State of the Art Arachidonic Acid Metabolism Implications of Biological Chemistry for Lung Function and Disease 1 - 3
MICHAEL J. HOLTZMAN Contents Introduction Biosynthetic and Degradative Pathways Cyclooxygenase Lipoxygenase Monooxygenase Phospholipase Oxygen Radicals Leukocyte Pathways Eosinophils Neutrophils Mast Cells Mononuclear Phagocytes Epithelial Cell Pathways Cyclooxygenase Activity Lipoxygenase Activity Potential Substrates Phospholipase Activity Novel Functional Aspects Regulation of Oxygenation Pathways CJrowth Factors Cytokines Pharmacology Substrate Availability Product Biosynthesis and Binding Perspective Introduction
Out of the myriad of biologically active mediators generated under normal physiologic conditions and during injury and inflammation, few compounds have receivedmore interest than those derived from oxygenation of arachidonic acid. The significance of altered arachidonic acid metabolism has been the focus of investigation in disorders as diverse as hydronephrosis, myocardial infarction, ulcerative colitis, and rheumatoid arthritis. In each case, the changes in eicosanoid formation are associated with an influx of inflammatory cells. Thus, a major focus toward understanding the pathophysiologic response to injury is the correlation of inflammatory cell influx and the resulting changes in cellular metabolism of arachidonic acid at the site of inflammation. 188
This same approach has been used successfully in studies of disease in the lung. For example, studies of patients with asthma and experimental models of asthma have correlated abnormalities in pulmonary airway function with the influx of leukocytes (1-4) and the generation of arachidonic acid products in the airway (5-8). To decipher the complex interaction of mediators detected in vivo, it has been advantageous to define the specific cellular sources of mediators. Consequently, a logical extension of studies carried out in vivo has been the characterization of arachidonic acid metabolism in the relevant purified cell types isolated in vitro. Studies of intact cells are in turn further extended to work on molecular mechanisms using isolated enzyme systems. This report boldly attempts to organize these different classesof studies (molecular, cellular, and whole organism) and to assess their implications for lung function and disease mechanisms. Coverage of the major biosynthetic and degradative pathways for arachidonic acid products is presented first. This information serves as a basis for describing the synthesis of compounds from two classes of cells (leukocytes and epithelial cells) that are conspicuous during inflammation and are likely to be an important source of arachidonate-derived mediators. Lastly, areas of recent fervor in the regulation and pharmacology of arachidonic acid metabolism are presented as well as a final perspective on metabolite effects. Biosynthetic and Degradative Pathways
Each of the three enzymatic pathways for oxygenation of fatty acids - cyclooxygenase, lipoxygenase,and monooxygenasegives rise to a distinct array of products
(figure 1), and each has the potential for differential stimulation and selective inhibition. The availability of arachidonic acid to oxygenation pathways is tightly controlled in most cells by the activity of phospholipases that release fatty acids from structural pools of glycerophospholipid in cell membranes. In this section, the generation of lipid mediators from each of these enzymatic cascades is described separately and then connected by the common thread of free radical oxidant mechanisms.
Cyclooxygenase Cyclooxygenase (prostaglandin endoperoxide synthase) catalyzes the conversion of arachidonic acid (and certain other polyunsaturated fatty acids) to prostaglandin endoperoxides, which are the precursors of a series of biologically active compounds (thromboxane, prostacyclin, and other prostaglandins). The cyclooxygenaseactivity of the enzyme inserts two molecules of oxygen into arachidonic acid to yield prostaglandin (PO) O 2 , and a peroxidase activity of the enzyme reduces P002 to its IS-hydroxy analogue, POH2 (figure 2). Nonsteroidal anti-inflammatory drugs inhibit the cyclooxygenase but not the hydroperoxidase activity of the enzyme. In particular, aspirin acetylates the enzyme, caus(Received for publication July 24, 1990) 1 From the Department of Medicine, Washington University School of Medicine, St. Louis, Missouri. 2 Portions of work described herein were supported by Grant No. HL-40078 from the National Institutes of Health and by the Schering Career Investigator Award from the American Lung Association. 3 Correspondence and requests for reprints should be addressed to M. J. Holtzman, Washington University School of Medicine, 660 South Euclid Avenue-Box 8052, St. Louis, MO 63110.
AM REV RESPIR DIS 1991; 143:188-203
STATE OF THE ART: ARACHIDONIC ACID METABOLISM
189
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Fig. 1 (left). Pathways for arachidonic acid metabolism. The firststep in the synthesis of oxygenation products is the appearance of unesterified arachidonic acid (5,8,11,14eicosatetraenoic acid). Availability is regulated by the activity of phospholipases that control release from storage sites in membrane phospholipids. After release, arachidonate can be oxygenated by three major pathways: cyclooxygenase, lipoxygenase, and cytochrome P-450 monooxygenase, each with a distinct enzymatic mechanism. Immediate products of these enzymatic pathways-prostaglandin endoperoxides, hydroxy- and hydroperoxyeicosatetraenoic acids (HETE and HPETE), leukotrienes, and epoxides (epoxyeicosatrienoic acids [EET))-have the capacity to act as specific mediators or may be further metabolized to other bioactive products by additional enzymatic steps. Fig. 2 (right). Generation of cyclooxygenase products from arachidonic acid. Cyclooxygenase activity results in initial hydrogen abstraction and radical formation at carbon-13 (closed dot). The intermediate fatty acid radical can be converted via oxygenation and peroxidation to PGH 2 , which is the precursor for other prostaglandins, prostacyclin, and thromboxane. Alternatively, in the absence of endoperoxidation, the initial step may be followed by radical migration and oxygenation to yield 11- and 15·HETE. The HETE generally represent only a small proportion of the final product amount, in contrast to lipoxygenase-catalyzed reactions in which they often are the major end products.
