We have already mentioned that thromboxane A2 and prostacyclin have very short half-lives in vivo. In addition, it was shown by Vane and Piper in the late 1960s that prostaglandins like PGE2 or PGF2a were rapidly catabolized and did not survive a
single pass through the circulation. The lung plays a major role in this inactivation process, which is usually initiated by oxidation of the hydroxyl group at C15. The A13 double bond is next attacked and further degradation involves P- and rn-oxida-tion (Sections 2.3.1 and 2.3.3) in peroxisomes. Concentrations of major active prostaglandin products in blood are less than 10-10 M and because of their rapid catabolism they can only act as local hormones or autocoids, which modify biological events close to their sites of synthesis. Moreover, in contrast to typical circulating hormones, prosta-noids are produced by practically every cell in the body. They exit the cell via carrier-mediated transport before being inactivated rapidly in the circulation.
2.4.6 Instead of cyclooxygenation, arachidonate can be lipoxygenated or epoxygenated
For over 40 years it has been known that plant tissues contain lipoxygenases, which catalyse the introduction of oxygen into polyunsaturated fatty acids (Section 2.3.6). In 1974 Hamberg and Samuelsson found that platelets contained a 12-lipoxygenase and since that time 5-, 8- and 15-lipoxygenases have also been discovered. The immediate products are hydroperoxy fatty acids, which for the arachidonate substrate are hydro-peroxy eicostatetraenoic acids (HPETEs).
The HPETEs can undergo three reactions (Fig. 2.44). The hydroperoxy group can be reduced to an alcohol, thus forming a hydroxyeicosatetraenoic acid (HETE). Alternatively, a second lipoxygena-tion elsewhere on the chain yields a dihydroxy-eicosatetraenoic acid (diHETE) or dehydration produces an epoxy fatty acid.
Epoxy fatty acids, such as leukotriene A4 (Fig. 2.44) can undergo non-enzymic reactions to various diHETEs, can be specifically hydrated to a given diHETE or can undergo ring opening with GSH to yield peptide derivatives. Epoxy eicosatrienoic acids and their metabolic products are called leu-kotrienes - the name being derived from the cells (leukocytes) in which they were originally recognized.
As mentioned above, four lipoxygenases (5-, 8-, 12- and 15-) have been found in mammalian tissues. The 5-lipoxygenase is responsible for leukotriene production and is important in neutrophils, eosi-nophils, monocytes, mast cells and keratinocytes as well as lung, spleen, brain and heart. Products of 12-lipoxygenase activity also have biological activity, e.g. 12-HPETE inhibits collagen-induced platelet aggregation and 12-HETE can cause migration of smooth muscle cells in vitro at concentrations as low as one femtomolar (10-15M). In contrast, few biological activities have been reported for 15-HETE, although this compound has a potentially important action in inhibiting the 5- and 12-lipox-ygenases of various tissues. The physiological role of the 8-lipoxygenase, which has only recently been discovered, is also obscure.
Arachidonic acid and other oxygenated derivatives of the arachidonate cascade can also be metabolized by cytochrome P450-mediated pathways of which the most important in animals appears to be the epoxygenase pathway.
The opening of the epoxy group of leukotriene LTA4 by the action of glutathione S-transferase attaches the glutathionyl residue to the 6 position (Fig. 2.44). Unlike most glutathione S-transferases, which are soluble, the LTC4 synthase is a micro-somal protein with a high preference for its LTA4 substrate. The product (LTC4) can then lose a y-glutamyl residue to give LTD4 and the glycyl group is released in a further reaction to give LTE4 (Fig. 2.45). Prior to their structural elucidation, leuko-trienes were recognized in perfusates of lungs as slow-reacting substances (SRS) after stimulation with cobra venom or as slow-reacting substances of anaphylaxis (SRS-A) after immunological challenge. LTC4 and LTD4 are now known to be major components of SRS-A.
Unlike the cylcooxygenase products, the formation of leukotrienes is not solely determined by the availability of free arachidonic acid in cells (Section 2.4.9). Thus for example, 5-lipoxygenase requires activation in most tissues and various immunolo-gical or inflammatory stimuli are able to cause this.
In some cells both cyclooxygenase and lipoxygenase products are formed from arachidonic acid. It is possible that the arachidonic acid substrate comes from separate pools. For example, macrophages release prostaglandins but not leukotrienes when treated with soluble stimuli. In contrast, when insoluble phagocytic stimuli (such as bacteria) are used, both prostaglandin and leukotriene formation is increased.
We have already mentioned that 15-HETE, a 15-lipoxygenase product, can inhibit 5- and 12-lipoxy-genases. Conversely, 12-HPETE has been shown to increase 5-HETE and LTB4 formation. Thus, the various lipoxygenase pathways have interacting effects as well as the interdependent actions of cyclooxygenase and lipoxygenase products.
Genes for various animal lipoxygenases have been isolated and their primary sequences compared. Various ds-acting elements have been found in the 5'-flanking regions and all the genes contain multiple GC boxes but no typical TATA boxes in their promoter regions. They can, therefore, be considered as housekeeping genes. Apart from various putative transcriptional regulatory elements, the lipoxygenases themselves can be subject to activation. For the 5-lipoxygenase, ATP, Ca2+ and various leukocyte stimulatory factors have been demonstrated. Moreover, in the absence of phos-
Fig. 2.45 Formation of peptidoleukotrienes.
cells. However, the mechanism of regulation is distinctly different with the FLAP gene containing a TATA box and other regulatory motives not found on the 5-lipoxygenase gene.
Because of the important role of FLAP in 5-lipoxygenase activity and, hence, leukotriene biosynthesis, pharmaceuticals that bind to FLAP have been developed in addition to those that inhibit the catalytic reaction. Many of the latter are redox-active and, although very effective 5-lipoxygenase inhibitors, they often show unwanted side-effects. Two separate classes of drugs, indoles (like compound HK-886) and quinolines, bind to FLAP and several members have been developed and tested for medical use.
Fig. 2.45 Formation of peptidoleukotrienes.
pholipid the purified enzyme has poor activity suggesting that it may need to act at a membrane interface.
Whereas, the key event for prostaglandin production is the release of substrate-free fatty acid by phospholipase A2 (Section 2.4.9), leukotriene synthesis only occurs in intact cells following exposure to certain stimuli of which the most important is a rise in intracellular calcium. Moreover, activation of 5-lipoxygenase seems to involve translocation of the enzyme from the cytosol to membrane surfaces, where it becomes active, makes leukotrienes and undergoes suicide inacti-vation.
Studies with a highly potent inhibitor of leuko-triene biosynthesis, MK-886, in intact cells led to the discovery of 5-lipoxygenase activating protein (FLAP). Although the details of FLAP's function are still being elucidated it seems most likely that the protein makes the 5-lipoxygenase reaction much more efficient by containing a binding site for ara-chidonic acid and facilitating transfer of the substrate to the enzyme. A possible model for FLAP-dependent synthesis and release of LTC4 is shown in Fig. 2.46.
Because both FLAP and 5-lipoxygenase are needed for leukotriene synthesis it might be expected that their genes would be regulated together. Indeed, this has been shown to be the case in some
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