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

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

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

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) http://www.caymanchem.com/app/template/scientificIllustrations%2CIllustration.vm/illustration/2018/a/z

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

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. http://themedicalbiochemistrypage.org/aspirin.html#

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

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) http://elfstrom.com/arthritis/nsaids/actions.html

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

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

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?