EICOSANOIDS mirka.rovenska@lfmotol.cuni.cz

Eicosanoids: 20-carbon compounds Comprise: prostaglandins thromboxanes leukotrienes lipoxins hydroxyeicosatetraenoic acids (HETEs) hepoxilins prostanoids

EICOSANOID BIOSYNTHESIS

Eicosanoid biosynthesis In polyunsaturated fatty acid metabolism, especially metabolism of linoleic and arachidonic acid:

In human, arachidonic acid is being synthesized from linoleic acid: Linoleic and linolenic acids are essential, i.e. must be supplied in food (plant oils, peanut, soybean, corn)

Principle sites of eicosanoid biosynthesis Endothelium Leukocytes Platelets Kidney Unlike histamine, eicosanoids are NOT synthesized in advance and stored in granules – in case of need, they can be produced very quickly from arachidonate released from membranes

Main steps of eicosanoid biosynthesis Activation of phospholipase A2 (PLA2) Release of arachidonate from membrane phospholipids by the action of PLA2 Eicosanoid synthesis: COX or LO pathway + subsequent modifications (e.g. conversion of the precursor PGH2 into another prostaglandin/ thromboxane, conversion of LTA4 into peptido-leukotrienes…)

Phospholipase A2 activation PLA2 is activated primarily by an increase in intracellular Ca2+: ligand binding to a receptor induces phospholipase C (PLC) activation → PLC cleaves PIP2 into DAG and IP3 that opens the Ca2+ channels in the ER. Some activation role can probably be attributed also to phosphorylation of PLA2 by kinases (MAPK). Activated PLA2 then translocates to membranes of GA, ER and/or nucleus from which it releases arachidonate for here residing COX / LO. GA, ER, or nucleus membrane NOS synthesis/ activation translocation The ligand can be also ATP released from dying cells activation Ca

PLA2 expression/activity is stimulated by: Interleukin-1 Angiotensin II Bradykinin TGF-β2 EGF (Epidermal Growth Factor) Thrombin Epinephrine Dexamethasone (synthetic glucocorticoid) Annexin 1 (lipocortin; protein) – glucocorticoid-inducible PLA2 expression/activation is blocked by:

Arachidonate release for eicosanoid biosynthesis From membrane phospholipids – mainly by the action of phospholipase A2: Arachidonate release from phospholipids can be blocked by the anti-inflammatory steroids!

Eicosanoid biosynthesis In almost all types of cells (except for red blood cells) 3 pathways: cyclooxygenase (COX) – produces prostaglandins and thromboxanes lipoxygenase (LO) – produces leukotrienes, lipoxins, and 12- and 15-HETEs (hydroxyeicosatetraenoic acids) cytochrome P450s (monooxygenases) – produce the other HETEs (20-HETE); principal pathway in the proximal tubules

Eicosanoid production - overview food linoleic acid PRINCIPAL arachidonic acid dihomo-γ-linolenic acid (8,11,14-eicosa- trienoic) 1…cyclooxygenase pathway 2…lipoxygenase pathway linolenic acid eicosapentaenoic acid food food – mainly fish oils  high intake in Eskimos may be the reason of their low incidence of heart diseases and prolonged clotting times

Prostaglandin nomenclature The three classes A, E, F (third letter) are distinguished on the basis of the functional groups about the cyclopentane ring The subscript numerals refer to the number of double bonds in the side chains The subscript  refers to the configuration of the 9–OH group (projects down from the plane of the ring) PGE2 E…β-hydroxyketone 2 double bonds

Structural features of individual types Prostaglandins – cyclopentane ring Thromboxanes – six-membered oxygen-containing ring Leukotrienes – 3 conjugated double bonds + one more non-conjugated Lipoxins – conjugated trihydroxytetraenes

1) Cyclooxygenase (COX) pathway Prostaglandin H synthase, present as two isoenzymes (PGHS-1, PGHS-2), each possessing two activities: cyclooxygenase – catalyzes addition of two molecules of O2 to arachidonic acid, forming PGG2 hydroperoxidase – mediates a glutathione-dependent reaction that converts the hydroperoxy function of PGG2 to an OH group (in the molecule of PGH2) Capable of self-catalyzed destruction!!! Particular cell type produces one particular type of prostanoid: platelets produce almost exclusively thromboxanes, vascular endothelial cells predominantly synthesize the prostacyclins, heart muscle makes PGI2, PGE2, PGF2

