PROSTANOIDS - PROSTAGLANDINS, PROSTACYCLINS and THROMBOXANES


Chemistry and Biology


1.  Nomenclature and Structures of Prostanoids

The prostanoids are part of a family of biologically active lipids derived derived from the action of cyclooxygenases or prostaglandin synthases upon the twenty-carbon essential fatty acids or eicosanoids. They can be further subdivided into three main groups, the prostaglandins, prostacyclins and thromboxanes, each of which is involved in some aspect of the inflammatory response. The prostaglandins were first isolated from semen and named from the prostate gland, thought to be their source, as long ago as the 1930s, but it was the 1960s before the biosynthetic relationship to specific essential fatty acids was described and intensive research into their biological properties began. The Nobel Prize for Medicine for 1982 was given to Professors Bengt Samuelsson, John Vane and Sune Bergstrom for their discoveries in this field (see the review by Samuelsson cited below). In general, prostaglandins occur at very low levels in tissues, of the order of nanomolar concentrations, but they have profound biological activities.

In structure, they are best considered as derivatives of a C20 saturated fatty acid, prostanoic acid, which does not itself occur in nature. A key feature is a five-membered ring encompassing carbons 8 to 12, as illustrated below. The thromboxanes are similar but have heterocyclic oxane structures. They are all synthesised by specific enzymes, which confer stereospecificity and chirality on every functional group, and are thus distinct from the isoprostanes, which are produced by non-enzymic means.

Structural formulae of prosanoids

In the approved nomenclature, each prostaglandin is named using the prefix 'PG' followed by a letter A to K depending on the nature and position of the substituents on the ring. Thus PGA to PGE and PGJ have a keto group in various positions on the ring, and are further distinguished by the presence or absence of double bonds or hydroxyl groups in various positions in the ring. PGF has two hydroxyl groups while PGK has two keto substituents on the ring. PGG and PGH are bicyclic endoperoxides. An oxygen bridge between carbons 6 and 9 distinguishes prostacyclin (PGI). Thromboxane A (TXA) contains an unstable bicyclic oxygenated ring structure, while thromboxane B (TXB) has a stable oxane ring. In addition, all prostaglandins have a hydroxyl group on carbon 15 and a trans-double bond at carbon 13 of the alkyl substituent (R2).

Further, a numerical subscript (1 to 3) is used to denote the total number of double bonds in the alkyl substituents, and a Greek subscript (α or β) is used with prostaglandins of the PGF series to describe the stereochemistry of the hydroxyl group on carbon 9. This is illustrated for prostaglandins PGE and PGFα of the 1, 2 and 3 series below, as examples.

Biosynthesis of prostaglandins - 1

The number of double bonds depends on the nature of the fatty acid precursor. Thus, the prostaglandins PGE1, PGE2 and PGE3 are derived from 8c,11c,14c-eicosatrienoic (dihomo-γ-linolenic), 5c,8c,11c,14c-eicosatetraenoic (arachidonic) and 5c,8c,11c,14c,17c-eicosapentaenoic acids, respectively. Of these, PGE2 is the most common and is involved in many physiological processes. Dihomo-prostaglandins derived from adrenic acid (22:4(n-6) have also been detected in cell preparations.


2.  Biosynthesis of Prostanoids

Eicosanoids, including the prostanoids, are not stored within cells, but are synthesised as required in response to hormonal stimuli. The prostaglandins PGE2 and PGF were first isolated and characterized from human seminal fluid in 1963 by Samuelsson. The first step in their synthesis is the release of the substrate fatty acid, such as arachidonic acid, from the cellular phospholipids, by the action of the enzyme phospholipase A2, and this is discussed in the Introductory document to this series. Next, the free acids are acted upon by one of two related enzymes, cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2) (alternatively termed prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and PGHS-2)), as is also discussed in the Introductory document. Both enzymes catalyse the same two reactions at different sites, i.e. a cyclooxygenase reaction in which two molecules of oxygen are added to arachidonic acid to form a bicyclic endoperoxide with a further hydroperoxy group in position 15, i.e. to form prostaglandin PGG2. The hydroperoxide is then reduced by a functionally coupled peroxidase reaction to form prostaglandin PGH2.

