Introduction

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Hydrolysis of arachidonic acid at the sn-2 position of the glycerol in membrane phospholipids is the rate-limiting step for eicosanoid production. Mammalian cells contain diverse types of PLA₂ that can play a key role in the release of arachidonic acid, leading to the generation of inflammatory mediators and the activation of signal transduction pathways (for review, see Glaser et al., 1993; Kudo et al., 1993; Dennis, 1994). Most cells contain at least two forms of PLA₂: a 14-kDa secretory enzyme, sPLA₂, and an 85-kDa cytosolic enzyme, cPLA₂, (Glaser et al., 1993; Kramer et al., 1989). The secretory enzymes can be classified mainly into group I and group II (Heinrikson et al., 1977), although sPLA₂ from bee venom is sometimes included in a separate group III (Glaser et al., 1993).

sPLA₂ has been found to induce release of arachidonic acid and eicosanoid production in several cell types (Pfeilschifter et al., 1993; Fonteh et al., 1994; Miyake et al., 1994). On the other hand, cPLA₂ exhibits a more selective preference for arachidonyl-containing phospholipids and plays an important role in arachidonic acid release (Clark et al., 1990; Ramesha and Ives, 1993). In fact, cPLA₂ is the main mediator in the hormonally regulated production of eicosanoids (Lin et al., 1992).

sPLA₂s can induce an inflammatory response in animals (Vadas and Pruzanski, 1986; Vishwanath et al., 1988; Neves et al., 1993), whereas in humans, group II sPLA₂ is present at high levels in synovial fluids, articular cartilage and blood from patients with rheumatic diseases (Pruzanski et al., 1987; Bomalaski and Clark, 1993), which suggests the participation of these types of enzymes in the inflammatory process.

Thus the inhibition of PLA₂ could result in the inhibition of inflammatory responses acting at an early step in the biosynthesis of inflammatory mediators such as prostaglandins, leukotrienes and platelet-activating factor. Marine organisms are an important source of PLA₂ inhibitors; some of them can be of interest either as pharmacological tools to establish the role of the different PLA₂ activities in disease or as anti-inflammatory agents (for review, see Potts et al., 1992). We have shown recently that a number of terpenoids present in sponges inhibit PLA₂ with some differences in potency and selectivity (Gil et al., 1995; Cholbi et al., 1996). The purpose of our studies was to examine the influence of variabilin (fig. 1), a sesterterpene isolated for the first time from the marine sponge Ircinia variabilis (Faulkner, 1973), on sPLA₂ activity of types I, II and III, as well as on cPLA₂ activity from U937 cells. We have also studied its influence on human neutrophil responses in vitro and its effect on inflammatory responses in mice and eicosanoid synthesis in vivo. Recently, a different compound has been named variabilin, a protein that is isolated from the hard tick Dermacentor variabilis and inhibits platelet aggregation (Wang et al., 1996).

View larger version (K): [in this window] [in a new window]   Fig. 1.   Structural formula of variabilin.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Reagents. The variabilin utilized in this work was isolated from Hemimycale columela following known procedures (Faulkner, 1973). Antibody against LTB₄, the 5-lipoxygenase inhibitor ZM230,487 and human synovial recombinant PLA₂ were kindly provided by Zeneca Pharmaceuticals, Macclesfield, Cheshire, UK. [9,10-³H]oleic acid and L-3-phosphatidylcholine 1-palmitoyl-2-arachidonyl [arachidonyl-1-¹⁴C] were purchased from Du Pont (Itisa, Madrid, Spain); ([5,6,8,11,12,14,15(n)-³H]PGE₂, [5,6,8,9,11,12,14,15(n)-³H]LTB₄ and [5,6,8,9,11,12,14,15(n)-³H]LTC₄ were from Amersham Iberica, (Madrid, Spain). PTK was purchased from Universal Biologicals Ltd. (London, UK). The rest of the reagents were from Sigma Chemical Co., St. Louis, MO. E. coli strain CECT 101 was a gift from Prof. Uruburu, Department of Microbiology, University of Valencia, Spain.

