lipid mediators: synthesis, metabolism, and function · –non-steroidal anti-inflammatory drugs...

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Lipid Mediators: Synthesis,Lipid Mediators: Synthesis,Metabolism, and FunctionMetabolism, and Function

Biochemistry 201Oct 23, 2007Electa Park

OverviewOverview

• 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

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• A protective response of the body

• Elicited by cell injury (microbes, toxins)– Mechanical, thermal, chemical, and bacterial insult

• Goal: eradication of harmful agents and initiationof the healing process

• Arachidonic acid (AA) metabolites participate inthe pathogenesis of inflammation and fever

InflammationInflammation• Hallmarks:

– Pain- PGE2, LTB4, LTC4, LTD4, PAF (hyperalgesia)– Fever- PGE2– Vasodilation- PGE2, PGI2– Increased vascular permeability- PGE2, PGD2, PGI2, LTC4,

LTD4, PAF– Chemotaxis and Leukocyte activation- LTB4, PAF– Cell Migration- LTB4– Tissue damage

• Anaphylaxis:– LTC4, LTD4

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Other Effects of AAOther Effects of AAMetabolitesMetabolites

• Bronchioconstrictor- LTs, PAF– Asthma, acute insult

• Platelet aggregation-– Promoted by TXA2

– Inhibited by PGI2

• Gastric cytoprotection-PGI2, PGE2, PGF2, PAF– Smooth muscle contraction, inhibit acid secretion in stomach,

enhance mucus secretion and mucosal blood flow

• Renal function-PGI2, PGE2– Modulate renal blood flow, urine formation

• Reproduction-PGE2, PGF2, PAF– Conception, labor, menstruation, male reproductive

mechanism

• Musculoskeletal-PGs– Bone remodelling (osteoclast and osteoblasts)– Rheumatoid Arthritis

Other Effects of AAOther Effects of AAMetabolitesMetabolites

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PLA2

Arachidonic Acid

Prostaglandins Leukotrienes

Prostacyclin Thromboxanes

COX 1/2 lipoxygenase

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Cellular Membranes:Cellular Membranes:Phospholipid BilayersPhospholipid Bilayers

Phosphatidylcholine majorsource of AA in body;enriched in membranes ofcells of myeloid origin andendothelium

• 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

Phospholipase Phospholipase AA22

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Phospholipase Phospholipase AA22

• Cytosolic– 85 kDa major isoform (cPLA2-α), also β (110 kDa)

and γ (61 kDa)• α, β Ca2+-dependent• γ Ca2+-independent

– Primary source of AA for PGs, etc.– Found in most tissues– Activated by Ca2+ 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 cPLAStructure of cPLA22

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(Ca(Ca2+2+))22

cPLAcPLA22 Active Site Serine Active Site Serine

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PLA2

Arachidonic Acid




Cyclooxygenase Cyclooxygenase 1 & 21 & 2(Cox 1/2)(Cox 1/2)

• Also known as Prostaglandin Endoperoxide HSynthase 1 & 2

• Bind AA with Km ≈ 5 µM, O2 ≈ 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• Km for 2-arachidonylglycerol ≈ 5 µM; for

arachidonylethanolamide ≈ 24 µM

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COX 1/2: One Enzyme, TwoCOX 1/2: One Enzyme, TwoActivitiesActivities

• Cyclooxygenase activity-– Dependent on active site tyrosyl radical– Results in PGG2

• Can activate other COX enzymes

• Peroxidase activity-– Heme dependent– Leads to production of PGH2

• Short t1/2• Rapidly metabolized to more stable PGs

Epidermal Growth Factor DomainEpidermal Growth Factor DomainMembrane Binding DomainMembrane Binding DomainCatalytic DomainCatalytic Domain

COX1 COX1 HomodimerHomodimer

FlurbiprofenFlurbiprofenHemeHeme

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COX1 MonomerCOX1 Monomer

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•• Cyclooxygenase Cyclooxygenase activity dependent on activity dependent on tyrosyl tyrosyl radicalradical

•• Peroxidase Peroxidase activity dependent on activity dependent on heme heme groupgroup

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11

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1315

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PGISynthase

PGI2 (prostacyclin)

