Lecture 2 Flashcards
Bradykinin synthesis pathway
- Hageman factor becomes activated by contacting neg charged surfaces e.g LPS when leaking out of vessels during inflammation
- Activated hageman factor converts plasma prekallikrein to kallikrein
- Kallikrein cleaves HMW kininogen to bradykinin
(LMW kininogen to kalidin)
Inactivation of bradykinin
- 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)
Bradykinin receptors
- B1 upregulated in infalmmation (activated by cytokines esp IL-1) reponds primarily to des-arg-bradykinin
- B2 constitutively expressed and potently activated by bradykinin and kallidan
Both Gq coupled GPCRs
Activation of bradykinin receptors
- 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)
Hereditary angioedema (HAE) pathology
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)
Treatment of HAE
- Recombinant C1-INH
- Kallikrien inhibitors
- B2 antagonist icatibant
Predominant treatment in the UK is icatibant
Chemokines
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
Colony stimulating factors
Stimulate formation of maturing colonies of leukocytes
Primarily used to overcome deficits in a persons WBC count following chemotherapy
Nerve growth factor and drugs that target it
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
Annexin-A1
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
Production of lipid mediators of inflammation
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)
leukotriene synthesis
- Arachodonic acid → 12-HETE via 12-lipoxygenase
- Arachodonic acid → leukotriene A4 (LTA4) via 5-lipoxygenase
- LTA4 →LTB4 via LTA4 hydrolase
- LTA4→LTC4, LTD4, LTE4 via LTC4 synthase
LTC-LTE4 known as the cysteinergic leukotrienes (CysLTs)
Leukotriene receptors
All LT receptors are GPCRs
LTB4 acts on BLT1 and BLT2 receptors (Gq or Gi)
CysLTs act at CysLT1 and CysLT2 receptors (always Gq)
Efficacy of CysLTs at different receptors
CysLT1: D > C > E
CysLT2: D = C > E
Actions of CysLTs
All cause bronchoconstriction, increase vascular permeability and mucus secretion
Montelukast
CysLT1 receptor antagonist used in the maintenance of asthma
CysLTs are released from mast cells and eosinophils in the airways of athmatics
Action of LTB4
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
Lipoxin synthesis
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
Actions of LXA4
Binds to formylpeptide receptor 2 (FPR2) which is Gi coupled
Reduced neutrophil chemotaxis and degranulation
Acts as an antagonist of CysLT1 receptors
Platelet activating factor (PAF)
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
Metabolism of arachodonic acid
Metabolised to prostaglandins by cyclooxygenases (COX)
COX catalyses 2 reactions:
- arachidonic acid is cyclised and oxygenated to form the endoperoxide PGG2
- peroxyl in PGG2 is reduced to form PGH2
Sources of prostanoids in inflammation
- 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
Prostanoid receptor
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
PGD2
- DP1 vasodilatation, inhibition of platelet aggregation and relaxation of GI/uterine smooth muscle
- TP = bronchoconstriction
PGE2
- 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
PGI2
Vasodilatation and inhibition of platelet aggregation
TXA2
Vasoconstriction, bronchoconstriction and platelet aggregation
PGF2a
Uterine contraction
Platelet aggregation
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
NSAID mechanism of action
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)
side effects of NSAIDs
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
COX-2 selectivity
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
Problems with cox-2 selectivity
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)
Capsaicin
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
anti-NGF drug trial
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
Naproxen and cardiovascular risk
Non selective NSAID
Relatively similar cardiovascular risk in naproxen, ibuprofen adn cox2 selective inhibitor
