment suppressed sEH expression in RAECs, suggesting that 4-HHE (derived from n-3 PUFAs) is involved in this phenomenon. The suppression of sEH was attenuated by the p38 kinase inhibitor (SB203580) and by treatment with the antioxidant N-acetyl-L-cysteine. In conclusion, sEH expression decreased after n-3 PUFAs treatment, potentially through oxidative stress and p38 kinase. Mild oxidative stress induced by n-3 PUFAs may contribute to their cardio-protective effect.

Cytochrome p450 enzymes in the bioactivation of polyunsaturated Fatty acids and their role in cardiovascular disease.

Various members of the cytochrome P450 (CYP) superfamily have the capacity of metabolizing omega-6 and omega-3 polyunsaturated fatty acids (n-6 and n-3 PUFAs). In most mammalian tissues, CYP2C and CYP2J enzymes are the major PUFA epoxygenases, whereas CYP4A and CYP4F subfamily members function as PUFA hydroxylases. The individual CYP enzymes differ in their substrate specificities as well as regio- and stereoselectivities and thus produce distinct sets of epoxy and/or hydroxy metabolites, collectively termed CYP eicosanoids. Nutrition has a major impact on the endogenous CYP-eicosanoid profile. "Western diets" rich in n-6 PUFAs result in a predominance of arachidonic acid-derived metabolites, whereas marine foodstuffs rich in n-3 PUFAs shift the profile to eicosapentaenoic and docosahexaenoic acid-derived metabolites. In general, CYP eicosanoids are formed as second messengers of numerous hormones, growth factors and cytokines regulating cardiovascular and renal function, and a variety of other physiological processes. Imbalances in the formation of individual CYP eicosanoids are linked to the development of hypertension, myocardial infarction, maladaptive cardiac hypertrophy, acute kidney injury, stroke and inflammatory disorders. The underlying mechanisms are increasingly understood and may provide novel targets for the prevention and treatment of these disease states. Suitable pharmacological agents are under development and first proofs of concept have been obtained in animal models.

Role of arachidonic acid-derived metabolites in the control of pulmonary arterial pressure and hypoxic pulmonary vasoconstriction in rats

BackgroundThe roles of arachidonic acid (AA) metabolites in hypoxia-induced pulmonary vasoconstriction (HPV), a critical physiological mechanism that prevents ventilation/perfusion mismatch, are still incompletely understood. MethodsPulmonary arterial pressure was measured in ventilated/perfused rat lungs. Isometric tones of rat intralobar pulmonary arteries were also measured, using a myograph. ResultsHypoxia (Po2, 3%)-induced pulmonary arterial pressure increases (ΔPAPhypox) were stable with blood-mixed perfusate, but decayed spontaneously. ΔPAPhypox was inhibited by 29%, 16%, and 28% by the thromboxane A2 (TXA2) antagonist SQ-29548, the 5-lipoxygenase inhibitor, MK886, and the leukotriene D4 antagonist, LY-171883, respectively. The prostacyclin synthase inhibitor tranylcypromine augmented ΔPAPhypox by 5%, whereas inhibition of cytochrome P450 did not affect ΔPAPhypox. Consistently, the TXA2 analogue U46619 increased ΔPAPhypox whereas prostacyclin abolished ΔPAPhypox. However, leukotriene D4 had no direct effect on ΔPAPhypox. In the isolated pulmonary arteries, pretreatment with U46619 was essential to demonstrate hypoxia-induced contraction. ConclusionsThe above results suggest that TXA2 and cysteinyl leukotrienes, other than leukotriene D4, are endogenous factors that facilitate HPV in rats. The indispensable role of TXA2-induced pretone in the HPV of isolated pulmonary arteries indicates that the signal from thromboxane receptors might be a critical component of oxygen sensation mechanisms.

