Lipid Mediators: Synthesis, Metabolism, and Function. Overview

Transcription

1 Lipid Mediators: Synthesis, Metabolism, and Function Biochemistry 201 Oct 23, 2007 Electa Park Overview Biological function of lipid mediators Arachidonic acid (AA) synthesis AA metabolites and related enzymes Prostaglandins, thromboxanes, leukotrienes EETS, Lipoxins, Isoprostanes Inhibiton of AA metabolizing enzymes Receptors and Metabolism 1

2 A protective response of the body Elicited by cell injury (microbes, toxins) Mechanical, thermal, chemical, and bacterial insult Goal: eradication of harmful agents and initiation of the healing process Arachidonic acid (AA) metabolites participate in the pathogenesis of inflammation and fever Inflammation Hallmarks: Pain- PGE 2, LTB 4, LTC 4, LTD 4, PAF (hyperalgesia) Fever- PGE 2 Vasodilation- PGE 2, PGI 2 Increased vascular permeability- PGE 2, PGD 2, PGI 2, LTC 4, LTD 4, PAF Chemotaxis and Leukocyte activation- LTB 4, PAF Cell Migration- LTB 4 Tissue damage Anaphylaxis: LTC 4, LTD 4 2

3 Other Effects of AA Metabolites Bronchioconstrictor- LTs, PAF Asthma, acute insult Platelet aggregation- Promoted by TXA 2 Inhibited by PGI 2 Gastric cytoprotection-pgi 2, PGE 2, PGF2, PAF Smooth muscle contraction, inhibit acid secretion in stomach, enhance mucus secretion and mucosal blood flow Other Effects of AA Metabolites Renal function-pgi 2, PGE 2 Modulate renal blood flow, urine formation Reproduction-PGE 2, PGF 2, PAF Conception, labor, menstruation, male reproductive mechanism Musculoskeletal-PGs Bone remodelling (osteoclast and osteoblasts) Rheumatoid Arthritis 3

4 PLA 2 Arachidonic Acid COX 1/2 lipoxygenase Prostaglandins Leukotrienes Prostacyclin Thromboxanes 4

5 Cellular Membranes: Phospholipid Bilayers Phosphatidylcholine major source of AA in body; enriched in membranes of cells of myeloid origin and endothelium Phospholipase A 2 Secretory - first isolated ~10 groups, only IIA, V, and X involved in PG synthesis Low molecular weight (~14 kda) Restricted expression IIA - small intestine V - eye, heart, pancreas X - testis, stomach Have specific cell surface receptors Are involved in signaling directly; can modulate PG production via receptor binding and internalization 5

6 Phospholipase A 2 Cytosolic 85 kda major isoform (cpla 2 -α), also β (110 kda) and γ (61 kda) α, β Ca 2+ -dependent γ Ca 2+ -independent Primary source of AA for PGs, etc. Found in most tissues Activated by Ca 2+ binding and membrane association Activation also regulated by phosphorylation, phosphoinositides, scaffold proteins Site of action: Nuclear membrane Also reported on ER, mitochondrial membranes Structure of cpla 2 6

7 Structure of cpla 2 Structure of cpla 2 7

8 Structure of cpla 2 (Ca 2+ ) 2 cpla 2 Active Site Serine 8

9 PLA 2 Arachidonic Acid COX 1/2 lipoxygenase Prostaglandins Leukotrienes Prostacyclin Thromboxanes Cyclooxygenase 1 & 2 (Cox 1/2) Also known as Prostaglandin Endoperoxide H Synthase 1 & 2 Bind AA with K m 5 µm, O 2 5 µm Cox 1 constitutive expression in most tissues Cox 2 Expressed in nervous, immune, renal tissue Expression inducible by inflammatory and proliferative signals May play a role in modulating endogenous cannabinoid signaling K m for 2-arachidonylglycerol 5 µm; for arachidonylethanolamide 24 µm 9

10 COX 1/2: One Enzyme, Two Activities Cyclooxygenase activity- Dependent on active site tyrosyl radical Results in PGG 2 Can activate other COX enzymes Peroxidase activity- Heme dependent Leads to production of PGH 2 Short t 1/2 Rapidly metabolized to more stable PGs COX1 Homodimer Epidermal Growth Factor Domain Membrane Binding Domain Catalytic Domain Flurbiprofen Heme 10

