Lecture 34: Eicosanoids

Which types of molecules can form eicosanoids?

20-carbon PUFAs

What is an example of a 20-carbon PUFA?

arachidonate

What are the major classes of eicosanoids?

• prostaglandins

• thromboxanes

• leukotrienes

Describe eicosanoids.

• result in strong hormone-like actions in the tissues where they are produced

• signaling molecules

• made from 1 cell and then targets another cell

• not stored

• very unstable

• act over short times and distances

• short half-life

What are the 3 processes for how arachidonic acid is released from membrane lipids?

Option 1:

1. stimulus binding to receptor

2. receptor binding activates phospholipase A₂ Ca²⁺

3. phospholipase A₂ Ca²⁺ cleaves phosphatidylcholine to form arachidonic acid

Option 2:

1. stimulus binding to receptor

2. receptor binding activates phospholipase C

3. phospholipase C cleaves phosphatidylinositol biphosphate to form 1,2-diacylglycerol

4. diacylglycerol lipase acts on 1,2-diacylyglycerol to form arachidonic acid

Option 3:

1. stimulus binding to receptor

2. receptor binding activates phospholipase C

3. phospholipase C cleaves phosphatidylinositol biphosphate to form 1,2-diacylglycerol

4. diacylglycerol lipase acts on 1,2-diacylyglycerol to form monoacylglycerol

5. monoacylglycerol lipase acts monoacylglycerol to form arachidonic acid

What does phospholipase C cleave, and what does it release?

• cleaves phosphatidylinositol

• releases IP3 and DAG

Where does phospholipase A₂ cleave phosphoacylglycerol?

at position 2

Where are PUFAs often present in cell membranes?

at the SN2 motion of phosphatidylinositol

When arachidonate is needed for eicosanoid synthesis, what happens?

phospholipase A₂ liberates arachidonate

State the three pathways for how arachidonic acid can synthesize different eicosanoids.

Option 1:

1. arachidonic acid is converted to PGG₂ by cylco-oxygenase

2. PGG₂ forms prostaglandins and thromboxanes

Option 2:

1. arachidonic acid is converted to HPETE by lipoxygenase

2. HPETE forms leukotrienes, HETE, and lipoxins

Option 3:

1. arachidonic acid is converted to epoxides by cytochrome P₄₅₀ 

2. epoxides form diHETE and HETE

What does cyclooxygenase make?

prostaglandins and thromboxanes

What does PGG₂ stand for?

prostaglandin G2

Describe the synthesis of prostacyclin/PGI₂

General:

1. arachidonic acid

   1. cyclo-oxygenase

2. PGG₂

3. PHG₂

4. PGI₂

Step 1:

• substrate(s): arachidonic acid

• coactivator(s): 2O₂

• enzyme: cyclo-oxygenase

• product(s): PGG₂

Step 2:

• substrate(s): PGG₂

• coactivator(s): 2 GSH (glutathione)

• enzyme: cyclo-oxygenase

• product(s): PGH₂

Step 3:

• substrate(s): PGH₂

• coactivator(s): none

• enzyme: PGI synthase

• product(s): prostacyclin/PGI₂

Describe the synthesis of PGF_2alpha.

General:

1. arachidonic acid

   1. cylco-oxygenase

2. PGG₂

3. PGH₂

4. PGE₂

5. PGF_2alpha

Step 1:

• substrate(s): arachidonic acid

• coactivator(s): 2O₂

• enzyme: cyclo-oxygenase

• product(s): PGG₂

Step 2:

• substrate(s): PGG₂

• coactivator(s): 2 GSH (glutathione)

• enzyme: cyclo-oxygenase

• product(s): PGH₂

Step 3:

• substrate(s): PGH₂

• coactivator(s): none

• enzyme: PGE synthase

• product(s): PGE₂

Step 4:

• substrate(s): PGE₂

• coactivator(s): none

• enzyme: PGE 9-keto reductase

• product(s): PGF_2alpha

Describe the synthesis of PGD₂.

General:

1. arachidonic acid

   1. cyclo-oxygenase

2. PGG₂

3. PGH₂

4. PGD₂

Step 1:

• substrate(s): arachidonic acid

• coactivator(s): 2O₂

• enzyme: cyclo-oxygenase

• product(s): PGG₂

Step 2:

• substrate(s): PGG₂

• coactivator(s): 2 GSH (glutathione)

• enzyme: cyclo-oxygenase

• product(s): PGH₂

Step 3:

• substrate(s): PGH₂

• coactivator(s): none

• enzyme: PGD synthase

• product(s): PGD₂

Describe the synthesis of TXA₂.