Assay of sPLA₂. sPLA₂ was assayed by using a modification of the method of Franson et al. (1974). E. coli strain CECT 101 were seeded in medium containing 1% tryptone, 0.5% NaCl and 0.6% sodium dihydrogen orthophosphate, pH 5.0, and grown for 6 to 8 hr at 37°C in the presence of 5 µCi/ml [³H]oleic acid (sp. act. 10 Ci/mmol). After centrifugation at 2,500 × g for 10 min, the cells were washed in buffer (0.7 M Tris-HCl, 10 mM CaCl₂, 0.1% BSA, pH 8.0), resuspended in saline and autoclaved for 30 to 45 min. At least 95% of the radioactivity was incorporated into phospholipids. Naja naja venom enzyme, bee venom enzyme and human recombinant synovial enzyme were diluted in 10 µl of 100 mM Tris-HCl, 1 mM CaCl₂ buffer, pH 7.5. Supernatants (10 µl) of exudates from zymosan-injected rat air pouch (Payá et al., 1996) were also used as a source of sPLA₂. Enzymes were preincubated at 37°C for 5 min with 2.5 µl of test compound solution or its vehicle in a final volume of 250 µl. Incubation proceeded for 15 min in the presence of 10 µl of autoclaved oleate-labeled membranes and was terminated by addition of 100 of µl ice-cold solution of 0.25% BSA in saline to a final concentration of 0.07% w/v. After centrifugation at 2,500 × g for 10 min at 4°C, the radioactivity in the supernatants was determined by liquid scintillation counting.

Preparation of human leukocytes. The citrated blood of healthy volunteers was centrifuged at 200 × g for 15 min at room temperature. The platelet-rich plasma was removed, and the leukocytes contained in the residual blood were isolated by sedimentation with 2% (w/v) dextran in 0.9% NaCl at room temperature. The supernatant was centrifuged at 1,200 × g for 10 min at 4°C. Contaminating erythrocytes were lysed by hypotonic treatment. The pellet was resuspended in PBS, and Ficoll-hypaque was layered under the cell mixture. The cell gradient mixture was centrifuged at 400 × g for 40 min at 20°C. Neutrophils were separated and resuspended in PBS containing 1.26 mM Ca⁺⁺ and 0.9 mM Mg⁺⁺ (Bustos et al., 1995). Viability was greater than 95% by the trypan blue exclusion test. The monocyte and lymphocyte layer was removed and pelleted by centrifugation. The cell pellet was resuspended in RPMI-1640 media, pH 7.4, with 10% fetal bovine serum, 2 mM L-glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin and was incubated at a cell density of 10⁷/ml in 60/15-mm tissue culture dishes. The cells were allowed to adhere for 2 hr at 37°C in a 5% CO₂ atmosphere incubator. The nonadherent cells were removed by vacuum suction of media followed by two washes with 1 ml of RPMI-1640. The adherent cells resulted in a greater than 90% pure monocyte population as assessed by differential staining.

Cell viability assays. LDH was determined by measuring the rate of oxidation of NADH (Bergmeyer and Bernt, 1974). Tubes containing 0.5% Triton X-100 were used for measurement of the total cellular content of LDH.

