Eicosanoids synthesis and biosignaling

Prostaglandins and related compounds are "local hormones" that are

synthesized from the polyunsaturated fatty acid arachidonate. They have
specific effects on target cells close to their site of formation. They are rapidly
degraded, so they are not transported to distal sites within the body.
Examples include prostaglandins, prostacyclins, thromboxanes,
leukotrienes, and epoxyeicosatrienoic acids. They have roles in inflammation,
fever, regulation of blood pressure, blood clotting, immune system modulation,
control of reproductive processes and tissue growth, and regulation of the
sleep/wake cycle. Prostaglandins and related compounds are collectively known
as eicosanoids. They are produced from arachidonic acid, a 20-carbon
polyunsaturated fatty acid (5,8,11,14-eicosatetraenoic acid).

Fig.1 Arachidonic acid

Nomenclature.
The numbering of the carbon atom is based on the numbering in prostanoic acid
below.

Fig 2.Prostanoic acid

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Fig 3.Nomenclature around the cyclopentane ring

The nomenclature of PGs is based on the letter component (PGA, PGB, PGC...) which
identifies the functional groups of the cyclopentane ring (see the previous picture) the
numerical subscript (PGA1, PGA2...) which recalls the number of double bonds in the
carbon chains. This number depends on the precursor fatty acid, PGE1 deriving from
20:3(n-6), PGE2 from 20:4(n-6) and PGE3 from 20:5(n-3).
To summarize the cyclooxygenase metabolism, prostaglandin G/H synthase catalyzes the
sequential formation of PGG2 and PGH2 (they are both named endoperoxides) by
addition of molecular oxygen at the C9, C11 and C15 positions..

Fig.4 Early products of the cyclooxygenase pathway

PGH2 is then enzymatically and non-enzymatically converted in several

molecules, biologically active but precursors of other active molecules. Thus, are
formed PGD2 (the principal cyclooxygenase product of the mast cells and the
nervous system, it inhibits platelet aggregation), PGE2 (produced in near all cell
types, elevates cAMP levels, stimulates bone resorption), PGF2a, PGI2
(prostacyclin, characterizes endothelial cells, induces vasodilation and inhibits
platelet aggregation), thromboxane A2, TXA2, (was first isolated from
thrombocytes or platelets, it has strong platelet-aggregating activity) and even a
17 -carbon hydroxylated fatty acid 12-HHT (12-hydroxyheptadeca-5,8,10-trienoic
acid), stimulates prostacyclin production and has chemotactic activity).

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Fig 5.Various eicosanoids

Eicosanoid biosynthesis in animal cells either results from agonist-stimulated

phospholipase activation (endogenous pathway) or from lipoprotein receptor-
mediated uptake and lysosomal lipid hydrolase-dependent release of AA
(exogenous pathway).LDL stimulates eicosanoid formation through delivery of
substrate AA to enzymes of oxidative AA metabolism. The classical LDL receptor
is a control point of the effects of LDL AA on eicosanoid formation in different
tissues: LDL AA metabolism occurs in several cell types of mesenchymal and
epithelial origin and generates the formation of distinct eicosanoid patterns in
each case. The LDL AA pathway does appear to couple directly to the PGH
synthase reaction, whereas it does not couple directly to the 5-lipoxygenase
reaction
The fatty acid arachidonate is frequently esterified at the hydroxyl on C-2 of
glycerophospholipids, especially phosphatidyl inositol, shown at right with
arachidonate in blue. Arachidonate is released from phospholipids by hydrolysis.

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Sites of hydrolytic cleavage by Phospholipase A2 hydrolyzes the ester linkage
between a fatty acid and the hydroxyl at carbon 2 of the glycerol backbone,
releasing the fatty acid (e.g., arachidonate) and a lysophospholipid as products.

Fig.6 Phosphatidyl inositol

There are multiple Phospholipase A2 enzymes, subject to activation via different

signal cascades. The inflammatory signal molecule platelet activating factor is
involved in activating some variants of Phospholipase A 2.Corticosteroids are
anti-inflammatory because they inhibit Phospholipase A 2, reducing arachidonate
release. Phosphatidyl inositol signal cascades may lead to release of
arachidonate. After phosphatidyl inositol is phosphorylated to PIP2, cleavage of
PIP2 via Phospholipase C yields diacylglycerol (and IP3). Arachidonate
release from diacylglycerol is then catalyzed by Diacylglycerol Lipase.

