'Antiflammins': two nonapeptide fragments of uteroglobin and lipocortin I have no phospholipase A2-inhibitory and anti-inflammatory activity

The 'antiflammin' nonapeptides P1 and P2 [(1988) Nature 335, 726-730] were synthesized and tested for inhibition of phospholipase A2 and release of prostaglandin E2 and leukotriene C4 in stimulated cells in vitro, and in vivo for anti-inflammatory activity in rats with carrageenan-induced paw oedema. Porcine pancreatic phospholipase A2 was not inhibited at concentrations of 0.5-50 microM. Prostaglandin E2 and leukotriene C4 release by mouse macrophages stimulated with zymosan or ATP was not affected up to a concentration of 10 microM, nor was prostaglandin release by interleukin 1 beta-stimulated mesangial cells and angiotensin II-stimulated smooth muscle cells. Both peptides exhibited no anti-inflammatory activity in carrageenan-induced rat paw oedema after topical (250 micrograms/paw) or systemic administration (1 or 4 mg/kg s.c.). These results do not support the claim of potent phospholipase A2-inhibitory and anti-inflammatory activity of the 'antiflammins' P1 and P2.     

Purification and characterization of a soluble phospholipase A2 from guinea pig lung

Guinea pig lung cytosolic phospholipase A2 was purified to near homogeneity by chromatography on a phosphocellulose column, followed by Q-Sepharose, S-Sepharose, gel filtration chromatography and reverse-phase HPLC. The purified enzyme exhibited an apparent molecular weight of 16,700 by SDS-polyacrylamide gel electrophoresis. Active enzyme eluted from the gel at an apparent molecular weight of 16,700. The purified enzyme exhibited a pH optimum of 9.0 and was calcium-dependent. Guinea pig lung phospholipase A2 hydrolyzed phosphatidylcholine and phosphatidylethanolamine equally well. Substrates containing unsaturated fatty acids in the sn-2 position were hydrolyzed preferentially to those containing saturated fatty acids. Anionic detergents stimulated enzyme activity while nonionic detergents inhibited the enzyme. Disulfide reducing agents dithiothreitol, glutathione and 2-mercaptoethanol modestly stimulated enzyme activity. The sulfhydryl aklylating agent n-ethylmaleimide had no effect on enzyme activity and only high concentrations of p-hydroxymercuribenzoic acid inhibited enzyme activity. The histidine modifying agent, bromophenacyl bromide did not inhibit guinea pig lung phospholipase A2 under conditions in which Crotalus adamanteus phospholipase A2 was inhibited 80%. Manoalide inhibited guinea pig lung phospholipase A2 in a concentration-dependent manner (IC50 = 2 microM). Antibodies prepared against porcine pancreatic phospholipase A2 specifically immunoprecipitated guinea pig lung phospholipase A2 suggesting that the major phospholipase A2 in guinea pig lung cytosol is immunologically related to pancreatic phospholipase A2 in agreement with the biochemical properties of the enzyme.     

Immunochemical relatedness between secretory phospholipase A2 and intracellular phospholipase A2

The immunochemical relationship between rat pancreatic phospholipase A2 and rat splenic phospholipase A2 was examined with the use of anti-rat pancreatic phospholipase A2 antibody as a probe. The immunoelectrophoretic patterns showed that the antibody cross-reacted with the splenic enzyme. The immuno-crossreactivity was also shown by counter immunoelectrophoresis. The splenic phospholipase A2, whether it was purified from the cytosolic fraction or the microsomal fraction, formed an immunoprecipitin band with the anti-pancreatic phospholipase A2 antibody. The antibody was shown to inhibit the activity of the pancreatic phospholipase A2 as well as that of the splenic phospholipase A2.     