Assay of cPLA₂. cPLA₂ was prepared from human monocytic U937 cells (Cell Collection, Department of Animal Cell Culture, C.S.I.C:, Madrid, Spain) grown in the above medium which were disrupted by sonication in 10 mM HEPES buffer, pH 7.4, containing 0.32 M sucrose, 100 µM EDTA, 1 mM dithiothreitol, 2 mM phenylmethylsulphonylfluoride and 100 µM leupeptin. The homogenated cells were centrifuged at 2,000 × g for 10 min at 4°C, and the resulting supernatant was further centrifuged at 100,000 × g for 100 min at 4°C to obtain the cytosolic fraction. cPLA₂ activity was measured as the release of radiolabeled arachidonic acid according to the method of Clark et al. (1990). 1-Palmitoyl-2-[¹⁴C] arachidonyl-sn-glycero-3-phosphocholine (57.0 mCi/mmol, 2 × 10⁶ cpm) was dried under nitrogen and suspended in 1 ml of 100 mM glycine buffer, pH 9.0, containing 200 µM Triton X-100, 10 mM CaCl₂, 0.25 mg/ml BSA and 40% v/v glycerol. The suspension was then sonicated to form mixed micelles of phospholipid and Triton X-100. The reaction was started by adding the enzyme solution (approximately 24 µg of protein of cytosolic fraction from human monocytes) to a final volume of 100 µl of the assay mixture, which contained 1 mM CaCl₂, 2 mM 2-mercaptoethanol, 150 mM NaCl, 40% glycerol, 1 mg/ml BSA and 50 mM HEPES, pH 9.0. The substrate consisted of 5 µl of micelles (10⁴ cpm) containing dioleoyl glycerol at the molar ratio 2:1 (Kramer et al., 1987). Test compounds were dissolved in methanol and added to the reaction mixture just before the addition of the enzyme solution. The final concentration of methanol in the reaction mixture was less than 1%, which showed no effect on the enzyme activity. The reaction was stopped after a 60-min incubation period at 37°C by mixing with 0.5 ml of isopropyl alcohol/heptane/0.5 M H₂SO₄ (10:5:1). Heptane (0.7 ml) and water (0.2 ml) were then added, and the solution was vigorously mixed for 15 sec. The heptane phase was mixed with 100 mg silica gel 60 (Merck, 70-230 mesh) and centrifuged, and the radioactivity in each supernatant was measured (Zhang et al., 1991).

Elastase release by human neutrophils. 2.5 × 10⁶ neutrophils/ml were preincubated with test compound or vehicle for 5 min and then stimulated with cytochalasin B (10 µM) and FMLP (10 nM or 10 µM), or PAF (0.5 µM) for 10 min at 37°C. In other experiments, calcium ionophore A23187 (1 µM) was used as stimulus. After centrifugation at 1,200 × g at 4°C, supernatants were incubated with N-tert-butoxy-carbonyl-L-alanine p-nitrophenyl ester (200 µM) for 20 min at 37°C (Barrett, 1981). The extent of p-nitrophenol release was measured at 414 nm in a microtiter plate reader. Possible direct inhibitory effects on elastase activity were assessed by preincubating variabilin for 5 min with supernatants of cytochalasin B + FMLP-stimulated human neutrophils, followed by addition of substrate and a 20-min incubation at 37°C. Direct effects on myeloperoxidase were also tested using aliquots of supernatants of cytochalasin B + FMLP-stimulated human neutrophils following published procedures (Suzuki et al., 1983; De Young et al., 1989).

Superoxide generation by human neutrophils. A neutrophil suspension (0.5 ml) containing 2.5 × 10⁶ cells/ml was preincubated for 5 min at 37°C with test compounds or vehicle (methanol, 1% final concentration) and TPA (1 µM) was added to induce superoxide generation, which was estimated as the superoxide dismutase-inhibitable reduction of cytochrome c at 550 nm (Payá et al., 1993).

Synthesis and release of LTB₄ by human neutrophils. A suspension of human neutrophils (5 × 10⁶/ml) in PBS was preincubated with test compounds or vehicle and then stimulated with 1 µM A23187 for 10 min at 37°C. After centrifugation at 1,200 × g for 10 min at 4°C, the supernatants were frozen at 80°C until the radioimmunoassay for LTB₄ was performed (Moroney et al., 1988).

Synthesis of LTB₄ by high-speed supernatants from human neutrophils. High-speed (100,000 × g) supernatants from sonicated human neutrophils were obtained as previously described (Tateson et al., 1988). Aliquots (50 µg of protein/tube) in PBS containing 2 mM CaCl₂ were incubated with 5 µM arachidonic acid at 37°C for 5 min, in the presence of test compounds or vehicle. The samples were then heated at 90°C for 5 min and centrifuged at 10,000 × g at 4°C for 30 min. The LTB₄ levels in supernatants were measured by radioimmunoassay (Moroney et al., 1988).

Cyclo-oxygenase-1. J774 cells (Cell Collection, Department of Animal Cell Culture, C.S.I.C:, Madrid, Spain) were sonicated at 4°C in an ultrasonicator at maximum potency. Microsomes were prepared by centrifugation at 2,000 × g for 5 min at 4°C followed by centrifugation of the supernatant at 100,000 × g for 100 min at 4°C. Microsomes (20 µg of protein/tube) were incubated for 30 min at 37°C in 50 mM Tris-HCl, pH 7.4, with 5 µM arachidonic acid and test compound or vehicle in the presence of 2 µM hematin and 1 mM L-tryptophan. The reaction was terminated by boiling the samples for 5 min, and PGE₂ levels were determined by radioimmunoassay (Moroney et al., 1988).