Involvement of Cytosolic Phospholipase A2, Calcium Independent Phospholipase A2 and Plasmalogen Selective Phospholipase A2 in Neurodegenerative and Neuropsychiatric Conditions

Enzymes belonging to the PLA(2) superfamily catalyze the hydrolysis of unsaturated fatty acids from the sn-2 position of glycerol moiety of neural membrane phospholipids. The PLA(2) superfamily is classified into cytosolic PLA(2) (cPLA(2)), calcium-independent PLA(2) (iPLA(2)), plasmalogen-selective PLA(2) (PlsEtn-PLA(2)) and secretory PLA(2) (sPLA(2)). PLA(2) paralogs/splice variants/isozymes are part of a complex signal transduction network that maintains cross-talk among excitatory amino acid and dopamine receptors through the generation of second messengers. Individual paralogs, splice variants and multiple forms of PLA(2) may have unique enzymatic properties, tissue and subcellular localizations and role in various physiological and pathological situations, hence tight regulation of all PLA(2) isoforms is essential for normal brain function. Quantitative RT-PCR analyses show significantly higher relative level of expression of iPLA(2) than cPLA(2) in all regions of the rat brain. Upregulation of the cPLA(2) family is involved in degradation of neural membrane phospholipids and generation of arachidonic acid-derived lipid metabolites that have been implicated in nociception, neuroinflammation, oxidative stress and neurodegeneration. In contrast, studies using a selective iPLA(2) inhibitor, bromoenol lactone, or antisense oligonucleotide indicate that iPLA(2) is an important "housekeeping" enzyme under basal conditions, whose activity is required for the prevention of vacuous chewing movements, a rodent model for tardive dyskinesia, and deficits in the prepulse inhibition of the auditory startle reflex, a common finding in schizophrenia. These studies support the view that PLA(2) activity may not only play a crucial role in neurodegeneration but depending on the isoform, could also be essential in prevention of neuropsychiatric diseases. The findings could open new doors for understanding and treatment of neurodegenerative and neuropsychiatric diseases.

Lipidomic metabolism analysis of the endogenous cannabinoid anandamide (N-arachidonylethanolamide)

Elucidation of pathways involved with lipid metabolism has been limited by analytical challenges associated with detection and structure identification. A discovery-based mass spectrometry lipidomic approach has been applied to identify metabolites of the endogenous cannabinoid anandamide (N-arachidonylethanolamide). Previously, a model system was established to show that anandamide can be recycled by cells to form new endocannabinoids suggesting recycling of the arachidonate carbon chain. We hypothesized that distinct cellular pathways exist to direct the anandamide-derived arachidonate chain into a specific set of metabolites, different from the metabolite pool that is comprised of non-anandamide-derived arachidonic acid. Using stable isotope encoding and liquid chromatography–mass spectrometry, we identified a distinct pool of lipid metabolites derived from exogenous anandamide or arachidonic acid in RBL-2H3 cells. We discovered that arachidonic acid-derived metabolites were primarily comprised of the eicosanoid lipid class, whereas anandamide-derived arachidonic acid, in addition to eicosanoids, was metabolized into diradylglycerols, fatty acid amides, sterols, and glycerophospholipids. From the list of anandamide metabolites of particular interest was 1- O-arachidonyl- sn-glycero-3-phosphocholine. Furthermore, we determined that while 1- O-arachidonyl- sn-glycero-3-phosphocholine may be a metabolite of anandamide, the sn-2 compound was more abundant in mouse brain tissue. Overall, our results provide a novel approach to study the metabolic fate of endocannabinoids and fatty acid-derived signaling molecules.

Oxymetazoline Inhibits Proinflammatory Reactions: Effect on Arachidonic Acid-Derived Metabolites