Biosynthesis of prostaglandins - 2

PGH2 is an unstable intermediate from which all other prostanoids are derived by a variety of different enzymic reactions. Some of these are illustrated above (for arachidonate as the primary precursor). The nature and proportions of the various enzymes and of the prostanoids produced differ according to cell type. Indeed different forms of some of the enzymes exist in cells; they may be functionally similar, but differ in amino acid sequence, structure and co-factor requirements. Thus, PGH2 is converted to PGE2 by prostaglandin E synthases. At least three distinct forms of this exist that are structurally and biologically distinct. The most important of these is a cytosolic enzyme, which is expressed constitutively in many different types of cell and is linked functionally to COX-1 to promote immediate PGE2 production. A second membrane-bound enzyme is induced by inflammatory stimuli and functions in concert with the inducible COX-2. Similarly, PGD2 is formed from PGH2 by the action of prostaglandin D synthases, which also exist in two forms and are evolutionarily distinct but functionally convergent. One is located in the central nervous system and the other in peripheral tissues.

Levuglandins,  such as LGE2 (also termed ‘isoketals’), are formed from PGH2 by a non-enzymic rearrangement. They have a very short half-life and react more rapidly than most lipid oxidation products with the free primary amine groups of proteins and phosphatidylethanolamine (see below) to form covalent adducts.

The most common stereochemical form of prostaglandin F (PGF) is synthesised by two main routes. For example, it can be produced directly from PGH2 by the action of prostaglandin H-endoperoxide reductase, using NADPH. Interestingly, this enzyme can also utilize PGD2 as a substrate for the synthesis of the second of the four stereochemical forms of PGF2α, 9α,11β-PGF2α. As an alternative, PGF2α is synthesised via PGE2 by the action of an enzyme prostaglandin E 9-ketoreductase.

The cyclopentenone prostaglandins A and J, with reactive α,β-unsaturated keto groups and high biological activity, are produced by spontaneous dehydration reactions from PGE and PGD, respectively, and further modifications can then occur. For example, PGA2 isomerizes to form the highly unstable PGC2, which rapidly undergoes a secondary isomerization to produce PGB2. Similarly, PGJ2 isomerizes to form Δ12-PGJ2 and then promotes a secondary dehydration of the C-15 hydroxyl with 15-deoxy-Δ12,14-PGJ2 as the end product. The highly electrophilic α,β-unsaturated carbonyl groups of these prostanoids react readily with the thiol moieties of cysteinyl residues on glutathione and cellular proteins.

2-Arachidonoylglycerol and anandamide can be substrates for enzymatic conversion to 2-prostanoylglycerols and prostanoylethanolamides (prostamides), respectively, by COX-2 specifically, and thence by further enzymic reactions to PGE2 and PGF analogues. While the physiological relevance of this is not yet clear, there is some evidence that 2-PGE2-glycerol has biological activity independent of that of the free prostanoid.

Prostacyclin (PGI2) and thromboxanes are also synthesised directly from PGH as illustrated below. Thus, a prostacyclin synthase converts PGH2 to PGI2, while a thromboxane A synthase catalyses the production of TXA2 from PGH2. These enzymes are related to the cytochrome P450 group of proteins and are located on the cytosolic face of the endoplasmic reticulum, so the precursor PGH must cross the membrane. PGI and TXA are the main prostanoids formed in endothelial and smooth muscle cells and in platelets and lung, respectively. Indeed, PGI2 and some other prostanoids can be produced by cell–cell interactions by using enzymes in adjacent cells, i.e. PGH2 of platelet origin is converted to PGI2 in the vascular epithelium.

Prostacyclin-thromboxane biosynthesis

The vinyl ether moiety in prostacyclin is unstable below pH 8.0, and PGI2 is rapidly deactivated non-enzymatically by a hydrolysis reaction to form 6-keto-PGF. Similarly, TXA2 contains an unstable ether linkage and is deactivated by non-enzymatic hydrolysis to from inert TXB2.