11 COX1 Monomer 11

12 Cyclooxygenase activity dependent on tyrosyl radical Peroxidase activity dependent on heme group

13 PGI Synthase TXA Synthase PGH 2 PGI 2 (prostacyclin) TXA 2 6-keto-PGF 1α PGD Synthase PGF Synthase TXB 2 PGE Synthase PGD 2 PGF 2 (9α, 11α-PGF 2α ) PGE 2 PGH2 Metabolizing Enzymes: Localization PGD 2 synthase (isomerase) Immune cells PGE 2 synthase (isomerase) Most cells PGF 2 synthase (reductase) Uterus, seminal vesicles PGI 2 (Prostacyclin) synthase Endothelial cells Thromboxane synthase Platelets 13

14 Thromboxane Synthase Thromboxane Synthase 14

15 Cell activation: Generic cell cytokines, growth factors, mechanical trauma VSMC Vasoconstriction Vasodilation Aggregation Platelets Declumping Uterine smooth muscle Th2 lymphocyte Contraction, parturition Chemotaxis Allergic asthma Lung epithelial cell Osteoclast Bone resorption Fever Generation Neurons of OVLT of PDA X = NSAIDS celecoxib rofecoxib Ovarian CO cells Spinal neurons Maturation for ovulation and fertilization Pain response PLA 2 Arachidonic Acid COX 1/2 lipoxygenase Prostaglandins Leukotrienes Prostacyclin Thromboxanes 15

16 Lipoxygenase 5-Lipoxygenase 5-HPETE Leukotrienes 5,12-DHETE 8-Lipoxygenase 8-HPETE 11-HPETE 8-HETE 11-HETE 12-lipoxygenase 12-HPETE 15-lipoxygenase 15-HPETE 12-HETE 5,12-DHETE 5S,12S-THETE 15-HETE 16,15-DHETE 8,15-DHETE DHETE, HETE - Leukocytes, eosinophils, platelets = muscle contraction, cell degranulation, superoxide production, chemotaxis AA Metabolism by 5-Lipoxygenase 16

17 Leukotriene (LT) Synthesis LTA 4-5-Lipoxygenase Neutrophils, basophils, eosinophils, mast cells, monocytes, macrophages LTB 4 - LTA 4 hydrolase Most cells LTC 4 - LTC 4 synthase (glutathione transferase) Basophils, eosinophils, mast cells, monocytes/macrophages, platelets LTD 4 - γ-glutamyl transpeptidase, γ-glutamyl leukotrienase Extracelluar metabolite of LTC 4 LTE 4 - aminopeptidase activity towards LTD 4 17

18 LTA 4 Synthesis Step LTA 4 Synthesis Step LTA 4 10 H H H 7 + H H 2 O H HO-O 6 O-OH 18

19 Leukotriene synthesis cpla 2α 5- LO LTA 4 -H 5- LO cpla 2α FLAP LTC 4 -S 5- LO FLAP 5- LO LTA 4 -H LT Synthesis LTA 4-5-Lipoxygenase Neutrophils, basophils, eosinophils, mast cells, monocytes, macrophages LTB 4 - LTA 4 hydrolase Most cells LTC 4 - LTC 4 synthase (glutathione transferase) Basophils, eosinophils, mast cells, monocytes/macrophages, platelets mgst 2 or mgst 3 (testis) LTD 4 - γ-glutamyl transpeptidase, γ-glutamyl leukotrienase Extracelluar metabolite of LTC 4 19

20 LTA 4 Hydrolase LTA 4 -H active site 20

21 LT Synthesis LTA 4-5-Lipoxygenase Neutrophils, basophils, eosinophils, mast cells, monocytes, macrophages LTB 4 - LTA 4 hydrolase Most cells LTC 4 - LTC 4 synthase (glutathione transferase) Basophils, eosinophils, mast cells, monocytes/macrophages, platelets mgst 2 or mgst 3 (testis) LTD 4 - γ-glutamyl transpeptidase, γ-glutamyl leukotrienase Extracelluar metabolite of LTC 4 LTA4 Metabolites Cell activation Neutrophil Chemotaxis Most Tissues LTA 4 Airway SMC, Postcapillary venul endothelium LTB 4 Bronchioconstriction, edema Heart, adrenal, brain, spleen 21

