1. Prostaglandins, Leukotrienes, and Eicosanoids
Matthew L. Fowler, Ph.D., OMS-II Class of 2015 Cell Biology and Physiology Block 6 Renal and Reproduction

2. Lecture Objectives
Define the term “eicosanoid” and list the main members of this group.
Describe the mechanism of action of eicosanoids including their effects on tissues, their half lives, and where they are produced.
Describe the structural features of prostaglandins, thromboxanes and leukotrienes. Explain the common starting material for these compounds.
Using figure 35.2, explain how cellular signals lead to the production of eicosanoids. In particular, include the appearance of arachidonic acid in response to the cellular stimulus, which is the primary signal.
List the three enzymes that convert arachidonic acid to the three major classes of eicosanoid (prostaglandin, HPETE and epoxides).
Describe the nomenclature of prostaglandins and thromboxanes. NOTE: it is not necessary to memorize the structures and the names. Just understand what each component of the name indicates.
Describe in general terms the synthesis of prostaglandins and thromboxanes from arachidonic acid.
Explain the relationship between the ω-6:ω-3 fatty acid ratio and inflammation.
Explain the production of series 1, 2 and 3 prostaglandins from ω-6 versus ω-3 fatty acids.
Explain the mechanism of degradation of prostaglandins and thromboxanes to inactivate these molecules and terminate signaling.
Explain the mechanisms by which steroidal and non-steroidal anti-inflammatory drugs block the formation of prostaglandins and thromboxanes.
Describe the production of leukotrienes and HETE and lipoxins from arachadonic acid. Note how the molecule is activated with the presence of the –OH group.

3. Arachidonic Acid (AA) Signaling
AA is the most common FA released by PLA2 following signaling
PLA2 cleaves the AA from position 2 of the phospholipid to form free AA OR
Diacyl Glycerol (DAG) lipase can cleave AA from position 2 on DAG after PLC cuts PIP2
AA enters the eicosanoid pathway.

4. Eicosanoids
Prostaglandins (PG), Thromboxanes (TX), and Leukotrienes (LT
Effects
Prostaglandins (think stomach flu)
Cause pain, inflammation, fever, nausea and vomiting
Normally in kidney lots of PGs produced. Therefore inhibitors of synthesis (normally) have little effect.
Characteristics
Produced in very small amounts
Very short half-life
Most potent signaling compounds we make
Synthesis, Degradation, Location of Action (Tissue dependent)
20 carbon FAs
Arachidonic acid most common source in humans
Formed in most tissues
Metabolized to inactive products at the site of synthesis
Act locally
The kidney produces lots of prostaglandins.

5. Eicosanoid Function is Diverse!
Exam questions and boards here. Will point out specifics. Questions will focus on those of the renal system.

6. PGs, TXs, and LTs PGs are FAs: 20 carbon atoms
Internal, saturated 5-carbon ring
Provides actual function
Hydroxyl group at carbon 15,
Double bond between carbons 13 and 14
Various substituents on the ring
TXs
Similar structure to PGs
6-membered ring
Some TXs have additional oxygen atom bridging carbons 9 and 11 of ring
LTs
Characterized by three consecutive double bonds (triene)
Also have an oxygen atom (or two) bound as an OH or as an epoxide
All
Synthesized from polyunsaturated fatty acids (PUFAs)
Containing 20 carbons
3-5 double bonds
MOST COMMON arachidonic acid

7. Synthesis of Eicosanoids
Eicosanoids Are Produced in Response to Cellular Signals
Pathway activation is triggered by:
Stimulus binding to membrane receptors = histamine or cytokines
PUFAs (AA below) usually attached to glycerol backbone of phospholipid
Phosphoatidylcholine (PC)
AA cleaved from PC by PLA2
Note: NSAIDs inhibit PLA2
Phosphatidylinositol bisphosphate (PIBP)
AA cleaved from PIBP by PLC and DAG
MAG lipase action yields additional AA
Focus: Worry mostly about the cyclooxygenase pathways with respect to NSAIDs for this exam.
NOTE THIS: Arachidonic acid is most common eicosanoid taught. There are other FAs that will enter into these eicosanoid synthesis pathways (covered later in lecture). HERE I will say PUFA meaning AA or one of the other fatty acids that will work.

8. Synthesis of Eicosanoids Three Pathways
Tissue dependent
Cyclooxygenase (COX)
PGG2
PGs and TXs
COX-1
Constitutive
Kidney
Also Gastric mucosa, platelets, vascular endothelium
COX-2
Inducible
Due to inflammation
Mainly expressed in activated macrophages
Inhibition of COX-1/COX-2 can lead to pathological process
Lipoxygenase
HPETE
LTs, HETE, and Lipoxins
Dioxygenase that inserts a peroxide
LT synthesis has a pathological process in the renal system
But less than PGs
CYP450
Epoxides
Synthesis of diHETE and HETE via epoxides
Inhibition of both the COX-1 and COX-2 can lead to problems.

