The Lipid Library

 

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 from 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 when 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 Flower 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.

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 PGF_α of the 1, 2 and 3 series below, as examples.

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 recently 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 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.

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 a prostaglandin E synthase. A number of distinct forms of this exist, but the main ones are a cytosolic enzyme that operates in conjunction with COX-1, and a membrane-bound enzyme, which is induced by inflammatory stimuli and which functions in concert with the inducible COX-2. PGE₂ is the principal prostanoid found in certain renal cells, for example. Similarly, PGD₂ is formed from PGH₂ by the action of prostaglandin D synthases, which also exist in two forms, one of which is located in the central nervous system and the other in peripheral tissues.

Levuglandins,  such as LGE₂, are formed from PGH₂ by a non-enzymic rearrangement.

The most common stereochemical form of PGF_2α is synthesised by via two routes. For example, it can be produced directly from PGH₂ by the action of prostaglandin H-endoperoxide reductase, using NADPH. Alternatively, it can be synthesised via PGE₂ by the action of an enzyme prostaglandin E 9-ketoreductase. A second of the four stereochemical forms of PGF_2α, 9α,11β-PGF_2α, is formed from PGD₂ by reduction of the keto group in position 9 by a PGD 11-ketoreductase. Prostaglandins A and J, with reactive α,β-unsaturated keto groups and high biological activity, are produced from PGE and PGD, respectively, by spontaneous dehydration reactions, and further modifications can then occur.

Prostacyclin and thromboxanes are also synthesised directly from PGH as illustrated above. 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. The vinyl ether moiety in prostacyclin is unstable below pH 8.0, and PGI₂ is deactivated non-enzymatically by a hydrolysis reaction to form 6-keto-PGF_1α. Similarly, thromboxane A is deactivated by non-enzymatic hydrolysis to from thromboxane B (TXB).

2-Arachidonoylglycerol and anandamide can be can be substrates for enzymatic conversion to 2-prostanoylglycerols and prostanoylethanolamides (prostamides), respectively, by COX-2 specifically. 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. There are also suggestions that the separate activities of COX-1 and COX-2 are functionally coupled in some way to the downstream production of prostanoids.

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 produces PGE₂ in vitro from exogenous arachidonate by a novel biochemical mechanism.

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.

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. Thus, a NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase oxidizes the hydroxyl to a keto group, before the double bond in position 13 is hydrogenated by a specific reductase. The resulting keto-prostanoids have little biological activity. Further catabolism involves beta- and omega-oxidation.

4.  The Functions of Prostanoids

Prostanoids are ubiquitous lipids 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.

In order to express their activity, prostanoids interact with specific G-protein-linked receptors mainly and can be considered as local hormones that coordinate the effects of other hormones in the circulation. A number of different receptors have been characterized from diverse cell types, and these 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 proliferators-activated receptors (PPARs). The picture 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.

Inflammation: 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. 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. 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, including inducing fever and enhancing pain, but it also has anti-inflammatory properties, such as suppressing lymphocyte proliferation and inhibiting the production of certain interleukins and interferons.

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 mainly the pro-inflammatory prostanoids 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.

PGD₂ has anti-inflammatory properties and may be involved in the resolution of inflammation. Similarly, PGJ₂, Δ¹²-PGJ₂ and 15-Deoxy-Δ^12,14-PGJ₂, the J series of prostaglandins that were once thought to be simply inactive degradation products of PGD₂, are now well established as an anti-inflammatory regulators and may function via an interaction with PPARs as discussed briefly above. 15-Deoxy-Δ^12,14-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.

Vascular 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. In contrast, prostacyclin is the main product of macro-vascular endothelial cells. It is a potent vasodilator, and also inhibits platelet aggregation and smooth muscle cell proliferation; 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 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). 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. Once more, polyunsaturated fatty acids of the omega-3 family are believed to have beneficial effects.

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.

Kidney 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 the 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_2α 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.

15-Deoxy-Δ^12,14-PGJ₂ (15-d-PGJ₂), a potent anti-inflammatory regulator that may function via its interaction with PPARs, 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.

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. 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.

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 PGE_2, 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 defense. 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.

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 gas chromatography 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.

7.  Suggested Reading

• Fetalvero, K.M., Martin, K.A. and Hwa, J. Cardioprotective prostacyclin signaling in vascular smooth muscle. Prostaglandins Other Lipid Mediators, 82, 109-118 (2007).
• Flower, R.J. Prostaglandins, bioassay and inflammation. Brit. J. Pharm., 147, S182-S192 (2006).
• Harris, S.G., Padilla, J., Koumas, L., Ray, D. and Phipps, R.P. Prostaglandins as modulators of immunity. Trends Immunol., 23, 144-150 (2002).
• Lee, S.H., Williams, M.V. and Blair, I.A. Targeted chiral lipidomics analysis. Prostaglandins Other Lipid Mediators, 77, 141-157 (2005).
• Nicolaou, A. Prostanoids. In: Bioactive Lipids. pp. 197-222 (edited by A. Nicolaou and G. Kokotos, Oily Press, Bridgwater) (2004).
• Parente, l. and Perretti, M. Advances in the pathophysiology of constitutive and inducible cyclooxygenases: two enzymes in the spotlight. Biochem. Pharmacol., 165, 153-159 (2003).
• Rowley, A.F. Vogan, C.L., Taylor, G.W. and Clare, A.S. Prostaglandins in non-insectan invertebrates: recent insights and unsolved problems. J. Exp. Biol., 208, 3-14 (2005).
• Salomon, R.G. Levuglandins and isolevuglandins: stealthy toxins of oxidative injury. Antioxid. Redox Signal., 7, 185-201 (2005).
• Smith, M.L. and Murphy, R.C. The eicosanoids: cyclooxygenase, lipoxygenase and epoxygenase pathways. In: Biochemistry of Lipids, Lipoproteins and Membranes. pp. 341-371 (Vance, D.E. and Vance, J. (editors), Elsevier, Amsterdam) (2002).
• Tapiero, H., Nguyen Ba, G., Couvreur, P. and Tew, K.D. Polyunsaturated fatty acids (PUFA) and eicosanoids in human health and pathologies. Biomed. Pharmacother., 56, 215-222 (2002).
• Tilley, S.L., Coffman, T.M. and Koller, B.H. Mixed messages: modulation of inflammation and immune responses by prostaglandins and thromboxanes. J. Clin. Invest., 108,15-23 (2001).
• Tsikas, D. Application of gas chromatography mass spectrometry and gas chromatography tandem mass spectrometry to assess in vivo synthesis of prostaglandins, thromboxane, leukotrienes, isoprostanes and related compounds in humans. J. Chromatogr. B, 717, 201-245 (1998).

Updated: 10/12/2007 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland