Mammalian non-pancreatic phospholipases A2

Biochimica et Biophysics Acta, 117 (1993) 217-231 0 1993 Elsevier Science Publishers B.V. All rights reserved

BBALIP

217 0005-2760/93/$06.00

Review

54288

Mammalian non-pancreatic

phospholipases

A,

Ichiro Kudo, Makoto Murakami, Shuntaro Hara and Keizo Inoue * Faculty of Pharmaceutical Sciences, Unicersity of Tokyo, 7-3-l Hongo, Bunkyo-ku, Tokyo 113 (Japan) (Received

Key words:

Phospholipase

1 June 1993)

A,; Enzyme

structure;

Cloning

Contents I.

Introduction

..............................................................

217

II.

Classification

..............................................................

218

III. Purified

isoforms

...........................................................

Type II PLA, ........................................................ Detection and purification ............................................... Structure and gene cloning ............................................... Properties .................................. ........................ Regulation ................................. ........................ Physiological functions ......................... ........................ ................ (i) Involvement in inflammation ........................ .......... (ii) Regulation of eicosanoid generation ........................ (iii) Antimicrobial activity in combination with bacteriocidal permeability-increasing tein ..................................... (iv) Possible activation in accordance with tissue injury (v) Degranulation .......................... III-B. cPLA, ..................................... Detection and purification ...................... Structure and gene cloning ...................... Properties and functions ....................... III-C. Other PLA,s ................................ Myocadium PLA 2 ............................ Phosphatidylserine-specific PLA, ................. RenalPLA* ................................ 14.3-3 Protein: a second cPLA,? ................. Human spermatozoa PLA, ..................... Membrane-associated PLA, ..................... III-D. Cellular proteins as possible regulators of PLA, .......

219

III-A.

IV. Conclusion References

* Corresponding

...............................................................

pro224 224 225 225 225 226 226 227 227 228 228 228 228 228 228 228

..................................................................

author. Fax: +81 3381 83173. Abbreviations: PLA,, phospholipase A,; cPLA,, cytosolic PLA,; PLA,, phospholipase A,; PG, prostaglandin; DTT, dithiothreitol; HWEC, human umbilical vein endothelial cells; BPI, bacteriocidal permeability-increasing protein; IL, interleukin; TNF, tumor necrosis factor; PDGF, platelet-derived growth factor; TGF, transforming growth factor; LPS, lipopolysaccharide; PLAP, PLA*-activating protein.

219 219 221 222 223 223 223 223

229

I. Introduction

Phospholipase A, (PLA,; EC 3.1.1.41, which hydrolyzes fatty acids bound at the sn-2 position of glycerophospholipids, has been detected universally in a variety of mammalian tissues and cells [l-4]. Liberation of arachidonate, which may be mediated by either

218

PLA, or phopsholipase C followed by lipase(s), is a rate-limiting step for a generation of eicosanoids. Understanding of the regulatory mechanism of cellular PLA, activity is therefore of cruciai importance for clarifying the molecular basis of signal transduction, including generation of lipid mediators from biological membranes. A large amount of information on PLA, isolated from mammalian pancreas has been accumulated because of the relatively high content of the enzyme in this organ. ~though the structure and properties of mammalian pancreatic PLA, have been well documented, mammalian PLA,s bearing different structures from the pancreatic enzyme have recently been isolated from various cells and tissues. The present review focuses on the characterization and possible functions of these new mammalian PLA, isoforms. II. Classification There are at present three types of mammalian PLA, for which the primary structure has been determined or deduced from the gene structure. To avoid confusion with the nomenclature of PLA, isoforms, these three groups will be referred to tentatively as types I, II and cPLA, (Table I). Snake venom PLA,s have been purified and characterized [3-51, and are divided into two groups on the

basis of primary structure; those from the Elapidae and ~yd~~~~idae are called type I, and the others from the C~~~al~daeand Eperidae are called type II [6]. It is well known that pancreatic PLA, isolated from a variety of animals has structural characteristics similar to those of type I snake venom PLA,s. The enzymes with a structure similar to those of type I snake venom PLA2s are called type I enzymes. As mentioned in a later section, various types of cell including platelets, mast cells, hepatocytes, and smooth muscle cells secrete PLA,s which resemble type II snake venom enzymes [7]. These mammalian enzymes (mammalian non-pancreatic secretory PLA,, PLA,-II, s-PLA,) are called type II enzymes. Both type I and type II PLA, bear molecular weight of about 14000 and they are separated on the basis of difference in disulfide bonds. Mammals possess both type I and type II enzymes, whereas snake venoms contain either the type I or type II enzyme, depending on subclass. Bee venom contains another type of PLA,, with a structure quite different from either type I or type II enzyme [8]. Although no enzyme showing high homology with this type of PLA, has yet been detected in mammals, Dennis refers to this enzyme as type III 191. New types of PLA2s have recently been identified from various sources on the basis of either biochemical or immunochemical properties. These enzymes have a relatively high molecular weight, and are activated by submicromolar concentrations of free Ca2+ ions. The

TABLE I ~haru~t~r~t~~s of PLA,

c~aracte~~ed

PE; phosphatidylethanolamine,

and cloned

PC, phosphatidyicholine.

* Hydrolysis rates for PE and PC in the absence of detergent are compared.

PLA,

Type I

Type II

cPLA Z

molecular weight origins mammalia

14000

14000

85 000

pancreatic juice

cytosol of various cells

snake venom

exudate fluid in inflamed sites inflammatory cells snake venom

( Elapidae, H~d~o~hidae)

(Crotalidae,

substrate specificity *

PE=PC

PE>PC

Ca*+ requirement possible function in mammalia

mM digestion of phospholipids in food. proliferative effect on cells.

unique characteristics

biosynthesized as inactive proenzyme and processed to mature enzyme by proteolytic cleavage.

mM inflammation. tissue damage. degranulation. destruction of bacterial membrane. induction of gene expression by infl~mato~ cytokines. high affinity for heparin.

other nomenclatures

group I PLA 2 PLA *-I pancreatic PLA Z secretory PLA 2 (sPLA 2)

group II PLA, PLAZII non-pancreatic PLA, secretory PLA 2 (sPLA 2)

non-mammalia

not detected

Yiperadae) PE and PC bearing

arachidonoyl residue I.LM receptor-coupled arachidonate release.

