Klinische Wochenschrift

Klin Wochenschr (1989) 67:153-t59

© Springer-Verlag1989

Phospholipase As- Regulation and Inhibition W. Scheuer Boehringer Mannheim GmbH, Forschung Biochemie, Penzberg

Summary. Phospholipase A 2 (PLA2) has been implicated in the pathogenesis of different diseases. Thus, the pharmacological intervention of PLA 2 activity by specific inhibitors is of great therapeutical value in ameliorating pathological conditions. Despite a great number of published data regarding PLA 2 inhibitors none has reached clinical application. Since enzyme activity can be greatly influenced by the experimental conditions of the test system used, a potent in vitro enzyme inhibitor does not indicate therapeutic effectiveness per se. In order to enhance the predictable value of an in vitro screening system for PLA 2 inhibitors, a battery of test systems each measuring certain parameters should be applied. Considering the complex mechanism(s) of PLA 2 it is extremely important to elucidate the exact inhibition mechanism of those compounds, which have passed these first filters. True inhibitors of PLA 2 should then be evaluated in suitable ex vivo, in vivo models. Key words: P h o s p h o l i p a s e A 2 - Test systems - Inhibitors - Inhibition mechanism(s)

Phospholipase A 2 ( P L A 2 , EC 3. 1.1.4.) is a lipolytic enzyme which hydrolyzes the fatty acyl ester at the sn-2 position producing equimolar amounts of lysophosphatides and free fatty acid. PLA 2 is ubiquitous and is found in a soluble form (extracellular as pancreatic and duodenal juice and intracellular in lysosomes and in the cytosol) and in a nonsoluble form associated with membranes. In biochemical terms it appears that all mammalian membrane-bound (nonsoluble) and pancreaticPLA2s (soluble) are Ca 2 + dependent with pH optima in the neutral to basic range, whereas those PLA2s found in cytosolic or lysosomal compart-

ments are Ca 2 + independent and are optimally active in the acid range. In the pancreas the PLA 2 serves a digestive function, lysosomal PLA2 probably plays a role in the catabolism of phagocytised organic material, and membrane-associated PLA 2 is involved in the metabolic turnover of phospholipids. Furthermore, it has been shown that the peroxidation of membrane phospholipids is accompanied by an increased activity of PLA 2 which excises oxidized fatty acids, indicating that PLA 2 plays a role in protecting membranes from oxidative injury [21]. Lately it has become evident that both membraneassociated PLA2s as well as lysosomal PLAas are involved in the pathogenesis of inflammatory processes and allergic diseases [4, 13, 29]. In this regard much attention has been focused on the role of PLA z in the release of arachidonic acid (AA) from membrane phospholipids which serves as a substrate for prostaglandins (PGS) and leukotrienes (LTS) [11, 12, 19] producing enzymes. It has also been postulated that PLA 2 is involved in the formation of platelet activating factor (PAF) by forming the acyl group from position two of 1-0 alkyllysophosphatidylcholine which is rapidly acetylated to form PAF [4]. Thus once activated in an unregulated fashion the enzymatic activity of PLA 2 contributes to the pathogenesis of diseases either through attacking structural or functional phospholipids leading to tissue injury and/or through subsequent transformation of its products (AA and lysophospholipids) to several potent biologically active substances such as PG, LT, and PAF. It has been shown that elevated PLA 2 levels in serum are associated with diseases like pancreatitis, septic shock, adult respiratory distress syndrome, rheumatoid arthritis, allergic shock, and cardiovascular diseases [28]. However, it has to be kept in mind that inflammatory diseases (like rheumatic ar-