22 Lipoxins Lipoxin Function Cell-cell interactions Endogenous antiinflammatory substance Inhibits neutrophil activation, migration, and chemotactic signals Inhibits TNF, NFκB, cytoprotective for enterocytes, stimulates macorphage phagocytosis of neutrophils 22

23 Other enzymes that metabolize AA Cytochrome P450 enzymes ER resident enzymes Main actions in renal and vascular systems Action via receptor binding and modulation of intracellular signaling pathways Epoxyeicosatrienoic acid Mechanism of Action: Activate smooth muscle large conductance Ca 2+ activated K + channels (Bk Ca ) which hyperpolarizes the smooth muscle causing vasorelaxation. Endothelium-derived hyperpolarizing factor. 23

24 Isoprostanes Non-cyclooxygenase isomers of prostaglandins: auto-oxidation products of polyunsaturated fatty acids. Stimulate the TP receptors Markers of lipid peroxidation, stimulate platelet aggregation, cell proliferation and vasoconstriction. PLA 2 NSAIDS Arachidonic Acid COX 1/2 lipoxygenase Prostaglandins Leukotrienes Prostacyclin Thromboxanes 24

25 Inhibitors of PG Synthesis COX inhibitors- Non-Steroidal Anti-Inflammatory Drugs (NSAIDS) Salicylates Arylpropionic acid derivatives Acetaminophen???? Selective COX 2 inhibitors History The bark of willow was prescribed as far back as 400B.C by Hippocrates as a pain reliever and fever reducer First purified in 1829 by Leroux Introduced as aspirin by Bayer and Co. of Germany as an antiinflammatory in

26 Mechanisms of Action NSAIDS were used for years without any knowledge of how it worked In 1971 Vane et al demonstrate that low concentrations of aspirin inhibit the enzymatic production of prostaglandins Salicylates O C CH 2 Aspirin Salycylic acids Diflunisal (arthritis) 26

27 Salicylates Mechanism of action NH 2 Acetylates proteins including COX-1 & 2 Irreversible inhibition O - CH O O C CH 2 salicylate NH 2 O C O CH CH 2 O Aspirin-Triggered Lipoxin (ATL) 27

28 Inhibitors of PG Synthesis COX inhibitors- Non-Steroidal Anti-Inflammatory Drugs (NSAIDS) Salicylates Arylpropionic acid derivatives Ibuprofen (Motrin, Nuprin, Advil) Naproxin (Aleve, Naprosyn, Anaprox) Flurbiprofen (Ansaid) Ketoprophen (Orudis) Oxaprozin (Daypro) - long half-life Acetaminophen???? Selective COX 2 inhibitors Arylproprionic Acid Derivatives Mechanism of Action Competitive inhibitors Block cyclooxygenase activity Reversibly bind to COX 1 & 2 inhibiting interaction with arachidonic acid COX 1 and Flurbiprofen COX 2 and SC

29 Proprionic Acid Derivatives: Competitive COX Inhibitors Ibuprofen in AA binding site of COX NSAID & Propionic acid derivative Activities Antiinflammatory Treatment of muskuloskeletal disorders (rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis) Symptomatic relief only Antipyretic Reduce body temperature in febrile states Analgesic Low to moderate pain caused by inflammation 29

30 Analgesic Antiinflammatory Acetominophen Not antipyrogenic Doesn t inhibit COX1/2; may inhibit COX3 No longer considered an NSAID Hepatotoxicity when combined with alcohol Glutathione depletion Inhibitors of PG Synthesis COX inhibitors- Non-Steroidal Anti-Inflammatory Drugs (NSAIDS) Salicylates Arylpropionic acid derivatives Acetaminophen???? Selective COX 2 inhibitors 30

31 COX-2 Inhibitors Celecoxib (Celebrex) Rofecoxib (Vioxx) Valdecoxib (Bextra) COX-2 Inhibitors Mechanism of action: Selectively inhibits COX-2 which is induced locally at the site of inflammation Does not interfere with constitutive form that protects the gastrointestinal tract Drug companies thought they had a safer drug to treat forms of osteoarthritis Vioxx recall- COX-2 metabolites improtant for platelet deaggregation and vascular healing Major cardiovascular complications» Heart attack» Stroke 31