Purification and properties of phospholipase A2 from human seminal plasma

The soluble Ca2+-dependent phospholipase A2 (EC 3.1.1.4) was purified 6500-fold with a yield of about 20% from human seminal plasma. The successive purification steps comprised gel filtration, affinity chromatographies and micropartition. The final preparation consisted of two proteins in about equal quantities with molecular weights of 12000 and 14000, according to SDS-polyacrylamide slab gel electrophoresis. As yet these two proteins can not be separated without complete loss of activity. Apparent kinetic parameters have been determined for the purified preparation with different substrates (Vmax = 494 U/mg, and Km = 1.25 X 10(-4) M long-chain phosphatidylethanolamine; Vmax = 7.4 U/mg, and Km = 2.5 X 10(-5) M long-chain phosphatidylcholine; Vmax = 7196 U/mg and Km = 8.32 X 10(-4) M dioctanoylphosphatidylcholine). The enzymatic activity was not affected by diisopropylfluorophosphate and thiol reagents but it was inhibited by higher concentrations of nonionic and ionic (except taurocholate) detergents and by the alkylating reagent p-bromophenacyl bromide. Although the seminal enzyme functionally strongly resembles the pancreatic phospholipase A2, no immunochemical relationship was observed; anti-pancreatic phospholipase A2 IgGs did not inhibit seminal phospholipase A2. Similarly, partially purified phospholipase A2 from horse seminal fluid was not affected by antibodies raised against horse pancreatic phospholipase A2.     

Inhibition of pancreatic phospholipase A2 activity by uteroglobin and antiflammin peptides: possible mechanism of action

We investigated the possible mechanism of inhibition of porcine pancreatic phospholipase A2 in vitro by rabbit uteroglobin and by the antiflammin peptides. We optimized the conditions of phospholipase A2 assay using a deoxycholate-phosphatidylcholine mixed micellar substrate and established the activity of these inhibitors under optimized conditions. The results of fluorescence studies and crosslinking experiments indicate that the inhibitors interact with the enzyme in solution and affect the increase in intrinsic fluorescence of phospholipase A2 observed upon interaction with a mixed micellar substrate. In addition, we identified a sequence similarity between the antiflammin peptides, the putative active region of uteroglobin and a region in pancreatic phospholipase A2. This region of phospholipase A2 has been previously identified as being involved in the regulation of dimerization of this enzyme, and is conserved in the pancreatic-type enzymes. Taken together, these observations suggest that uteroglobin and antiflammins interact with porcine pancreatic phospholipase A2 and this may, at least in part, explain the enzyme inhibitory effect of these molecules observed in vitro. One possible mechanism of this effect may be an interference with the dimerization process of phospholipase A2 which is associated with interfacial activation.     

The efficacy of anti-inflammatory agents with respect to extracellular phospholipase A2 activity

The effects of steroidal and non-steroidal anti-inflammatory agents on extracellular phospholipase A2 (PLA2) activity were investigated. Enzyme release was inhibited by 5 x 10(-8) M dexamethasone but not by indomethacin, whereas the soluble extracellular enzyme was inactivated by mepacrine but not by dexamethasone or indomethacin. PLA2, released into the interstitium by activated macrophages is both pro-inflammatory and vasoactive. The anti-inflammatory efficacy of steroidal and non-steroidal drugs may partially reside in their ability to inhibit the release of PLA2, or inactivate preformed extracellular PLA2 in chronically inflamed sites.     

Changes of phospholipase A2 inhibitory activity in the K+-sensitive actin gelation factor during the differentiation of myeloid leukemia cells

The phospholipase A2 inhibitory activity of a 38 kDa K+-sensitive actin gelation factor in a murine leukemia cell line (M1) was examined. A specific antibody against 38 kDa protein was found to cross-react with 37 kDa protein (lipocortin) in rat peritoneal exudates. Although the native 38 kDa protein from M1 cells did not block phospholipase A2 activity, pretreatment with alkaline phosphatase produced a form that did inhibit this enzyme. However, a purified 38 kDa protein from differentiated M1 cells blocked phospholipase A2 activity without pretreatment with alkaline phosphatase. Phospholipase A2 inhibitory activity of the 38 kDa protein was not altered by addition of actin. These findings suggest that the phospholipase A2 inhibitory of our 38 kDa protein was induced during differentiation. We also proposed that our 38 kDa protein has the same epitope as lipocortin.     