6-keto-PGF1α

PGD2 PGF2(9α, 11α-PGF2α)

PGDSynthase

PGFSynthase

PGESynthase

PGE2

TXB2

TXA2

TXASynthase

PGH2

PGH2 Metabolizing Enzymes:PGH2 Metabolizing Enzymes:LocalizationLocalization

• PGD2 synthase (isomerase)– Immune cells

• PGE2 synthase (isomerase)– Most cells

• PGF2 synthase (reductase)– Uterus, seminal vesicles

• PGI2 (Prostacyclin) synthase– Endothelial cells

• Thromboxane synthase– Platelets

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Thromboxane SynthaseThromboxane Synthase

Thromboxane SynthaseThromboxane Synthase

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VSMC

Vasoconstriction

Vasodilation

Aggregation

PlateletsDeclumping

Uterine smoothmuscle

Contraction,parturition

Chemotaxis

Th2 lymphocyte

Allergic asthma

Lung epithelial cell

Bone resorptionOsteoclast

Fever Generation

Neurons of OVLT of PDA

Maturation for ovulationand fertilization

OvarianCO cells

Pain response

Spinal neurons

Cell activation: Generic cellcytokines, growth factors, mechanical trauma

X = NSAIDS celecoxib rofecoxib

PLA2

Arachidonic Acid




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LipoxygenaseLipoxygenase• 5-Lipoxygenase 5-HPETE

• 8-Lipoxygenase

• 12-lipoxygenase 12-HPETE

• 15-lipoxygenase 15-HPETE

Leukotrienes

5,12-DHETE

8-HPETE

11-HPETE

8-HETE

11-HETE

12-HETE5,12-DHETE5S,12S-THETE

15-HETE16,15-DHETE8,15-DHETE

DHETE, HETE - Leukocytes, eosinophils,platelets = muscle contraction, cell degranulation,superoxide production, chemotaxis

AA Metabolism byAA Metabolism by5-5-LipoxygenaseLipoxygenase

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Leukotriene Leukotriene (LT) Synthesis(LT) Synthesis

• LTA4 - 5-Lipoxygenase– Neutrophils, basophils, eosinophils, mast cells, monocytes,

macrophages• LTB4 - LTA4 hydrolase

– Most cells• LTC4 - LTC4 synthase (glutathione transferase)

– Basophils, eosinophils, mast cells, monocytes/macrophages,platelets

• LTD4 - γ-glutamyl transpeptidase, γ-glutamylleukotrienase– Extracelluar metabolite of LTC4– LTE4 - aminopeptidase activity towards LTD4

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5

6

10

7

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LTA4 Synthesis Step 1

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5

5

6

10

7

7

10

6

10

H

6

LTA410

H

6

H

O-OHHO-O

H2O

7

10

H

6

H+

LTA4 Synthesis Step 2

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Leukotriene Leukotriene synthesissynthesis

cPLA2α

cPLA2α

5-LO

5-LO

5-LO

5-LO

FLAP

FLAP LTC4-S

LTA4-H

LTA4-H

LT SynthesisLT Synthesis





– mGST 2 or mGST 3 (testis)• LTD4 - γ-glutamyl transpeptidase, γ-glutamyl

leukotrienase– Extracelluar metabolite of LTC4

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LTALTA44 HydrolaseHydrolase

LTALTA44-H active site-H active site

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LT SynthesisLT Synthesis





– mGST 2 or mGST 3 (testis)• LTD4 - γ-glutamyl transpeptidase, γ-glutamyl

leukotrienase– Extracelluar metabolite of LTC4

LTA4 MetabolitesLTA4 MetabolitesNeutrophil

Chemotaxis

Most Tissues

Heart, adrenal,brain, spleen

Bronchioconstriction,edema

Airway SMC,Postcapillary venul endotheliumLTA4

LTB4

Cell activation

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LipoxinsLipoxins

Lipoxin Lipoxin FunctionFunction

• Cell-cell interactions• Endogenous anti-

inflammatory substance• Inhibits neutrophil activation,

migration, and chemotacticsignals

• Inhibits TNF, NFκB,cytoprotective for enterocytes,stimulates macorphagephagocytosis of neutrophils

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Other enzymes thatOther enzymes thatmetabolize AAmetabolize 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 Epoxyeicosatrienoic acidacid• Mechanism of Action: Activate smooth

muscle large conductance Ca 2+ activatedK + channels (BkCa) which hyperpolarizesthe smooth muscle causingvasorelaxation.

