Biochemistry - 40 - Eicosanoid Metabolism 2023.pdf

Eicosanoids Lecture 40 Reference: Lieberman and Peet, Chapter 31 (pp 688 – 695) Bluefield University – VCOM Campus MABS Program Biochemistry Jim Mahaney, PhD 1 Lecture Objectives a. Relate the term eicosanoid and identify the main members of this group. b. Recall the general mechanisms of action...

Eicosanoids Lecture 40 Reference: Lieberman and Peet, Chapter 31 (pp 688 – 695) Bluefield University – VCOM Campus MABS Program Biochemistry Jim Mahaney, PhD 1 Lecture Objectives a. Relate the term eicosanoid and identify the main members of this group. b. Recall the general mechanisms of action of eicosanoids, including their effects on tissues, their half lives, and where they are produced. c. Compare and contrast the direct versus indirect method of phospholipase A2 (PLA2) activation that leads to the release of arachidonic acid and the subsequent production of eicosanoids. d. Identify the characteristic structural features of prostaglandins, thromboxanes and leukotrienes. Identify the common starting material for these compounds. e. Identify the three major enzyme pathways that convert arachidonic acid to the major classes of eicosanoid (prostaglandin/thromboxane, HETE/leukotriene and epoxides). f. Recall the general nomenclature of prostaglandins and thromboxanes to the level of what different letters, subscripts and Greek letters indicate about these molecules. g. Recall the synthesis pathway of prostaglandins and thromboxanes from arachidonic acid, including the central role of the cyclooxygenase enzyme in the process. h. Identify the primary structural modification of prostaglandins and thromboxanes that inactivates these molecules and terminates their signaling. i. Compare and contrast the mechanisms by which steroidal and non-steroidal anti-inflammatory drugs block the formation of prostaglandins and thromboxanes. j. Recall the production of leukotrienes and HETE and lipoxins from arachidonic acid. 2 2 Objective A Introduction: Eicosanoids • Eicosanoids = class of signaling compounds synthesized from 20 carbon FAs • arachidonic acid most common source in humans • consist primarily of prostaglandins (PG), thromboxanes (TX) and leukotrienes (LT) • elicit a wide variety of effects • Commonly prostaglandins result in symptoms like pain, inflammation, fever, nausea and vomiting • normally produced in very small amounts, and they have a very short half-life • among the most potent signaling compounds we make. • metabolized to inactive products at the site of synthesis • Formed in most tissues and act locally (compare to hormones which are produced in specialized tissues and act system wide. ** Many exhibit autocrine signaling: eicosanoid is produced by the cell it affects - and neighboring cells too 3 3 Objective B Eicosanoid Function is Diverse! Synthesis, lifetime and function of eicosanoids is tissue-dependent…more later… 4 4 Example: Thromboxane A2 and Platelet Activation • Platelets: Adhere to site of damage and become activated, provide a scaffold for fibrin binding and clot formation, also provide factors and Ca2+ to drive the clot formation process. • Early signaling event leads to the production of TXA2 from arachidonic acid. Autocrine signaling. • TXA2 activates platelet cells, stimulates other platelet cells to aggregate, is prothrombotic … and is a potent vasoconstrictor. 5 5 Objective C Arachidonic Acid (AA) Signaling: Big Picture Arachidonic acid is the most common FA released by PLA2 following signaling PLA2 cleaves the arachidonic acid from position 2 of the phospholipid to form free arachidonic acid … ** OR ** … DAG lipase can cleave arachidonic acid from position 2 on DAG after PLC cleaves PIP2 Arachidonic acid enters the eicosanoid pathway. See note below 6 6 Objective C Arachidonic Acid (AA) Liberation: Direct Pathway Arachidonic acid is bound to position 2 of a glycerophospholipid in the inner half of the plasma membrane. Excitation of a G-protein coupled receptor leads to the activation of the enzyme Phospholipase A2, which cleaves the glycerol-fatty acid ester bond at Carbon 2. Free arachidonic acid enters the eicosanoid pathway within the membrane environment. Disregard the blue rectangles in this part of the diagram. These are part of the “indirect pathway” 7 7 Arachidonic Acid (AA) Signaling: Indirect Pathway Objective C In the indirect pathway, G-protein receptor mediated activation of phospholipase C results in the liberation of IP3, which activates the IP3 channel of the ER, resulting in an increase in the cell Ca2+ concentration. The increased cell Ca2+ stimulates PLA2, which cleaves the arachidonic acid from position 2 of the phospholipid to form free arachidonic acid. Arachidonic acid bound to the product diacylglycerol (DAG) is liberated by DAG lipase. Arachidonic acid enters the eicosanoid pathway. 