1. EICOSANOIDS

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

3. EICOSANOID BIOSYNTHESIS

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

5. 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)

6. 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

7. 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…)

8. 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

9. 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:

10. 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!

11. 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

12. 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

13. 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

14. 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

15. 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

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

17. 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

18. 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

19. 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

20. Peptidoleukotriene biosynthesis:
Requires glutathione!!!

21. 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…)

22. 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

23. BIOLOGICAL ROLES OF EICOSANOIDS

24. 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

25. 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

26. 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

27. 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)

29. 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 +

30. 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

31. 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

32. 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)

33. 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

34. 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

35. 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

36. 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

37. Phases of inflammation and their mediators

38. 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

39. 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)

40. 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