The nasal decongestant oxymetazoline effectively reduces rhinitis symptoms. We hypothesized that oxymetazoline affects arachidonic acid-derived metabolites concerning inflammatory and oxidative stress-dependent reactions. The ability of oxymetazoline to model pro- and anti-inflammatory and oxidative stress responses was evaluated in cell-free systems, including 5-lipoxygenase (5-LO) as proinflammatory, 15-lipoxygenase (15-LO) as anti-inflammatory enzymes, and oxidation of methionine by agglomerates of ultrafine carbon particles (UCPs), indicating oxidative stress. In a cellular approach using canine alveolar macrophages (AMs), the impact of oxymetazoline on phospholipase A(2) (PLA(2)) activity, respiratory burst and synthesis of prostaglandin E(2) (PGE(2)), 15(S)-hydroxy-eicosatetraenoic acid (15-HETE), leukotriene B(4) (LTB(4)), and 8-isoprostane was measured in the absence and presence of UCP or opsonized zymosan as particulate stimulants. In cell-free systems, oxymetazoline (0.4-1 mM) inhibited 5-LO but not 15-LO activity and did not alter UCP-induced oxidation of methionine. In AMs, oxymetazoline induced PLA(2) activity and 15-HETE at 1 mM, enhanced PGE(2) at 0.1 mM, strongly inhibited LTB(4) and respiratory burst at 0.4/0.1 mM (p < 0.05), but did not affect 8-isoprostane formation. In contrast, oxymetazoline did not alter UCP-induced PLA(2) activity and PGE(2) and 15-HETE formation in AMs but inhibited UCP-induced LTB(4) formation and respiratory burst at 0.1 mM and 8-isoprostane formation at 0.001 mM (p < 0.05). In opsonized zymosan-stimulated AMs, oxymetazoline inhibited LTB(4) formation and respiratory burst at 0.1 mM (p < 0.05). In conclusion, in canine AMs, oxymetazoline suppressed proinflammatory reactions including 5-LO activity, LTB(4) formation, and respiratory burst and prevented particle-induced oxidative stress, whereas PLA(2) activity and synthesis of immune-modulating PGE(2) and 15-HETE were not affected.

The spinal basis of opioid tolerance and physical dependence: Involvement of calcitonin gene-related peptide, substance P, and arachidonic acid-derived metabolites

Chronic opioid use in the management of pain is limited by development of analgesic tolerance and physical dependence. The mechanisms underlying tolerance-dependence are not entirely clear, however, recent evidence suggests that spinal adaptations leading to increased activity of sensory neuropeptides (calcitonin gene-related peptide (CGRP), substance P) and their downstream signaling messengers derived from metabolism of arachidonic acid: prostaglandins (PG), lipoxygenase (LOX) metabolites, and endocannabinoids, plays an important role in this phenomenon. In this communication we review the evidence implicating these factors in the induction and expression of opioid tolerance and physical dependence at the spinal level.

Inhibition of COX activity by NSAIDs or ascorbate increases cAMP levels and enhances differentiation in 1α,25-dihydroxyvitamin D 3-induced HL-60 cells

Arachidonic acid metabolism is modulated during differentiation induced by 1α,25(OH) 2D 3 in HL-60 cells. Antioxidants that affect arachidonic acid metabolism enhance this differentiation program. Ascorbate also enhances differentiation in 1α,25(OH) 2D 3-induced cells depending on the induction of cAMP. The aim of this work was to study if this cAMP rise depends on modulation of arachidonic acid metabolism by ascorbate. Cyclooxygenase inhibitors, indomethacin and aspirin, increased cAMP levels and also enhanced 1α,25(OH) 2D 3-induced differentiation in HL-60 cells. Ascorbate did not affect the release of arachidonic acid-derived metabolites but decreased the levels of TXB 2 and PGE 2, suggesting the inhibition of cyclooxygenase. On the other hand, free arachidonic acid increased both cAMP levels and differentiation in the absence or presence of 1α,25(OH) 2D 3. Neither cyclooxygenase inhibitors nor ascorbate modified AA effect. Then, inhibition of cyclooxygenase activity by ascorbate could accumulate free arachidonic acid or other metabolites that increase cAMP levels and enhance differentiation in 1α,25(OH) 2D 3-induced HL-60 cells.