8 8 Objective C A Simpler Version Direct Indirect • PUFAs usually attached to glycerol backbone of phospholipid. • Phosphoatidylcholine or phosphatidylinositol. • PUFA is cleaved from phospholipids by phospholipase A2 • Triggered by stimulus binding to membrane receptors • histamine or cytokines • Steroidal anti-inflammatory agents inhibit phospholipase A2 Alternatively, phosphatidyl inositol bisphosphate can be cleaved by phospholipase C liberating a 1,2diacylglycerol containing the PUFA, which can be cleaved directly, or in two steps by other lipases. 9 9 Prostaglandins (PG), Thromboxanes (TX) and Leukotrienes (LX) Objective D • Prostaglandins are fatty acids: • • • • • 20 carbon atoms an internal, saturated 5-carbon ring a hydroxyl group at carbon 15, a double bond between carbons 13 and 14 various substituents on the ring • Thromboxanes structure similar to PG, but they contain a 6membered ring • Some TXs have an additional oxygen atom bridging carbons 9 and 11 of the ring. • Leukotrienes are characterized by three consecutive double bonds (triene) • Also have an oxygen atom (or two) bound as an OH or as an epoxide • All synthesized from polyunsaturated fatty acids (PUFAs) containing 20 carbons and 3-5 double bonds (typically arachidonic acid) 10 10 Three Pathways • Once released, PUFA is converted to eicosanoids by three major pathways • Tissue dependent • Cyclooxygenase (COX): leads to prostaglandins and thromboxanes via PGG2 • COX-1 is a constitutive enzyme in gastric mucosa, platelets, vascular endothelium, and kidney • COX-2 is inducible and is generated in response to inflammation. Mainly expressed in activated macrophages but other tissues also Objective E • Lipoxygenase: leads to leukotrienes, HETE and lipoxins via HPETE • lipoxygenase is a dioxygenase that inserts a peroxide • Cytochrome P450: leads to diHETE and HETE via epoxides 11 11 Objective F PG and TX: Nomenclature • PG and TX designate type • All have a capital letter that designates ring substituents • most common classes are A,E, F • Some have a subscript, which designates the number of double bonds in the linear portion of the hydrocarbon chain (but not within the ring) See Figure 35.6 for example. • compounds in the 2-series are of the greatest significance in humans • The PGF series has two hydroxyl groups on the ring. A Greek subscript is used to denote the position of the hydroxyl group at carbon 9 12 12 Objective G Synthesis of PG and TX from Arachidonic Acid: The Cyclooxygenase Pathway • Oxygen is added and a 5-carbon ring is formed by a cyclooxygenase (COX) that produces the initial prostaglandin, which is then converted to other classes of PGs or TXs. • PGH2 may be converted to TXA2 in the thromboxane synthesis pathway • Next step is tissue specific: function and then degradation 13 13 Objective F Series • Notice the positions and configurations of double bonds in the non-ring portion of the hydrocarbon: • This determines the “series” of prostaglandin molecule • Different series have different functions (inflammatory versus non-inflammatory, etc). 14 14 Diversity of Function: Prostaglandins http://www.sciencemag.org/content/vol294/issue5548/images/large/se4619990001.jpeg 15 15 Objective H Degradation of PG and TX • PG and TX designed to elicit a short-lived signal: must degrade them to stop signal • Rapidly inactivated by oxidation of the 15-hydroxyl group critical for activity active • Hydroxyl is oxidized to a ketone • Half life is seconds to minutes • Breakdown products are excreted in urine ││ O inactive 16 16 Objective E A Word on COX-1 and COX-2 • Cyclooxygenase Enzymes: three isoforms • COX 1: constitutive, found in nearly all tissues • COX 2: inducible, near zero level in tissues, mainly in macrophages and other cells related to inflammation • COX 3: splice variant of COX 1 – same function • Arachidonic Acid is the main substrate, PGH2 is the main product: leads to the “Series 2” prostanoids = more inflammatory • DGLA and EPA (covered in later slides) are also substrates of COX • DGLA (Dihomo-gamma-linolenic acid) à “Series 1” prostanoids • EPA (Eicosapentanoic acid) à “Series 3” prostanoids • Dietary