Cyclo-oxygenase-2. Human monocytes or J774 cells were resuspended in RPMI1640 culture medium containing aspirin (300 µM) and incubated at 37°C for 2 hr. The cells were washed twice, resuspended in RPMI1640 with 10% fetal bovine serum and incubated with E. coli lipopolysaccharide (10 µg/ml) at 37°C for 24 hr (Grossman et al., 1995). After centrifugation the cells were sonicated at 4°C in an ultrasonicator at maximum potency, and microsomes were prepared as above. Microsomes (40 µg of protein/tube) were used as a source of cyclo-oxygenase-2, and reactions were carried out in the same conditions as above. PGE₂ synthesis was determined by radioimmunoassay (Moroney et al., 1988).

Release of PGE₂ by rat cecum. Male Wistar rats weighing between 140 and 180 g were fasted overnight but allowed water ad libitum. The animals were sacrificed, and the intestine was removed. Cecum was cleaned with saline and cut with scissors. Fragments of rat cecum weighing 37.8 ± 1.4 mg (mean ± S.E.M., n = 47) were placed in 0.4 ml of 50 mM Tris-HCl buffer, pH 7.5. Drugs were dissolved in the medium at appropriate concentrations. After preincubation for 60 min at 4°C, the fragments were transferred to tubes containing fresh medium with the same concentrations of drugs and incubated for 15 min at 37°C. Aliquots of the solution were used for PGE₂ radioimmunoassay as above.

Mouse ear edema. The protocols were approved by the institutional Animal Care and Use Committee. All studies were performed in accordance with European Union regulations for the handling and use of laboratory animals. TPA (5 µg) or arachidonic acid (2.0 mg) dissolved in 20 µl of acetone was applied in 10-µl volumes to both inner and outer surfaces of the right ear of Swiss mice (20-25 g). Test compounds were applied topically in acetone before TPA administration or 20 min before arachidonic acid. The left ear (control) received only acetone. The animals were killed by cervical dislocation after 4 hr (TPA) or 1 hr (arachidonic acid), and equal sections of both ears were punched out and weighed. The increase in the weight of the right ear punch over that of the left indicated the edema (Carlson et al., 1985). The ear sections were homogenized in 750 µl of saline, and after centrifugation at 10,000 × g for 15 min at 4°C, the PGE₂ and LTC₄ content in supernatants was determined by radioimmunoassay (arachidonic acid edema). Direct inhibitory effects on myeloperoxidase activity were assessed by preincubating variabilin for 5 min with supernatants of homogenized control TPA-treated ears (Suzuki et al., 1983; De Young et al., 1989).

Mouse paw edema. Swelling was induced by a modification of the technique of Sugishita et al. (1981). Female Swiss mice (20-25 g) were fasted for 12 hr with free access to water. Drug or vehicle (ethanol/Tween 80/distilled water, 5:5:90, v/v/v) was administered p.o. (0.5 ml) 1 hr before the injection of carrageenan (0.05 ml; 3% w/v in saline) into the subplantar area of the right hind paws of groups of six animals. The volumes of injected and contralateral paws were measured at 1, 3 and 5 hr after induction of edema by using a plethysmometer (Ugo Basile, Comerio, Italy). The volume of edema was expressed for each animal as the difference between the carrageenan-injected and contralateral paws.

Mouse air pouch. Male Swiss mice (25-30 g) were anesthetized with ethyl ether, and 10 ml of sterile air was injected into the s.c. tissue of the back, and 3 days later, 5 ml of sterile air was injected into the same cavity. Another 3 days later, mice were administered, into the air pouch, 1 ml of 1% w/v zymosan in saline + vehicle (10 µl of ethanol: control group) or 1 ml of 1% w/v zymosan in saline + test drug (dissolved in 10 µl of ethanol) at the concentrations indicated in the results (treated groups). Another group received only 1 ml of saline + vehicle (saline group). Four hours after administration, the animals were killed by cervical dislocation, and the exudate in the pouch was collected with 1 ml of saline (Edwards et al., 1981). Leukocytes present in exudates were measured using a Coulter counter. After centrifugation of exudates at 1,200 × g at 4°C for 10 min, the supernatants were used to measure LTB₄ and PGE₂ levels by radioimmunoassay (Moroney et al., 1988).