• Endothelium-derived hyperpolarizingfactor.

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IsoprostanesIsoprostanes

• Non-cyclooxygenase isomers ofprostaglandins: auto-oxidation products ofpolyunsaturated fatty acids.

• Stimulate the TP receptors• Markers of lipid peroxidation, stimulate

platelet aggregation, cell proliferation andvasoconstriction.

PLA2

Arachidonic Acid




NSAIDS

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Inhibitors of PG SynthesisInhibitors of PG Synthesis• COX inhibitors-

– Non-Steroidal Anti-Inflammatory Drugs(NSAIDS)

• Salicylates• Arylpropionic acid derivatives• Acetaminophen????

– Selective COX 2inhibitors

HistoryHistory• The bark of willow was prescribed as far back as

400B.C by Hippocrates as a pain reliever andfever reducer

• First purified in 1829 byLeroux

• Introduced as “aspirin” byBayer and Co. of Germanyas an antiinflammatory in 1900

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Mechanisms of ActionMechanisms of Action

• NSAIDS were used for years without anyknowledge of how it worked

• In 1971 Vane et al demonstrate that lowconcentrations of aspirin inhibit theenzymatic production of prostaglandins

SalicylatesSalicylates

• Aspirin• Salycylic acids• Diflunisal (arthritis)

CH2CO

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SalicylatesSalicylates• Mechanism of action

– Acetylates proteins including COX-1 & 2COX-1 & 2– Irreversible inhibition

CH

O-

NH2 O

CH2CO

salicylate CHNH2 OCH2C

OO

Aspirin-Triggered Aspirin-Triggered Lipoxin Lipoxin (ATL)(ATL)

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– 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 Arylproprionic Acid DerivativesAcid Derivatives• Mechanism of Action

– Competitive inhibitors– Block cyclooxygenase activity– Reversibly bind to COX 1 & 2COX 1 & 2 inhibiting

interaction with arachidonic acid

COX 1 andFlurbiprofen

COX 2 andSC-588

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Proprionic Proprionic Acid Derivatives:Acid Derivatives:Competitive COX InhibitorsCompetitive COX Inhibitors

Ibuprofen in AAbinding site ofCOX

NSAID & NSAID & Propionic Propionic acidacidderivative Activitiesderivative Activities

– Antiinflammatory• Treatment of muskuloskeletal disorders

(rheumatoid arthritis, osteoarthritis, andankylosing spondylitis)

• Symptomatic relief only– Antipyretic

• Reduce body temperature in febrile states– Analgesic

• Low to moderate pain caused by inflammation

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AcetominophenAcetominophen

• Analgesic• Antiinflammatory• Not antipyrogenic• Doesn’t inhibit COX1/2; may inhibit COX3• No longer considered an NSAID• Hepatotoxicity when combined with alcohol

– Glutathione depletion


– Non-Steroidal Anti-Inflammatory Drugs(NSAIDS)

• Salicylates• Arylpropionic acid derivatives• Acetaminophen????

– Selective COX 2inhibitors

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COX-2 InhibitorsCOX-2 Inhibitors

• Celecoxib (Celebrex)• Rofecoxib (Vioxx)• Valdecoxib (Bextra)

• Mechanism of action:– Selectively inhibits COX-2COX-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 deaggregationand vascular healing

– Major cardiovascular complications» Heart attack» Stroke

COX-2 InhibitorsCOX-2 Inhibitors

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Merck Announces Voluntary WorldwideWithdrawal of VIOXX®WHITEHOUSE STATION, N.J., Sept. 30, 2004—Merck & Co., Inc. today announced a voluntaryworldwide withdrawal of VIOXX® (rofecoxib), its arthritis and acute pain medication.The company’s decision, which is effective immediately, is based on new, three-year data from a prospective, randomized, placebo-controlled clinical trial, theAPPROVe (Adenomatous Polyp Prevention on VIOXX) trial.