32 Merck Announces Voluntary Worldwide Withdrawal of VIOXX WHITEHOUSE STATION, N.J., Sept. 30, 2004 Merck & Co., Inc. today announced a voluntary worldwide withdrawal of VIOXX (rofecoxib), its arthritis and acute pain medication. The company s decision, which is effective immediately, is based on new, threeyear data from a prospective, randomized, placebo-controlled clinical trial, the APPROVe (Adenomatous Polyp Prevention on VIOXX) trial. PLA 2 Arachidonic Acid PAF COX 1/2 lipoxygenase Prostaglandins Leukotrienes Prostacyclin Thromboxanes 32

33 Platelet Activating Factor Lipid mediator derived from phosphatidylcholine Platelet and leukocyte activation Cell polarization Integrin activation Priming (for degranulation) Redistribution of surface ligands Displayed on cell surface of endothelial cells PAF Synthesis 33

34 Lipid Mediators elicit their effects through GPCRs G Protein-Coupled Receptors Also called 7-transmembrane (TM) receptors Couple to trimeric G-proteins that are activated by ligand binding Have complex biology; not simply on-off switches PAF Receptor 34

35 TXA 2 Receptor PGE Receptor & Isoforms 35

36 Prostaglandin Signaling: Receptors and 2nd Messengers PGD 2 DP G s camp PGE EP 1 IP 3 /DAG/Ca 2+ PGE EP 2 G s camp PGE EP 3,4 camp PGF 2 FP IP 3 /DAG/Ca 2+ PGI IP G o K +, TXA2 TP IP 3 /DAG/Ca 2+ 36

37 EET Receptor system Metabolism of Lipid Mediators Cytochrome P450 4A, 4F ω-oxidation β-oxidation Peptidase activity Make compounds less hydrophobic excretion in urine 37

38 References Phospholipases Diaz BL, JP Arm Phospholipase A2, Prostaglands Leukot and Essent Fatty Acids 69 (2003) Dessen A Structure and Mechanism of human cytosolic phospholipase A2, Biochim Biophys Acta 1488 (2000) COX1/2 & PGs Rouzer CA, LJ Marnett Structural and functional differences between cyclooxygenases: Fatty acid oxygenases with a critical role in cell signaling Biochem Biophys Res Comm 338 (2005) Smith WL, I Song The enzymology of prostaglandin endoperoxide H synthases 1- and 2, Prostaglands other lipid mediat (2002) Garavito RM, MG Malkowski, DL DeWitt the structures of prostaglandin endoperoxide H synthases-1 and -2, Prostaglanding other lipid mediat (2002) Gupta K, BS Selinsky, CJ Kaub, AK Katz, PJ Loll The 2.0 A Resolution Crystal Structure of Prostaglandin H2 Synthase-1: Structural Insights into an Unusual Peroxidase, J Mol Biol 335 (2004) Thromboxane Wang L-H, RJ Kulmacz Thromboxane synthase: structure and function of protein and gene Prostaglandins other lipid mediat (2002) Lipoxygenases and LTs Murphy RC, MA Gijon Biosynthesis and metabolism of leukotrienes Biochem J 405 (2007) PAF Prescott SM, GA Zimmerman, DM Stafforini, TM McIntyre Platelet-Activating Factor and Related Lipid Mediators Annu Rev Biochem 69 (2000) Inhibitors Bertolini A, A Ottani, M Sandrini Selective COX-2 Inhibitors and Dual Acting Anti-inflammatory Drugs: Critical Remarks Curr Med Chem 9 (2002) Rajakariar R, MM Yaqoob, DW Gilroy COX-2 in Inflammation and Resolution Mol Interv 6 (2006) Receptors Narumiya S Prostanoid Receptors: Structure, Function, and Distribution Ann N Y Acad Sci 744 (1994) CYP 450 Kalsotra A, HW Strobel Cytochrome P45 4F subfamily: At the crossroads of eicosanoid and drug metabolism Pharmacol Ther 112 (2006) PG/LT/TX function Miller SB Prostaglandins in Health and Disease: An overview J Semarthrit 3 (2006) William KI, GA Higgs Eicosanoids and Inflammation J Pathol 156 (1988)