Prostaglandin H synthase cyclic 9,11-endoperoxide 15-hydroperoxide is being formed PGH2 – precursor of all series-2 prostaglandins and thromboxanes

Products of the COX pathway Platelets contain thromboxane synthase which mediates formation of TXA2, TXB2 Vascular endothelial cells contain prostacyclin synthase which catalyzes the synthesis of prostacyclin PGI2

Inhibition of the COX pathway Aspirin inhibits the COX activity of both PGHS-1 and PGHS-2 (by acetylation of a distinct Ser of the enzyme) Other nonsteroidal anti- inflammatory drugs (NSAIDs) also inhibit the COX activity (ibuprofen – competes with arachidonate) Transcription of PGHS-2 can be blocked by anti-inflammatory corticosteroids

2) Lipoxygenase (LO) pathway 3 different lipoxygenases insert oxygen into the 5, 12, or 15 position of arachidonate; the first product is the hydroperoxy-eicosatetraenoic acid (HPETE) Only 5-lipoxygenase produces leukotrienes; its activation requires protein called FLAP hepoxilins (HXA3) 5-lipoxygenase Gly-Cys-γGlu S leukotriene D4 leukotriene E4 peptidoleukotrienes -Glu -Gly

Peptidoleukotriene biosynthesis: Requires glutathione!!!

3) Eicosanoid synthesis by CYP450s Cytochrome P450s – monooxygenases: RH + O2 + NADPH + H+ ROH + H2O + NADP+ Two main groups of compounds are formed: epoxygenases catalyze the formation of epoxyeicosatrienoic acids (EETs) that are further metabolized by epoxide hydrolases to dihydroxyeicosatrienoic acids (DiHETEs) which are almost inactive: hydroxylases catalyze the formation of HETEs, e.g. -hydroxy-lase catalyzes formation of 20-hydroxyeicosatetraenoic acid (20-HETE); other produce 13-HETE, 10-HETE…)

Summary of the products arachidonic acid CYP450s lipoxygenases cyclooxygenases 19-, 20-, 8-, 9-, 10-, 11-, 12-, 13-, 15-, 16-, 17-, 18-HETE EETs prostacyclins thromboxanes lipoxins DiHETEs prostaglandins leukotrienes hepoxilins 5-, 8-, 12-, 15-HETE

BIOLOGICAL ROLES OF EICOSANOIDS

Eicosanoids, like hormones, have profound effects at extremely low concentrations They have a very short half-life; thus, they act in an autocrine or paracrine manner (unlike hormones) Biological effects depend not only on the particular eicosanoid but also on the local availability of receptors that it can bind to

In general, eicosanoids mediate: inflammatory response, notably as it involves the joints (rheumatoid arthritis), skin (psoriasis), and eyes production of pain and fever regulation of blood pressure regulation of blood clotting regulation of renal function control of several reproductive functions, such as the induction of labor

Biological roles of prostaglandins Mediate inflammation – they: cause vasodilation  redness, heat (PGE1, PGE2) increase vascular permeability  swelling Produce pain and fever: PGE2 enhance the intensity and the duration of pain caused by histamine and bradykinin PGE2, PGF2 induce parturition Prostaglandins of the PGE series inhibit gastric acid secretions (synthetic analogs are used to treat gastric ulcers) Regulation of blood pressure: vasodilator prostaglandins PGE, PGA, and PGI2 lower systemic arterial pressure Influence platelet aggregation: PGI2 (produced by vascular endothelial cells) inhibits platelet aggregation, whereas PGE2 promotes it PGE2 inhibits reabsorption of Na+ in kidney, influences reabsorption of water, and stimulates renin secretion; PGD2 increases diuresis and excretion of Na+ and K+. PGI2 produced in kidney causes vasodilation and has a protective effect

Mechanism of prostanoid action Through G-protein-linked receptors: a) Gs stimulate adenylate cyclase (AC), i.e. formation of cAMP which activates protein kinase A (PKA) b) Gi inhibit adenylate cyclase (e.g. PKA)

c) Gq activates phospholipase C (Ca2+ also necessary) that cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), yielding inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG); DAG together with Ca2+ activates protein kinase C, IP3 opens Ca2+ channels of the ER +

Biological roles of thromboxanes Thromboxanes are synthesized by platelets and, in general, cause vasoconstriction and platelet aggregation TXA2 is also produced in kidney (by podocytes and other cells) where it causes vasoconstriction and mediates the response to ANGII Thus, both thromboxanes and prostaglandins regulate blood coagulation; in Eskimos, the increased intake of eicosapentaenoic acid and group 3 prostanoids may be responsible for their low incidence of heart diseases and prolonged clotting times since TXA3 is a weaker aggregator than TXA2 and both PG3 and TXA3 inhibit arachidonate release and formation of TXA2