The metabolic effects of inhibitors of 5-lipoxygenase and of cyclooxygenase 1 and 2 are an advancement in the efficacy and safety of anti-inflammatory therapy

Chronic treatment of inflammatory diseases with non-steroidal anti-inflammatory drugs is effective but not always devoid of serious side effects. In particular, the use of traditional non-steroidal aspirin-like drugs has been associated with a high incidence of gastrointestinal bleedings. The development of a new class of drugs, the selective cyclooxygenase type 2 (COX-2) inhibitors, has generated much expectation on the possibility to have safer compounds. After the initial enthusiasm of the scientific community, a re-evaluation of some large, randomized double-blind clinical studies performed with two of these compounds, has disclosed that the late serious gastrointestinal complications are not significantly reduced in comparison with non-selective inhibitors and that cardiovascular concerns might arise particularly if theses drugs are utilized in patients with underlying heart diseases. A new promising class of drugs to control inflammatory diseases is in advanced clinical development. The balanced inhibitors of 5-lipoxygenase (5-LOX) and of cyclooxygenase (both types 1 and 2) block the formation of all the enzymatically arachidonic acid-derived metabolites, both prostaglandins (like COX inhibitors) and leukotrienes (LT); these drugs have been shown to possess a very good anti-inflammatory efficacy without serious side effects. Licofelone, previously known as ML3000, is the molecule in the most advanced phase of clinical development (phase III) among this class of compounds; it is a potent, competitive, and well balanced inhibitor of 5-LOX and COX pathways. The drug has been shown to possess analgesic, anti-inflammatory, antipyretic antibronchocostrictory and antiplatelet properties at doses which are safe for the gastrointestinal tract. Moreover, the newly performed preclinical studies, here briefly reviewed, appear to indicate that the compound seems particularly suitable to protect the articular cartilage and the synovial space in degenerative joint disease and to exert a relevant antithrombotic activity. Preliminary results of clinical studies of licofelone in osteoarthritis indicate that the drug has a comparable or slightly better efficacy than that of naproxen but possesses a much better gastrointestinal safety. This latter important aspect has been also evaluated by an endoscopic study in normal volunteers randomly assigned to a 4-week treatment with licofelone, placebo or naproxen. The results indicate that no ulcers occurred in either licofelone group or the placebo group, while ulcers with unequivocal depth were present in 20% of the naproxen-treated subjects.

Activation of the arachidonic acid cascade by interleukin-1 in human chondrocytes

The arachidonic acid cascade in interleukin-1-stimulated chondrocytes was investigated by studying the activation of phospholipase A2 and the regulation of cyclooxygenase. IL 1 stimulated the [3H]-arachidonic acid release and analyses of the [3H]-arachidonic acid-derived metabolites stressed that most of the released radioactivity after IL 1 activation was associated with prostaglandin E2. Human IL 1-stimulated chondrocytes also synthesized prostaglandin F2α and 6-keto-prostaglandin F1α. Using transcription and translation inhibitors it was shown that protein synthesis was required for PG synthesis. Aspirin, a specific inhibitor of cyclooxygenase, was also used and confirmed that the production of PG induced by IL 1 is imputable to ade novo synthesis of cyclooxygenase. In human chondrocytes, cyclooxygenase is thus a key enzyme in the AA cascade in addition of PLA2.

Cholestasis and changes of portal pressure caused by chlorpromazine in the perfused rat liver

Chlorpromazine (10 mumol/L) causes a marked increase in portal pressure in perfused rat liver. Simultaneously, oxygen consumption, hepatic clearance of taurocholate and bile flow are diminished. These effects are prevented by the cyclooxygenase inhibitors indomethacin (15 mumol/L), acetylsalicylate (3 mmol/L) or ibuprofen (200 mumol/L). On addition of chlorpromazine the liver releases increased amounts of prostaglandin D2; this increase does not occur in the presence of indomethacin. At higher concentrations of chlorpromazine (100 mumol/L) the inhibition of taurocholate clearance and bile flow is accompanied by only a moderate increase of portal pressure, and indomethacin is without effect. At this high concentration, substantial cell damage, as indicated by the release of lactate dehydrogenase, is present. We conclude that arachidonic acid-derived metabolites, notably prostanoids, are involved in the inhibition of bile flow and of taurocholate clearance observed at low concentrations of chlorpromazine. The data suggest that changes in the microcirculation are responsible for the impairment of the liver functions. At higher concentrations of chlorpromazine the cell toxicity of the drug becomes prominent.