Fig.7 Phospholipase C cleavage

Cyclic pathway:
Prostaglandin H2 Synthase (PGH2 Synthase) catalyzes the committed step in
the "cyclic pathway" that leads to production of prostaglandins, prostacyclins, and

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thromboxanes. Different cell types convert PGH2 to different compounds.
Prostaglandin H2 Synthase is a heme-containing dioxygenase, bound to
endoplasmic reticulum membranes. (A dioxygenase incorporates O 2 into a
substrate.) PGH2 Synthase exhibits two catalytic activities, Cyclooxygenase and
Peroxidase. The enzyme expressing both activities is sometimes referred to as
Cyclooxygenase, abbreviated COX.

Fig 8. Various pathways for eicosanoid synthesis Fig. 9 Portion of the cyclooxygenase pathway

The interacting cyclooxygenase and peroxidase reaction pathways are complex.

A peroxide (such as that generated later in the reaction sequence) oxidizes the
heme iron. The oxidized heme accepts an electron from a nearby tyrosine
residue (Tyr385). The resulting tyrosine radical is proposed to extract a hydrogen
atom from arachidonate, generating a radical species that reacts with O 2.
The signal molecule NO (nitric oxide) may initiate prostaglandin synthesis by
reacting with superoxide anion (O2-) to produce peroxynitrite, which oxidizes the
heme iron enabling electron transfer from the active site tyrosine. Prostaglandin
synthesis in response to some inflammatory stimuli is diminished by inhibitors of
Nitric Oxide Synthase.
Non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin and
derivatives of ibuprofen, inhibit Cyclooxygenase activity of PGH 2 Synthase. They
inhibit formation of prostaglandins involved in fever, pain and inflammation. They
inhibit blood clotting by blocking thromboxane formation in blood platelets.
Ibuprofen and related compounds act by blocking the hydrophobic channel by
which arachidonate enters the Cyclooxygenase active site. An iodinated analog
of ibuprofen is seen in the structural diagram above, between the membrane-
binding domain and the heme.

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Fig.10 Ibuprofen

Aspirin acetylates a serine hydroxyl group near the active site, preventing
arachidonate binding. The inhibition by aspirin is irreversible. However, in most
body cells re-synthesis of PGH2 Synthase would restore cyclooxygenase activity.
Thromboxane A2 stimulates blood platelet aggregation, essential to the role of
platelets in blood clotting. Many people take a daily aspirin for its anti-clotting
effect, attributed to inhibition of thromboxane formation in blood platelets. This
effect of aspirin is long-lived, because platelets lack a nucleus and do not make
new enzyme.

Fig.11 Salicylic acid

Two isoforms of PGH2 Synthase are designated COX-1 and COX-2

(Cyclooxygenase 1 and 2).
COX-1 is constitutively expressed at low levels in many cell types. COX-1 is
essential for thromboxane formation in blood platelets, and for maintaining the
integrity of the gastrointestinal epithelium.
COX-2 expression is stimulated by growth factors, cytokines, and endotoxins.