154 thritis) are complex multimediator diseases and it has been extremely difficult to ascertain a potential role tbr one mediator (like PLA2) through measurements of levels in joint fluids. Nevertheless, because of the serious consequences that could result from the pathological conditions, it is highly desirable to modulate or block PLA 2 activity in order to ameliorate the progression of the disease [12, 29, 30]. It has been hoped that after elucidating the biochemical and molecular events of PLA 2 regulation, a model would emerge thus offering a rationale which would allow the establishment of a screening system for PLA 2 inhibitors. However, several mechanisms are involved in the regulation of PLA z and to date it is not known which of them plays the primary role in the activation of the enzyme [2, 7, 22]. Pancreatic PLA 2 is the only mammalian PLA 2 known to occur in a zymogenic form. The pancreatic proenzyme is irreversible activated by tryptic cleavage of the N-terminal heptapeptide. This kind of regulation seems inappropriate since once activated the enzyme will exert its cleavage activity unless efficient product inhibition [3] or other control mechanism(s) take place (on a cautionary note it is not known whether the enhanced PLA2 activity is caused by a conversion of a zymogen into an active enzyme or to secondary effects, such as digestion of endogenous inhibitory proteins or changes in membrane structure). Calcimn is one candidate for consideration as PLA 2 regulator and a Ca 2 ÷ binding site has been identified on PLA 2. Recent studies suggest that a signal transduction pathway involving inositol phospholipid hydrolysis is important in PLA 2 activation. In this pathway receptor occupation by a ligand stimulates phosphoinositide-specific phospholipase C (PLC). The hydrolysis product diacylglycerol (DAG) activates protein kinase C (PKC) by increasing its affinity for Ca 2÷, while inositol 1,4,5-trisphosphate (IP 3) mobilizes Ca 2 ÷ from the endoplasmatic reticulum, thereby increasing free intracellular Ca 2+ concentration [5]. The mechanism(s) through which PKC regulates PLA 2 activity may be complex. One possible participant is lipocortin, the endogenous inhibitor of PLA2. Phosphorylation of lipocortin by PKC neutralizes the endogenous inhibition of PLA 2 , resulting in activation of the enzyme. The discovery that steroids induce the synthesis of PLA 2 inhibitory proteins (lipocortins), raises the possibility that endogenous proteins play a role in controlling PLA 2 activity [8, 23]. However, a recent study has cast doubt on this role for the tipocortins and leaves open the identity of the factor that mediates the

M. Bfichler and H. G. Beger (eds) PhospholipaseA action of antiinflammatory steroids [1, 10, 16]. Recent research has revealed that lipocortins belong to a family of proteins termed annexins (lipocortin I and II, calpactin I and II, endonexin, calelectrin, calcimedin, chromobindin and tyrosine kinase substrate p35 and p36) that undergo C a 2 +-dependent binding to phospholipids [25, 26]. Lipocortin is a substrate for the protein-tyrosine kinase activities associated with the epidermal growth factor receptor (EGF-R) which raises the possibility that lipocortin is involved in the regulation of cellular proliferation [16, 24]. Rous sarcoma virus transforms cells by the production of a 60 kD phosphoprotein (pp60 v-st*) which acts as a tyrosine kinase, p36 (which represents the same gene product as lipocortin II) appears to be the substrate for pp60 ~-sr¢ kinase, suggesting that the members of the tipocortin family may play a role in oncogenic transformation. Substantial evidence has been presented to indicate that guanine nucleotide binding proteins (G-proteins) regulate PLA 2 activity [2]. Taken together, regulation of PLA 2 activity seems to be a complex mechanism involving different control entities on the cellular and subcellular level. Because of the central importance of PLA 2 in the liberation of AA and tissue injury much work has been directed toward the discovery of specific physiologically acceptable inhibitors [7, 11, 12, 18, 20, 30]. One approach is the synthesis of phospholipid analogues with high affinity for PLA2, thus preventing the cleavage of its natural substrate [7, 32]. Another approach is to test known drugs which have been implicated in phospholipid storage disorders (accumulation of polar lipids) and drugs with anti-inflammatory activity [18, 28, 29]. Further strategies imply the prevention of PLA 2 activation (since the activation mechanism has not been clarified in detail this approach has not been followed), the stimulation of enzymes which mediate fatty acid incorporation and, in the case of soluble extracellular PLA 2, the elimination of this enzyme by extracorporal affinity techniques. One strategy mostly employed in pharmaceutical research is a large-scale in vitro screening of inhibitors for certain PLA2s. In this work we have tested inhibitors of PLA 2 and their effect on PLA2s from different sources. The efficacy of these inhibitors was compared using three different PLA 2 assays. Materials and Methods

