1. Do you administer NSAIDs or steroids to these patients?
A 54-year-old woman presents to her family physician's office with a 2 week history of pain and numbness in her left hand
A 19-year-old man, lying flat on a stretcher and wearing a hard cervical collar, arrives in the Emergency Room
Do you administer NSAIDs or steroids to these patients?
Eric Niederhoffer
SIU-SOM

2. Prostaglandin, Leukotriene, Lipoxin, and Thromboxane Synthesis
Pathway overview
Prostaglandin receptors
Pathway details
Differential actions of cyclooxygenases
COX-1 and COX-2 comparison
Tissue comparison
Role of ω-3 fatty acids

3. Anti-inflammatory steroids
Pathway Overview
Linoleic acid
Zileuton
Anti-inflammatory steroids
Glucocorticoids
Arachidonic acid
NSAIDs
aspirin
Prostaglandins (PG)
Lipoxins (LX)
Lipoxygenase
Leukotrienes (LT)
Prostaglandin H2 synthase
Lipoxygenase
Thromboxanes (TXA)
Thromboxane A2 synthase
There are series 1 prostaglandins derived from dihomo-γ-linolenic acid (DGLA; series 2 derived from arachidonic acid (AA); series 3 derived from eicosapentaenoic acid (EPA), start with α-linolenic acid (ALA)
Series 3 (ω3) can interfere with (competitive) series 2 (ω6) production (decrease inflammation)
ω6 from corn oil; ω3 from canola oil, engineered rapeseed oil, Krill oil
PG: prostaglandins - PGG2, PGH2 (constriction), PGD2 (constriction or vasodilation), PGE1 (vasodilation), PGE2 (vasoconstriction/dilation), PGF2a (constriction), PGI2 (prostacyclin, dilation, inhibition of platelet adhesion)
LT: leukotrienes - LTB4, LTC4, LTD4, LTE4 (multiple roles, microvascular vasoconstriction); zileuton inhibits 5-lipoxygenase
LX: lipoxins - LXA4, LXB4
TBX: thromboxanes - TXA2 (constriction, platelet adhesion), TXB2 (constriction)
NSAIDs: nonsteroidal anti-inflammatory drugs, aspirin, ibuprofen; anti-inflammatory steroids work by boosting levels of lipocortin (an annexin, Ca2+-dependent inhibitor protein/enzyme that inhibits phospholipase A2); lipocortin-1 = annexin-1. Salicylate directly activates AMP kinase.
Most of the enzymes are located in the smooth endoplasmic reticulum
Brain/nerves - PGD2, PGE2, and PGF2 Kidneys - PGE2 and PGI2 Lungs - PGD2
Synovial cells - PGE2 and PGI2 when stimulated by interleukin-1 Vascular beds - PGE2 and PGI2 & PGH2 and TXA2
COX-1 deficiency: Type 1 - complete absence in platelets; Type 2 - normal protein, impaired activity; bleeding disorders

4. Prostaglandin Receptors
Signal Transduction
Distribution
DP1 (PGD2)
AC↑, [cAMP]↑
Platelets, VSM, nervous tissue, retina, small intestine, ileum, lung, stomach, uterus
DP2 (PGD2)
Mobilize intracellular [Ca2+]
Eosinophils, basophils, Th2 cells
EP1 (PGE2)
phosphoinositol turnover↑, [Ca2+]↑
Kidney, lung, spleen, skeletal muscle, testis uterus
EP2 (PGE2)
Lung, placenta, heart?
EP3 (PGE2)
Most receptors AC↓, [cAMP]↓, some AC↑ and [cAMP]↑
Kidney, stomach, uterus, pancreas, adrenal, testis, ovary, small intestine, brain, spleen, colon, heart, liver, skeletal muscle, lung, thymus, ileum
EP4 (PGE2)
Small intestine, lung, thymus, kidney, uterus, pancreas, spleen, heart, stomach, brain, ileum, peripheral blood mononuclear cells
FP (PGF2)
Corpus luteum, uterus, stomach, kidney, heart, lung, eye, liver
IP (PGI2) (IP1, IP2)
[PGI2 = prostacyclin]
Platelets, VSM, kidney, thymus, liver, lung, spleen, skeletal muscle, heart, pancreas
TP (TXA2)
Platelets, VSM, thymus, spleen, lung, kidney, heart, uterus
PG: prostaglandin AC: adenylyl cyclase
cAMP: cyclic adenosine monophosphate VSM: vascular smooth muscle
TXA2: thromboxane A2
Leukotrienes bind to LT receptors (I.e., ALT1, BLT1, CLT1)