ing irreversible inactivation, and this action is responsible for both therapeutic effects of the drug and idiosyncratic reactions to it. Cyclooxygenase is found on the endoplasmic reticulum and nuclear membranes, and also undergoes self-inactivation by oxidants generated during catalysis (9). Its primary sequence has been recently determined by molecular cloning from a sheep seminal vesicle complementary DNA (cDNA) library (10) and from human genomic DNA (11), and these events have initiated studies oftranscriptional regulation of the enzyme. In addition to its capacity to generate prostaglandins, cyclooxygenase may also catalyze the formation of 11- and 15hydroxy acids from arachidonic acid (figure 2) or 9- and 13-hydroxy acids from linoleic acid. These compounds may even become the predominant enzymatic products under some circumstances (12). Because identical products can be formed by lipoxygenase or monooxygenase activities, finding these products in biologic material does not specify the enzymatic mechanism of formation. Thromboxane. POH2 is converted predominantly to thromboxane (1X) A 2 by thromboxane synthase in platelets and perhaps other cell types. The compound is a potent constrictor of vascular and airway smooth muscle and may be solely responsible for some states of pulmonary hypertension in rabbits (13) and airway hyperreactivity in dogs (14). 1XA2
spontaneously hydrolyzes to the hemiacetal1XB 2 , which unlike the unstable parent compound is not active in causing smooth muscle contraction or platelet aggregation. Purification of the thromboxane synthase by affinity chromatography with an immobilized inhibitor of the enzyme suggests that it may be a cytochrome P-450. Inhibitors of thromboxane synthase include imidazole and substituted derivatives, certain pyridine derivatives, and prostaglandin analogues. Inhibition of 1XA2 synthesis or binding does not prevent effects of arachidonic acid on platelet aggregation, however, because POH 2 may still be formed and stimulate the same or a closelyrelated receptor (15). The receptor has recently been purified from platelets (16), and aspects of its mechanisms for signal transduction have been determined (17), but the characteristics of a putative POH2/1XA2 receptor on pulmonary cells remains less certain. Current evidence suggests that a receptor classified as a thromboxane TP receptor mediates the bronchial smooth muscle contraction induced by 1XA2 , POD2 , and POF2a and may be distinct from the receptor on platelets and mast cells (18). Prostacyclin. Vascularendothelial cells and vascular and nonvascular smooth muscle cells convert POH2 to pro stacyclin (POI 2 ) , an enol-ether that spontaneously hydrolyzes to 6-keto-POF 1a • POl 2 synthase has been purified and localized to the plasma and nuclear mem-
branes, and it may also be a cytochrome P-450 (19). POl 2 is responsible for arachidonate-induced vasodilation and for an antiaggregatory influence on platelets. Recent studies carried out in vivo indicate that POl2 (and other prostaglandins) is overproduced after thromboxane synthase inhibition. These studies indicate that POl 2 is formed locally at the site of vessel injury and provide evidence that supports transcellular metabolism of prostaglandin endoperoxides in humans (20). POl 2 production declines as cultured endothelial cells reach confluence and increases when the cells are seeded into fresh medium (21). POl2 also inhibits, whereas indomethacin promotes differentiation of adipocytes (22). The compound (as well as other prostaglandins) may therefore serve to modulate the proliferation and differentiation of some cells. In addition, POl 2 elevates cAMP levels, and this action may represent a general mechanism for prostaglandin functional effects on responsive cells. PGD2 • Conversion of POH2 to POD 2 can be catalyzed by serum albumin but occurs more efficiently by the action of POD isomerase. The primary structure of the rat brain enzyme has recently been deduced from cDNA sequence (23). The high concentrations of the enzyme in the central nervous systemsuggestthat POD2 may have neuromodulatory actions, and these have been demonstrated in a variety of systems. Other effects of the com-
MICHAEL J. HOLTZMAN
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pound include inhibiting platelet aggregation, increasingplatelet cAMP content, and causing peripheral vasodilation, pulmonary vasoconstriction, bronchoconstriction, and airway hyperreactivity. POD 2 is the principal cyclooxygenase product of human and rat mast cells (24, 25). After release, it (like other prostaglandins) can be metabolized to compounds reflecting various combinations of ll-keto reduction, dehydrogenation of the 15-hydroxylgroup, reduction of the f,. 13 double bond, p-oxidation, and m-oxidation. Overproduction of the compounds may be the cause of hypotensive episodes of patients with systemic mastocytosis. A novel metabolite of POD2 has been identified that is a diastereoisomer of POF2U (9a, II P-POF2 u ) and is formed from POD 2 by an NADPHdependent, cytosolic activity in the liver and by POF synthase in the lung (26). It is equipotent to POF2U as a vasopressor and possesses activity for airway smooth muscle constriction in vitro and in vivo (27). Levelsof this compound (as well as POD 2 and POF2U) are elevated in lavage fluid from the lungs of asthmatics (8). PGE2 • Isomerization of POH2 to POE2 is catalyzed by a microsomal enzyme that has been partially purified from bovine seminal vesicle. POE2 is the predominant arachidonic acid product in a variety of cell types, and its potential role in cell function has been reviewed (28). Release of POE2 is decreased when epithelial cellsbecome confluent (29)and increased when they are stimulated by growth factors (see below), suggesting a role for the compound in regulating cell growth. It is also produced by macrophages, and the evidence that the compound may mediate effects of macrophages on neighboring cells as wellas on macrophages themselves has been reviewed (30). POE2 (and POD2 ) can potentiate the effects of histamine and bradykinin on vascular permeability and can cause hyperalgesia to the same agents. A similar mechanism may explain the compound's capacity to sensitize the cough reflex (31). Prostaglandins of the E series and their analogs inhibit gastric acid secretion and are cytoprotective to the gastric and intestinal mucosa, inhibiting the formation and promoting the healing of ulcers. Such a role in the pulmonary airway remains unexplored, although POE2 may decrease mucus secretion in some systems (32). POE2 is also able to induce relaxation of airway
smooth muscle by a direct effect on the muscle and by an inhibitory effect on acetylcholine release (33). POEt inhibits neutrophil adherence and neutrophilmediated injury to endothelial cells (34), indicating its potential to inhibit some aspects of inflammation. PGF;a. Although defined isomerases are responsible for POE 2 and POD 2 generation, the mechanism for formation of POF2 U in tissues is less certain. Possibilities include enzymatic or nonenzymatic 9,11 endoperoxide reduction of POH 2 , ll-keto reduction of POD 2 , and 9-keto reduction of POE2 (35). Both POH2 and POF2U are potent constrictors of airway smooth muscle (36). POF2U may also cause heightened reflex bronchoconstriction by sensitizing airway nerve endings (37), and plasma levels of its metabolite are reported to be elevated in acute asthma (38). The compounds also directlystimulate airway sensory nerve endings (39), an effect that might cause reflex bronchoconstriction and cough in vivo.