9. PG and TX Nomenclature All have a capital letter
Designates ring substituents
MOST COMMON classes are A,E, F
Important: PGE, PGFα, PGI, TXA2
Some have subscript
Designates # of double bonds in linear portion of the hydrocarbon chain
Does not include ring
Compounds in 2-series are of the greatest significance
Example: The PGF series has two hydroxyl groups on the ring. A Greek subscript is used to denote the position of the hydroxyl group at carbon 9
Ring structure is important.

10. Cyclooxygenase Pathway Synthesis of PG and TX
PG synthesis from AA by COX
O2 is added
5-carbon ring is formed
PGG2 now formed
Peroxidase and GSH
PGH2 now formed
Used to synthesize PGs and TXs
Specifically TXA2
Next step is tissue specific
Function
Degradation
Don’t need to know the specifics about the structures. Understand the physiological activation and inhibition of PLA2. Also understand the consequences of COX action to form PGs and LTs.
Arachidonic acid undergoes activation by COX to form the ring structure.

11. Prostaglandins 1, 2, and 3 series and Precursors
PGX1 = 1 double bond
Derived from eicosatrienoic acid
Synthetic source
C 13 and 14
PGX2 = 2 double bonds
Derived from arachidonic acid
Meat source
C 13 and 14
C 5 and 6
PGX3 = 3 double bonds
Derived from eicosapentanoic acid
Fish source
C 13 and 14
C 5 and 6
C 17 and 18
Arachidonic acid comes from ingested meats.
The PGE”2”: the “2” indicates that this has come from arachidonic acid. The “1” is an analog. The “3” series will become TXA3 and plays a role in platelet aggregation.
Note: Two double bonds are used up in the generation of the ring in prostaglandins

12. Degradation of PG and TX
Must degrade them to stop signal
Short t½ (seconds to minutes)
Rapidly inactivated
Oxidation of the 15-hydroxyl (required for activity)
Oxidized to a ketone
Renal excretion
Example
TXA2 (mediates thrombosis)
Converted to TXB2 = inactive

13. NSAIDs Covalently Modify COX
NSAIDs block PG formation by irreversibly inhibiting COX
Aspirin is the only COX inhibitor that covalently modifies COX
Serine acetylation
Aspirin is more potent against COX-1 than COX-2
Note: NSAIDs don’t block AA formation so AA can form other eicosanoids
Basis of anti-thrombotic activity associated with low dose aspirin (80 mg/day)
Irreversibly blocks COX-1 in platelets
Lowers TXA2 levels
Lasts for the lifetime of the platelets (~10 days)

14. Steroids Inhibit PLA2
Steroidal anti-inflammatory drugs block PG formation by inhibiting PLA2
Blocks release of arachidonic acid for PG synthesis
Most effective anti-inflammatory
Ex. hydrocortisone, prednisone
Steroids

15. NSAIDs The Good and the Bad
PGs produced by COX-1 provide gastric cytoprotection
Partly by  intracellular [cAMP]
Partly by stabilizing platelets
Protecting blood vessels from inflammatory agents
NSAIDs block synthesis of PGG2
Same action on both COX-1 and COX-2
Good
Inhibits the inflammatory effects of PGs
Bad
May inhibit the cytoprotective effects of constitutive COX-1

16. Lipoxygenase Pathway Synthesis of LTs, HETEs and Lipoxins
Lipoxygenase incorporates an oxygen molecule onto a carbon of one of several double bonds of AA
Activity of enzyme is tissue dependent
Forms HPETEs (hydroperoxyeicosatetraenoic acids)
Reduced to the corresponding hydroxyl metabolites (HETEs) OR
Metabolized to form leukotrienes or lipoxins

17. Eicosanoid Receptors Both cytosolic and nuclear signaling
Majority are cytosolic
Involves G-protein signaling process
 intracellular cAMP
 intracellular IP3
 intracellular Ca2+
PGE, PGD, PGI via cAMP
PGF2α, TXA2 via Ca2+ resulting in SM contraction

18. Clinical Functions of Eicosanoids
PGE2
Pregnancy, abortion, erection
PGI2
Bronchial dilation (scleroderma) pulmonary hypertension
TXA2
Platelet aggregation
LTB4
Acute respiratory distress
LTD4
Asthma, inflammatory bowel disease
HETE’s
Release Ca2+ stores and promote cell proliferation