Before they can function, prostanoids that have been newly synthesised must be transported from the cytosol and cross various membranes. This is accomplished by active transporter systems.

Certain pathogenic fungi and yeasts produce 3-hydroxy-eicosanoids from host arachidonic acid and they can hijack the host’s COX-2 enzymes to produce 3-hydroxy-prostaglandins from these that are as active biologically as the normal compounds. In addition, the yeast Candida albicans and other pathogenic fungi produce PGE2 in vitro from exogenous arachidonate by a novel biochemical mechanism, which does not involve the COX enzymes.


3.  Prostanoid Catabolism

Prostanoids function close to the site of synthesis, and they are deactivated before they are exported into the circulation as inactive metabolites. Some, such as PGI and TXA, are deactivated spontaneously as described above. However, active enzyme systems also operate, and these function primarily by reaction with the 15(S)-hydroxyl group as discussed in the Introductory webpage. A significant portion of the thromboxanes undergoes dehydrogenation at C-11 by an 11-dehydroxythromboxane B2 dehydrogenase to form 11dh-TXB2, a metabolite found in human blood plasma and urine.


4.  The Functions of Prostanoids

Prostanoids are ubiquitous lipids in animal tissues that coordinate a multitude of physiologic and pathologic processes, either within the cells in which they are formed or in closely adjacent cells (they are deactivated too readily to be transported far) in response to specific stimuli. Under normal physiologic conditions, they have essential homeostatic functions in the cytoprotection of gastric mucosa, renal physiology, gestation, and parturition, but they are also implicated in a number of pathological conditions, such as inflammation, cardiovascular disease and cancer.

Prostanoids are sometimes described as local hormones that act in an autocrine fashion close to the site of their synthesis to coordinate the effects of other hormones in the circulation, although some can undergo facilitated transport from the cell via specific transporters to exert paracrine actions. In order to express their activity, they interact with specific G-protein-linked receptors mainly, , which have been subdivided into five classes in the mouse and man. These are specific for PGE2 (designated EP or four subclasses EP1–EP4), PGD2 (DP or two subclasses DP1-DP2), PGF (FP), PGI2 (IP) and TXA2 (TP). The immediate result is an increase or decrease in the rate of generation of cytosolic second messengers (cAMP or Ca2+), a change in membrane potential or activation of a specific protein kinase. The different receptors characterized from diverse cell types tend to have high, but not absolute, specificity for particular prostanoids with characteristic functions in each cell. Certain of the cyclopentanone prostanoids (PGA and PGJ series) interact with peroxisome proliferator-activated receptors (PPARs), especially PPARγ, which is a nuclear hormone receptor or ligand-activated transcription factor involved in adipogenesis, glucose homeostasis and lipid metabolism.

The picture of prostanoid actions is complicated by the fact that a given prostanoid can have a number of different biological functions, sometimes opposing, according to the cell type, the nature of the stimulatory response and the type of receptor. For example, PGE2 can have either pro- or anti-inflammatory effects depending on its interactions with one of four receptors in different cell types. The relative activities of the two iso-enzymes COX-1 and COX-2 are also essential to an understanding of the activity of prostanoids in any given circumstance. The complexity of the various interactions can only be hinted at here.

cartoonInflammation and immune responses: Arguably the best known of the functions of prostaglandins and thromboxanes in cells is that they modify the inflammatory response, affecting symptoms, such as pain, fever and swelling. It should be recognized that inflammation is an intrinsically beneficial event that leads to removal of offending molecules and restoration of tissue structure and function. The main cause for concern is when acute inflammation fails to resolve leading to excessive tissue damage. In the early days of prostaglandin research, it was evident that prostaglandins injected into tissues could induce all the symptoms of inflammation. However, it is now recognized that the interactions are complex, and prostanoids can act both in a pro- and anti-inflammatory manner according to the nature of the inflammatory stimulus and the specific prostanoid produced, together with the profile of prostanoid receptors in a given type of cell. For example, EP3 receptors are involved in the development of fever, while EP2 and EP4 function in allergy and bone resorption.