General:

1. arachidonic acid

   1. cyclo-oxygenase

2. PGG₂

3. PGH₂

4. TXA₂

Step 1:

• substrate(s): arachidonic acid

• coactivator(s): 2O₂

• enzyme: cyclo-oxygenase

• product(s): PGG₂

Step 2:

• substrate(s): PGG₂

• coactivator(s): 2 GSH (glutathione)

• enzyme: cyclo-oxygenase

• product(s): PGH₂

Step 3:

• substrate(s): PGH₂

• coactivator(s): none

• enzyme: TXA synthase

• product(s): thromboxane/TXA₂

What is cyclooxygenase also called?

prostaglandin H₂ synthase 

Where are the enzymes needed for prostaglandin and thromboxane synthesis?

in the smooth ER

What is PGI₂ produced by?

vascular endotherlial cells

What does PGI₂ do?

• inhibits platelet aggregation

• causes vasodilation

What is TXA₂ produced by?

platelets

What does TXA₂ do?

• stimulates platelet aggregation

• causes vasoconstriction

What are some common structural features of prostaglandins?

• 20 carbons

• carboxyl group at carbon 1

• carbons 8-12 form a five-membered ring with substituents at carbons 9 and 11

• carbon 15 contains a hydroxyl group

• trans double bond between 13 and 14

• double bonds may be present between carbons 5 and 6 and between carbons 17 and 18

Why do prostaglandins have such a short half-life?

there’s a hydroxyl group at carbon 15 that is immediately converted to a ketone

Thromboxane A₂ is rapidly converted to what?

its inactive form, thromboxane B₂

What are the two isoforms of cyclooxygenase (COX)?

COX-1 and COX-2

Describe COX-1.

• constitutively expressed at low levels in many cell types

• essential for maintaining the integrity of the GI epithelium

• important for gastric acid production

Describe COX-2.

• expression is highly regulated

• transcription of COX-2 is stimulated by growth factors, cytokines, and endotoxins

• expression may be enhanced by cAMP

What is often produced as a result of COX-2 activity and changes in cAMP levels?

PGE₂

COX-2 is induced in what?

in inflammation

What type of drugs are COX inhibitors?

NSAIDs

What do NSAIDs stand for?

non-steroidal anti-inflammatory drugs

What do NSAIDs do?

• antipyretic (reduce fever)

• analgesic

• anti-inflammatory

Where does aspirin come from?

• bark of willow trees (used to treat fever)

• from Spiraea ulmaria

What does aspirin do to COX?

acetylates active-site serine residue of COX to prevent arachidonate binding

What type of regulatory effect does aspirin have on COX?

irreversibly inactivation 

What does acetylation of aspirin form?

salicylate

What are examples of other NSAIDs?

• ibuprofen/advil

• naproxen/aleve

What type of inhibitors are other NSAIDs?

reversible competitive inhibitors

True or False: Aspirin and other NSAIDs inhibit both COX-1 and COX-2.

true

What can aspirin and other NSAIDs cause as side effects?

stomach bleeding and ulceration

Why were specific COX-2 inhibitors developed?

to decrease the gastric bleeding side-effects of NSAIDs

What are some examples of selective COX-2 inhibitors?

• Vioxx/rofecoxib

• Bextra/valdeocoxib

• Celebrex/celecoxib

True or False: Vioxx and Bextra were withdrawn from the market due to the increased risk of heart attack and stroke, but Celebrex is still on the market but with warnings.

true

Low doses of aspirin can act as what kind of agent?

an anti-thrombotic agent

What can TXA₂ lead to?

thrombus formation at sites of vascular injury (or a ruptured atherosclerotic plaque) in the lumen of coronary arteries causing acute myocardial infarction

How does aspirin act as an anti-thrombotic agent?

inhibition of COX—blocking the production of TXA₂ from arachidonic acid (major precursor)

Describe the synthesis of LTE₄.

General:

1. arachidonic acid

   1. 5-lipoxygenase

2. 5-HPETE

3. LTA₄

4. LTC₄

5. LTD₄

6. LTE₄

step 1:

• substrate(s): arachidonic acid

• coactivator(s): none

• enzyme: 5-lipoxygenase

• product(s): 5-HPETE

step 2:

• substrate(s): 5-HPETE

• coactivator(s): GSH

• enzyme: ?