Novel peptides derived from a region of local homology between uteroglobin and lipocortin-1 inhibit platelet aggregation and secretion

The active site for uteroglobin inhibition of phospholipase A2 has been localized to a nonapeptide (P1) which is partially homologous to a nonapeptide (P2) in lipocortin, which also inhibits phospholipase A2. P1 and P2 share an identical tetrapeptide (P4) which is required for inhibition, although P4 alone does not inhibit this enzyme. We found the mechanism of inhibition of platelet aggregation and secretion by the nonapeptides and P4 varied depending on whether platelets were thrombin- or ADP-activated. All three peptides decrease thrombin esterolytic activity and thereby inhibit thrombin-induced platelet activation. P1 decreases ADP-induced aggregation and serotonin secretion by inhibiting phospholipase A2 whereas P4 decreases only aggregation by blocking fibrinogen binding to activated platelets. The P4 sequence in P1 may affect the interaction of P1 with platelets since the presence of P4 potentiates P1 inhibition of platelet activation.     

Effect of sulfatide and gangliosides on phospholipase C and phospholipase A2 activity. A monolayer study

The effect of sulfatide and gangliosides GM1, GD1a and GT1b on the activity of phospholipase C from Clostridium perfringens on dilauroylphosphatidylcholine and of porcine pancreatic phospholipase A2 on dilauroylphosphatidic acid was studied in lipid monolayers containing different proportions of glycolipids under zero-order kinetics at various constant surface pressures. The presence of sulfatide in the monolayer increases the activity of phospholipase C at high surface pressures. Gangliosides shift the cut-off pressure to lower values and inhibit the action of phospholipase C. In mixed monolayers with dilauroylphosphatidic acid, sulfatide at a molar fraction of 0.5 increases the activity of phospholipase A2 at surface pressures below 18 mN/m and shows an inhibitory effect at higher pressures. Ganglioside GM1 at a molar fraction of 0.25 completely inhibits the enzyme above 20 mN/m and markedly reduces its activity at lower pressures. Gangliosides GD1a and GT1b abolish the enzyme activity at all pressures at molar fractions of 0.25 and 0.15, respectively. The modified velocity of the enzymatic reaction in the presence of glycosphingolipids is not due to an irreversible alteration of the catalytic activity.     

Interfacial catalysis by phospholipase A2: dissociation constants for calcium, substrate, products, and competitive inhibitors.

Interpretation of the kinetics of interfacial catalysis in the scooting mode as developed in the first paper of this series [Berg et al. (1991) Biochemistry 30 (first paper of six in this issue)], was based on the binding equilibrium for a ligand to the catalytic site of phospholipase A2. In this paper, we describe direct methods to determine the value of the Michaelis-Menten constant (KMS) for the substrate, as well as the equilibrium dissociation constants for ligands (KL) such as inhibitors (KI), products (KP), calcium (KCa), and substrate analogues (KS) bound to the catalytic site of phospholipase A2 at the interface. The KL values were obtained by monitoring the susceptibility to alkylation of His-48 at the catalytic site of pig pancreatic PLA2 bound to micellar dispersions of the neutral diluent 2-hexadecyl-sn-glycero-3-phosphocholine. The binding of the enzyme to dispersions of this amphiphile alone had little effect on the inactivation rate. The half-time for inactivation of the enzyme bound to micelles of the neutral diluent depended not only on the nature of the alkylating agent but also on the structure and the mole fraction of other ligands at the interface. The KL values for ligands obtained from the protection studies were in excellent accord with those obtained by monitoring the activation or inhibition of hydrolysis of vesicles of 1,2-dimyristoyl-sn-glycerophosphomethanol. Since only calcium, competitive inhibitors, and substrate analogues protected phospholipase A2 from alkylation, this protocol offered an unequivocal method to discern active-site-directed inhibitors from nonspecific inhibitors of PLA2, such as local anesthetics, phenothiazines, mepacrine, peptides related to lipocortin, 7,7-dimethyleicosadienoic acid, quinacrine, and aristolochic acid, all of which did not have any effect on the kinetics of alkylation nor did they inhibit the catalysis in the scooting mode.     

Melanocyte stimulating hormone peptides inhibit TNF-alpha signaling in human dermal fibroblast cells

Alpha-melanocyte stimulating hormone (alpha-MSH) has been identified as a potent anti-inflammatory in various tissues including the skin. It has previously been shown in skin cell keratinocytes and melanocytes/melanoma cells that MSH peptides inhibit TNF-alpha stimulated NF-kappaB activity and intercellular adhesion molecule-1 (ICAM-1) upregulation. However, the precise anti-inflammatory role of MSH peptides in dermal fibroblasts is unclear. Some studies report on pro-inflammatory responses, while others on anti-inflammatory responses. The present study confirms MC1R expression in cultured human dermal fibroblasts and reports that the MSH peptides alpha-MSH and KP(-D-)V inhibit TNF-alpha stimulated NF-kappaB activity and ICAM-1 upregulation, consistent with an anti-inflammatory role. However, involvement of IkappaB-alpha regulation by either peptide was not confirmed, supporting a mechanism independent of the NF-kappaB inhibitor. In conclusion, alpha-MSH and KP(-D-)V peptides have an anti-inflammatory action on dermal fibroblast signaling by inhibiting the pro-inflammatory activity of TNF-alpha in vitro.     

Antigenic relatedness between human lecithin-cholesterol acyltransferase and phospholipases of the A2 family

A monoclonal antibody, B10, generated against pure human lecithin-cholesterol acyltransferase (EC 2.3.1.43) caused the inhibition of the esterolytic and cholesterol esterifying activities of the enzyme. This antibody also reacted with a number of pancreatic and snake venom phospholipases A2 species but not phospholipase A1. A concentration-dependent inhibition of phospholipase A2 was also seen in the presence of B10. Treatment of lecithin-cholesterol acyltransferase or B10-reacting phospholipases with phenacyl bromide, a reagent known to interact with the active site of phospholipase A2, inhibited both their esterolytic activity and their capacity to bind to B10. A dimeric phospholipase A2 species with a known occluded active site did not cross-react with B10. Thus, lecithin-cholesterol acyltransferase and some enzymes of the phospholipase A2 family share a common antigenic determinant which is probably located near or at their esterolytic active site.     

Lipocortin I and lipocortin II inhibit phosphoinositide- and polyphosphoinositide-specific phospholipase C. The effect results from interaction with the substrates

Lipocortins I and II, known to inhibit phospholipase A2, have been purified from bovine lung and tested with respect to their ability to affect the enzymatic activities of phosphoinositide- and polyphosphoinositide-specific phospholipase C from human platelets, rat liver cytosol or rat brain membranes. At 0.67 microM, both lipocortins led to complete inhibition of phospholipase C activity with either phosphatidylinositol or phosphatidylinositol 4,5-bisphosphate as substrate. The inhibition could be overcome by increasing the substrate concentration. Ultracentrifugation studies with lipocortin II showed a direct interaction between phosphatidylinositol and the lipocortin, indicating that the lipocortins inhibit phospholipase C not directly but by interacting with the substrate. In experiments with plasma membranes from [3H]inositol-labeled HL-60 cells, lipocortin II did not affect PI-specific phospholipase C activity in the absence or presence of calcium plus or minus GTP-gamma-S.     

Dissociation of Ca2+ mobilization from breakdown of phosphatidylinositol 4,5-bisphosphate in activated human platelets

The early breakdown of phosphatidylinositol 4,5-bisphosphate in human platelets stimulated by a threshold concentration of either collagen or thrombin was inhibited by 5 mM NaF through its inhibition of phospholipase C activity. However, 5 mM NaF did not inhibit Ca2+ mobilization due to the stimuli from internal stores, but it did inhibit the influx of extracellular Ca2+ through its suppression of thromboxane A2 formation.     

Phospholipase activation during monocyte adherence and spreading.

Phospholipase activation is an important element in cellular signal transduction. In our study we investigated the role and regulation of phospholipase activation during human monocyte adherence and spreading. In human monocytes, phospholipase inhibition (with bromophenacyl bromide (BPB) or manoalide) impaired cell adherence and spreading. In contrast, neither cyclooxygenase/lipoxygenase inhibition nor platelet activating factor receptor blockade affected these responses. The impaired adherence and spreading induced by phospholipase inhibition with BPB could be partially reversed by the addition of nM levels of arachidonate (20:4(n - 6)). Dihomogammalinolenic acid (20:3(n - 6)) could substitute for arachidonate, but other polyunsaturated fatty acids were ineffective in this regard. The phospholipase inhibitor, BPB was selective in its effects on cellular phospholipase activities. BPB inhibited adherence/spreading-related and PMA-stimulated phospholipase activities, but not Ca2+ ionophore-stimulated phospholipase activity. To further probe for the role of Ca2+ in monocyte adherence and spreading, monocytes were loaded with MAPTAM (bis-(2-amino-5-methylphenoxy)-ethane-N,N,N',N', tetraacetic acid tetraacetoxymethyl ester), an EGTA analog. In contrast to phospholipase inhibition, intracellular Ca2+ chelation with MAPTAM did not affect monocyte adherence but did inhibit monocyte spreading. MAPTAM partially inhibited adherence/spreading-stimulated phospholipase activity, but did not inhibit PMA-stimulated phospholipase activity. These data suggest that human monocyte adherence and spreading may sequentially activate Ca(2+)-independent and then Ca(2+)-dependent phospholipases to release arachidonate. The activation of phospholipase and the release of arachidonate appear to be integral parts of the adhesion process.     

Facilitation of phospholipase A2 activity by mastoparans, a new class of mast cell degranulating peptides from wasp venom

The effect of mastoparan, Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-LeuNH2, and related peptides on the release of arachidonic acid from egg yolk lecithin liposomes, rat peritoneal mast cells, and cultured human fibroblasts was studied. In unsonicated liposomes, labeled with 1-stearoyl-2[1-14C]arachidonyl-sn-glycero-3-phosphocholine, 5 X 10(-5) M mastoparan caused a 12-, 15-, and 50-fold increase in the production of arachidonic acid catalyzed by phospholipase A2 from bee venom, eastern diamondback rattlesnake and porcine pancreas, respectively. The stimulant effect of mastoparan and related peptides was dose-dependent and further enhanced by sonication of liposomes. In contrast, melittin, while stimulating the production of arachidonic acid by phospholipase from bee venom, was inactive with the rattlesnake and pancreatic enzymes. Melittin was also only weakly active with liposomes containing stearic acid in place of arachidonic acid. Like melittin, mastoparans stimulated phospholipase activity in tissue homogenates and caused a dose-dependent release of arachidonic acid from rat peritoneal mast cells and cultured human fibroblasts prelabeled with [14C]arachidonic acid. The heptapeptide fragments mastoparan 1-7 and mastoparan 8-14, and succinylated mastoparan were ineffective. The results suggest that mastoparan and related peptides in insect venoms act, at least in part, by stimulating phospholipase activity.     