Under normal conditions, prostanoid levels in cells are low, but during inflammation both the nature and concentration of prostanoids can change dramatically. For example, macrophages produce both PGE2 and TXA2, but the ratio changes to an excess of PGE2 with an inflammatory stimulus. In these actions, prostanoids are best viewed as part of complex regulatory networks that modulate the actions of immune cells. PGE2 in particular has potent pro-inflammatory effects and is involved in all the processes leading to the classic signs of inflammation, including inducing fever and enhancing pain, On the other hand, it has anti-inflammatory properties also, such as suppressing lymphocyte proliferation and inhibiting the production of certain interleukins and other cytokines. It inhibits the action of 5-lipoxygenase, which is involved in the synthesis of pro-inflammatory leukotrienes, and stimulates the activity of the anti-inflammatory lipoxins. Therefore, PGE2 has a role in initiating the inflammatory response and in its eventual resolution. There is a particular interest in findings that in its pro-inflammatory role PGE2 promotes the growth of colorectal tumors (see below). It is also involved in the pathology of rheumatoid arthritis. Similarly, prostaglandin PGF is an important pro-inflammatory mediatormediator, especially in patients with chronic inflammatory diseases such as rheumatoid arthritis.

PGI2 an important mediator of the oedema and pain that accompany acute inflammation and it is produced rapidly following tissue injury or inflammation. For example, it is the most abundant prostanoid in synovial fluid in human arthritic knee joints.

The high levels of prostanoids found in inflammation are presumed to be due to the recruitment of leukocytes and the induction of the COX-2 enzyme (COX-1 appears to have a minor role only), which then produces the pro-inflammatory prostanoids mainly in many tissues. This explains the interest in COX-2 inhibitors for treating arthritis and other chronic inflammatory diseases. Inhibition of cyclooxygenases also explains the role of non-steroidal drugs, such as aspirin, in reducing the symptoms of fever. In the brain, COX-2 is present in neurons and has been implicated in the progression of Alzheimer's disease.

Immune responses are initiated and coordinated by T lymphocytes. Prostanoids are known to interact with T cells in a variety of ways, and appear to modify their development and maturation. Thus, PGE2 inhibits lymphocyte activation and proliferation, while TXA2 has opposing effects. Again, the actions of COX-2 (and COX-1) may be the key to triggering antigen-specific inflammation. However, this view may be too simplistic, and there is evidence that COX-2 is pro-inflammatory in the early stages of inflammation, but is beneficial at later stages by generating anti-inflammatory prostanoids. COX-1 derived prostanoids may sustain the inflammatory response.

Structural formula for 15-deoxy-PGJ2Although PGD2 has pro-inflammatory properties in allergic responses and in brain in the perception of pain, it is also recognized to be a key anti-inflammatory prostanoid that may be involved in the resolution of inflammation. Similarly, PGJ2, Δ12-PGJ2 and the short-lived 15-deoxy-Δ12,14-PGJ2, the J-series of prostaglandins produced by dehydration of PGD2, are now well established as anti-inflammatory regulators, which function mainly if not solely via an interaction with PPARγ as discussed briefly above. They may also be involved in the immune response as they are produced in antigen-presenting cells such as activated T lymphocytes. 15-Deoxy-Δ12,14-PGJ2 is important as an inhibitor of tumorigenesis (see below).

Polyunsaturated fatty acids of the omega-3 family are known to have anti-inflammatory properties. One explanation is that they inhibit the release of arachidonate from membrane phospholipids for eicosanoid production, or they may compete with arachidonate for the same enzymes of eicosanoid biosynthesis. Another reason may be that the 3-series prostanoids derived from eicosapentaenoic acid (EPA) have different biological activities from those of the 2-series. The resolvins and protectins must also be considered in this context.

Cardiovascular effects: Two prostanoids are especially important and have essential but opposing functions in the maintenance of vascular homeostasis, i.e. thromboxane TXA2 and prostacyclin PGI2 (PGE2 and PGD2 are also relevant). TXA2 is synthesised mainly in platelets (which express only COX-1), production being enhanced during platelet activation, and it promotes platelet aggregation, vasoconstriction, and smooth muscle proliferation, even though it has a half-life of only 20-30 seconds. In contrast, PGI2 is the main product of macro-vascular endothelial cells. It is produced as required and exerts its effects a potent vasodilator locally through a specific IP receptor; it also inhibits platelet aggregation and smooth muscle cell proliferation. Thus, it contributes substantially to myocardial protection. COX-2 is the enzyme that provides the main source of prostacyclin. Both TXA2 and PGI2 are therefore important mediators of pathological vascular events including thrombosis and atherogenesis, and it is evident that the correct balance between the two prostanoids is essential to good cardiovascular health. The ratio of TxA2:PGI2 seems to be more important than the absolute amounts of these mediators that are produced in vivo. Further relevant factors are increased expression and activation of the TP receptor (for TXA2) in atherosclerotic lesions, which can directly accelerate atherogenesis and plaque growth.

The cardio-protective effect of aspirin, established by clinical trials, is exerted by the irreversible long-term inhibition of platelet COX-1 and thence of TXA2 biosynthesis for the lifetime of a platelet in the circulation (aspirin has little effect on PGI synthesis). Indeed, aspirin appears to be the only COX inhibitor with proven cardioprotective activity. In contrast, there is some concern that specific COX-2 inhibitors may have pro-thrombotic effects by inhibiting prostacyclin synthesis relative to that of thromboxanes. In clinical practice, such potential adverse effects of these drugs have to be balanced against positive effects in other tissues since only 1-2% of patients are believed to be at risk. Once more, polyunsaturated fatty acids of the omega-3 family are believed to have beneficial effects.

Lung: PGE2 can have anti-inflammatory and anti-asthmatic effects by activating the EP3 receptor. The role of PGD2 is more complex, but it may be pro-inflammatory.

Gastrointestinal system: COX-1 is always present throughout the human gastrointestinal tract, and produces PGI2 and PGE2, which have protective effects on the gastrointestinal mucosa. Both of these prostanoids reduce acid secretion from parietal cells, while increasing blood flow and stimulating the secretion of mucus. In this instance, the non-steroidal anti-inflammatory drugs, such as aspirin, have negative effects, while the COX-2 inhibitors can be beneficial. On the other hand, these findings are challenged by studies showing that COX-2 is expressed in the intestinal mucosa, and is induced in ulceration, for example, when large amounts of prostaglandins are produced that assist in healing.

Scottish thistleKidney function: Prostaglandins generated by both COX-1 and COX-2, especially PGE2, assist in the regulation of kidney function by maintaining vascular tone, blood flow, and salt and water excretion. PGE2 is required for the regulation of sodium re-absorption, while PGI2 (and possibly PGE2) increases potassium secretion. In addition, this PGI2 with its well-known vasodilatory properties increases renal blood flow and the flow of fluids through the kidney. These actions are mediated via specific receptors, four of which have been identified for PGE2, for example.

Reproductive system: Prostaglandins produced both by COX-1 and COX-2 are involved in many aspects of reproduction, from ovulation and fertilization through to labour. They are produced in the fetus and in the placenta as well as in other reproductive tissues. In particular, the synthesis of PGE2 and PGF is increased appreciably during labour, and these prostaglandins are in fact used as drugs to induce labour.

Cancer: COX-2 is over-expressed in many cancers, including those of the breast, colon and prostate. In particular, PGE2 produced by the enzyme occurs at much higher concentrations in tumor than in normal tissues. It promotes survival of tumor cells by inhibiting apoptosis and inducing proliferation, and by increasing cell motility and migration. In addition, via its effect on the immune system and inflammation, it has adverse effects in relation to the destruction of tumors. In consequence both the non-steroidal anti-inflammatory drugs, such as aspirin, and the COX-2 inhibitors have been found to have beneficial effects towards some types of cancer. Also, it is established that EP1 receptors are involved in chemically induced colon cancer. In contrast, both pro- and anti-tumorigenic activities have been demonstrated for PGD2 depending on the experimental model.