Colorimetric PLA Assay

This method has been published [17] and was used with minor modifications. Briefly, enzymes (PLA z

M. Bfichler and H. G. Beger (eds) Phospholipase A

from human pancreas and human duodenal fluid kindly provided by Dr. G. Hoffmann, Institute for Clinical Chemistry, City Hospital, Munich-Bogenhausen; PLA 2 from porcine pancreas, Boehringer Mannheim, Mannheim) were diluted with PBS buffer (Boehringer Mannheim, Mannheim) to an appropriate activity. Enzyme solution (20 gl) was incubated at 37°C with 5 ~tl inhibitor solution (chloroquine, chlorpromazine, 4-bromophenacylbromide, from Sigma) in inhibitor buffer (125mmol Tris, 1% DMSO in distilled water pH 7.5). After 30 min 20 ~tl substrate (soy bean phosphatidylcholine, Roth, Karlsruhe) in substrate buffer (250 mmol Tris, 8 mmol CaC12, 4 mmol sodium desoxycholate, 0.5% Triton X100 in distilled water pH 7.5) was added and incubated for another 60 rain. The liberated free fatty acids were quantitated using the test combination free fatty acids (Boehringer Mannheim, Mannheim) according to the manufacturers instructions (see paper by Drs. Hoffmann and Neumann in this issue). All tests were performed in microtiter plates (Nunclon, Nunc, Denmark). The plates were scanned in a microplate reader (MR 700 Dynatech Laboratories, Alexandria) at 500/630 nm, set to correct for buffer controls. Results are expressed as percent inhibition compared with enzyme activity without inhibitor. Radiometric PLA 2 Assay with Labelled Synthetic Phosphatidylcholine The same standard procedure as in the colorimetric assay was used except that L-3 phosphatidylcholine,-l-stearoyl-2-(1-14C)arachidonyl (58.3 mCi/ retool, Amersham, Braunschweig) was used as substrate. Assays were performed in Eppendorf cups. The lipid (20 gl) was dried under N 2 and suspended in 800 gl substrate buffer by sonication at room temperature. PLA 2 solution (20 gt at appropriate dilutions) was incubated at 37°C with or without 5 gl inhibitor solution in inhibitor buffer for 30 rain. Thereafter, 20 gl substrate solution was added to give a final concentration of 0.2 nmol. The reaction was terminated after 60 min by adding 50 gl chloroform. After vortexing for 20 s (the lipids were quantitatively recovered in the organic phase since no labelled products could be detected in the aqueous phase) the free arachidonic acid was separated from the uncleaved substrate by TLC (DC plates SI F, Riedel de Haen, Seelze) using a solvent system consisting of chloroform/methanol/ acetic acid/water 70/30/4/i (v/v/v/v). Radioactive spots were detected by scanning with an automatic TLC-linear analyzer (Berthold, Munich) and quart-