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Inflammation is associated with up-regulation of COX-2 and increased formation
of prostaglandins. COX-2 levels increase in inflammatory disease states such as
arthritis.
Increased expression of COX-2 is seen in some cancer cells. Angiogenesis
(blood vessel development) essential to tumor growth requires COX-2.
Overexpression of COX-2 leads to increased expression of VEGF (vascular
endothelial growth factor). Regular use of NSAIDs has been shown to decrease
the risk of developing colorectal cancer.
Most NSAIDs inhibit both COX-1 and COX-2. More selective COX-2 inhibitors
have been developed, e.g., Celebrex and Vioxx. COX-2 inhibitors are anti-
inflammatory and block pain, but are less likely to cause the gastric toxicity often
associated with chronic use of less specific NSAIDs.However, cardiovascular
side effects have curtailed use of some of these drugs.
Some evidence suggests the existence of a third isoform of PGH 2 Synthase,
designated COX-3, with roles in mediating pain and fever, and subject to
inhibition by acetaminophen (Tylenol). Acetaminophen has little effect on COX-1
or COX-2, and thus lacks anti-inflammatory activity.
Prostaglandin receptors: Prostaglandins and related compounds are
transported out of the cells that synthesize them. Most affect other cells by
interacting with plasma membrane G-protein coupled receptors. Depending on
the cell type, the activated G protein may stimulate or inhibit formation of cAMP,
or may activate a phosphatidylinositol signal pathway leading to intracellular Ca++
release. Another prostaglandin receptor, designated PPAR, is related to a
family of nuclear receptors with transcription factor activity.
Prostaglandin receptors are specified by the same letter code. For example:
Receptors for E-class prostaglandins are designated EP.
Thromboxane receptors are designated TP.
Multiple receptors for a prostaglandin are specified by subscripts (e.g., EP 1,
EP2, EP3, etc.). Different receptors for a particular prostaglandin may activate
different signal cascades. Effects may vary in different tissues, depending on
which receptors are expressed
Linear Pathway:
The first step of the linear pathway for synthesis of leukotrienes is catalyzed by
Lipoxygenase. Mammalian organisms have a family of Lipoxygenase enzymes
that catalyze oxygenation of various polyunsaturated fatty acids at different sites.
Many of the products have signal roles.
5-Lipoxygenase, found in leukocytes, catalyzes conversion of arachidonate to
5-HPETE (5-hydroperoxyeicosatetraenoic acid). 5-HPETE is then converted to
various leukotrienes that induce inflammation and asthmatic constriction of the
bronchioles.
Anti-asthma medications include inhibitors of 5-Lipoxygenase, such as Zyflo
(zileuton), and drugs that interfere with leukotriene-receptor interactions. For
example, Singulair (montelukast) and Accolate (zafirlukast) block binding of
leukotrienes to receptors on the plasma membranes of airway smooth muscle. A
non-heme iron is the prosthetic group of Lipoxygenase enzymes. Ligands to the
iron include 4 histidine nitrogen atoms and the C-terminal carboxylate oxygens.

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The structure has been described as resembling an octahedron with two
unoccupied vertices. Presumably O2 binds at one of these open ligand positions.
5-Lipoxygenase is overexpressed in cancer cells. As in the case of COX-2, 5-
Lipoxygenase has a role in promoting angiogenesis essential to tumor growth

Linear Pathway
Cytochrome P450 epoxygenase pathways:
Epoxyeicosatrienoic acids (EETs) and hydroxyeicosatrienoic acids are formed
from arachidonate by enzymes of the cytochrome P450 family. Other members of
the cytochrome P450 family participate in a variety of oxygenation reactions,
including hydroxylation of sterols (to be discussed in the section on cholesterol
synthesis and metabolism).
An example of an EET (14,15-epoxyeicosatrienoic acid), produced from
arachidonate by activity of a cytochrome P450 epoxygenase, is shown at right.
EETs are modified by additional enzyme-catalyzed reactions to produce many
distinct compounds. They may be incorporated into phospholipids, and
released by action of phospholipases. EETs have roles in regulating cellular
proliferation, inflammation, peptide hormone secretion, and various cellular signal
pathways relevant to cardiovascular and renal functions.

Fig.13 14,15-epoxyeicosatrienoic acid(14,15EET)

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It has been shown that selenium deficiency alters the formation of eicosanoids
and signal transduction in rat lymphocytes

PUFAs, particularly arachidonic acid (AA), and their metabolites (eicosanoids)

as well as cytokines belong to the most important natural factors with non-
genotoxic (epigenetic) mode of action involved in promotion of malignances. The
PUFAs are the important components of membrane phospholipids and their
availibility can vary considerably depending on the interaction between
endogenous synthesis and external supply. This modulation of membranes can
have profound effects on the operation of membrane-associated proteins, the
affinity of receptors for their specific ligands, the release and action of cytokines
and change the composition of the second messenger system. PUFAs and
eicosanoids thus play an important role in regulating the transmission of signals
from the extracellular environment, via a complex intracellular network of relays
to the nucleus and the genome, and modulate the biological response

Fig.14. Eicosanoids play an important role in regulating the transmission of

signals from the extracellular environment
It has been demonstrated that some eicosanoids, i. e. leukotrienes (LTs) can act
as second messengers of some "positive" growth factors.There is some sketcy

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information about the role of eicosanoids in the effects of "negatively" acting
cytokines such as transforming growth factor beta 1 (TGF-1) and tumour
necrosis factor alfa (TNF).These cytokines, which are considered to be
important regulators of proliferation, differentiation and apoptosis of mammalian
cells

Fig.15.Well documented and undocumented actions of eicosanoids

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