translation to membrane in a Ca’+-dependent manner. exhibition of lysophospholipase, phospholipase 5 and transacylase activities. activation by phosphorylation. arachidonoyl-preferential PLA,. high molecular weight PLAZ

219 most remarkable characteristics of these enzymes commonly observed are that they preferentially hydrolyze phospholipid molecular species containing arachidonate. The amino acid sequence of one such enzyme deduced from its cDNA structure exhibits no homology with those of type I, type II and type III enzymes [lO,ll]. This new type of PLA,s has been called arachidonoyl-preferential PLA *, cytosolic or cPLA Zt or high-molecular-weight PLA,. In the present paper, these enzymes will be referred to as cPLA,s ‘. Detection of cPLA, can also be performed immunologically [12], since antibodies against the cPLA, do not react appreciably with either type I or type II enzyme. cPLA, has not yet been detected in non-mammalian species. Other types of PLA,, with properties distinctly different from those of type I, type II and cPLA,s, have also been purified from mammalian sources, although their primary structures have not yet been determined. These enzymes are classified under ‘others’ in the present paper. III. Purified isoforms

Detection and ~~r~~cut~on.In 1982, Verger and coworkers [I31 isolated a novel PLA, with a molecular weight of about 14~0 from porcine intestine. The NH,-terminal 48 amino acid residues of this enzyme were rather different from those of porcine pancreatic PLA,. From ascitic fluid of rabbit administered glycogen intraperitoneally, Weiss and Elsbach detected and purified an extracellular PLA,, with a molecular weight of about 14000 [14]. Amino acid sequence analysis of both the NH2 and COOH terminal portions of the enzyme showed that the presence of cysteine residues was similar to that in Crotulidae venom PLA,, a group II enzyme. Horigome et al. [lS] found that upon application of appropriate stimuli (thrombin, for instance) rat platelets released more than 80% of the PLA, detected in intact cells. The release was energy-dependent, and both the dose-dependence and time-course of enzyme release were slightly different from those of either serotonin (a dense-granule marker) or p-glucuronidase (a lysosome marker). Like platelet-derived growth factor (PDGF) and platelet factor 4, this PLA, had high affinity for heparin. These findings indicate that the PLA, might be stored in n-granules. It is noteworthy that heparin-Sepharose column chromatography was effective for puri~ing this platelet secretory PLA,, whose molecular weight was again about 14000

’ cPLA, is classified as a non-pancreatic PLA, in the present paper, although its absence on pancreatic tissue has not been proven.

[16]. Partial amino acid sequence analysis of the purified enzyme revealed that, like rabbit ascitic fluid PLA, [14], the rat platelet-derived enzyme belongs to type II [17]. Rabbit platelets also secreted an extracellular type II PLA, upon stimulation [18]. Kramer et al. [191 showed later that human platelets also secreted an extracellular PLA, upon stimulation, and concluded that it was a type II enzyme. It should be emphasized that no apparent secretion of type II enzyme was detected from human platelets under the same conditions as those we employed for detection of rat or rabbit platelet enzymes (unpublished data). This discrepancy has not yet been fully explained, but might be due to differences in assay conditions. They used radioactive autoclaved E. coli as a substrate, whereas we used a suspension of radioactive phosphatidylethanolamine. It had been shown previously that the use of autoclaved E. co& as a substrate for the type II PLA, assay facilitated the measurement of a higher specific activity than that with a purified phospholipid suspension [201. Indeed, the activity of the type II PLA, was not measurable in human platelet lysate when purified phospholipid liposome was used as a substrate. Chang et al. 121,221 detected and purified an extracellular PLA, from peritoneal fluid of rats that had been injected intraperitoneally with casein. The peritoneal enzymes from both rat and rabbit exhibited properties similar to those of platelet secretory enzymes. Antibodies raised against rat platelet secretory PLA, reacted equally with the peritoneal enzyme [23,24], and the partial amino acid sequences of the two enzymes were identical [22]. Extracellular PLA, activities have also been detected at inflamed sites and in circulating blood in patients with various diseases [25-281. Relatively high enzymatic activity was detected in synovial fluid of patients with rheumatoid arthritis [19,27,28]. The molecular weight of the purified enzyme was again calculated to be about 14000. The partial NH, terminal amino acid sequence of this enzyme showed high homology with the corresponding portion of both the rat and rabbit enzymes 1291, suggesting that humans also have secretory type II PLA,. As mentioned above, Kramer et al. [19l found that human platelets also secreted the type II PLA, upon stimulation. The fact that the NH, terminal amino acid sequence of the purified platelet enzyme was the same as that of PLA, isolated from human synovial fluid strongly suggested that the enzymes were identical. Type II PLA, has purified not only from platelets and inflammatory exudate but also from other cells and tissues. Type II PLA, was also purified and characterized from rat E30] and human 1311 spleen as well as from rat liver [32]. A human hepatocyte cell line, HepG2, was shown to express type II PLA, [33]. Es-

220 tablishment of specific antibodies against type II PLA, has enabled us to detect the immuno-crossreactive enzymes in various cells [23,241. Granulocytes found at inflamed sites contained relatively high type II PLAz activity [34]. Mast cells were also shown to have the type II PLA, [35]. As shown in the later section (III-A, Structure and gene coding), some types of cells such as chondrocytes [36,37], vascular smooth muscle cells [38], and renal mesangial cells [39,40] express type II PLA, by treatment with inflammatory cytokines, such as interleukin (IL)-1 or tumor necrosis factor (TNF). These cells appeared to secrete the type II enzyme extracellularly. Relatively large amounts of type II PLA, were also found in human seminal plasma obtained from healthy donors and purified to near homogeneity [41]. The detection of type II PLA, is summarized in Table II. One of the most important considerations is the source of type II PLA, detected in the extracellular space. It was reported previously that the seminal vesicles and prostate secrete PLA, into the seminal plasma of several animal species [42,431. However, the

TABLE

source of the type II PLA, detected at inflamed sites has not been determined conclusively. Platelets are one possible source, as pointed out by Kramer et al. [19]. However, platelets might not be a main source on the basis of the following facts: (1) A sufficient number of platelets cannot be detected at inflamed sites showing high type II PLA, activity. (2) No appreciable reduction of PLA, activity is observed at inflamed sites in rats, even when platelets have been depleted by pretreatment of the animals with anti-platelet antibody [44]. Cells which are stimulated with the inflammatory cytokines, such as chondrocytes, vascular smooth muscle cells or granulocytes infiltrating inflamed sites, may synthesize the enzyme and secrete it extracellularly. The subcellular localization of the type II enzyme is somewhat unclear. Although several reports have described type II PLA, as a membrane-bound enzyme, such as in spleen [30,31] and liver mitochondria [32], the deduced cDNA sequence revealed that the type 11 PLA, is a typical secretory protein [19,28,48,49]. In latent platelets [15,16,18,19] and mast cells 1451, the type II PLAz appears to be stored in secretory gran-

II

Delection of type II PLA, * Arai et al., unpublished

data.

Sources

Animal

(A) normal cells or tissues platelets

rat

neutrophils mast cells megakaryocytes intestine spleen liver lung thymus bone marrow tonsil kidney seminal plasma placenta (B) inflamed sites or tissue injury glycogen-induced ascitic fluid caseinate-induced ascitic fluid synovial fluid of rheumatoid arthritis adjuvant arthritis carrageenan-induced hind paw edema plasma of endotoxin shock pleural fluid of tuberculosis ischemic heart zymosan-induced pleural fluid Ccl,-induced liver disorder

species

47,4x

rabbit human guinea rabbit rat s5 mouse rat 24 porcine rat 4y human rat 32 rat 24 rat ‘e rat 24 human human human human

‘s ” pig *s s4 ss ‘s 3’

I’) ty 4’ Is2

rabbit t4 rat 22 human ‘y,2s rat 77 rat 77 rat “’ human 2h rat ‘s rat ‘Ob rat *

Method

for detection

sequence, cDNA cloning N-terminal sequence N-terminal sequence immunoblotting N-terminal sequence immunoprecipitation, immunoprecipitation, immunostaining N-terminal sequence cDNA cloning sequence N-terminal sequence immunoblotting immunoprecipitation immunoprecipitation Northern blotting Northern blotting N-terminal sequence cDNA cloning N-terminal sequence N-terminal sequence cDNA cloning immunoprecipitation immunoprecipitation immunoprecipitation immunoprecipitation immunoprecipitation immunoprecipitation immunoprecipitation

221 signal sequence

and then processed to a mature enzyme during translocation from the cytosolic to luminal side of the endoplasmic reticulum. It should be noted that, unlike the type I enzyme which is expressed as the proenzyme, the type II enzyme does not bear a zymogen form. The type II PLA, is probably stored intracellularly as a form inactivated by some unknown mechanism, and acts as an extracellular enzyme. The refined, three-dimensional crystal structure at 2.2 A resolution of recombinant human rheumatoid arthritic synovial PLA, was reported recently [50,51]. The data again indicate that this human enzyme is closer to Crotalus atrox PLA, (type II enzyme) than the pancreatic enzyme (type I enzyme). However, the human enzyme still has notable unique structures; for instance, the hydrophobic channel is narrower in the human type II PLA, than in bovine type I PLA, or Crotalus atrox PLA 2. The human ]19,28] and rat [52,53] genes coding for type II PLA, (genomic clones) have been cloned and sequenced. Southern blotting analysis revealed that only a single gene copy exists per haploid in both species, suggesting that enzymes discovered in various cells and tissues are identical. The exon-intron structures of the genomic clones of the human and rat enzymes resembled each other, indicating the close relationship of the two genes. The exon-intron structures of genes for type II PLA, also exhibited some resemblance to those for type I enzyme [54,55]. When rabbit chondrocytes 136,371,rat mesangial cells [39,40], rat vascular smooth muscle cells [38] or rat astrocytes 1461were incubated in the presence of proinflammatory cytokines, such as TNF and IL-l, lipopolysaccharide or DAMP-elevating reagents, gene expression of type II PLA, was induced, and newly generated PLA, was released into the extracellular medium (Ta-

ules and, upon stimulation, it is secreted rapidly (Fig. 1). By contrast, newly generated type II enzymes are readily secreted extracellularly without intracellular storage in some types of cells, such as mesangial cells 139,401, vascular smooth muscle cells 1381, astrocytes [46] and hepatocytes [33]. Structure and gene cloning. The amino acid sequence of type II PLA, purified from activated rat platelets was determined 1471. It contained 14 cysteine residues and their relative positions corresponded exactly with those of the enzyme from Crotalidae and Viperidae venom. The platelet enzyme differed from the pancreatic one in possessing an extra segment at the carboxy terminal portion, These observations confirmed that the rat platelet secretory PLA, can be included in the type II family. The human type II enzyme isolated from a spleen membrane fraction has also been sequenced 1311. Cloning and sequencing of both human [19,28] and rat [48,49] cDNA has shown that their primary structures have about 71% (nucleotide) and 67% (amino acid) homology with each other. Human type II PLA, is composed of 124 amino acid residues, whereas the rat enzyme contains 125 amino acid residues. ~though the primary structure of type II enzymes shows low homology with that of type I enzymes over all (30% and 31% amino acid homolo~ for human and rat, respectively), type II enzymes contain some well conserved residues which have been postulated to represent the ‘active site’, ‘Ca2+-binding site’, and ‘surface recognition site’ in the type I enzyme. Judging from the gene structure, both human and rat type II PLA,s carry a typical signal sequence composed of 21 amino acid residues and located adjacent to the NH, terminal portion of the mature enzyme structure. Type II PLA, may be synthesized as a precursor form containing this

1001

I

Thrombin (units/ml)

Dilution-of Anti-IgE

Fig. 1. Release of type II PLA, from activated platelets (A) and mast cells (B). (A) Rat platelets (1. lo9 cells/ml) were incubated in the presence of various ~n~entrations of thrombin at 37°C for 15 min. PLA, f@l and serotonin (0) released into the supernatant were quantified. (B) Mouse bone marrow-derived mast cells (1. IO6 cefls/mII were sensitized with IgE and then stimulated with the indicated concentrations of antigen for 15 min at 37°C. PLA, <*I and histamine (01 released into the supernatant were quantified. A, Ref. 15; B, Ref. 45.

222

ble III). interestingly, dexamethasone was shown to suppress the gene expression of type II PLA, [46,56,57]. It was argued that the well known activity of anti-inflammatory steroids might be partly explained by this suppression. In the case of rat mesangial celis, expression of the type 11 PLA, was also suppressed significantly by PDGF [SS] or transforming growth factor (TGFI-/3 [591. It was found that cultured human hepatocyte HepG2 cells treated with IL-l, IL-6 TNF or their combinations expressed the gene for type II PLA, [33]. Therefore, it was suggested that type II PLA, might be an acute phase protein. These findings seem to be in line with the findings that administration of lipopolysaccharide (LPS) to rats led to an increment of type II enzyme in various tissues, including liver [60]. High levels of type II PLA, in plasma of patients with systemic in~ammation 125-281 might also be explained by continuous generation and secretion of type II PLA, from various tissues into blood circuIation by inflammatory stimuli. When purified rat type II PLA, was injected into rats intravenously, the intravascular level of the enzyme fell rapidly, due to active degradation in the liver to an acid-soluble fraction within several minutes 1611. Therefore, the elevated type II PLA, level in plasma might be balanced by its secretion from PLA,-generating cells and by its clearance in the liver. Properties. The activity of type II PLA, is detected in the basic pH range, and shows an optimum at around pH 8.0-9.0. Although the apparent substiate specificity is known to be dependent on the assay condition used, hydrolysis rate of type II PLA, for major phospholipids in the absence of detergent exhib-

ited some unique specificities as follows. Type II enzyme hydrolyzes phosphatidylethanolamine much more effectively than phosphatidylcholine [18,22,27]. Rat and rabbit type II enzymes hydrolyze phosphatidylserine as effectively as phosphatidylethanolamine [16,18], whereas the human type II enzyme hydrolyzes phosphatidylserine poorly [27]. Unlike the cPLA,, no fatty acid selectivity was observed with the type II PLA,; it hydrolyzes phospholipid with an arachidonoyl residue and that with other fatty acid such as linoleate, almost equally [62]. When assayed using mixed micelles as a substrate, phosphatidylglycerol was shown to be the best substrate for rat type II PLA, [30]. The type II enzyme has an absolute requirement for Ca2+ ions for expressing of full activity, the maximum activity occurring at 4 mM Cazt [16-18,22,27]. Ca2+ ions can not be replaced by other divalent cations. The addition of monovalent cations partially suppresses the activity of the purified human and rat type II enzymes [22,27]. The purified enzyme is fully active on phosphatidylethanolamine in the absence of detergents. Addition of either sodium deoxycholate at 0.05% or Triton X-100 at 0.1% abolishes the enzyme activity. p-Bromophenacylbromide, a known active site-directed histidine reagent for the type I PLA,, has been found to inhibit both rat [22] and human 1271type II enzymes. This inhibition was partially weakened by the presence of Ca 2c ions. The activity was also inhibited by dithiothreitol (DTT) [22,27], which reduces disulfide linkages. These observations are in good agreement with the structural features; type II enzymes bear an active site similar to that of type I enzymes and seven disuIfide linkages are expected to be formed to stabi-

TABLE 111 Regulation of type II PLA 2 expression by carious agents Cells or organs

Stimulus

Effect on type II PLA 2 expression

rabbit chondrocytes 36.37 rat vascular smooth muscle cells ss

IL-1 IL- 1, TNF, LPS CAMP-elevating agent glucocorticoid IL-l, TNF, LPS phorbol ester CAMP-elevating agent glu~o~i~id IL-l, TNF CAMP-elevating agent glucocorticoid PDGF, TGF-@ IL-l, IL-6, TNF

induction induction induction suppression induction induction induction suppression induction induction suppression suppression induction

TNF LPS glu~ocorticoid

induction induction suppression

rat astrocytes 4h

rat mesangial cells 3y~Kis.58S9

human hepatoma HepG2 cells 33 human umbilical vein endothelial cells ” rat aorta, spleen, lung, thymus, brain m

223 lize the tertiary structure. It should be noted that a serine-reagent, diisopropylfluorophosphate, does not affect the activity appreciably. Type II enzyme was also inhibited by mepacrine [63], by a low-molecular-weight bacterial metabolite (thielocin A,) [64] and by a complement degradation product (see section III, Regulation) [65]. The inhibitory effect of both compounds was quite specific for type II PLA,. Manoalide, the marine natural product, or some chemically-synthesized inhibitors inhibit the activity of type II PLA, [66,67]. Type II enzyme shows a high affinity to heparin [16,27]. Haparan sulfate proteoglycan of human endothelial cells was recently found to act as an anchor for type II PLA, to be retained on the cell surface [68]. Because heparan sulfate is expressed on a wide variety of cells, similar interaction of type II enzyme on cell surface might occur ubiquitously. Re~lutjon. Mammalian pancreatic PLA, (type I> is known to be synthesized as the proenzyme (zymogen form) [69], which exhibits only limited activity [70,71] and is cleaved proteolytically to express its full activity, whereas the type II enzyme does not exist as a pro-form. Therefore, once the signal sequence is processed properly, the type II enzyme can exhibit its full enzymatic activity. As discussed already, type II enzymes are detected in variety of extracellular fluids in which a sufficient concentration of free Ca2+ ions for activation is available. The active type II enzyme might damage cells present in the vicinity by hydrolysis of plasma membrane phospholipids. Some regulatory mechanism(s) may, therefore, operate to avoid such a nonspecific membrane damage. This might include inhibitory factors such as masking protein(s) or inhibitory component(s) present in the extracellular fluid. The susceptibility of substrate phospholipid on the membranes could be another factor regulating the reactivity of type II PLA, secreted extracellularly. Two proteinaceous factors which inhibit the activity of type II PLAz were isolated from the peritoneal cavity of rats administered dexamethasone 1651. The purified inhibitors had molecular weights of 37K and 33K, respectively. It was deduced from amino acid sequence analysis that these inhibitors were degradation products of rat complement component 3 (C3). Intact C3 did not appreciably affect the activity of type II PLA, (Suwa et al., unpublished data). The inhibitory activity of the C3 degradation product against PLA, was further confirmed by constructing a recombinant rat C3a protein. Complement activation takes place under various pathological and physiological conditions, including inflammation. During the process of complement activation, several proteases such as factor I, kallikrein and elastase cleave C3 to yield various C3 degradation products, some of which might function as a specific regulatory factors for type II PLA,. It is

noteworthy, however, that the factor(s) did not affect the activity of either type I or cPLA,s. Recombinant lipocortin also inhibited the activity of PLA, under certain conditions, but no inhibition was observed in the presence of an excess amount of phospholipid substrates, with which lipocortin interacted [72,73]. Physiological functions (i) In~u~~e~ent in in~~~~at~o~. Type II PLA,s have been detected in exudative fluid at various sites of inflammation [25-281 and thus it is postulated that they play some critical roles in the process of inflammation. This idea is supported by observations that inflammation can be induced in animals by injection of PLA, 174-761. However, induction of inflammation has not always been observed with a purified enzyme. Rats given an intradermal injection of Freund’s complete adjuvant develop chronic arthritis with remarkable paw edema [77]. This experimental system has been widely used as a model of human rheumatoid arthritis. The level of type II PLA, is greatly elevated in the paw edema of rat adjuvant arthritis. Injection of purified type II PLA, exacerbated the paw edema in this model system, whereas no appreciable formation of paw edema was observed with normai rats under the same conditions. It was suggested, therefore, that some pathological conditions may be required in order for the type II enzyme to exhibit a progressive effect on the inflammation. It should be noted that, unlike rats, normal rabbits develop inflammation after an intraderma1 injection of purified recombinant human type II PLA z [78]. The molecular mechanism for such a species difference should be clarified in future. The mechanism whereby the type II enzyme potentiates the inflammatory response in vivo is not fully explained. It was found in in vitro experiments that extracellular type II enzyme might be involved in arachidonate metabolism in inflammatory responsive cells under certain conditions (see below). Inflammatory eicosanoids produced from liberated arachidonate might be responsible for potentiation of the inflammation. Putative functions and pharmacological modulations of type II PLA, were recently reviewed [78b]. (ii) Regulation of eicosanoid generation. Under certain conditions, exogenously added type II PLA, stimulates arachidonate metabolism. HL-60, a human Ieukemic cell Iine, differentiates to human peripheral neutrophil-like cells by treatment with retinoic acid [79]. When retinoic acid-treated HL-60 cells were incubated in the presence of a suboptimal concentration of the Ca2+ ionophore A23187 and purified rat type II PLA,, the amount of prostaglandin (PC) E, produced was dependent on the amount of purified enzyme added exogenously [80]. By contrast, no appreciable

224 PGE, production was observed with the cells were treated with the purified enzyme in the absence of A23187. Extracellular PLA, might hydrolyze phosphoIipids translocated to the outer leaflet of the plasma membrane of HL-60 cells, which had been primed in the eliciting state by exposure to A23187. Because A23187 is a non-physiological stimulus, it is important to identify its physiological counterparts, which make cell surfaces susceptible to type II PLA,. Exudating leukocytes prepared from rats with experimental pleurisy synthesized appreciable amounts of PGE, by in vitro exposure to the type II PLA, even in the absence of A23187 (Sob). The cells at inflamed sites might have already been elicited by some factor(s), When human umbilical vein endothelial cells (HUVEC) were treated with TNF, they expressed type 11 PLA, [68]. PGI 2 synthesis stimulated by TNF was suppressed appreciably when the cells were cultured in the presence of an antibody neutralizing type II PLA,, demonstrating that newly generated type II PLA, plays a role in generation of PGI, in TNF-stimulated HUVEC. Furthermore, exogenously added type II PLA, enhanced synergistically PGI 2 generation in TNFstimulated HUVEC, whereas such augmentation of PGI, generation was not observed with cells treated with PLA, alone. TNF might produce unique signals, which not only enhance the expression of type 11 PLA, but also render the membranes of target cells much more susceptible to type II PLA, of either endogenous or exogenous origin. Another example for the role of extracellular type II PLA, in PG synthesis was observed with activated mast cells [81]. Addition of exogenous type II PLA, to rat peritoneal mast celIs sensitized with IgE and an appropriate antigen increased the amount of PGD, produced in a dose-dependent manner. The type II enzyme did not promote arachidonate metabolism by mast cells when the cells were not sensitized with IgE and antigen. Taken together, extracellular type II PLA, may attack phospholipids in membranes at a certain stage of activation. Such ‘membrane rearrangement’ might be induced by some cell stimulators, such as TNF or cross-linking of IgE receptors (Fig. 21. Although the precise mechanism of rearrangement is not fully understood yet, these stimulators may modify the asymmetrical distributions of plasma membrane phospholipids, resuiting a translocation to the outer leaflet of amino phospholipids, which are rich in arachidonate. Alternatively, extracellular type II PLA, may function as a kind of ligand-stimulating metabolism of arachidonate through binding to specific receptor. (iii) Antimicrobial activity in combination with bacteriocidal permeability-increasing protein (BPI). Type II PLA, cannot directly degrade the phospholipid in intact bacterial cells to manifest antimicrobial activity.

Phospholipd Eicosanoid

Degradation Synlhesls

Fig. 2. Schematic model for eicosanoid biosynthesis by endogenous (upper) or exogenous (lower) type II PLA,. Inflammatory cytokine such as TNF stimulates induction and secretion of type II PLA?. Either type 11 PLA, generated endogenously or added exogenously associates with cell surface via heparan sulfate proteoglycan. TNF also produce a unique signal which renders plasma membrane phospholipids susceptible to the type II PLA,. Thus, membrane-hound type If PLAZ becomes capable of hydrolyzing membrane phospholipids, resulting in liberation of arachidonic acid.

Weiss and Elsbach found that the type II enzyme decreased the viability of microorganisms in combination with a neutrophil”derived protein called bacteriocidat pe~eability-increasing protein, BP1 182,831. When BP1 was included in the reaction mixture, phosphoiipids in intact bacteria were hydrolyzed by type II PLA,. BP1 does not merely pave the way for PLA,, since there was a high degree of specificity in the interaction between BP1 and the enzyme [84]. Thus it appears that a certain structure of the enzyme is required for interaction with BPI. The cluster of basic residues in the NH, terminal region may account at least in part for the ability of the type II PLA, but not most other PLA, to act on BPI-treated bacterial menbrane. It is therefore proposed that antimicrobial activity could be one of the physiological roles of type II PLA,. (iu) Possible activation in accordance with tissue injury. It is widely accepted that, during tissue ischemia, phospholipids are abnormally degraded and free fatty acids and lysophospholipids are generated. The accumulation of these abnormal lipids is of great importance, since they are potentially cytotoxic. Since it has been considered that the activation of PLA, is involved in the generation of abnormal lipids to cause tissue damage upon ischemia, numerous attempts have been made to characterize the PLA,s present in the

225 myocardium. We found that the breakdown of endogenous phosphatidyiethanolamine by type II PLA,, which was detected during incubation of rat heart homogenates, was accelerated markedly by ischemic treatment of the tissue in vivo [SS]. Similar activation of type II PLA, was observed in homogenate of liver from Ccl,-treated rats (Arai et al., unpublished data); when liver homogenate from rats treated with CCI, intraperitoneally was incubated at 37°C endogenous phosphatidylethanolamine was selectively hydrolyzed. Enhanced activity of PLA, was also observed after brain or renal ischemia [86,87]. (v) Degranulution. Mast cells play important roles in allergic reactions of the immediate type. Upon stimulation, mast cells release histamine stored in secretory granules. The released histamine then potentiates a variety of bioactions involved in the allergic response. Mast cells contain three types of PLA, including the type II enzyme [35]. Specific inhibitors of type II PLA, (antibody [24], recombinant rat C3a [65], and thielocin A, [64]) inhibited the release of histamine from both activated rat and mouse mast cells [45,63]. None of the inhibitors tested was able to permeate the plasma membrane of intact mast cells. it was observed that mast cell type II PLA, was expressed transiently on the external surface and secreted eventually into the medium upon cell activation [45]. Moreover, release of histamine from mast cells was induced by exposure of cells to exogenous type II PLA 2 (Murakami et al., unpublished data). These findings indicate that the ‘ecto-form’ of type II PLA, may play a crucial role in the degranulation process. Several investigators have postulated the possible involvement of PLA, in degranulation in various secretory cells. Treatment of ceils with either pbromophenacylbromide or mepacrine was shown to prevent various types of degranulation such as histamine release from mast cells [881, neurotransmitter release from neuronal cells [891, and release of catecholamine from chromaffin cells [901. Certain secretory cells are known to contain the type II PLA,. Verger et al. 1131 purified the type II PLA, from porcine intestine, and the location of the enzyme was later identified immunochemically in Paneth’s cells, the function of which are known to be secretion [91]. The molecular mechanisms involved in the action of type II PLA, in the degranulation process have yet to be elucidated. HI-B. CPU

2

Detection and purification. Cellular PLA, is believed to play an important role as a key enzyme during stimulus-coupled arachidonate metabolism. When human platelets are activated by collagen, phospholipids containing arachidonate show predominant degradation [92]. This observation suggests the presence of a

cellular PLA, which is activated during the process of stimulus-coupled signal transduction to hydrolyze arachidonate residue preferentially. Existence of such a cellular PLA, was doccumented by Bills et al. [92bl. The first biochemical indication of preferential hydrolysis of arachidonate by PLA, was reported by Alonso et al. [93] who detected arachidonate-preferential hydolysis activity in a human neutrophil lysate. Similar activities with arachidonate preference were thereafter detected in mouse peritoneal macrophages [941, the mouse macrophage-like cell line RAW 264.7 [95], 5774 [96], rat kidney [97], rat brain [981, the human mononuclear leukemic cell line U937 [99-1011, and platelets from ox 11021, rat [12], rabbit 11031 and human [1041071. It has been often difficult to detect the cPLA, in crude cell or tissue lysates. One of the reasons is the low activity of the enzyme. Another reason is the co-existence of other PLA,s such as type I and type II enzymes, both exhibiting no fatty-acid selectivity. For example, no arachidonate preference of hydrolysis could be detected in homogenates of rabbit platelets, since they contained both type II and cPLA, activities [ 1081. Effective experimental protocols have, therefore, been devised to eliminate the activity of PLA,s other than the cPLA,. For example, addition of DTT to the ceil lysate enabie us to measure the activity of cPLA, [99], since the activity of the cPLA, is DIT-insensitive whereas those of the type I and II enzymes are sensitive to DTI. Alternatively, heparin-Sepharose affinity column chromatography is used effectively to separate the activity of type II PLA, from the cPLA,. When a soluble fraction of rabbit platelets was applied to the column, the ffow-through fraction exhibited arachidonate-preferential activity [103]. cPLA, is mostly detected in the soluble fraction of cells and tissues. With human platelets, about one-tenth of the total activity in the membrane fraction prepared by sonication followed by ultracentrifugation preferentally hydolyzes arachidonate 11071. Evidence has accumulated to suggest the translocation of cPLA, in accordance with increased Ca2+ ion concentration. Higher arachidonate-preferentially hydrolyzing activities detected in various kinds of cells and tissues, including rat brain [98], mouse RAW 264.7 cells [109], and human U937 cells [loll, were associated with membrane fractions when the free Ca*’ concentration was about lo-’ M, whereas they behaved as soluble enzymes in the presence of a sufficient of Ca2+ chelator. In activated cells, arachidonate-preferential hydrolyzing activity in the cytosolic fraction was significantly reduced, whereas that bound to the membrane tended to increase. Thus, an increase in the free Ca*+ ion concentration might induce translocation as well as stimulation of the enzyme activity. The membrane-associated form of the enzyme might be in a ‘physio-

226 logically active’ state, and the cytosolic enzyme might constitute a reservoir of potential activity. cPLA, has been purified to near homogeneity from several cellular sources, such as mouse RAW 264.7 cells [1101, 5774 cells [961, human U937 cells [99-1001, rat kidney [971, rabbit platelets [103], ox platelets [102], and human platelets [107]. Although the apparent molecular masses of the purified enzymes were diverse (ranging from 60 kDa to 140 kDa), recent immunochemical studies [12] and/or molecular cloning experiments [lO,ll] have revealed that their real molecular masses are about 85 kDa. Specific antibodies raised against cPLA, isolated from rabbit platelets cross-reacted with the enzyme from human U937 cells and vice versa. These observations are not contradictory with findings that the genes encoding cPLA, exhibit high homology among different species. Establishment of specific antibodies has further enabled us to detect immuno-crossreactive PLA, activity in various cells and tissues [12]. Neutrophils, mast cells, brain, lung and liver were shown to contain cPLA,s (Table IV>. Structure and gene cloning. The cDNA for human cPLA, was recently cloned from U937 cells [lO,lll. It was shown that the human cPLA, is composed of 749 amino acids, with an estimated molecular mass of approx. 85.5K. The deduced amino acid sequence of human cPLA, did not contain any region showing high homology with known 14-kDa type I or type II PLA,s. Sequencing analysis revealed the presence of a region showing high homology with the motif identified as the Ca2+-dependent membrane-binding domain in several proteins, such as protein kinase C, phospho-

TABLE

lipase C-7, GAP and ~53. The existence of such a motif in cPLA, is in good agreement with previous observations that an increase in free Ca*+ ions induced translocation of the cPLA, from the soluble to the membrane-bound form [98,101,109]. The molecular mechanism of the change in subcellular localization is not yet clear. Properties and functions. Purified cPLA, hydrolyzes phosphatidylcholine and phosphatidylethanolamine, both of which contain an arachidonate at the m-2 position of the glycerol backbone, much more efficiently (about IO-fold) than phospholipids containing linoleate and other fatty acids [107]. It is of great interest to examine whether the cPLA, preferentially releases arachidonate from natural membrane. It was reported that pure recombinant cPLA, selectively hydrolyzed phospholipids containing arachidonate in natural membranes [lo]. In an experiment where a crude enzyme fraction was used as an enzyme source, incorporation of phosphatidic acid into liposomes composed of a mixture of arachidonoyl and linoleoyl phospholipids diminished the apparent substrate specificity [20]. One of the most remarkable features of cPLA, is its Ca2+ sensitivity. The catalytic activity observed with either phosphatidylcholine or phosphatidylethanolamine as a substrate increases sharply with an increase in the Ca*+ ion concetration from lo-’ to 10mh M [96-102,104,107] (Fig. 3). The Ca2+ ion concentration in the cytosol generally increases to arround the micromolar level upon cell stimulation [llll. The activity of cPLA, may be regulated by the concentration of Ca2+ ions in the cytosol during stimulus-coupled reactions. cPLA, binds to natural membrane vesicles in a Ca2+-

IV

Detection of cPL.A, * cDNA

cloning

from RAW264.7

cells was also reported

(Ref. 10).

Sources

Animal

RAW264.7 cells (macrophage cell line) UY37 cells (monoblasts) 5774 cells (macrophage cell line) platelets

mouse human mouse human rabbit ox lo2 rat I2 rabbit rat ss mouse human rabbit rat 9X rat a’ rabbit rabbit

neutrophils mast cells umbilical brain kidney lung liver

vein endothelial

cells

soecies ” * “-rr Ye lo7 ‘OS

” 35 ‘s ”

” I2

Method

for detection

purification cDNA cloning purification purification purification purification immunoprecipitation immunoprecipitation immunoprecipitation immunoprecipitation immunoprecipitation immunoprecipitation purification purification immunoprecipitation immunoprecipitation

227

dependent fashion, resulting in the selective release of arachidonic acid, thus implicating cPLA, in the hormonally regulated production of eicosanoids. The treatment of Chinese Hamster ovary (CHO) cells overexpressing cPLA, with ATP or thrombin resulted in an increase of arachidonic acid as compared with parental CHO cells, demonstrating the hormonal coupling of cPLA, [112]. The activation of cPLA, with a wide variety of agents such as ATP, PDGF and epidermal growth factor stimulated the phosphorylation of cPLA, on serine (not tyrosine) residues. The PLA, activity was increased about 2-fold by the phosphorylation, and pretreatment of cells with staurosporin blocked agonist-induced phosphorylation as well as activation of cPLA,. When cPLA, purified from 5774 cells was phosphorylated by protein kinase C, no activation of PLA, activity was observed [96]. site cPLA, contains a consensus phosphorylation (around Ser-505) for the MAP kinase. It was recently shown that cPLA, is phosphorylated and activated by MAP kinase [ 1131. The possible scheme for cPLA, activation is shown in Fig. 4. It was reported that IL-l stimulated cell-associated PLA,, which required submicromolar Ca2+ for activity in human rheumatoid synovial fibroblasts [114] and in rat mesangial cells [114b]. Glucocorticoids inhibited the TNF-induced increase in cPLA,-like activity in human epithelial carcinoma cell line HEp-2 cells [115]. It was also shown that glucocorticoids blocked the IL-l-mediated increases in both cPLA, level and PGE, production without affecting the cyclooxygenase level in human lung fibroblast cell line WI-38 [116]. Thus, not only type II but also cPLA, might be associated with

Fig. 4. A schematic model for activation of cPLA,. cPLA, is activated by raise in cytosolic Ca*+ concentration and by phosphorylation. PKC, protein kinase C; PLC, phospholipase C; PIP,, phosphatidylinositol 1,4,5triphosphate; IPs, inositol 1,4,5_triphosphate; DC, diacylglycerol; PL, phospholipid; AA, arachidonic acid; TX, thromboxane; LT, leukotriene; PAF, platelet-activating factor.

increased eicosanoid production in cytokine-stimulated cells. cPLA, exhibits lysophospholipase activity in addition to PLA, activity [110,116b1. It was also reported that PLA, prepared from mouse RAW 264.7 cells showed relatively weak phospholipase A 1 (PLA ,) activity. cPLA, purified from rabbit platelets also exhibited lysophospholipase activity and the PLA, activity (Fujimori et al., unpublished data). It was recently shown that human recombinant cPLA, also exhibited a tansacylase activity [116b] III-C. Other PLA 2s Myocardium PLA,.

4

3

2

EGTAIEOTA

on the purified rabbit platelet Fig. 3. Effect of Cazf concentration cPLA,. The purified enzyme (10 ng) was incubated with phosphatidylethanolamine co), phosphatidylchohne (0) or phosphatidylinositol (A 1, all of bearing [‘4C]arachidonoyl residue at sn-2 position, in the presence of various concentrations of CaCI, at pH 7.4. [‘4C]Arachidonic acid liberated was extracted and the radioactivity was counted. Ref. 103.

Gross and co-workers [117] purified an unique PLA, from canine myocardium soluble fraction. This PLA, was Ca*+-independent and selective for a plasmalogen substrate. Separation on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis revealed that the purified enzyme had a molecular weight of about 40000. The PLA, possessed a pH optimum of 6.4 and had an absolute requirement for DTT for enzymatic activity. The purified enzyme selectively hydrolyzed ether-containing glycerophospholipids (subclass rank order; 1-alkenyl-2-acyl glycerophosphocholine > l-alkyl-2-acyl glycerophosphocholine > 1,2-diacyl glycerophosphocholine). Among fatty acids at the sn-2 position of phosphatidylcholine, arachidonate was preferentially hydrolyzed, as was the case with cPLA,. It was recently shown that rabbit [118] and human 11191 myocardiac cytosol PLA,s existed as a high molecular weight complex comprised of catalytic and regulatory polypeptides whose activity and stability were influenced by specific interactions with ATP.

228 In rabbit myocardium, a membrane-bound PLA, with similar characteristics, i.e., Ca*+-independent and plasmaIogen-selective, is also present [120]. This membrane-bound PLA, activity may be involved in the pathogenesis of myocardial disease, since it was recently shown that the enzyme increased immediately after ischemic myocardial injury [ 1211. Phosphatidylserine-specific PM,. A novel PLA, was partially purified from rat mastocytoma RBL-2H3 cells 1351. This PLA, exhibited an unique substate specificity; it hydrolyzed phosphatidylserine much more effectively than phosphatidylethanolamine. No appreciable hydrolyzing activity was observed when phosphatidylcholine or phosphatidylinositol was used as a substrate. The optimal pH was between 5.5 and 7.4, and relatively low concentration of Ca2+ ions (lo-’ M) was required for expression of enzymatic activity. Renew PLA,s. Kidney is one of the tissues that generate prostanoids in large amounts. As mentioned above, cPLA, was detected previously in rat kidney [97]. From rabbit renal cortex, another PLA,, which was selective for phosphatidylethanolamine, was purified. This enzyme appeared to be a tetramer with a 23-kDa monomeric structure 11221.It displayed an obligatory requirement for Ca’+. From rat kidney, PLA, with a molecular weight of about 60000 was also purified. It did not exhibit either selective hydrolysis of fatty acid nor cross-reactivity with antibodies raised against type I, type II or cPLA, (Hara et al., unpublished data). 14-3-3 Protein; a second cPLA,?. PLA, isolated from sheep platelets was shown to be a dimeric polypeptide (58K) composed of chromatographically resolvable isoforms of nearly identical monomers (30K) [ 1231. Like cPLA *s, this isoform was fully activated by physiological concetrations of Ca2+ ions [124]. Recently, human equivalent of one such isoform was cloned, which catalyzed the cleavage of the sn-2 fatty acid of substrates through the formation of a stable acyl-enzyme intermediate [ 1251. Transesterifi~ation of the sn-2 fatty acyl group of the substrate to the 30-kDa recombinant polypeptide was over SO-foId seiective for arachidonic acid and was augmented by Ca2+. Homology analysis demonstrated that the cloned enzyme mediating this transesterification was one member of a family of proteins collectively designated as 14-3-3 proteins 11261.The isolation of an arachidonate-enzyme intermediate demonstrates the potential of the thioesterified arachidonate to be specificalIy transferred to putative nucleophilic acceptors including water (PLA, activity), lipids (transacylase activity) or proteins in oxidative cascades (cyclooxygenase or lipoxygenase). Human spermutozoa PLA,. A novel PLA 2, whose molecular mass was 16.7 kDa, was isolated from human spermatozoa 11271. Sequence analysis of the NH2-terminal portion of the molecule revealed the

identitiy of the first 19 amino acids to be YNYQFGLMIVITKGHFAMV. Although the PLA, resembled type II PLA, in view of tack of Cys-11, it is evident that this PLA, represents a new sequence. Of interest is the location of residues Gin-4, Phe-5, Met-8 and Ile-9, which are highly conserved throughout evolution of secretory PLAzs. Membrane-associuted PLA2. A PLA,, which was tightly bound to sheep erythrocytes membrane and as solublized by treatment with SDS, was purified [128,129]. The PLA, required Ca** for effective hydrolysis of phosphatidylcholine in particles where the enzyme existed. Treatment of the erythrocytes with protease decreased the activity of the PLA,, indicating the localization of the PLA? in the external side of the plasma membrane. Purification of another membrane-b~~und PLA?, with a moIecuiar mass of about 18 kDa, was reported from butanol-extract of the membrane fraction of mouse macrophage-like cell line P388Dl [130]. The activity was assessed with dipalmitoylphosphatidylcholine as a substrate [131]. III-D. Cellular proteins as possible regulators of PLA L. It is widely accepted that guanine nucleotide-binding proteins (G proteins) are involved in linking receptor activation to a variety of intraceIlular responses. In particular, it has become evident that, in several experimental systems, receptor-mediated PLA, activation is mediated by G proteins [132-1361. Pertussis toxin inhibited the release of arachidonate from ligand-stimulated CHO cell expressing human recombinant cPLAz (Knopf, J.L., unpublished data). It was also reported that pretreatment of homogenates of HL-60 granutocytes with specific antibodies against cPLA, attenuated the basal as well as GTP-yS- and Ca”-stimulated arachidonate reIease [ 1371.The activity of cPLA, might thus be regulated by G proteins either directly or indirectly. Recently, PLA, activating protein, PLAP, has been characterized 11381 and cloned f1391. PLAP was originaIIy found as a cellular protein which cross-reacted with an anti-mellitin (a protein which is known to activate PLA, [140]) antibody. Although it has been suggested that PLAP acted as a regulator of type II PLA, [139,141-1431, further studies must be carried out to conclude PLAP as a a key regulatory component in the inflammatory process. There must be a number of PLAP-independent pathways for increasing eicosanoid synthesis. IV. Conclusion

During the last several years, technological progress in protein chemistry and gene engineering has promoted the study of mammalian non-pancreatic ‘regulatory’ PLA2. Little information was previously avail-

229

able on this type of PLA,, since it was present in only small amounts in cells relative to the “digestive’ pancreatic PLA,. It is notable that a recent careful study has revealed the expression of pancreatic (type I) PLA, in non-digestive tissues and cells [144-1471. Thus, the type I enzymes might play a physiological function(s) other than digestion. Arita et al. recently found that type I PLA, possessed unique cell proliferating and chemotactic activities [148-1501. A receptor-like moiecule recognizing the mature type 1 PLA,, but not proenzyme or type II PLA,, was found on some cell surfaces [ 15 11. It is thus clear that there are several types of ‘nonpancreatic’ PLA, in the mammalian body, and that they may play different roles under physiological or pathological conditions. The structural characteristics as we11as the mechanisms of regulation of some PLA,s (types II PLA, and ePLA,) have also been partially clarified. The findings indicate the possible role of type II PLA, and cPLA, in the production of chemical mediators such as eicosanoids and platelet-activating factor. Some PLA,s may be involved in a deacylation-reacylation cycle. Identification of the en~e(s) involved has not yet been made. Using the tools currentfy available such as specific inhibitors and antibodies may promote the clarification of the functions and dynamics of each PLA, isoform. References 1 van den Bosch, H. (lY821 in Phospholipi~s (Hawthorne, J.N. and

2

3 4 5 6 7 8 9 10

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