5. Pathway Details (PG, TX, LT)
IL-1 (inflammation)
IL-1R
Membrane phospholipids
Anti-inflammatory steroids
Glucocorticoids
(mediated by lipocortin-Ca2+)
Arachidonic acid
Phospholipase A2
(or PLC)
LTA4
NSAIDS (aspirin)
PGG2
LTC4
Glutathione S-transferase
LTB4
PGH2 synthase
Cyclooxygenase O2
LTD4
LTE4
PGH2
2GSH
GSSG
PG hydroperoxidase
PGJ2
PGD2 synthase
TXA2
TXA2 synthase
PGD2
PGI2 (PC)
PGF2a
PGE2
IL: interleukin-1 IL-1R: interleukin-1 receptor. The pathway also activated by bradykinin with its receptor
NSAIDS: nonsteroidal anti-inflammatory drugs, aspirin (irreversible inhibitor of COX-1), ibuprofen (lesser ratio of COX-1/COX-2)
Acetaminophen (an analgesic, does not affect COX-1 or COX-2 but may indicate presence of a COX-3 or PCOX-1a or PCOX-1b isoforms that are not involved in PG synthesis but address fever and pain)
PG: prostaglandin GSH: glutathione (reduced form)
GSSG: glutathione disulfide (oxidized form) PC: PGI2 or prostacyclin
PGE2 synthase is also denoted PG endoperoxidase E isomerase, microsomal form is key enzyme
PLA2: cytosolic associated with COX-1, secretory associated with COX-2
PGH2 can rearrange to form levuglandin, which forms histone H4 adducts and destabilizes nucleosomes
Leukotriene receptor antagonists – zafirlukast (DLT4 and ELT4); montelukast (DLT4)
PGI2 synthase
PGE2 synthase
PGF2
synthase

6. Pathway Details (LX) Arachidonic acid 15S-H(p)ETE 15S-ETT 15R-HETE
15-LOX
5-LOX
15S-H(p)ETE
15S-ETT
Airway epithelia
Leukocytes
15R-HETE
5-LOX
15R-ETT
15 epi-LXA4
15 epi-LXB4
LTA4
12-LOX
5-LOX
LXA4
LXB4
Platelets
Leukocytes
Acyl-COX-2
Aspirin
Epithelia
Endothelia
Monocytes
Anti-Inflammatory Effects
COX: cyclooxygenase LOX: lipoxygenase HETE: hydroxyeicosatetraenoic acid
LX: lipoxin LT: leukotriene ETT: epoxytetraene
The production of LX and epi-LX are transcellular pathways. Epi-lipoxins are also referred to as aspirin-triggered lipoxins (ATL), which interact with ATL receptors (ATLR). ATLR also bind to small peptide derived signaling molecules involved in the immune response.

7. Differential Actions of Cyclooxygenases
Housekeeping
Unwanted side-effects
Endothelial integrity
Vascular patency
Gastric mucosal integrity
PGI2
COX1
Constitutive
PGE2
TXA2
Bronchodilation
Renal function
Platelet function
NSAIDs
PGE2
PGF2a
COX2
Inducible
Inflammatory
COX: cyclooxygenase, COX1 constitutive (endoplasmic reticulum), COX2 inducible (perinuclear envelope), COX3 brain
NSAIDs: nonsteroidal anti-inflammatory drugs, aspirin, ibuprofen
PG: prostaglandins (PGI2 = prostacyclin, endothelial cells)
TX: thromboxane (TXA2 = thromboxane, platelets)
Acetaminophen is an analgesic.
COX-1 and COX-2 serve identical functions in catalyzing the conversion of arachidonic acid to prostanoids. The specific prostanoid(s) generated in any given cell is determined by which distal enzymes in the prostanoid synthetic pathways are expressed. For example, stimulated human synovial cells synthesize small amounts of PGE2 and prostacyclin but not thromboxane or PGD or PGF2a. Following exposure to interleukin-1, synovial cells make considerably more PGE2 and prostacyclin, but they still do not synthesize PGD, TXB2 or PGF2a. The IL1-induced increase in PGE2 and prostacyclin is mediated through COX-2.
Thus, while the species of prostanoid synthesized in a cell is dependent upon the specific distal synthetic enzyme(s) expressed, the amount synthesized is determined by the amount of COX —1 and —2 activities expressed. COX-1 is expressed in nearly all cells (except red cells) in their basal (unstimulated) state. COX-1 mediated production of thromboxane in platelets promotes normal clotting. And COX-1 mediated synthesis of prostaglandins in the kidney appears to be responsible for maintaining renal plasma flow in the face of vasoconstriction.
Aspirin appears to have a role (through action of CREB) in increasing myelin-associated proteins in oligodendrocytes and protecting the cells from TNFα insult.
Inflammation
Proteases
Therapeutic anti-inflammatory effects

