Download Metabolism of Leukotrienes: The Linear Biosynthetic Pathway

Metabolism of Leukotrienes: The Linear Biosynthetic Pathway
David Goad
Department of Infectious Diseases and Physiology, Oklahoma State University
Introduction
Leukotrienes (LT) are short-lived, highly biologically active eicosanoid products of the linear pathway of
arachidonic acid (AA) metabolism, originating with the 5-lipoxygenase (5-LO)-catalyzed catalyzed oxidation of
arachidonate. LT biosynthesis, metabolism, and degradation have been extensively reviewed (1-5). LT are
synthesized in a variety of cell types including neutrophils, eosinophils, monocytes, mast cells, and lymphocytes and
in a number of tissues, most notably the lung, as well as brain, heart, and spleen. Two distinct types of
enzymatically-produced LT have been identified: Cysteinyl (or peptidyl) leukotrienes LTC4, LTD4, and LTE4
(originally identified as the slow reacting substances of anaphylaxis), and the dihydroxy leukotriene LTB 4 (4). The
pathophysiological effects of LT have been reviewed (1) and the biological effects of both classes of LT are
summarized in table 1. In addition, recent data suggests LTD4 and LTE4 may have substantial effects upon the
histamine H1 receptor, with the potential to exacerbate the physiological effects of histamine (6). Biosynthesis of LT
is not inhibited by non-steroidal antiinflammatory drugs (NSAIDS) which decrease production of the bioactive
products of cyclic arachidonate metabolism, the prostaglandins, prostacyclins, and thromboxanes. However, the
physiological relationship between cyclic and linear AA metabolites is demonstrated by recent evidence suggesting
inhibition of LT production may alleviate gastric ulceration associated with the administration of NSAIDS (7).
When one considers the bioactivity of LT with respect to pathophysiological processes it is not surprising that the
biosynthesis of LT, and the search for inhibitors of LT synthesis or receptor function remains an area of highly active
research (8). This review will outline the linear biosynthetic pathway of bioactive LT, with particular emphasis on
recent experimental observations.
Table 1. Biological effects of LT.
Cysteinyl LT
Vasoconstriction
Increase of vascular permeability in
postcapillary venules
Bronchoconstriction
Stimulation of mucus secretion
Intestinal contraction (ileum)
Plasma extravasation
Decrease of blood pressure
Reduction of myocardial contractility
and coronary blood flow
Decrease of renal blood flow
LTB4
Aggregation; chemokinesis
Chemotaxis; release of lysosomal
enzymes; stimulation of
superoxide anion production
Adhesion and transendothelial
migration of neutrophils
Increase of vascular permeability
Modulation of lymphocyte function
Affector of the production and
action of cytokines
Release of intracellular calcium
Increase of cAMP and cGMP synthesis
(Adapted from (1).)
Mobilization of Arachidonic Acid and the 5-Lipoxygenase Reaction
LT production originates with a phospholipase A2 (PLA2) catalyzed cleavage of AA from membrane
phospholipids (figure 1)(8). PLA2 may exist as either a cytosolic 85 kDa enzyme, or in an extracellular or cellassociated 14 kDa form (9). Activity of neutrophilic 85 kDa PLA2 has been shown to be stimulated by a downstream
product of the linear pathway, LTB4. A positive feedback model was proposed wherein increased AA mobilization
in concert with LTB4 receptor stimulation and increased Ca2+ levels helped drive activation of PLA2 via enzyme
phosphorylation (10). However, in monocytes which display both the 85 and 14 kDa forms of PLA2, inhibition or
depletion of the 85 kDa form did not significantly decrease the production of LT, suggesting the 14 kDa form may
be more important in LT formation (9).
Figure 1. 5-Lipoxygenase pathway to leukotriene generation. The key enzymatic and biochemical steps in the
synthesis of products of the 5-lipoxygenase pathway are shown. LTA4 is the common intermediate in both
branches of this pathway. The heavy arrows indicate unidirectional steps by which LTB4 and LTC4 are exported
from cells. FLAP denotes the 5-lipoxygenase activating protein. (From Lewis et al. (2).)
Following AA mobilization the next step in LT biosynthesis, indeed the determining step, involves the
membrane-associated 5-LO oxidation of AA to 5S-hydroperoxy-6,8-trans-11,14-cis-eicosatetraenoic acid (5HPETE) followed by 5-LO catalyzed dehydration to the epoxide form LTA4. The activity and function of 5-LO has
been comprehensively reviewed (11). 5-LO pools may be cytosolic or nuclear depending upon cell type and are
translocated to the nuclear envelope where the reaction occurs (12), with 5-LO in complex with a required 5lipoxygenase activating protein (FLAP)(11,13). The 5-LO reactions are the determining factors committing AA to
linear metabolism to LT which, in combination with the requirement of FLAP for 5-LO activity, have made both of
these proteins the subject of intensive research to generate inhibitors of LT biosynthesis (8). Transient LTA4 is
rapidly converted to LTB4 by LTA4 hydrolase, or to peptidyl LTC4 by LTC4 synthase (figure 1). In addition to
intracellular metabolism, LTA4 may also be secreted allowing transcellular metabolism by homologous or
heterologous cell types, irrespective of native 5-LO activity (6,8,14,15). Mobilization of AA, 5-LO production of
LTA4, and intracellular and transcellular metabolism is shown in a simplified schematic representation (figure 2).