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titated using an integration program. Radioactive spots were identified with standards run in a separate experiment. Blank values (substrate control) were determined by the reaction without enzyme and subtracted from the values determined by enzyme (enzyme control). Results are expressed as percent inhibition compared with enzyme activity without inhibitor. Pulse Labelling of RBL-2H3 Cells with 1-14C-Arachidonic Acid (Cellular PLA 2 Test) Arachidonic acid (AA) release was measured using a modification of a method described previously [14, 27]. RBL-2H3 cells (kindly provided by Dr. R. Siraganian, National Institutes of Health, Bethesda) were maintained in Dulbecco's medium supplemented with 15% heat-inactivated fetal calf serum, 1% glutamine and penicillin/streptomycin (50,000 IU/50 mg in 500 ml). Cells were harvested with trypsin-EDTA (Boehringer Mannheim, Mannheim) and plated as monolayers at 5 × 105 cells/ml in 24-well plates (Costar, Cambridge, Mass.). Cells were used 24 h later, when they had become firmly attached. After washing with Pipes-ACM buffer (119 m M NaC1, 5 m M KC1, 25 m M Pipes, 40 m M NaOH, 5 . 6 m M glucose, 1raM CaCI2, and 0.4 m M MgC12, pH 7.4) the cells were pulsed with 1-t4C-arachidonic acid (Amersham, 58.3 mCi/ mmol) at a final concentration of 4 nM for 30 rain at 37°C and 5% CO2. Then the medium was aspirated and cells were washed three times with PipesACM buffer to remove nonincorporated arachidonic acid. After incubation with PLA2 inhibitors (in 100 ~tl inhibitor buffer) for 30 rain, cells were challenged with 100 ~tl A23187 (10 ~tg/ml; Boehringer Mannheim, Mannheim) and incubated for another 15 rain. The reaction was stopped with 100 gl EDTA (10 mM), the plates were centrifuged (1,000g, 15 rain, 4°C) and the supernatant was removed. An aliquot (200 gl) containing 800 gl "readysafe cocktail" (Beckman, Munich) was used to quantitate the radioactivity in a liquid scintillation counter. Buffer control was subtracted from the positive control (A23187 alone) and from values with inhibitors (A23187 plus inhibitors). Results are expressed as percent inhibition compared with arachidonic acid release without inhibitor. Results and Discussion

The inhibitory effects of three reagents on PLA 2 from three different sources were examined (Fig. 1). Whereas chloroquine and 4-bromophenylbromide (4-BPB) show only a marginal inhi-

156

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bition up to 100 gg/ml inhibitor, chlorpromazine inhibits all three PLA2s in a concentrationdependent manner. Duodenal PLA 2 seems to be less susceptible to chlorpromazine and chloroquine, whereas 4-BPB exerts its optimal effect (at 10 gg/ml) on this enzyme. These results indicate that an inhibitory profile for a given drug is greatly influenced by the enzyme used. Figure 2 reveals that both assay systems for PLA 2 inhibition (cotorimetric vs radiometric assay) produced comparable results, again showing that chlorpromazine is a potent inhibitor. With chloroquine no inhibition was observed using duodenal PLA2 in the radiometric assay. Whereas the inhibitory effect of chlorpromazine has been established firmly, chloroquine exerts its inhibitory action only at high concentration (IC 50 >300 gg/ml) which is in agreement with the studies presented here [29]. Since both systems represent the same mechanism (release of fatty acids from soy bean phosphatidylcholine or labelled phosphatidylcholine) a third method was applied which consisted of a cellular assay. It has been shown recently that the stimulation of cells of the RBL-2H3 celt line [14, 27] or from rat mast cells [31] by Ca ionophore A23187 or antigen resulted in

the activation of PLA 2 as measured by the release of arachidonic acid preincorporated mainly in phosphatidylcholine. Figure 3 reveals and confirms that chtorpromazine was the best inhibitor tested and again no inhibition was observed with chloroquine. Taken together, chlorpromazine is the most effective inhibitor for PLA2s from three different sources. The efficacy of chlorpromazine could be confirmed by using three different assays. However, it has been reported that this drug interferes with the substrate-enzyme interface [7]. Thus, care has to be taken by indicating this drug as a direct enzyme inhibitor. Furthermore, drugs which show a diminished arachidonic release in a cellular system may act by reducing arachidonic acid availability by enhancing the incorporation of this fatty acid into triglycerides. Moreover, PLA 2 activity is not only a function of substrate and Ca 2 ÷ concentration and pH, but is greatly influenced by the physiochemical state of the phospholipid structure [7, 20, 22]. Changes in lipid composition, protein composition, or the presence of detergents alter these physical characteristics and, in turn, enzyme activities. In addition PLA 2 activity increases expo-