Statistical analysis. The results are presented as mean ± S.E.M. IC₅₀ values and their 95% CL were calculated from at least four significant concentrations (n = 6). The approximate ID₅₀ value was estimated from three significant doses (n = 6). The level of statistical significance was determined by analysis of variance followed by Dunnett's t test for multiple comparisons.

	Results

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Inhibition of PLA₂ activities. Variabilin significantly inactivated sPLA₂ and cPLA₂ in a concentration-dependent manner (fig. 2). As shown in table 1, this marine product was somewhat less potent on human synovial PLA₂ than the reference sPLA₂ inhibitor, scalaradial. Inhibition of cPLA₂ gave an IC₅₀ value and 95% CL of 84.2 (47.0-170.0) nM for the selective inhibitor PTK. Variabilin inhibited in vitro human synovial sPLA₂ and U937 cPLA₂ with IC₅₀ values in the µM range and showed less inhibition of nonhuman sPLA₂ activities from bee venom, porcine pancreas or zymosan-injected rat air pouch. This last activity, which does not show selectivity for arachidonyl phospholipids, has recently been reported by us (Payá et al., 1996). In contrast, variabilin was inactive on Naja naja venom sPLA₂. To determine whether the effects on PLA₂ were reversible, we used the dilution method (Lister et al., 1989) and bee venom and human synovial enzymes. No significant difference in the degree of inactivation was observed; variabilin (100 µM) inhibited bee venom sPLA₂ by 81.6 ± 1.0% (mean ± S.E.M, n = 6) and after a 25-fold dilution, the observed inhibition was 73.0 ± 1.1% (mean ± S.E.M, n = 6). On human synovial sPLA₂, variabilin (10 µM) exhibited 92.6 ± 0.6% inhibition (mean ± S.E.M, n = 6), and after a 25-fold dilution, the value was 89.5 ± 0.2% inhibition (mean ± S.E.M, n = 6). An analysis of drug influence on enzyme activity vs. enzyme concentration was also performed. Figures 3 and 4 illustrate the concentration-response relationship for bee venom and human synovial sPLA₂, respectively, as a function of enzyme concentration. The results were similar with both enzymes. The regression line for variabilin-treated samples was shifted to the right of control values at a given velocity, and there was no significant difference between the slopes of these lines, which suggests an irreversible inhibition (Segel, 1975). The loss of enzyme activity was progressive with time and linear on a semilogarithmic plot (fig. 5). The rate of inactivation increased as the concentration of variabilin increased from 5 to 50 µM, with appreciable inhibitory activity at t = 0 (without preincubation of variabilin with the enzyme), a result that suggests an initial quick binding to the enzyme. Time-dependent bee venom PLA₂ inactivation has been reported for irreversible inhibitors such as manoalide and scalaradial (Glaser and Jacobs, 1986; de Carvalho and Jacobs, 1991).

View larger version (K): [in this window] [in a new window]   Fig. 2.   Dose-response curves showing the effect of variabilin on several PLA2 activities. Data are mean ± S.E.M. (n = 6). () Bee venom sPLA2. () RAP + zymosan sPLA2. () Human recombinant synovial sPLA2. () Porcine pancreatic sPLA2. () U937 cPLA2. The inhibitory effect of variabilin was measured in relation to control enzyme activity in tubes containing enzyme and the inhibitor vehicle (methanol).

View larger version (K): [in this window] [in a new window]   Fig. 3.   Activity of bee venom sPLA2 as a function of enzyme concentration in the absence and presence of variabilin. () Control. () 10 µM variabilin. Data are mean ± S.E.M. (n = 6). Different enzyme concentrations were preincubated with vehicle (control) or variabilin (10 µM) for 5 min at 37°C, and after addition of substrate, incubation proceeded for 15 min.

