Lecture 2

Question 1

Bradykinin synthesis pathway

Answer 1

1. Hageman factor becomes activated by contacting neg charged surfaces e.g LPS when leaking out of vessels during inflammation
2. Activated hageman factor converts plasma prekallikrein to kallikrein
3. Kallikrein cleaves HMW kininogen to bradykinin

(LMW kininogen to kalidin)

Question 2

Inactivation of bradykinin

Answer 2

• Kininase 1 removes C terminal arg to form des-arg-bradykinin (an agonist at B1 receptors). Mediated by several peptidases including serum carboxypeptidase
• Kininase 2 is a peptidyl dipeptidase. Removes 2 C terminal amino acids (aka ACE)

Question 3

Bradykinin receptors

Answer 3

1. B1 upregulated in infalmmation (activated by cytokines esp IL-1) reponds primarily to des-arg-bradykinin
2. B2 constitutively expressed and potently activated by bradykinin and kallidan

Both Gq coupled GPCRs

Question 4

Activation of bradykinin receptors

Answer 4

• Activation causes inc Ca2+ which activates cystolic phospholipase A2 (cPLA₂)
• Prostacyclin (PGI2) and NO diffuse to vascular smooth muscle layer and induce vasodialtation by inc cAMP and cGMP respectively
• Also results in nociceptor activation since Gq activation leads to PKC activation which phosphorylates numerous ion channels involved in the sensation of pain (dry cough)

Question 5

Hereditary angioedema (HAE) pathology

Answer 5

Caused by mutation in the gene encoding c1-INH (a c1 esterase inhibitor which inhibits kallirein). Mutation reduces inhibition so kallirein is over-expressed and patients present with severe and painful swelling.

• Type I HAE from mutations that compromise CI-INH synthesis/secretion
• Type II from mutations that poduce inactive C1-INH
• ACE inhibitor associated angioedema may be linked to variation in genes that regulate the immune system (higher prevalence in African americans)

Question 6

Treatment of HAE

Answer 6

• Recombinant C1-INH
• Kallikrien inhibitors
• B2 antagonist icatibant

Predominant treatment in the UK is icatibant

Question 7

Chemokines

Answer 7

Chemoattractant cytokines

• CCL3 induces mast cell degranulation by acting at CCR1 receptors
• Chemokine receptors are GPCRs (differ from cytokine receptors which are tyr kinases)
• Nomenclature governed by N terminus cysteine e.g CXC = single amino acid between 2 cysteines

Question 8

Colony stimulating factors

Answer 8

Stimulate formation of maturing colonies of leukocytes

Primarily used to overcome deficits in a persons WBC count following chemotherapy

Question 9

Nerve growth factor and drugs that target it

Answer 9

NGF is a potent sensitising agent released from mast cells and macrophages. It activates signalling pathways that result in a lesser stimulus causing pain (5 degree decrease in pain threshold following an NGF injection)

• Effects mediated by high affinity TrkA (receptor tropomyosin-related kinase A)
  
  • Theoretically NGF mABs that sequester NGF should reduce pain. TANEZUMAB has been developed but i still in trials

Question 10

Annexin-A1

Answer 10

Anti-inflammatory

• Produced by many cells and downregulates both inflammatory cell activation and mediator release
• Actions mediated by binding to the GPCR formylpeptide receptor 2 (FPR2)
  
  • FPR2 is also the receptor for lipoxin

Question 11

Production of lipid mediators of inflammation

Answer 11

e.g. leukotrienes, platelet activating factor and prostanoids (prostaglandins and thromboxane)

cPLA2 activation by Ca2+ and phosphorylation

• Bradykinin at B2 = Ca2+ release
• TNFa at TNFR1 promotes MAPK phos of cPLA2 at ser 505 and ser727
• TNFa at TNFR2 = Ca2+ release

Liberation of arachodonic acid from phospholipid membrane (rate limiting step) producing leukotrienes and prostaglandins

PLA2 action on membrane also produces lysoglycerlphosphorylcholine (precursor of PAF)

Question 12

leukotriene synthesis

Answer 12

• Arachodonic acid → 12-HETE via 12-lipoxygenase
• Arachodonic acid → leukotriene A₄ (LTA₄) via 5-lipoxygenase
• LTA4 →LTB₄ via LTA₄ hydrolase
• LTA₄→LTC_4, LTD₄, LTE₄ via LTC₄ synthase

LTC-LTE₄ known as the cysteinergic leukotrienes (CysLTs)

Question 13

Leukotriene receptors

Answer 13

All LT receptors are GPCRs

LTB4 acts on BLT1 and BLT2 receptors (Gq or Gi)

CysLTs act at CysLT1 and CysLT2 receptors (always Gq)

Question 14

Efficacy of CysLTs at different receptors

Answer 14

CysLT1: D > C > E

CysLT2: D = C > E

Question 15

Actions of CysLTs

Answer 15

All cause bronchoconstriction, increase vascular permeability and mucus secretion

Question 16

Montelukast

Answer 16

CysLT1 receptor antagonist used in the maintenance of asthma

CysLTs are released from mast cells and eosinophils in the airways of athmatics

Question 17

Action of LTB4

Answer 17

Potent chemoattractant and activator of neutrophils and macrophages

-upregulates neutrophil adhesion molecule expression and promotes macrophage cytokine release