PLA2

Arachidonic Acid




PAF

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Platelet Activating FactorPlatelet 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 endothelialcells

PAF SynthesisPAF Synthesis

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Lipid Mediators elicit theirLipid Mediators elicit theireffects through effects through GPCRsGPCRs

• G Protein-Coupled Receptors– Also called 7-transmembrane (TM) receptors

• Couple to trimeric G-proteins that areactivated by ligand binding

• Have complex biology; not simply on-offswitches

PAF ReceptorPAF Receptor

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TXATXA22 Receptor Receptor

PGE Receptor & PGE Receptor & IsoformsIsoforms

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Prostaglandin Signaling:Prostaglandin Signaling:Receptors and 2nd MessengersReceptors and 2nd Messengers

• PGD2 DP Gs ↑cAMP• PGE EP1 IP3/DAG/Ca2+

• PGE EP2 Gs ↑cAMP• PGE EP3,4 ↑↓cAMP• PGF2 FP IP3/DAG/Ca2+

• PGI IP Go K+,• TXA2 TP IP3/DAG/Ca2+

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EET Receptor systemEET Receptor system

Metabolism of Lipid MediatorsMetabolism of Lipid Mediators

• Cytochrome P450 4A, 4F– ω-oxidation– β-oxidation

• Peptidase activity• Make compounds less hydrophobic →

excretion in urine

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ReferencesReferences• Phospholipases

– Diaz BL, JP Arm Phospholipase A2, Prostaglands Leukot and Essent FattyAcids 69 (2003) 87-97

– Dessen A Structure and Mechanism of human cytosolic phospholipase A2,Biochim Biophys Acta 1488 (2000) 40-47

• COX1/2 & PGs– Rouzer CA, LJ Marnett Structural and functional differences between

cyclooxygenases: Fatty acid oxygenases with a critical role in cell signalingBiochem Biophys Res Comm 338 (2005) 34-44

– Smith WL, I Song The enzymology of prostaglandin endoperoxide H synthases1- and 2, Prostaglands other lipid mediat 68-69 (2002) 115-128

– Garavito RM, MG Malkowski, DL DeWitt the structures of prostaglandinendoperoxide H synthases-1 and -2, Prostaglanding other lipid mediat 68-69(2002) 129-152

– Gupta K, BS Selinsky, CJ Kaub, AK Katz, PJ Loll The 2.0 A Resolution CrystalStructure of Prostaglandin H2 Synthase-1: Structural Insights into an UnusualPeroxidase, J Mol Biol 335 (2004) 503-518

• Thromboxane– Wang L-H, RJ Kulmacz Thromboxane synthase: structure and function of

protein and gene Prostaglandins other lipid mediat 68-69 (2002) 409-422

• Lipoxygenases and LTs– Murphy RC, MA Gijon Biosynthesis and metabolism of leukotrienes Biochem J

405 (2007) 379-395• PAF

– Prescott SM, GA Zimmerman, DM Stafforini, TM McIntyre Platelet-ActivatingFactor and Related Lipid Mediators Annu Rev Biochem 69 (2000) 419-445

• Inhibitors– Bertolini A, A Ottani, M Sandrini Selective COX-2 Inhibitors and Dual Acting

Anti-inflammatory Drugs: Critical Remarks Curr Med Chem 9 (2002) 1033-1043– Rajakariar R, MM Yaqoob, DW Gilroy COX-2 in Inflammation and Resolution

Mol Interv 6 (2006) 199-207• Receptors

– Narumiya S Prostanoid Receptors: Structure, Function, and Distribution Ann NY Acad Sci 744 (1994) 126-138

• CYP 450– Kalsotra A, HW Strobel Cytochrome P45 4F subfamily: At the crossroads of

eicosanoid and drug metabolism Pharmacol Ther 112 (2006) 589-611• PG/LT/TX function

– Miller SB Prostaglandins in Health and Disease: An overview J Semarthrit 3(2006) 37-49

– William KI, GA Higgs Eicosanoids and Inflammation J Pathol 156 (1988) 101-110

lipid mediators: synthesis, metabolism, and function · –non-steroidal anti-inflammatory drugs...

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