Lipoxygenase Lipoxygenases are a group of ironcontaining dioxygenasesthat catalyze insertion of one oxygenmolecule into polyunsaturated fatty acids containing a 1,4cis,cis-pentadiene structure (figure 3). The initial catalytic step is stereospecific removal of a hydrogen atom followed by
antarafacial addition of molecular oxygen. Mammalian lipoxygenasesalso possess regional specificity during interaction with substrate and on that basis have been designated as arachidonate 5-, 12-, of 15-lipoxygenase. The three distinct enzyme types insert oxygen at carbon 5, 12, or 15 of arachidonic acid, so the immediate product is a 58-, 128-, or 158-hydroperoxyeicosatetraenoic acid (5-, 12-, or 15-HPETE). The enzymes may also catalyze the formation of unstable epoxide intermediates that can be further metabolized to a variety of dihydroxy acids, including leukotrienes, epoxyhydroxy and trihydroxy acids, aldehydes, and glutathione adducts (40, 41). 12-Lipoxygenase products. The 12lipoxygenase was the first to be discovered in animal or human tissues. The enzyme was described initially in platelets (42) and then later in leukocytes (43). Platelet 12-lipoxygenase activity is contained in both cytosolic and membrane fractions, and membrane translocation of the enzyme may be mediated by increasing calcium concentration (44). The leukocyte enzyme has been purified and can be differentiated immunologically and catalytically from the purified platelet enzyme (45). Neither form of the enzyme has cofactor requirements, but the recent cloning of the leukocyte 12-lipoxygenase (46) may soon permit other aspects of its regulation to be determined.
ARACHIDONIC ACID 14
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12S-HPETE Fig. 3. A model for stereo- and regiospecificity of arachidonate Iipoxygenases. In mammalian cells, the 15-, 12-, and 5-lipoxygenases stereospecifically remove hydrogen at the 13-, 10-, or 7-carbon, respectively, and then promote radical migration and insertion of molecular oxygen into the 155-, 125-, and 55- positions (Pathways 1, 2, and 3, respectively). The activity leads to the formation of the corresponding HPETE, which is often reduced to the corresponding HETE and released from the cell. Although targeting of the lipoxygenase for a specific region of the fatty acid substrate is not absolute, it forms the basis for useful categorization of this enzyme class. Further degrees of heterogeneity within each subclass have been demonstrated for epithelial, leukocyte, and platelet forms of the enzymes.
191
STATE OF THE ART: ARACHIDONIC ACID METABOLISM
12-HPETE can be converted to a variety of metabolites, and reduction of 12HPETE to the alcohol (12-HETE) occurs rapidly in most cells. In addition, 8- and 10-hydroxy-ll,12-epoxyeicosatrienoic acid (hepoxilins) may be formed by intramolecular rearrangement and further metabolized to triols. An analogous set of reactions may occur after 15HPETE formation (see below) and raises the same set of questions for biologic significance. A variety of studies have offered evidence that 12-HETE or 12-HPETE may act as specific mediators: 12-HETE induces secretion of neutrophil-specific granules and augments immunoglobulin (lg) E-mediated mast cell release (47). Reacylation of the compound into the membrane occurs and may modify granule to plasma membrane fusion or inhibit cellular prostaglandin production (48). 12-HETE also has chemotactic properties, enhances tumor cell adhesion to the subendothelial matrix (49), and stimulates migration of aortic smooth muscle cells (50). 12-HPETE may act as a second messenger for presynaptic inhibition (51). With few exceptions, effects of 12lipoxygenase products occur at relatively high concentrations, leading to the frequent impression that the products are biologically inactive. Critical proof of the products as specific mediators will likely rest on evidence of specificity and potency,and this combination has not yet been demonstrated in mammalian cells. 15-Lipoxygenase products. The 15lipoxygenase of leukocytes and reticulocytes has been purified (52-54), and comparison of its primary structure to other lipoxygenases has been reviewed recently (55). The complete gene for the rabbit reticulocyte 15-lipoxygenaserecently has been isolated and sequenced (56). The 15-lipoxygenase converts arachidonic acid predominantly to 15-HPETE and can use both unesterified fatty acid and fatty acid bound in phospholipid, raising the possibility that it has a role in the modification of membrane composition and function. For example, reticulocyte membranes contain increased levels of the oxygenated derivatives of linoleic and arachidonic acids (57), and the formation of these products may destabilize membrane structure and predispose it to proteolysisduring erythrocytematuration. The metabolism of 15-HPETE is analogous to that for 12-HPETE and includes conversion to, 15-HETE and keto-, hydroxyepoxy-and trihydroxy-acids. 15HPETE can also undergo a second
hydrogen abstraction that results in the formation of 8,15- and 14,15-dihydroxy acids. Radical migration and interaction with the adjacent 15-hydroperoxy group can also produce a 14,15-oxido compound analogous to leukotriene (LT) Aa, and hydrolysisof this intermediate results in the generation of dihydroxy acids containing a conjugated triene structure ("8,15-leukotrienes"). In addition, oxygenation and reduction of 15-HPETE or 15-HETE by the 5-lipoxygenase (from the same or a neighboring cell) yields 5,15-dihydroxy or 5,6,15- and 5,14,15trihydroxy acids (lipoxins A and B). Levels of lipoxin A are elevated in bronchoalveolar lavage fluid from patients with selected pulmonary diseases (58). Biologic function of the 15-lipoxygenase takes on special interest because it is the predominant pathway for arachidonic acid metabolism in human lung tissue and may even be increased in lung tissue from asthmatic patients (59, 60) and in lavage fluid from airways of asthmatics (5). It is also a major oxygenation pathway in both eosinophils and airway epithelial cellsisolated from humans (see below), two cell types that are implicated in the development of asthmatic airway inflammation. Most investigations of 15-lipoxygenase function have focused on the possibility that the enzymatic products are specific mediators (61). 15-HPETE andlor 15-HETE modify a host of cellular immune functions (62), may act as a mitogen (63), and may modulate other oxygenation enzymes (64,65). Effects of 15-HPETE metabolites include neuronal hypersensitivity due to 8,15-diHETE (66), inhibition of natural killer cell activity by 14,15diHETE (67), and neutrophil and protein kinase C activation by lipoxins (68). 5-Lipoxygenase products. The 5-lipoxygenase converts arachidonate to 5HPETE and also catalyzes the formation of an unstable allylic epoxide (LTAa) from 5-HPETE (40). LTAa can be hydrolyzed enzymatically to LTB4 or converted to LTC4 upon addition of glutathione. Removal of glutamic acid by y-glutamyl transpeptidase generates LTD4 , and subsequent removal of glycine by a dipeptidase yields LTE4 • The 5-lipoxygenaseis specifically stimulated by calcium (69), and other enzymes in the cascade to leukotriene formation do not exhibit this requirement (70). In addition, the 5-lipoxygenase activity is enhanced by ATP, and to a lesser extent by other nucleotides (69), and is sensitive to inhibition by peroxides (71).