• product(s): LTA₄

step 3:

• substrate(s): LTA₄

• product(s): LTC₄

step 4:

• substrate(s): LTC₄

• product(s): glutamate and LTD₄

step 5:

• substrate(s): LTD₄ 

• product(s): LTE₄ and glycine

How many active sites does COX have?

2 sites

What does glutathione do?

prevents free radicals

Describe the synthesis of LTB₄.

General:

1. arachidonic acid

   1. 5-lipoxygenase

2. 5-HPETE

3. LTA₄

4. LTB₄

step 1:

• substrate(s): arachidonic acid

• coactivator(s): none

• enzyme: 5-lipoxygenase

• product(s): 5-HPETE

step 2:

• substrate(s): 5-HPETE

• coactivator(s): GSH

• enzyme: ?

• product(s): LTA₄

step 3:

• substrate(s): LTA₄

• product(s): LTB₄

What does zilleuton inhibit?

5-lipoxygenase

What does FLAP inhibit?

5-HPETE

What are the cysteinyl-LT antagonists?

• Zafirlukast

• Montelukast 

What does 5-HETE lead to?

neutrophils

What does 12-HETE lead to?

platelets

What does 15-HETE lead to?

eosinophils

Describe the synthesis of 5-HETE.

General:

1. arachidonic acid

   1. 5-lipoxygenase

2. 5-HPETE

3. 5-HETE

step 1:

• substrate(s): arachidonic acid

• enzyme: 5-lipoxygenase

• product(s): 5-HPETE

step 2:

• substrate(s): 5-HPETE

• product(s): 5-HETE

Describe the synthesis of 12-HETE.

General:

1. arachidonic acid

   1. 12-lipoxygenase

2. 12-HPETE

3. 12-HETE

step 1:

• substrate(s): arachidonic acid

• enzyme: 12-lipoxygenase

• product(s): 12-HPETE

step 2:

• substrate(s): 12-HPETE

• product(s): 12-HETE

Describe the synthesis of 15-HETE.

General:

1. arachidonic acid

   1. 15-lipoxygenase

2. 15-HPETE

3. 15-HETE

step 1:

• substrate(s): arachidonic acid

• enzyme: 15-lipoxygenase

• product(s): 15-HPETE

step 2:

• substrate(s): 15-HPETE

• product(s): 15-HETE

What are lipoxins?

trihydroxy derivatives of arachidonic acid

What do lipoxins do?

• induce chemotaxis

• stimulate superoxide anion production in leukocytes

Describe the synthesis of lipoxin.

1. arachidonic acid

2. 15-lipoxygenase

3. 5-lipoxygenase

4. reductions

5. lipoxin A₄ (LXA₄)

Describe the synthesis of dihydroxide/5,6-diHETE.

• substrate(s): epoxide (5,6-EET)

• enzyme: cytochrome P450s

• process: reduction

• product(s): dihydroxide (5,6-diHETE)

What type of drugs are corticosteroids?

anti-inflammatory drugs

What do corticosteroids do?

decrease transcription of COX genes by:

• increasing lipocortin

• inhibiting phospholipase A₂

What do glucocorticoids inhibit?

phospholipase A₂

Which unsaturated fatty acids can mammals synthesize?

palmitoleate (omega-7) and oleate (omega-9)

Which PUFAs can mammals not synthesize and thus must require from the diet?

linolenate (omega 3) and linoleate (omega-6)

True or False: Mammals cannot introduce C=C bonds beyond carbon 9, but plants can.

true

What are some major PUFAs?

• linoleate

• linolenate

• arachidonate 

  • omega-6

  • eicosanoid precursor

• eicosapentanoate (EPA)

  • omega-3

  • eicosanoid precursor

• docosahexanoate (DHA)

  • omega-3

  • eicosanoid precursor

True or False: EPA and DHA are precursors for different eicosanoids than arachidonate. Properties of these eicosanoids differ.

true

True or False: When we were evolving, dietary ratio of omega-6 fatty acid (linoleate) to omega-3 (fatty acid (linolenate) was about 1:1 to 2:1. Currently, it is about 10:1 to 20:1 in Western diets.

true

Fish oils have a high content of what?

omega-4 fatty acids

What is high in omega-3 fatty acids?

flaxseed oil

Linoleic acid is metabolized into what?

arachidonic acid

Linolenic acid is metabolized into what?

EPA and DHA

What is the starting point of synthesizing thromboxane A₃?

EPA