• LTB4 found in the exudate in many conditions making LTA4 hydrolase the target of many anti inflammatory drugs
• One agent with this function was withdrawn due to causing dermatitis

Question 18

Lipoxin synthesis

Answer 18

2 main pathways:

• 12 lipoxygenase conversion of LTA₄ to LXA₄
• 15 lipoxygenase conversion of arachodonic acid to 15-S-HETE then converted to LXA4 by 5-lipoxygenase

Question 19

Actions of LXA4

Answer 19

Binds to formylpeptide receptor 2 (FPR2) which is Gi coupled

Reduced neutrophil chemotaxis and degranulation

Acts as an antagonist of CysLT1 receptors

Question 20

Platelet activating factor (PAF)

Answer 20

Formed throught the action of acetyltransferase on lyso-PAF

Acts on various GPCRs to increase thromboxane production in platelets

Chemotactic for neutrophils and can activate PLA2

Question 21

Metabolism of arachodonic acid

Answer 21

Metabolised to prostaglandins by cyclooxygenases (COX)

COX catalyses 2 reactions:

1. arachidonic acid is cyclised and oxygenated to form the endoperoxide PGG₂
2. peroxyl in PGG₂ is reduced to form PGH₂

Question 22

Sources of prostanoids in inflammation

Answer 22

• PGE₂ and PGI₂ tend to be produced locally by tissues and blood vessels
• PGD2 released from mast cells
• PGE2 and TXA2 released by macrophages in chronic inflammation

Question 23

Prostanoid receptor

Answer 23

Prostanoids act at GPCRs and some PGs can act at multiple receptors

• DP1, EP2, EP4 and IP3 are Gs coupled and therefore inc cAMP
• EP1, FP, TP are Gq coupled therefore inc Ca2+
• DP2, EP3 = Gi coupled, dec cAMP

Question 24

PGD₂

Answer 24

• DP₁ vasodilatation, inhibition of platelet aggregation and relaxation of GI/uterine smooth muscle
• TP = bronchoconstriction

Question 25

PGE₂

Answer 25

* EP1 = bronchial/GI muscle contraction * EP2 = bronchodilatation, vasodilatation and relaxation of GI muscle * EP3 = GI contraction, fever, reduced gastric acid secretion, increased gastric mucus * EP4 sensitisation of nociceptors

Question 26

PGI2

Answer 26

Vasodilatation and inhibition of platelet aggregation

Question 27

TXA2

Answer 27

Vasoconstriction, bronchoconstriction and platelet aggregation

Question 28

PGF2a

Answer 28

Uterine contraction

Question 29

Platelet aggregation

Answer 29

Balance between pro-aggregation (TXA2) and anti-aggregation (PGI2) In endothelial damage balance shifts towards TXA2 * use of anti-platelet agents e.g low dose aspirin in secondary prevention of stroke * fish oil reduces risk of MI as it is high in eicosapentanoic acids e.g. PGI3 and TXA3 * PGI3 more potent than PGI2 * TXA3 less potent than TXA2

Question 30

NSAID mechanism of action

Answer 30

Inhibit COX by entering a hydrophobic channel on the enzyme and forming hydrogen bonds with an arginine residue at position 120 This prevents entrance of fatty acids (e.g. arachidonic acid) into the catalytic domain thus inhibiting PG production Inhibition is reversible (except for aspirin)

Question 31

side effects of NSAIDs

Answer 31

Most common side effec tis GI bleeding (approx 5000 deaths pa) People at risk of GI bleeding (age, history of gastric ulceration) given NSAIDs in combo with PPIs e.g. omeprazole or PGE1 inhibitor misopristol

Question 32

COX-2 selectivity

Answer 32

It was thought that cox2 selectivity would reduce inflammation without the side effects The hydrophobic channel in the enzyme that NSAIDs enter is narrower in cox1 than in cox 2 Drugs were developed with bulky side chains that could pass through cox2 but not cox1 most NSAIDs e.g. ibuprofen are small and non-selective drugs ending in -coxib tend to be cox2 selective eg etoricoxib

Question 33

Problems with cox-2 selectivity

Answer 33

Although cox2 is upregulated in inflammation it is also constitutively expressed in some endothelial and vascular mooth muscle cells * Inhibition = reduced PGI2 = reduced vasodilatation/ increased platetlet aggregation * also inhibits eNOS = less NO = less vasodilatation Some associated with myocardial risk - ROFECOXIB withdrawn from the market Still associated with GI risk. possibly due to interference of healing of pre-existing ulcers (NB risk is reduced with selectivity)

Question 34

Capsaicin

Answer 34

Applied topically as a cream 3/4 times a day. Activated TRPV1 on nociceptors. Constant presence of agonist after an initial warming sensation induces a depolarising block of nociceptors as well as nocicpetor degeneration leading to pain relief

Question 35

anti-NGF drug trial

Answer 35

TANEZUMAB mAB against NGF. High levels of NGF found in CSF of OA patients. Tanezumab used in trial for OA but trial uspended due to some subjects developing rapdily progressing OA and requiring joint replacement It was subsuquently shown that the rate of joint replacement was the same in placebo so trial has been allowed to continue

Question 36

Naproxen and cardiovascular risk

Answer 36

Non selective NSAID Relatively similar cardiovascular risk in naproxen, ibuprofen adn cox2 selective inhibitor