Kinetic characterization of phospholipase A2 modified by manoalogue

Manoalogue, a synthetic analogue of the sea sponge-derived manoalide, has been previously shown to partially inactivate the phospholipase A2 from cobra venom (Reynolds, L. J., Morgan, B. P., Hite, E. D., Mihelich, E. D., & Dennis, E. A. (1988) J. Am. Chem. Soc. 110, 5172) by reacting with enzyme lysine residues. In the present study, the inactivation of the phospholipases A2 from pig pancreas, bee venom, and cobra (Naja naja naja) venom by manoalogue was studied in detail. Manoalogue-treated enzymes were examined in the scooting mode on vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol. Here the native enzymes bound irreversibly to the vesicles and hydrolyzed all of the phospholipids in the outer monolayer without leaving the surface of the interface. All three manoalogue-treated enzymes showed reduced catalytic turnover for substrate hydrolysis in the scooting mode, and the modified enzymes did not hop from one vesicle to another. Thus, inactivation by manoalogue is not due to the decrease in the fraction of enzyme bound to the substrate interface. This result was also confirmed by fluorescence studies that directly monitored the binding of phospholipase A2 to vesicles. A chemically modified form of the pig pancreatic phospholipase A2 in which all of the lysine epsilon-amino groups have been amidinated was not inactivated by manoalogue, indicating that the modification of lysine residues and not the amino-terminus is required for the inactivation. Several studies indicated that the manoalogue-modified enzymes contain a functional active site. For example, studies that monitored the protection by ligands of the active site from attack by a alkylating agent showed that manoalogue-modified pig phospholipase A2 was capable of binding calcium, a substrate analogue, lipolysis products, and a competitive inhibitor. Furthermore, relative to native enzymes, manoalogue-modified enzymes retained significantly higher catalytic activities when acting on water-soluble substrates than when acting on vesicles in the scooting mode. Intact manoalogue had no affinity for the catalytic site on the enzyme as it did not inhibit the enzyme in the scooting mode and it did not protect the active site from alkylation. Pig pancreatic phospholipase A2 bound to micelles of 2-hexadecyl-sn-glycero-3-phosphocholine was resistant to inactivation by manoalogue, suggesting that the modification of lysine residues on the interfacial recognition surface of the enzyme was required for inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of a soluble phospholipase A2 from synovial fluid in rheumatoid arthritis

A soluble phospholipase A2 (PLA2) was purified 4,500-fold from human rheumatoid synovial fluid. Preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded two bands of PLA2 activity of molecular weights 15,000 and 17,000 and pl 4.2-5.0. Purified PLA2 had absolute 2-acyl specificity, and hydrolyzed phosphatidylcholine with optimal activity at pH 7.5-8.0 and phosphatidylethanolamine with optimal activity at pH 7.0. Human synovial fluid PLA2 did not cross-react with anti-human pancreatic PLA2, as tested by radioimmunoassay.     

Calmodulin-independent inhibition of platelet phospholipase A2 by calmodulin antagonists

We tested the effects of calmodulin, two types of calmodulin antagonists, and various phospholipids on the phospholipase A2 activities of intact platelets, platelet membranes, and partially purified enzyme preparations. Trifluoperazine, chlorpromazine (phenothiazines) and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide (W-7), at concentrations which antagonize the effects of calmodulin, significantly inhibited thrombin- and Ca2+ ionophore-induced production of arachidonic acid metabolites by suspensions of rabbit platelets and Ca2+-induced arachidonic acid release from phospholipids of membrane fractions, but not phospholipase A2 activity in purified enzyme preparations. The addition of acidic phospholipids, but not calmodulin, stimulated phospholipase A2 activity in purified enzyme preparations while decreasing its Km for Ca2+. The dose-response and kinetics of inhibition by calmodulin antagonists of acidic phospholipid-activated phospholipase A2 activity in purified preparations were similar to those of Ca2+-induced arachidonic acid release from membrane fractions. Calmodulin antagonists were also found to inhibit Ca2+ binding to acidic phospholipids in a similar dose-dependent manner. Our results suggest that the platelet phospholipase A2 is the key enzyme involved in arachidonic acid mobilization in platelets and is regulated by acidic phospholipids in a Ca2+-dependent manner and that calmodulin antagonists inhibit phospholipase A2 activity via an action on acidic phospholipids.