8. COX-1 and COX-2 Comparison
Parameter
COX-1
COX-2
Regulation
usually constitutive
inducible
Range of gene induction
2 to 4-fold
10 to 80-fold
Rate of gene activation
24 h
0.5 to 4 h
Effect of glucocorticosteroids
inhibits activity*
Relative size of active site
smaller
larger
Rate of arachidonic acid consumption
34 nmol/min/mg
39 nmol/min/mg
Effect of aspirin on COX activity
Inhibited
Affected**
Aspirin will inactivate COX-1. Its effects take longer to wear off because it takes 24 hours for new enzyme synthesis.
Aspirin will acylate COX-2, but the larger active site can still bind arachidonic acid and will produce other mediators. **Aspirin modifies active site to produce lipoxins.
Glucocorticosteroids have effects at both the gene and protein level. *Best to look at this as a reduction in arachidonic acid precursor (think lipocortin) for COX-1 or COX-2 to act on.
There is recent work suggesting that inducible NO synthase activates COX-2 (no effect on COX-1). NO synthase and COX-2 enhances cPLA2 activity.
Oxidized low-density lipoprotein (LDL) appear to increase gene expression of COX-2.
Class I NSAIDs - simple, competitive COX inhibitors (ibuprofen, naproxen)
Class II NSAIDs - competitive, time-dependent COX inhibitors (indomethacin)
Class III NSAIDs - irreversible, competitive, time-dependent COX inhibitors (aspirin)

9. Tissue Comparison Brain/nerve Vascular beds Synovial cells Ar PGH2
PGF2a
PGD2
PGE2
PGI2 (PC)
PGE2
PGI2 (PC)
PGE2
TXA2
So what would happen if we gave a patient a large dose of aspirin or Coxib to reduce inflammation/pain in these tissues?
Ar: arachidonic acid PG: prostaglandin
PC: prostacyclin TX: thromboxane

10. Transcellular processes
Role of 𝛚-3 Fatty Acids
GPR120
βArr2
TAB1
DHA
Neutrophil
Platelet
COX/LOX
Resolvins
Protectins
anti-inflammatory
Transcellular processes
Macrophage
LPS
TNFα
TNFR
TAK1
TLR4
IKKβ
MKK4
NF𝛋B
JNK
Cytokines
inflammatory
Nucleus
DHA: docosahexanoic acid LPS: lipopolysaccharide TNFα: tumor necrosis factor α
TNFR: TNFα receptor GPR120: G protein receptor 120
βARR2: β-arrestin 2 TAB1: tumor β-activated kinase 1
TAK1: transforming growth factor β-activated kinase 1 TLR: Toll-like receptor
IKKβ: inhibitor of nuclear factor 𝛋-B-kinase subunit β NF𝛋B: nuclear factor 𝛋 B
MKK4: mitogen-activated protein kinase kinase 4 JUN: c-JUN N-terminal kinase
COX: cyclooxygenase LOX: lipoxygenase

11. Review Questions
How are prostaglandins, leukotrienes, lipoxins, and thromboxanes synthesized (substrates, enzymes, cofactors)?
What is the nomenclature for prostaglandin, leukotriene, lipoxin, and thromboxane receptors?
How do NSAIDs work?
How do steroids work?
What are important characteristics of COX-1 and COX-2?
How do ω-3 fatty acids affect the inflammatory response?