Biological roles of leukotrienes LTs are produced mainly in leukocytes that also express receptors for LTs coupled with G-proteins and AC or PLC Leukotrienes are very potent constrictors of the bronchial airway musculature: a mixture of LTC4, LTD4, and LTE4 is called the slow-reacting substance of anaphylaxis (SRS-A) They increase vascular permeability They cause attraction (LTB4) and activation of leukocytes (primarily eosinophils and monocytes), promote diapedesis to the site of injury (i.a. increase expression of integrins on the leukocyte surface and vascular permeability), enhance phagocytosis They regulate vasoconstriction they participate in inflammation, hypersensitivity (asthma) as well as host defense

LTs in host defense (receptors of LTs) (induction of gene expression) (activation of NADPH oxidase) (synthesis of iNOS) (release from neutrophils) (LTs support diapedesis, delay apoptosis of leukocytes)

BUT: Overproduction of LTB4 was demonstrated in: Crohn´s disease rheumatoid arthritis psoriasis cystic fibrosis Leukotrienes are also suspected of participating in atherosclerosis development Excessive bronchoconstriction can be found in some forms of asthma

Lipoxins Lipoxins are produced mainly by leukocytes and platelets stimulated by cytokines (IL-4, TGF-β): a) 5-lipoxygenase (5-LO) of neutrophils produces leukotriene LTA4 which enters platelets where it is converted by 15-LO into LXA4 or LXB4 b) 15-LO of epithelial cells and monocytes forms 15-HPETE which becomes a substrate of 5-LO and epoxid hydrolase of leukocytes …transcellular biosynthesis Principal products: LXA4, LXB4

Biological roles of lipoxins Unlike pro-inflammatory eicosanoids, lipoxins attenuate the inflammation and appear to facilitate the resolution of the acute inflammatory response Hypothesis: in the first phase of the inflammatory response, leukotrienes are produced (e.g. chemoattractive LTB4) → then, the level of PGs (PGE2) rises and PGs „switch“ the eicosanoid synthesis from the 5-LO pathway (LTs) to the 15-LO pathway which, in the second phase, produces lipoxins promoting the resolution of inflammation and preventing its transformation into chronic inflammation That is why potential therapeutic use of LXs in the treatment of distinct inflammatory diseases (glomerulonephritis, asthma) is being extensively studied Function via G-protein-coupled receptors

Effects of LXs that mediate the resolution of inflammation LXs inhibit chemotaxis of neutrophils and eosinophils Inhibit formation of ROS (neutrophils, lymphocytes) and ONOO- (neutrophils) Inhibit diapedesis of neutrophils Inhibit production of specific cytokines by leukocytes Stimulate non-inflammatory phagocytosis (of apoptotic neutrophils, cell debris…) They are also capable of antagonizing LT receptors Anti-inflammatory effects of LXs were demonstrated in the lung (asthma), kidney, epithelium of colon, inflammation of skin (psoriasis, contact dermatitis) Nezánětlivá fagocytóza – roste produkce TGF-β1 a klesá produkce IL-8

Phases of inflammation and their mediators

LXs affect not only the cells of the myeloid line: LXs inhibit the contraction of the bronchial smooth muscle Inhibit production of distinct cytokines by the cells of colon, fibroblasts, and dendritic cells In kidney, they cause vasodilation Inhibit the interaction between leukocytes and endothelial cells

Biological roles of HETEs 5-HETE participates in host defense against bacterial infection (chemotaxis and degranulation of neutrophils and eosinophils) 20-HETE causes vasoconstriction (by the effect on the smooth muscle of vessels) and plays a crucial role in the regulation of the vascular tonus; moreover, in kidney it regulates Na+ excretion, diuresis, and blood pressure 12- a 15-HETE are produced in kidney, too, and participate in the regulation of the renin-angiotensin system (they probably mediate feed-back inhibition of renin; 12-HETE mediates also synthesis of aldosteron induced by ANGII)

Biological roles of hepoxilins They participate in the regulation of inflammatory response HXA3 stimulates insulin secretion by pancreatic β cells Under oxidative stress, HXA3 upregulates the expression of glutathione peroxidase and heat-shock proteins…compensatory defense response to protect the cell viability