In contrast to the cyclooxygenase pathway where the first step is rate limiting, the rate limiting step in leukotriene biosynthesis occurs at the levels of the LTA hydrolase and LTC synthase (glutathione transferase) (72). Subcellular fractionation of enzymatic activity indicates that the 5-lipoxygenaseand LTAhydrolase are soluble cytosolic enzymes (72, 73). However, isolating the 5-lipoxygenase in the presence of excess calcium may increase its degree of membrane association (74), and this effect may mimic physiologic activation and translocation of the enzyme (75). Inhibitors of the translocation process may be useful in blocking leukotriene formation. In fact, at least one such inhibitor has been identified and also used to isolate and clone a membraneactivating protein necessary for leukotriene synthesis (76). The 5-lipoxygenase has been purified (73), and its full sequence determined by molecular cloning (77). Primary structural features have been reviewed (55). Expression of the 5-lipoxygenase cDNA in a baculoviruslinsect cell system (78) and in osteosarcoma cells (79) and isolation of the complete 5-lipoxygenase gene from genomic DNA libraries (80) have now been accomplished and are likely to lead to further insight into transcriptional and posttranscriptional mechanisms for lipoxygenase regulation. The LTA hydrolase has also been purified (81), and its cDNA has been isolated by immunoscreening a human lung cDNA expression library (82) and by screening a spleen cDNA library with an oligonucleotide probe based on peptide sequence of the purified hydrolase (83). The enzyme appears to be expressed in virtually all tissues and a variety of cell types, including those that lack 5-lipoxygenase activity (84). For example, the enzymatic capacity is present in erythrocytes so that LTAa released from leukocytes can be converted to LTB4 in the blood (85). The glutathione transferase, y-glutamyl transpeptidase, and dipeptidase are particulate enzymes (70).The glutathione transferase is therefore distinct from conventional glutathione transferases, which are generally soluble enzymes. The location of the enzymes implies that LTAa formed in a cytosolic location must be transported to the granule or other particulate portion for conversion to LTC4 • The transpeptidase and dipeptidase have been localized to the plasma membrane (86) and may therefore serve to convert LTC4 to LTD4 as it is released from the
192
cell or approaches the outside of another cell. The metabolism of the sulfidopeptide-containing leukotrienes has been reviewed (87). LTB4 is metabolized via cytochrome P-450 to 20-hydroxy-LTB4 and then to 20-carboxy- LTB4 in neutrophils but to little extent in other cell types (see below). Metabolic studies of radiolabeled LTB4 as well as studies of isolated hepatocytes (88) suggest that the compound is also extensively degraded by 13-oxidation. In contrast to the 12- and 15-lipoxygenases, the substrate specificity of the 5-lipoxygenase pathway is quite restricted. The double bonds at carbons 5, 8, 11,and 14 are required for optimal LTB formation, and this requirement helps explain the marked decrease in LTB4 formation in essential fatty acid deficiency. Reports of decreased inflammation in this condition may also be due in part to this biochemical deficiency. The addition of a double bond at carbon 17 yielding eicosapentaenoic acid (EPA) still permits formation of the corresponding LTBs but may decrease the efficiency of conversion significantly and have effects on leukocyte function (89). The leukotrienes derived from 5-lipoxygenase activity have been implicated as critical mediators for a variety of inflammatory diseases, and their effects on the airways and on the microcirculation have been reviewed recently (87, 90). LTB4 is a potent chemoattractant for human neutrophils and eosinophils (91, 92) as well as a lymphocyte activator (93). The compound binds with high affinity, stereospecificity, and saturability to receptors on cell membrane from polymorphonuclear neutrophils (94). Binding studies suggest the presence of high- and low-affinity receptor populations that are distinct from the receptors for other chemotactic substances such as C5a or FMLP. Leukotriene C 4 and D 4 , but to a lesser extent E 4 , are contractors of airway, vascular, and intestinal smooth muscle. Evidence that these products might specifically be important in the development of asthma includes production of the compounds by both human and animal lungs after antigen challenge (60)and increases in their concentration in airways of asthmatic patients (95). Administration of the compounds to animals or to humans causes airway constriction (60, 96-98), hyperreactivity (14, 99), and increased mucus secretion (100), and these functional alterations are each essential features of the disease.
MICHAEL J. HOLTZMAN
Even for 5-lipoxygenasepathway products, the importance of the compounds for pathophysiologic events in vivo is uncertain. As is the case for understanding 12-and 15-lipoxygenase function, the unavailability of specific inhibitors of the pathway has hindered better definition of pathway regulation in vitro and securing more convincing evidence of the role of the pathway products in vivo. The availability of purified enzymes and expression systems may aid in the development and testing of suitable inhibitors.
Monooxygenase Hydroxylation reactions in animal cells use a special class of complex microsomal monooxygenases designated as cytochrome P-450 (P for pigment) (101). The enzymes were first identified by the fact that the carbon monoxide derivative of its reduced form absorbs maximally at 450 nm. This class of enzymes is most active in the liver but is widely distributed in animal tissues, including the lung (102), and is highly active in monooxygenation of a variety of lipophilic compounds, including arachidonic acid and its metabolites. Metabolism of arachidonic acid by cytochrome P-450monooxygenase leads to
MICHAEL J. HOLTZMAN Contents Introduction Biosynthetic and Degradative Pathways Cyclooxygenase Lipoxygenase Monooxygenase Phospholipase Oxygen Radicals Leukocyte Pathways Eosinophils Neutrophils Mast Cells Mononuclear Phagocytes Epithelial Cell Pathways Cyclooxygenase Activity Lipoxygenase Activity Potential Substrates Phospholipase Activity Novel Functional Aspects Regulation of Oxygenation Pathways CJrowth Factors Cytokines Pharmacology Substrate Availability Product Biosynthesis and Binding Perspective Introduction
Out of the myriad of biologically active mediators generated under normal physiologic conditions and during injury and inflammation, few compounds have receivedmore interest than those derived from oxygenation of arachidonic acid. The significance of altered arachidonic acid metabolism has been the focus of investigation in disorders as diverse as hydronephrosis, myocardial infarction, ulcerative colitis, and rheumatoid arthritis. In each case, the changes in eicosanoid formation are associated with an influx of inflammatory cells. Thus, a major focus toward understanding the pathophysiologic response to injury is the correlation of inflammatory cell influx and the resulting changes in cellular metabolism of arachidonic acid at the site of inflammation. 188
This same approach has been used successfully in studies of disease in the lung. For example, studies of patients with asthma and experimental models of asthma have correlated abnormalities in pulmonary airway function with the influx of leukocytes (1-4) and the generation of arachidonic acid products in the airway (5-8). To decipher the complex interaction of mediators detected in vivo, it has been advantageous to define the specific cellular sources of mediators. Consequently, a logical extension of studies carried out in vivo has been the characterization of arachidonic acid metabolism in the relevant purified cell types isolated in vitro. Studies of intact cells are in turn further extended to work on molecular mechanisms using isolated enzyme systems. This report boldly attempts to organize these different classesof studies (molecular, cellular, and whole organism) and to assess their implications for lung function and disease mechanisms. Coverage of the major biosynthetic and degradative pathways for arachidonic acid products is presented first. This information serves as a basis for describing the synthesis of compounds from two classes of cells (leukocytes and epithelial cells) that are conspicuous during inflammation and are likely to be an important source of arachidonate-derived mediators. Lastly, areas of recent fervor in the regulation and pharmacology of arachidonic acid metabolism are presented as well as a final perspective on metabolite effects. Biosynthetic and Degradative Pathways
Each of the three enzymatic pathways for oxygenation of fatty acids - cyclooxygenase, lipoxygenase,and monooxygenasegives rise to a distinct array of products
(figure 1), and each has the potential for differential stimulation and selective inhibition. The availability of arachidonic acid to oxygenation pathways is tightly controlled in most cells by the activity of phospholipases that release fatty acids from structural pools of glycerophospholipid in cell membranes. In this section, the generation of lipid mediators from each of these enzymatic cascades is described separately and then connected by the common thread of free radical oxidant mechanisms.
Cyclooxygenase Cyclooxygenase (prostaglandin endoperoxide synthase) catalyzes the conversion of arachidonic acid (and certain other polyunsaturated fatty acids) to prostaglandin endoperoxides, which are the precursors of a series of biologically active compounds (thromboxane, prostacyclin, and other prostaglandins). The cyclooxygenaseactivity of the enzyme inserts two molecules of oxygen into arachidonic acid to yield prostaglandin (PO) O 2 , and a peroxidase activity of the enzyme reduces P002 to its IS-hydroxy analogue, POH2 (figure 2). Nonsteroidal anti-inflammatory drugs inhibit the cyclooxygenase but not the hydroperoxidase activity of the enzyme. In particular, aspirin acetylates the enzyme, caus(Received for publication July 24, 1990) 1 From the Department of Medicine, Washington University School of Medicine, St. Louis, Missouri. 2 Portions of work described herein were supported by Grant No. HL-40078 from the National Institutes of Health and by the Schering Career Investigator Award from the American Lung Association. 3 Correspondence and requests for reprints should be addressed to M. J. Holtzman, Washington University School of Medicine, 660 South Euclid Avenue-Box 8052, St. Louis, MO 63110.
AM REV RESPIR DIS 1991; 143:188-203
STATE OF THE ART: ARACHIDONIC ACID METABOLISM
189
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Fig. 1 (left). Pathways for arachidonic acid metabolism. The firststep in the synthesis of oxygenation products is the appearance of unesterified arachidonic acid (5,8,11,14eicosatetraenoic acid). Availability is regulated by the activity of phospholipases that control release from storage sites in membrane phospholipids. After release, arachidonate can be oxygenated by three major pathways: cyclooxygenase, lipoxygenase, and cytochrome P-450 monooxygenase, each with a distinct enzymatic mechanism. Immediate products of these enzymatic pathways-prostaglandin endoperoxides, hydroxy- and hydroperoxyeicosatetraenoic acids (HETE and HPETE), leukotrienes, and epoxides (epoxyeicosatrienoic acids [EET))-have the capacity to act as specific mediators or may be further metabolized to other bioactive products by additional enzymatic steps. Fig. 2 (right). Generation of cyclooxygenase products from arachidonic acid. Cyclooxygenase activity results in initial hydrogen abstraction and radical formation at carbon-13 (closed dot). The intermediate fatty acid radical can be converted via oxygenation and peroxidation to PGH 2 , which is the precursor for other prostaglandins, prostacyclin, and thromboxane. Alternatively, in the absence of endoperoxidation, the initial step may be followed by radical migration and oxygenation to yield 11- and 15·HETE. The HETE generally represent only a small proportion of the final product amount, in contrast to lipoxygenase-catalyzed reactions in which they often are the major end products.
ing irreversible inactivation, and this action is responsible for both therapeutic effects of the drug and idiosyncratic reactions to it. Cyclooxygenase is found on the endoplasmic reticulum and nuclear membranes, and also undergoes self-inactivation by oxidants generated during catalysis (9). Its primary sequence has been recently determined by molecular cloning from a sheep seminal vesicle complementary DNA (cDNA) library (10) and from human genomic DNA (11), and these events have initiated studies oftranscriptional regulation of the enzyme. In addition to its capacity to generate prostaglandins, cyclooxygenase may also catalyze the formation of 11- and 15hydroxy acids from arachidonic acid (figure 2) or 9- and 13-hydroxy acids from linoleic acid. These compounds may even become the predominant enzymatic products under some circumstances (12). Because identical products can be formed by lipoxygenase or monooxygenase activities, finding these products in biologic material does not specify the enzymatic mechanism of formation. Thromboxane. POH2 is converted predominantly to thromboxane (1X) A 2 by thromboxane synthase in platelets and perhaps other cell types. The compound is a potent constrictor of vascular and airway smooth muscle and may be solely responsible for some states of pulmonary hypertension in rabbits (13) and airway hyperreactivity in dogs (14). 1XA2
spontaneously hydrolyzes to the hemiacetal1XB 2 , which unlike the unstable parent compound is not active in causing smooth muscle contraction or platelet aggregation. Purification of the thromboxane synthase by affinity chromatography with an immobilized inhibitor of the enzyme suggests that it may be a cytochrome P-450. Inhibitors of thromboxane synthase include imidazole and substituted derivatives, certain pyridine derivatives, and prostaglandin analogues. Inhibition of 1XA2 synthesis or binding does not prevent effects of arachidonic acid on platelet aggregation, however, because POH 2 may still be formed and stimulate the same or a closelyrelated receptor (15). The receptor has recently been purified from platelets (16), and aspects of its mechanisms for signal transduction have been determined (17), but the characteristics of a putative POH2/1XA2 receptor on pulmonary cells remains less certain. Current evidence suggests that a receptor classified as a thromboxane TP receptor mediates the bronchial smooth muscle contraction induced by 1XA2 , POD2 , and POF2a and may be distinct from the receptor on platelets and mast cells (18). Prostacyclin. Vascularendothelial cells and vascular and nonvascular smooth muscle cells convert POH2 to pro stacyclin (POI 2 ) , an enol-ether that spontaneously hydrolyzes to 6-keto-POF 1a • POl 2 synthase has been purified and localized to the plasma and nuclear mem-
branes, and it may also be a cytochrome P-450 (19). POl 2 is responsible for arachidonate-induced vasodilation and for an antiaggregatory influence on platelets. Recent studies carried out in vivo indicate that POl2 (and other prostaglandins) is overproduced after thromboxane synthase inhibition. These studies indicate that POl 2 is formed locally at the site of vessel injury and provide evidence that supports transcellular metabolism of prostaglandin endoperoxides in humans (20). POl 2 production declines as cultured endothelial cells reach confluence and increases when the cells are seeded into fresh medium (21). POl2 also inhibits, whereas indomethacin promotes differentiation of adipocytes (22). The compound (as well as other prostaglandins) may therefore serve to modulate the proliferation and differentiation of some cells. In addition, POl 2 elevates cAMP levels, and this action may represent a general mechanism for prostaglandin functional effects on responsive cells. PGD2 • Conversion of POH2 to POD 2 can be catalyzed by serum albumin but occurs more efficiently by the action of POD isomerase. The primary structure of the rat brain enzyme has recently been deduced from cDNA sequence (23). The high concentrations of the enzyme in the central nervous systemsuggestthat POD2 may have neuromodulatory actions, and these have been demonstrated in a variety of systems. Other effects of the com-
MICHAEL J. HOLTZMAN
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pound include inhibiting platelet aggregation, increasingplatelet cAMP content, and causing peripheral vasodilation, pulmonary vasoconstriction, bronchoconstriction, and airway hyperreactivity. POD 2 is the principal cyclooxygenase product of human and rat mast cells (24, 25). After release, it (like other prostaglandins) can be metabolized to compounds reflecting various combinations of ll-keto reduction, dehydrogenation of the 15-hydroxylgroup, reduction of the f,. 13 double bond, p-oxidation, and m-oxidation. Overproduction of the compounds may be the cause of hypotensive episodes of patients with systemic mastocytosis. A novel metabolite of POD2 has been identified that is a diastereoisomer of POF2U (9a, II P-POF2 u ) and is formed from POD 2 by an NADPHdependent, cytosolic activity in the liver and by POF synthase in the lung (26). It is equipotent to POF2U as a vasopressor and possesses activity for airway smooth muscle constriction in vitro and in vivo (27). Levelsof this compound (as well as POD 2 and POF2U) are elevated in lavage fluid from the lungs of asthmatics (8). PGE2 • Isomerization of POH2 to POE2 is catalyzed by a microsomal enzyme that has been partially purified from bovine seminal vesicle. POE2 is the predominant arachidonic acid product in a variety of cell types, and its potential role in cell function has been reviewed (28). Release of POE2 is decreased when epithelial cellsbecome confluent (29)and increased when they are stimulated by growth factors (see below), suggesting a role for the compound in regulating cell growth. It is also produced by macrophages, and the evidence that the compound may mediate effects of macrophages on neighboring cells as wellas on macrophages themselves has been reviewed (30). POE2 (and POD2 ) can potentiate the effects of histamine and bradykinin on vascular permeability and can cause hyperalgesia to the same agents. A similar mechanism may explain the compound's capacity to sensitize the cough reflex (31). Prostaglandins of the E series and their analogs inhibit gastric acid secretion and are cytoprotective to the gastric and intestinal mucosa, inhibiting the formation and promoting the healing of ulcers. Such a role in the pulmonary airway remains unexplored, although POE2 may decrease mucus secretion in some systems (32). POE2 is also able to induce relaxation of airway
smooth muscle by a direct effect on the muscle and by an inhibitory effect on acetylcholine release (33). POEt inhibits neutrophil adherence and neutrophilmediated injury to endothelial cells (34), indicating its potential to inhibit some aspects of inflammation. PGF;a. Although defined isomerases are responsible for POE 2 and POD 2 generation, the mechanism for formation of POF2 U in tissues is less certain. Possibilities include enzymatic or nonenzymatic 9,11 endoperoxide reduction of POH 2 , ll-keto reduction of POD 2 , and 9-keto reduction of POE2 (35). Both POH2 and POF2U are potent constrictors of airway smooth muscle (36). POF2U may also cause heightened reflex bronchoconstriction by sensitizing airway nerve endings (37), and plasma levels of its metabolite are reported to be elevated in acute asthma (38). The compounds also directlystimulate airway sensory nerve endings (39), an effect that might cause reflex bronchoconstriction and cough in vivo.
Lipoxygenase Lipoxygenases are a group of ironcontaining dioxygenasesthat catalyze insertion of one oxygenmolecule into polyunsaturated fatty acids containing a 1,4cis,cis-pentadiene structure (figure 3). The initial catalytic step is stereospecific removal of a hydrogen atom followed by
antarafacial addition of molecular oxygen. Mammalian lipoxygenasesalso possess regional specificity during interaction with substrate and on that basis have been designated as arachidonate 5-, 12-, of 15-lipoxygenase. The three distinct enzyme types insert oxygen at carbon 5, 12, or 15 of arachidonic acid, so the immediate product is a 58-, 128-, or 158-hydroperoxyeicosatetraenoic acid (5-, 12-, or 15-HPETE). The enzymes may also catalyze the formation of unstable epoxide intermediates that can be further metabolized to a variety of dihydroxy acids, including leukotrienes, epoxyhydroxy and trihydroxy acids, aldehydes, and glutathione adducts (40, 41). 12-Lipoxygenase products. The 12lipoxygenase was the first to be discovered in animal or human tissues. The enzyme was described initially in platelets (42) and then later in leukocytes (43). Platelet 12-lipoxygenase activity is contained in both cytosolic and membrane fractions, and membrane translocation of the enzyme may be mediated by increasing calcium concentration (44). The leukocyte enzyme has been purified and can be differentiated immunologically and catalytically from the purified platelet enzyme (45). Neither form of the enzyme has cofactor requirements, but the recent cloning of the leukocyte 12-lipoxygenase (46) may soon permit other aspects of its regulation to be determined.
ARACHIDONIC ACID 14
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12S-HPETE Fig. 3. A model for stereo- and regiospecificity of arachidonate Iipoxygenases. In mammalian cells, the 15-, 12-, and 5-lipoxygenases stereospecifically remove hydrogen at the 13-, 10-, or 7-carbon, respectively, and then promote radical migration and insertion of molecular oxygen into the 155-, 125-, and 55- positions (Pathways 1, 2, and 3, respectively). The activity leads to the formation of the corresponding HPETE, which is often reduced to the corresponding HETE and released from the cell. Although targeting of the lipoxygenase for a specific region of the fatty acid substrate is not absolute, it forms the basis for useful categorization of this enzyme class. Further degrees of heterogeneity within each subclass have been demonstrated for epithelial, leukocyte, and platelet forms of the enzymes.
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STATE OF THE ART: ARACHIDONIC ACID METABOLISM
12-HPETE can be converted to a variety of metabolites, and reduction of 12HPETE to the alcohol (12-HETE) occurs rapidly in most cells. In addition, 8- and 10-hydroxy-ll,12-epoxyeicosatrienoic acid (hepoxilins) may be formed by intramolecular rearrangement and further metabolized to triols. An analogous set of reactions may occur after 15HPETE formation (see below) and raises the same set of questions for biologic significance. A variety of studies have offered evidence that 12-HETE or 12-HPETE may act as specific mediators: 12-HETE induces secretion of neutrophil-specific granules and augments immunoglobulin (lg) E-mediated mast cell release (47). Reacylation of the compound into the membrane occurs and may modify granule to plasma membrane fusion or inhibit cellular prostaglandin production (48). 12-HETE also has chemotactic properties, enhances tumor cell adhesion to the subendothelial matrix (49), and stimulates migration of aortic smooth muscle cells (50). 12-HPETE may act as a second messenger for presynaptic inhibition (51). With few exceptions, effects of 12lipoxygenase products occur at relatively high concentrations, leading to the frequent impression that the products are biologically inactive. Critical proof of the products as specific mediators will likely rest on evidence of specificity and potency,and this combination has not yet been demonstrated in mammalian cells. 15-Lipoxygenase products. The 15lipoxygenase of leukocytes and reticulocytes has been purified (52-54), and comparison of its primary structure to other lipoxygenases has been reviewed recently (55). The complete gene for the rabbit reticulocyte 15-lipoxygenaserecently has been isolated and sequenced (56). The 15-lipoxygenase converts arachidonic acid predominantly to 15-HPETE and can use both unesterified fatty acid and fatty acid bound in phospholipid, raising the possibility that it has a role in the modification of membrane composition and function. For example, reticulocyte membranes contain increased levels of the oxygenated derivatives of linoleic and arachidonic acids (57), and the formation of these products may destabilize membrane structure and predispose it to proteolysisduring erythrocytematuration. The metabolism of 15-HPETE is analogous to that for 12-HPETE and includes conversion to, 15-HETE and keto-, hydroxyepoxy-and trihydroxy-acids. 15HPETE can also undergo a second
hydrogen abstraction that results in the formation of 8,15- and 14,15-dihydroxy acids. Radical migration and interaction with the adjacent 15-hydroperoxy group can also produce a 14,15-oxido compound analogous to leukotriene (LT) Aa, and hydrolysisof this intermediate results in the generation of dihydroxy acids containing a conjugated triene structure ("8,15-leukotrienes"). In addition, oxygenation and reduction of 15-HPETE or 15-HETE by the 5-lipoxygenase (from the same or a neighboring cell) yields 5,15-dihydroxy or 5,6,15- and 5,14,15trihydroxy acids (lipoxins A and B). Levels of lipoxin A are elevated in bronchoalveolar lavage fluid from patients with selected pulmonary diseases (58). Biologic function of the 15-lipoxygenase takes on special interest because it is the predominant pathway for arachidonic acid metabolism in human lung tissue and may even be increased in lung tissue from asthmatic patients (59, 60) and in lavage fluid from airways of asthmatics (5). It is also a major oxygenation pathway in both eosinophils and airway epithelial cellsisolated from humans (see below), two cell types that are implicated in the development of asthmatic airway inflammation. Most investigations of 15-lipoxygenase function have focused on the possibility that the enzymatic products are specific mediators (61). 15-HPETE andlor 15-HETE modify a host of cellular immune functions (62), may act as a mitogen (63), and may modulate other oxygenation enzymes (64,65). Effects of 15-HPETE metabolites include neuronal hypersensitivity due to 8,15-diHETE (66), inhibition of natural killer cell activity by 14,15diHETE (67), and neutrophil and protein kinase C activation by lipoxins (68). 5-Lipoxygenase products. The 5-lipoxygenase converts arachidonate to 5HPETE and also catalyzes the formation of an unstable allylic epoxide (LTAa) from 5-HPETE (40). LTAa can be hydrolyzed enzymatically to LTB4 or converted to LTC4 upon addition of glutathione. Removal of glutamic acid by y-glutamyl transpeptidase generates LTD4 , and subsequent removal of glycine by a dipeptidase yields LTE4 • The 5-lipoxygenaseis specifically stimulated by calcium (69), and other enzymes in the cascade to leukotriene formation do not exhibit this requirement (70). In addition, the 5-lipoxygenase activity is enhanced by ATP, and to a lesser extent by other nucleotides (69), and is sensitive to inhibition by peroxides (71).
In contrast to the cyclooxygenase pathway where the first step is rate limiting, the rate limiting step in leukotriene biosynthesis occurs at the levels of the LTA hydrolase and LTC synthase (glutathione transferase) (72). Subcellular fractionation of enzymatic activity indicates that the 5-lipoxygenaseand LTAhydrolase are soluble cytosolic enzymes (72, 73). However, isolating the 5-lipoxygenase in the presence of excess calcium may increase its degree of membrane association (74), and this effect may mimic physiologic activation and translocation of the enzyme (75). Inhibitors of the translocation process may be useful in blocking leukotriene formation. In fact, at least one such inhibitor has been identified and also used to isolate and clone a membraneactivating protein necessary for leukotriene synthesis (76). The 5-lipoxygenase has been purified (73), and its full sequence determined by molecular cloning (77). Primary structural features have been reviewed (55). Expression of the 5-lipoxygenase cDNA in a baculoviruslinsect cell system (78) and in osteosarcoma cells (79) and isolation of the complete 5-lipoxygenase gene from genomic DNA libraries (80) have now been accomplished and are likely to lead to further insight into transcriptional and posttranscriptional mechanisms for lipoxygenase regulation. The LTA hydrolase has also been purified (81), and its cDNA has been isolated by immunoscreening a human lung cDNA expression library (82) and by screening a spleen cDNA library with an oligonucleotide probe based on peptide sequence of the purified hydrolase (83). The enzyme appears to be expressed in virtually all tissues and a variety of cell types, including those that lack 5-lipoxygenase activity (84). For example, the enzymatic capacity is present in erythrocytes so that LTAa released from leukocytes can be converted to LTB4 in the blood (85). The glutathione transferase, y-glutamyl transpeptidase, and dipeptidase are particulate enzymes (70).The glutathione transferase is therefore distinct from conventional glutathione transferases, which are generally soluble enzymes. The location of the enzymes implies that LTAa formed in a cytosolic location must be transported to the granule or other particulate portion for conversion to LTC4 • The transpeptidase and dipeptidase have been localized to the plasma membrane (86) and may therefore serve to convert LTC4 to LTD4 as it is released from the
192
cell or approaches the outside of another cell. The metabolism of the sulfidopeptide-containing leukotrienes has been reviewed (87). LTB4 is metabolized via cytochrome P-450 to 20-hydroxy-LTB4 and then to 20-carboxy- LTB4 in neutrophils but to little extent in other cell types (see below). Metabolic studies of radiolabeled LTB4 as well as studies of isolated hepatocytes (88) suggest that the compound is also extensively degraded by 13-oxidation. In contrast to the 12- and 15-lipoxygenases, the substrate specificity of the 5-lipoxygenase pathway is quite restricted. The double bonds at carbons 5, 8, 11,and 14 are required for optimal LTB formation, and this requirement helps explain the marked decrease in LTB4 formation in essential fatty acid deficiency. Reports of decreased inflammation in this condition may also be due in part to this biochemical deficiency. The addition of a double bond at carbon 17 yielding eicosapentaenoic acid (EPA) still permits formation of the corresponding LTBs but may decrease the efficiency of conversion significantly and have effects on leukocyte function (89). The leukotrienes derived from 5-lipoxygenase activity have been implicated as critical mediators for a variety of inflammatory diseases, and their effects on the airways and on the microcirculation have been reviewed recently (87, 90). LTB4 is a potent chemoattractant for human neutrophils and eosinophils (91, 92) as well as a lymphocyte activator (93). The compound binds with high affinity, stereospecificity, and saturability to receptors on cell membrane from polymorphonuclear neutrophils (94). Binding studies suggest the presence of high- and low-affinity receptor populations that are distinct from the receptors for other chemotactic substances such as C5a or FMLP. Leukotriene C 4 and D 4 , but to a lesser extent E 4 , are contractors of airway, vascular, and intestinal smooth muscle. Evidence that these products might specifically be important in the development of asthma includes production of the compounds by both human and animal lungs after antigen challenge (60)and increases in their concentration in airways of asthmatic patients (95). Administration of the compounds to animals or to humans causes airway constriction (60, 96-98), hyperreactivity (14, 99), and increased mucus secretion (100), and these functional alterations are each essential features of the disease.
MICHAEL J. HOLTZMAN
Even for 5-lipoxygenasepathway products, the importance of the compounds for pathophysiologic events in vivo is uncertain. As is the case for understanding 12-and 15-lipoxygenase function, the unavailability of specific inhibitors of the pathway has hindered better definition of pathway regulation in vitro and securing more convincing evidence of the role of the pathway products in vivo. The availability of purified enzymes and expression systems may aid in the development and testing of suitable inhibitors.
Monooxygenase Hydroxylation reactions in animal cells use a special class of complex microsomal monooxygenases designated as cytochrome P-450 (P for pigment) (101). The enzymes were first identified by the fact that the carbon monoxide derivative of its reduced form absorbs maximally at 450 nm. This class of enzymes is most active in the liver but is widely distributed in animal tissues, including the lung (102), and is highly active in monooxygenation of a variety of lipophilic compounds, including arachidonic acid and its metabolites. Metabolism of arachidonic acid by cytochrome P-450monooxygenase leads to