View larger version (K): [in this window] [in a new window]   Fig. 4.   Activity of human synovial sPLA2 as a function of enzyme concentration in the absence and presence of variabilin. () Control. () 1 µM variabilin. Data are mean ± S.E.M. (n = 6). Different enzyme concentrations were preincubated with vehicle (control) or variabilin (1 µM) for 5 min at 37°C, and after addition of substrate, incubation proceeded for 15 min.

View larger version (K): [in this window] [in a new window]   Fig. 5.   Time dependence of PLA2 inactivation for three concentrations of variabilin. Data represent mean ± S.E.M. (n = 3). () 5 µM variabilin. () 10 µM variabilin. () 50 µM variabilin. Variabilin was preincubated with µU bee venom PLA2 for different times, followed by a 15-min incubation with substrate.

Effect on degranulation, superoxide and LTB₄ generation by human neutrophils. No cytotoxic effects of variabilin were observed at the concentrations used in our study, because only at the high concentration of 100 µM did we observe a release of 24.4 ± 4.1% (mean ± S.E.M, n = 9, P < .01) LDH. Variabilin exerted direct inhibitory effects on human neutrophil enzymes, showing an IC₅₀ value of 1.2 (0.6-2.0) µM for myeloperoxidase and a lower effect on elastase with an inhibition of 36.5 ± 3.8% (mean ± S.E.M., n = 6, P < .05) at the highest concentration tested in human neutrophils (50 µM). Thus we chose elastase instead of myeloperoxidase for degranulation assays. The degranulation of neutrophils activated with FMLP or PAF in cytochalasin-pretreated neutrophils was blocked by variabilin in a concentration-dependent manner, as shown in figure 6. Variabilin was more potent against neutrophils treated with a submaximal concentration of FMLP, with IC₅₀ values of 0.4 (0.1-0.9) µM and 9.6 (4.0-26.0) µM for 10 nM FMLP and 10 µM FMLP, respectively. Inhibition of degranulation induced by PAF or A23187 was also observed for variabilin, which exhibited IC₅₀ values of 1.5 (1.2-1.9) µM and 7.4 (5.6-9.5) µM, respectively. Scalaradial was also an inhibitor of degranulation with IC₅₀ values of 2.2 (0.8-5.3) µM and 1.3 (0.8-17.9) µM for the response induced by PAF and by 10 µM FMLP, respectively. Generation of superoxide anion by human neutrophils was inhibited by variabilin with a lower potency (IC₅₀ = 33.1; 24.4-43.6 µM), whereas scalaradial showed an IC₅₀ value of 3.1 (2.8-3.9) µM, a value similar to that observed for inhibition of degranulation (fig. 7). Variabilin caused a concentration-dependent suppression of neutrophil LTB₄ production induced by the calcium ionophore A23187 (fig. 8), with an IC₅₀ value of 1.4 (1.2-1.6) µM. Total inhibition of LTB₄ production was observed with 5 µM. Scalaradial potently inhibited this response with an IC₅₀ value of 0.1 (0.1-0.3) µM. Because the rise in intracellular calcium concentration induced by A23187 activates PLA₂ and 5-lipoxygenase, leading to the release of arachidonic acid and the synthesis of LTB₄ in neutrophils, inhibition of one or both enzyme activities can result in a reduction in LTB₄ production. In order to distinguish between these possibilities, variabilin was incubated with a high-speed supernatant of neutrophils in the presence of substrate, arachidonic acid. In this case, variabilin at concentrations up to 10 µM failed to inhibit LTB₄ synthesis (table 2). Thus it appears that the effects of variabilin on LTB₄ production are not due to the inhibition of 5-lipoxygenase.

View larger version (K): [in this window] [in a new window]   Fig. 6.   Concentration dependence of the inhibitory effect of variabilin on elastase release by human neutrophils. Data represent mean ± S.E.M. (n = 6-10). () Cytochalasin B + 0.5 µM PAF. () Cytochalasin B + 10 nM FMLP. () Cytochalasin B + 10 µM FMLP.

View larger version (K): [in this window] [in a new window]   Fig. 7.   Concentration dependence of the inhibitory effect of variabilin and scalaradial on superoxide generation by human neutrophils stimulated by TPA. Data are mean ± S.E.M. (n = 6). () Variabilin. () Scalaradial.