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nentially when the substrate concentration reaches or exceeds the critical micelles concentration. This makes kinetic studies problematic and classical Michaelis-Menten kinetics do not apply. Another peculiarity of PLA 2 is that it exhibits the phenomenon of surface dilution kinetics. This means that the activity of the enzyme depends not only on the bulk concentration of the substrate but also on its surface concentration after the enzyme has bound to the surface [11]. Thus, when dealing with inhibitors, their effect on the physical state of the substrate must be taken into account. A routine screen developed for drug candidates is unlikely to be universally applicable to all types of potential inhibitors and may have to be adapted to the specific type of inhibitor under study. Despite a substantial body of evidence suggesting that PLA 2 plays an important role in certain diseases, further studies of the regulation process of PLA2 activation are required. In the context of recent observations that lymphokines (interleukin-1) [6] and cytokines (tumor necrosis factor) [9, 15] are able to activate PLA2, it is of special interest to elucidate precisely the link between inflammatory/immunological reactions and PLA2. Acknowledgements: I am grateful to Wolfgang Kinle and HansPeter Lohnert for excellent technical assistance and thoughtful comments.

References 1. Aarsman AJ, Mynbeek G, van den Bosch H, Rothut B, Prieur B, Comera C, Jordan L, Russo-Marie F (1987) Lipocortin inhibition of extracetlular and intracellular phospholipases A z is substrate concentration dependent. FEBS Lett 219:176 2. Axelrod J, Burch RM, Jelsema CL (1988) Receptormediated activation of phospholipase A 2 via GTP-binding proteins: arachidonic acid and its metabolites as second messengers. Trends Neurosci 11: 214 3. Ballou LR, Cheung WY (1985) Inhibition of human platetet phospholipase A 2 activity by unsaturated fatty acids. Proc Natl Acad Sci USA 82:371 4. Barnes PJ, Chung KF, Page CP (1988) Platelet-activating factor as a mediator of allergic disease. J Allergy Clin Immunol 81:919 5. Berridge MJ (1987) Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem 56:159 6. Chang J, Gilman SC, Lewis AJ (1986) Interteukin 1 activates phospholipase A 2 in rabbit chondrocytes: a possible signal for ILi action. J Immunol 136:1283 7. Chang J, Musser JH, McGregor H (1987) Phospholipase A2: function and pharmacological regulation. Biochem Pharmacol 36:2429 8. Cirino G, Flower RJ, Browning JL, Sinclair LK, Pepinsky RB (1987) Recombinant human lipoeortin I inhibits thromboxane release from guinea pig isolated perfused lung. Natm-e 328:270