Enzymes involved in the biosynthesis of leukotriene B4

Ten years have elapsed since the discovery and structural elucidation of the leukotriene compounds (1-5). The leukotrienes constitute a group of potent biologically active arachidonic acid-derived metabolites which are implicated as mediators of immediate hypersensitivity reactions and inflammation. They were first discovered in leukocytes and were determined to contain a conjugated triene structure, thus the derivation of their name (6). The cysteinyl-containing leukotrienes (LTC,, LTD4, and LTE,),' which together make up the slow reacting substance of anaphylaxis, are synthesized primarily by eosinophils, mast cells, and macrophages. They stimulate contraction of various smooth muscle cells, promote bronchial mucous secretion, and induce extravasation of plasma proteins by increasing microvascular permeability. Leukotriene B, (LTB,), synthesized predominantly by neutrophils, is capable of stimulating circulating leukocytes to adhere to the vascular endothelium and induces extravasation and chemotaxis of polymorphonuclear leukocytes in nanomolar concentrations. At higher concentrations, LTBI induces polymorphonuclear leukocyte degranulation, superoxide generation, and can augment natural killer cell activity. Several reviews on the structure, biosynthesis, and biological actions of the leukotrienes have been written (7-13). It is the aim of the present review to concentrate on the advances made in recent years, primarily through molecular biology studies, on the reaction pathway leading to production of LTB,. 5-Lipoxygenase

Recombinant interleukin-1β stimulates eicosanoid production in rat primary culture astrocytes

Astrocytes may play a prominent role in the initiation of immunoinflammatory responses in the central nervous system. They can be induced to synthesize eicosanoids but how immunologically relevant molecules modulate this process is not known. We examined the influence of recombinant interleukin-1 (rIL-1), an immunomodulating monokine on the release of arachidonic acid metabolites. IL-1 (1–30 U) induced a dose-related elaboration predominantly of the cyclo-oxygenation products prostaglandin E and throm☐ane B 2. Preincubation of rIL-1 with a specific antibody abrogated and heat-inactivation destroyed this activity. Both mepacrine and the isoquinolinesulfonamide H7 blocked the stimulatory effect dose-dependently, indicating involvement of protein kinase C in this novel biologic activity of IL-1. In central nervous system inflammation, IL-1-evoked release from astrocytes of arachidonic acid-derived metabolites may influence the severity of phlogistic responses and modulate local immune reactivity.

Effects of Chronic Intracutaneous Administration of Arachindonic Acid and Its Metabolites. Induction of Leukocytoclastic Vasculitis by Leukotriene B4 and 12-Hydroxyeicosatetraenoic Acid and Its Prevention by Prostaglandin E2

Despite the postulated role of arachidonic acid-derived metabolites in the pathophysiology of chronic inflammatory dermatoses such as psoriasis and atopic or contact dermatitis, the cutaneous effects of their chronic application have not yet been investigated. We therefore studied systematically the effects of chronic intracutaneous administration of arachidonic acid, prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and 12-hydroxyeicosatetraenoic acid (12-HETE) in guinea pigs, and describe previously unrecognized findings partly different from those reported in the past for short-term or topical application of these inflammatory mediators. Leukotriene B4 and 12-HETE led to massive histologic changes characteristic of leukocytoclastic vasculitis. These changes could be prevented by concomitant PGE2 administration. In epidermis, LTB4 and 12-HETE caused some spongiosis as well as hyperplasia and increased tritiated thymidine autoradiographic labeling index. Arachidonic acid and PGE2 alone had little effect. These data suggest that in addition to other inflammatory or hyperproliferative dermatoses, arachidonic acid metabolites formed via lipoxygenase pathways could play a major role in leukocytoclastic vasculitis, whereas PGs could exert a tissue-protective effect.