Production of LTB4, Isoleukotrienes, and Bioactive LTB4 Metabolites
Stereospecific hydration of LTA4 by LTA4 hydrolase yields the dihydroxy-AA metabolite LTB4. The active
site of LTA4 hydrolase and LTA4 hydrolase-targeted inhibitors have been recently reviewed (16). LTA4 hydrolase
has been shown to be activated by dephosphorylation in endothelial cells, although specific LTA4 hydrolase kinases
or phosphatases have not been identified (17). Polymorphonuclear leukocytes (PMN) are the major LTB4-producing
cells in blood (18), and adenosine has recently been shown to down-regulate LTB4 production in PMN cultures.
This effect was minimized by red blood cell-mediated removal of adenosine from these cultures (15). These results
taken together with transcellular LT metabolism and the bioactivity of LTs hint at the complexity of the LT cell
signalling system. The occurrence of biologically active structural isomers of LTB 4, termed B4-isoleukotrienes,
resulting from nonenzymatic peroxidation of LTA4 has been reported (19). These iso-LTs may have similar yet less
potent activity compared to LTB4. However, precise measurements of bioactivity and metabolism of these isomers
remains to be determined.
C.
Figure 2. A model for cellular leukotriene synthesis and transcellular metabolism. A. In a model insect
cell system a FLAP/5-LO membrane-bound complex metabolizes exogenously-supplied AA to LTA4 and
HPETE. B. In a leukocyte model the addition of membrane-bound phospholipase allows mobilization of AA
from membrane phospholipids. AA presentation via FLAP to 5-LO allows cellular production of LTB4 which
can be secreted from cell. C. In transcellular metabolism LTA4 is transferred from cells expressing 5-LO to
cells containing LTA4 hydrolase or LTC4 synthase activity. Recipient cells need not express 5-LO for LT
production to occur. Nonenzymatic breakdown of LTA4 also occurs. (A, B from Abramovitz et al. (13), C
from Lewis et al. (2).)
Production of Peptidyl Leukotrienes
LTA4 can also follow another enzymatic pathway to create the major bioactive peptidyl leukotrienes LTC 4,
LTD4, and LTE4. The reaction sequence begins with the conjugation of reduced glutathione to form LTC 4 (figure 1),
catalyzed by LTC4 synthase. The glutathione S-transferase (GST) function of LTC4 synthase is unusual in its high
specificity for LTA4 and glutathione as reaction substrates (4), as well as high amino acid sequence homology of
LTC4 synthase with FLAP (20). Further similarities to FLAP include LTC4 synthase sensitivity to FLAP inhibitor
MK-886, and both are integral membrane proteins (20). However, because no catalytic function has been assigned
to FLAP it is assumed that the primary FLAP function is to direct substrate in 5-LO reactions (21). The FLAP-like
moiety of LTC4 synthase is likely responsible for binding LTA4 during the glutathione transferase reaction (20). In
addition to the originally characterized LTC4 synthase, another human microsomal glutathione S-transferase (MGSTII) sharing similar properties has been identified and partially characterized (22). Experiments utilizing proteinspecific polyclonal antibodies have indicated substantial expression in liver tissue and endothelial cells.
Furthermore, MGST-II expression in endothelial cells may be responsible for substantial production of LTC 4 in this
cell type (23).
Production of LTD4 involves the -glutamyl transpeptidase catalyzed removal of glutamate from LTC4. In
addition to -glutamyl transpeptidase, a similar LTC4 cleavage activity has been reported in humans (24). Recent
studies using recombination-derived totally -glutamyl transpeptidase-deficient mice indicate that alternative
pathways for LTC4-specific glutamyl cleavage exist for this species as well (25). The biosynthesis of the final LT of
bioactive importance involves the cleavage of the glycine moiety from LTD4 to produce LTE4. This reaction is
catalyzed by a variety of dipeptidases which are likely membrane-bound (2, 4, 5). An additional leukotriene, LTF4,
has been demonstrated as an in vitro product of -glutamyl transpeptidase glutamyl addition to LTE4, although no in
vivo evidence for this reaction exists.
Further Metabolism of Leukotrienes
Although LT are rapidly metabolized to inactivity, some metabolites retain a fraction of original bioactivity
(typically more than 100-fold activity decrease compared to precursor molecules). LTB 4 is converted within
neutrophils to 20-OH LTB4 via -oxidation by LTB4-20-hydroxylase, a cytochrome p450-like enzyme (2, 4, 5).
Subsequent oxidation yields an inactive product. LTB4 may also be converted to less bioactive12-oxo-LTB4 by
LTB4 12-dehydrogenase (26). In addition to the peptidyl LT biosynthetic conversions, LTE 4 may become Nacetylated. Peptidyl LT are also subject to peroxidative inactivation by conversion to diasteromeric sulfoxides, and
LTC4 may be converted to 6-trans-LTB4 isomers which are degraded by -oxidation (2, 27) (figure 3).
Figure 3. Metabolism of leukotrienes. Rapid, localized metabolism of LTB4 and LTC4 is shown in the
right-hand panel. The progressive inactivation of LTB4 by the sequential oxidation of its terminal
carbon is shown on the far right. The left-hand panel shows the peptidolytic cleavage of LTC4 and the
potential acetylation of LTE4. (From Lewis et al. (2).)
Future Directions
Intensive research involving LT metabolism over the last 10 years has hastened the development of
synthetic inhibitors of LT formation. The application of recombinant DNA technology to cloning and characterizing
LT biosynthetic enzymes has been of tremendous importance, and it has logically followed that these enzymes are
ideal targets for specific inhibitors to allow switching off a metabolic pathway. The further application of these
techniques to allow mutagenesis studies of enzyme active sites or potential allosteric or inducer sites is likely to be
an area of highly active research for years to come.
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