consumption of DGLA and EPA can lead to reduced inflammation. • These molecules will compete for arachidonic acid binding to COX • Block Series 2 prostanoid formation but favor Series 1 and 3 prostanoids 17 17 Steroidal and Non-Steroidal Anti-inflammatory Agents Objective I • NSAIDs inhibit prostaglandin formation…decrease pain and inflammation • Aspirin acetylates an active site serine • • • Mechanism based inhibition à destroys active site. Aspirin is more potent against COX-1 than COX-2 There are specific COX-2 inhibitors (more later in Pharmacology) • Ibuprofen binds non-covalently to COX to inhibit • Competitive inhibition • Acetaminophen (not an NSAID) also inhibits COX by competitive inhibition mechanism. • Good for pain, fever but not a significant anti-inflammatory drug. • Note: These drugs stop AA conversion to PG by COX…but they don’t inhibit AA liberation from the phospholipid in the membrane. So free AA can form other eicosanoids by other routes • Steroidal anti-inflammatory drugs (hydrocortisone, prednisone) block prostaglandin formation by a more global mechanism, primarily by down regulating or inhibiting immune responses that drive inflammation. 18 18 Key Biochemical Principles: Cyclooxygenase 1 and 2 • Biochemical etiology: NSAIDs primarily inhibit COX-1, and here the COX-1 of the stomach lining that synthesizes prostaglandins that have a protective effect on the gastric mucosa. • COX-1 is constitutive and present in most tissues • COX-2 is inducible and is nearly absent under normal conditions but elevated with inflammation • How can coxib drugs help? Traditional NSAIDs will inhibit both COX1 and COX-2. The coxib drugs are NSAIDs that preferentially inhibit COX-2. So a coxib drug can take care of the inflammation pain (COX-2) while leaving constitutive COX-1 unaffected. • How is aspirin different from ibuprofen? • Aspirin covalently modifies COX-1 of platelet cells, thus irreversibly inhibiting thromboxane A2 formation for the lifespan of the platelet cell. • Ibuprofen will inhibit this COX-1 noncovalently, and so as the blood level ibuprofen drops over time, the inhibitory action of ibuprofen will decrease. The enzyme is reversibly inhibited. 19 19 Synthesis of Leukotrienes, HETE and Lipoxins: Lipoxygenase Pathway Objective J • arachidonic acid is starting material • Lipoxygenase incorporates an oxygen molecule onto a carbon of one of several double bonds : activity of enzyme is tissue dependent • Starts with the formation of HPETEs (hydroperoxyeicosatetraenoic acids) • HPETEs are reduced to the corresponding hydroxyl metabolites (HETEs) or metabolized to form leukotrienes or lipoxins 20 20 Diversity of Function: Leukotrienes http://www.sciencemag.org/content/vol294/issue5548/images/large/se4619990002.jpeg 21 21 Epoxides: the Cytochrome P450 Pathway • Note cytochrome P450 pathway. Allows for mono-oxygenase chemistry needed for this reaction • Produces epoxides, HETEs, diHETEs. • Prevalent in ocular, vascular, endocrine and renal systems – and cell proliferation. 22 22 Review The Alpha and “Omega” of Fats ω-9 • Double bonds can be counted from carboxyl carbon (α) or relative to the terminal (ω) carbon – more common clinically. ω-6 ω-3 • In humans, ω-3, ω-6 and ω-9 fatty acids predominate Recall: Linoleic acid is used to make arachidonic acid (an ω-6 fat) α-linolenic acid is used to make eicosapentanoic acid (EPA), which is a precursor for docosapentaonic acid (DPA) then docosapentanoic acid (DHA) – all ω-3 fats 23 23 Objective F Other Eicosanoid Pathways Series 1 and 3 are good, Series 2 is inflammatory DHA, EPA and DPA all inhibit inflammation (i.e., “soften the inflammatory role of arachidonic acid) DGLA and EPA are competitive inhibitors of COX enzymes 24 24 Question A patient has an enzyme deficiency that prevents the conversion of the peroxyl group at C-15 on Prostaglandin G to an –OH group during the metabolism of this molecule. How will this affect the biologic function of the PGG molecule? A. The peroxyl-bearing molecule will have a much longer half-life and be far more potent. B. The peroxyl-bearing molecule will behave identically to the - OH bearing molecule. C. The peroxyl bearing molecule will be completely inactive. 25 25 Question A patient has an enzyme deficiency that completely blocks production of ALL PG, TX and LT molecules. Which of the following enzyme deficiencies can account for this observation? a. phospholipase A2 b. cyclo-oxygenase c. lipoxygenase d. Cytochrome P450 26 26 Thank you! 27 27