Thromboxane TXA4 is a pro-carcinogenic mediator that affects a number of tumor cell survival pathways, including cell proliferation, apoptosis and metastasis, and its activity is again balanced by that of prostacyclin.

15-Deoxy-Δ12,14-PGJ2 (15-d-PGJ2), a potent anti-inflammatory regulator that functions via its interaction with PPARγ, also regulates adipogenesis and tumorigenesis and is produced by a variety of cells. An active transport system may carry it to the cells where it is required, and thence it is transported into the nucleus, where it affects gene transcription. Unlike PGE2, 15-d-PGJ2 is a potent anti-tumor agent, inhibiting tumor growth both in vitro and in vivo in many tissues. It appears to act in a number of ways, for example directly by inhibiting proliferation and stimulating apoptosis. Also, it can interact indirectly to inhibit migration of tumor cells, and it can affect surrounding cells to reduce the expression of key receptors. However, some experimental conditions have been identified in which it exerts contrary effects.

In general, PGE2 and 15-d-PGJ2 have profound but opposing effects on tumorigenesis. It is evident that the prostaglandin synthases that are responsible for their biosynthesis are likely to be key targets for the development of anticancer drugs.

Protein metabolism: γ-Keto aldehydes such as the levuglandins (see above) and isolevuglandins, the latter produced in an analogous manner to the isoprostanes, have a remarkable reactivity towards proteins, forming adducts with greatly modified biological functions. Thus, these di-aldehydes react with lysyl residues on proteins to form first Schiff base adducts and thence pyrrole derivatives, which are able to form intra- and intermolecular protein-protein cross-links. Pyrrole adducts are in turn sensitive to oxygen and are further oxidized in vivo to stable lactam and hydroxylactam products. Protein adducts of this type are not at all easy to analyse, but those in brain have been correlated with the severity of Alzheimer’s disease, for example. Indeed, levuglandins and isolevuglandins are believed to be among the most potent neurotoxic products of lipid oxidation.

Levuglandins also react with phosphatidylethanolamine to form hydroxy-lactam derivatives, which may be better markers of oxidative injury from a practical standpoint, as they are more easily analysed.

Parasitic infections: It has been established that a number of parasitic organisms produce prostaglandins in the same way as their mammalian hosts, and by similar enzymic mechanisms. They may play a part in the pathogenesis of parasitic diseases.


5.   Some Exotic Prostanoids

Marine invertebrates, including sponges, corals, and molluscs, contain a wide range of prostaglandins, many of which are of the conventional type such as PGE2, PGF2 and so forth. They are presumed to perform similar functions as in mammals, and are also involved in the regulation of oogenesis and spermatogenesis, ion transport and defence. One species of coral (Plexaura homomalla) contains up to 8% of its dry mass as PG esters, and for many years this was a primary source of material for experimental work. In many marine invertebrates, the prostaglandins exist largely in esterified form rather than the free state.

Some exotic marine prostanoids

In addition, a number of novel prostanoids have been discovered, some of which are illustrated above, which differ in stereochemistry from the typical prostanoids, or contain acetyl groups, or are substituted with halogen atoms, such as chlorine or bromine. Little is known of the biochemistry or function of the clavulones, bromovulones or punaglandins in marine organisms, but there is increasing interest in them because of reported anti-tumor activities.


6.  Prostanoid Analysis

Analysis of prostanoids is not a simple task, because they occur at such low levels in tissues and because of their high reactivity. Extraction must be carried out under mild conditions as rapidly as possible, and solid-phase extraction methods are now available that set the standard. Subsequent analysis usually involves derivatization to improve volatility, followed by analysis by GC or HPLC linked to mass spectrometry. Immunoassays are available that may be suitable for some clinical applications, but they are not sensitive to minor differences in prostanoid structure.


Suggested Reading


W.W. Christie

James Hutton Institute (and Mylnefield Lipid Analysis), Invergowrie, Dundee (DD2 5DA), Scotland.

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AOCS Credits/disclaimer Updated: January 7th, 2014 Author