View larger version (K): [in this window] [in a new window]   Fig. 8.   Concentration dependence of the inhibitory effect of variabilin and scalaradial on LTB4 biosynthesis by human neutrophils stimulated by A23187. Data are mean ± S.E.M. (n = 6). () Variabilin. () Scalaradial.

Effect of variabilin on cyclo-oxygenase 1, cyclo-oxygenase 2 and PGE₂ release by rat cecum. Variabilin at concentrations up to 10 µM did not affect the generation of PGE₂ by cyclo-oxygenase 1 or cyclo-oxygenase 2 present in microsomal fractions from human monocytes or J774 cells (data not shown). To determine the influence of variabilin on prostaglandin synthesis in the digestive tract, we performed experiments using rat cecum with indomethacin as reference. Tissue fragments released 470.0 ± 40.0 pg PGE₂/mg (mean ± S.E.M, n = 12) in control tubes. Incubation with 10 µM indomethacin significantly inhibited this release (75%, n = 6, P < .01), whereas variabilin at the same concentration showed no effect (4% inhibition, n = 6, P > .05).

Effect of variabilin on mouse ear edema. Both topical application of variabilin and that of indomethacin profoundly affected ear edema induced by TPA in mice, compared with control animals, inhibiting this inflammatory response at 200 µg/ear by 65% and 53%, respectively (fig. 9), with ID₅₀ values of approximately 133.6 µg/ear (variabilin) and 183.5 µg/ear (indomethacin). A high level of myeloperoxidase was noted in TPA-treated ears 4 hr after induction of inflammation. Variabilin incubated in vitro with supernatants of ear homogenates from the control group inhibited myeloperoxidase activity, showing an IC₅₀ value of 32.9 (26.0-44.3) µM. As expected in a PLA₂ inhibitor, variabilin at 250 or 500 µg/ear failed to modify arachidonic acid-induced ear edema or eicosanoid levels in ear homogenates, whereas indomethacin caused 30% inhibition of edema at the dose of 500 µg/ear, accompanied by a marked reduction of PGE₂ levels in ear homogenates (table 3).

View larger version (K): [in this window] [in a new window]   Fig. 9.   Effect of variabilin and indomethacin on TPA-induced edema. Drugs were topically administered at the time of TPA application. Data are mean ± S.E.M. from six animals. ** P < .01. Control (solid bars); variabilin (hatched bars); indomethacin (open bars).

Effect of variabilin on mouse paw edema. Variabilin at p.o. doses of 30 or 45 mg/kg demonstrated the ability to inhibit swelling early (1 hr after carrageenan) and exhibited continuously significant suppression of hind paw swelling between 1 and 5 hr after carrageenan administration, achieving a maximal response at the determination at 3 hr (fig. 10). Indomethacin exerted a higher effect and decreased edema values to 36.0 ± 12.0 µl (1 hr), 36.0 ± 5.0 µl (3 hr) and 56.0 ± 2.5 µl (5 hr) at the dose of 10 mg/kg p.o.

View larger version (K): [in this window] [in a new window]   Fig. 10.   Effect of variabilin on mouse paw edema induced by carrageenan. Data represent mean ± S.E.M. (n = 6). * P < .05, ** P < .01. Compounds were administered p.o. 1 hr before the injection of carrageenan.

Effect of variabilin on mouse air pouch. Inhibition of leukocyte migration into the zymosan-injected mouse pouch seems to be related to the inhibition of 5-lipoxygenase metabolites, because the selective inhibitor ZM230,487 was the only compound able to inhibit this parameter (table 4). Variabilin was less effective than indomethacin or ZM230,487 in decreasing the levels of PGE₂ or LTB₄, respectively, in mouse air pouch exudates. However, it is interesting to note that this marine product decreased the levels of both eicosanoids with the same potency (ID₅₀ values of about 0.028 and 0.029 µmol/pouch for PGE₂ and LTB₄, respectively). This activity profile suggests the inhibition of a previous step common to cyclo-oxygenase and 5-lipoxygenase pathways.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

The results of the studies presented here demonstrate the inhibitory activity of variabilin on PLA₂ from different sources. This marine compound did not display in vitro preference toward a type of PLA₂, although is more active on human enzymes, with IC₅₀ values in the µM range for sPLA₂ and cPLA₂.