19. Renal Effects of Eicosanoids
Renal medulla and cortex synthesize PGs
Medulla makes more
COX-1 effects GFR and RBF
Expression
Cortical and medullary CDs
Mesangial cells
Arteriolar endothelium
Podocytes of BC (epithelium)
COX-2 induced by  [Na+]
Medullary interstitial cells
Macula densa
Cortical TA LOH
Major Renal Eicosanoids
PGE2 + PGI2 > TXA2 > PGF2

20. Renal Effects of Eicosanoids Hemodynamics and ARF
Normal
Minimal influence of PGs on RBF and GFR
i.e. NSAIDs have no effect on renal function
Pathological
Renal function maintenance becomes dependent on PGs (rate-limiting)
Volume contracted state
CHF
Terminal LF (Cirrhosis w/ascites)
Nephrotic syndrome
PGE2 and PGI2 are vasodilators
Maintain RBF and GFR

21. Renal Effects of Eicosanoids NSAIDs in RF Patients
NSAIDS can cause a striking decrease in RBF and GFR in patients with renal problems.
Effect attributed to inhibition of constitutively expressed COX-1
Vasodilation cannot be maintained
COX-2 inhibitors also significantly reduce the GFR and RBF in patients with renal problems.

22. Renal Effects of Eicosanoids Regulation of BP and Na+
Inhibition of endogenous PG synthesis by NSAIDs may result in systemic HTN.
Can also compromise well-controlled blood pressure in subjects with preexisting Na+-sensitive HTN.
Relates role of PGs in modulating renal Na+ excretion and BP maintenance.

23. Renal Effects of Eicosanoids Regulation of BP and Na+
Medullary COX-2 expression  with  [Na+]
PGE2 and PGI2 synthesized
 Renin
 GFR
 Na+ reabsorption
 BP
COX-1 derived products
 Medullary blood+ flow
 Na+ excretion
Increased water clearance probably results result from attenuation of the action of antidiuretic hormone (ADH)
COX-1/COX-2 inhibited by  medullary RBF
Associated with Na+ retention leading to HTN

24. Renal Effects of Eicosanoids Regulation of BP and Na+
Cortical COX-2 expression  with  [Na+]
PGE2 and PGI2 synthesized
 Renin
 GFR
 Na+ reabsorption
 BP
May contribute to renovascular HTN.
Note differences here.

25. Renal Effects of Eicosanoids Vasoactive and Inflammatory Properties
TXA2 causes intrarenal vasoconstriction
Result:  Renal Function
Observed in conditions involving inflammatory cell infiltration GN
Renal transplant rejection
Note: The normal kidney synthesizes only small amounts of TXA2

26. Effects on Reproductive Organs
Female Reproductive Organs
PGE2 and PGF2 have a role in ovulation, luteolysis, and fertilization
Uterine muscle is contracted by PGF2, TXA2, and low concentrations of PGE2
Important in dysmenorrhea (blocked by NSAIDs)
PGI2 and high concentrations of PGE2 cause relaxation
PGF2 together with oxytocin is essential for onset of parturition.
Male Reproductive Organs
PGs found in seminal fluid (unclear role)
Men with a low seminal fluid concentration of PGs are relatively infertile.
Smooth muscle relaxing prostaglandins such a PGE1 enhance penile erection by relaxing the smooth muscle of the corpora cavernosa.

27. Question Prostaglandin structure is characterized by a
Five-member ring in the hydrocarbon chain.
Six-member ring in the hydrocarbon chain.
An OH group attached to the ω-carbon.
Set of three consecutive double bonds on the hydrocarbon chain.

28. Question
Which of the following is a cellular signaling mechanism that results in free arachidonic acid for eicosanoid formation?
Arachidonic acid is one of the hydrocarbon chains of glycerophospholipids and is cleaved by the action of phospholipase A2 in response to the cellular signal.
Cellular signals stimulate arachidonic acid synthesis using linoleate as starting material and specific desaturase enzymes and fatty acid elongation techniques.
The cAMP level rises in response to key cellular signals, which results in protein phosphorylation and activation of the three eicosanoid synthesis pathways.
Cellular signals stimulate phospholipase C, which cleaves arachidonic acid from 1,2-diacyl glycerol, resulting in free arachidonic acid for eicosanoid formation.

29. Question Which statement about eicosanoids is NOT correct?
The three major eicosanoid synthesis pathways involve cyclooxygenase, lipoxygenase and cytochrome P450 as a first step.
Non-steroidal anti-inflammatory drugs inhibit cyclooxygenases by covalently modifying a serine residue in the active site of the enzyme.
Steroidal anti-inflammatory drugs inhibit eicosanoid formation by inhibiting phospholipase A2, which blocks the liberation of free arachadonic acid (or other 20-carbon PUFA).
The ketone group on carbon 15 of prostaglandins and thromboxanes is a crucial structural requirement for these molecules to be active.