M. Bfichler and H. G. Beger (eds) Phospholipase A 9. Clark MA, Chen MJ, Crooke ST, Bomalaski JS (1988) Tumour necrosis factor (cachectin) induces phospholipase A 2 activity and synthesis of a phospholipase A2-activating protein in endothelial cells. Biochem J 250:125 10. Davidson FF, Dennis EA, Powell M, Glenney JR (1987) Inhibition of phospholipase A2 by "lipocortins" and calpactins. An effect of binding to substrate phospholipids. J Biol Chem 262:1698 11. Dennis EA (1987) Phospholipase A 2 mechanism: inhibition and role in arachidonic acid release. Drug Devel Res 10:205 12. Dennis EA (1987) Regulation of eicosanoid production: role of phospholipases and inhibitors. No/Technoi 5:1294 13. Garcia-Gil M, Siraganian RP (1986) Phospholipase A 2 stimulation during cell secretion in rat basophilic leukemia cells. J Immunol 136:259 14. Garcia-Gil M, Siraganian RP (1986) Source of the arachidonic acid released on stimulation of rat basophilic leukemia cells. J Immunol 136:3825 15. Godfrey RW, Johnson WJ, Hoffstein ST (1987) Recombinant tumor necrosis factor and interleukin-1 both stimulate human synovial cell arachidonic acid release and phospholipid metabolism. Biochem Biophys Res Commun 142:235 16. Haigler HT, Schlaepfer DD, Burgess WH (1987) Characterization of lipocortin I and an immunologically unrelated 33-kDa protein as epidermal growth factor/kinase substrates and phospholipase A 2 inhibitors. J Biot Chem 262:6921 17. Hoffmann GE, Schmidt D, Bastian B, Guder WG (1986) Photometric determination of phospholipase A. J Clin Chem Clin Biochem 24:871 18. Hostetler KY, Matsuzawa Y (1981) Studies on the mechanism of drug-induced lipidosis. Cationic amphiphilic drug inhibition of lysosomal phospholipases A and C. Biochem Pharmacol 30:1121 19. Irvine RF (t982) How is the level of free arachidonic acid controlled in mammalian cells? Biochem J 204:3 20. Jain MK, Streb M, Rogers J, DeHaas GH (1984) Action of phospholipase A 2 on bilayers containing lysophosphatidylcholine analogs and the effect of inhibitors. Biochem Pharmacol 33:2541 21. Van Kuljk FJGM, Sevanian A, Handelman GJ, Dratz EA (1987) A new role for phospholipase Az: protection of membranes from lipid peroxidation damage. Trends Biochem Sci 12:31 22. Lenting HBM, Neys FW, van den Bosch H (1988) Regulatory aspects of mitochondrial phospholipase A 2 from rat liver: effects of proteins, phospholipids and calcium ions. Biochem Biophys Acta 96•: 129 23. Lundgren JD, Hirata F, Marom Z, Logun C, Steel L, Kaliner M, Shelhamer J (1988) Dexamethasone inhibits respiratory glycoconjugate secretion from feline airways in vitro by the induction of lipocortin (lipomodulin) synthesis. Am Rev Respir Dis 137:353 24. Pepinsky BR, Sinclair LK (1986) Epidermal growth factor dependent phosphorylation of lipocortin. Nature 321:81 25. Schlaepfer DD, Mehlman T, Burgess WH, Haigler HT (1987) Structural and functional characterization of endonexin II, a calcium- and phospholipid-bindingprotein. Proc Natl Acad Sci USA 84:6078 26. Sfidhof TC, Slaughter CA, Leznieki I, Barjou P, Reynolds GA (1988) Human 67-kDa calelectrin contains a duplication of four repeats found in 35-kDa lipocortins. Proc Natl Acad Sci USA 85:664 27. Urata C, Siraganian RP (1985) Pharmacologic modulation of the IgE or Ca 2+ ionophore A23187 mediated Ca 2+ influx, phospholipase activation, and histamine release in rat

M. Bfichter and H. G. Beger (eds) Phospholipase A basophilic leukemia cells. Int Arch Allergy Appl Immunol 78:92 28. Vadas P, Pruzanski (t 986) Biology of disease. Role of secretory phospholipases A 2 in the pathobiology of disease. Lab Invest 55:391 29. Vadas P, Stefanski E, Pruzanski W (1986) Potential therapeutic efficacy of inhibitors of human phospholipase A 2 in septic shock. Agents Action 19:194 30. Wallach DP, Brown VJR (1981) Studies on the arachidonic acid cascade. I. Inhibition of phospholipase A 2 in vitro and in vivo by several novel series of inhibitor compounds. Biochem Pharmacol 30:1315

159 31. Yamada K, Okano Y, Miura K, Nozawa Y (1987) A major role for phospholipase A 2 in antigen-induced arachidonic acid release in rat mast cells. Biochem J 247:95 32. Yuan W, Berman RJ, Gelb MH (1987) Synthesis and evaluation of phospholipid analogues as inhibitors of cobra venom phospholipase A z. J Am Chem Soc 109:8071 Dr. Werner Scheuer Boehringer Mannheim GmbH Forschung Biochemie Nonnenwald 2 D-8122 Penzberg