PLA₂ enzymes may be involved in cell proliferation and signal transduction as well as in the pathogenesis of disease processes such as inflammation (Mukherjee et al., 1994). sPLA₂ could induce the release of arachidonic acid and PGE₂ production in neutrophils, HL-60 granulocytes treated with calcium ionophore (Hara et al., 1991), rat mesangial cells (Pfeilschifter et al., 1993), mast cells (Fonteh et al., 1994) and mouse peritoneal macrophages (Miyake et al., 1994). Nevertheless, PLA₂ secreted by guinea pig peritoneal macrophages does not participate in the synthesis of PGE₂ accumulating in the media (Marshall et al., 1994a). sPLA₂ could also play a role in cellular defense against infection, because this enzyme activity is bactericidal against E. coli (Weiss et al., 1994), L. monocytogenes (Weiss et al., 1994; Harwig et al., 1995) and S. aureus (Weinrauch et al., 1996). It has also been suggested that the roles sPLA₂ plays in inflammation may include production of cell damage by hydrolysis of membrane phospholipids in activated cells (Wright et al., 1990; Kudo et al., 1993; Weiss et al., 1994) and participation in a proliferative response and regulation of cytokine synthesis (Bomalaski and Clark, 1993).

On the other hand, cPLA₂ but not sPLA₂ is involved in arachidonic acid release in thrombin-stimulated human platelets (Bartoli et al., 1994) and calcium ionophore-challenged platelets (Riendeau et al., 1994) and is widely accepted as the main mediator of agonist-regulated production of eicosanoids (Lin et al., 1992). There is evidence that arachidonic acid release in response to zymosan or TPA is dependent on cytosolic PLA₂ stimulation through protein kinase C and MAP kinase activation in mouse peritoneal macrophages (Qiu and Leslie, 1994).

Recent investigations suggest that both type II PLA₂ and cPLA₂ are responsible for eicosanoid synthesis. Thus arachidonic acid mobilization seems to be dependent on both types of PLA₂ in P388D1 macrophages (Balsinde et al., 1994) and human umbilical vein endothelial cells (Murakami et al., 1993). In addition, exocytosis of sPLA₂ could modulate the activity of the cPLA₂ by initiating the formation of LTB₄, which after release would stimulate its own receptor, resulting in activation of the cPLA₂ in neutrophils (Wijkander et al., 1995). Assuming that both enzymes are involved in the production of inflammatory mediators, it is reasonable to expect that a compound with dual inhibitory activity could control inflammatory responses efficiently.

Increases in type II PLA₂ have been shown in various inflammatory processes, including rheumatoid arthritis, although whether this is the cause or the consequence of the disease has not been established (Glaser et al., 1993). In fact, administration of sPLA₂ of different types can induce or amplify inflammatory responses in animals (Vadas and Pruzanski, 1986; Vishwanath et al., 1988; Neves et al., 1993; Cirino et al., 1994). In contrast, there are no data on potent cPLA₂ inhibitors and their pharmacological effects in vivo, so variabilin appears to be a novel dual sPLA₂ and cPLA₂ inhibitor that shows anti-inflammatory activity after either topical or p.o. administration to laboratory animals. We have not determined the mechanism of PLA₂ inactivation by variabilin. Nevertheless, this marine compound shares some structural features with manoalide, and thus it may interact with sPLA₂ at the -hydroxybutenolide ring and hydrophobic region of the compound (Glaser et al., 1989).

We have also demonstrated that variabilin inhibits cellular functions in human neutrophils in vitro. During inflammation, neutrophils stimulated by various agents release reactive oxygen species and granular enzymes that mediate tissue injury (Smith, 1994). Neutrophil proteases and specifically elastase mediate damage to endothelium (Westlin and Gimbrone, 1993). In addition, myeloperoxidase is necessary to form the strong oxidant HOCl, which by reaction with superoxide can in turn generate the reactive hydroxyl radical (Ramos et al., 1992). Therefore, inhibition of cell-mediated responses could be considered an additional mechanism for attenuating inflammation. In this regard, variabilin potently inhibited in vitro some functions that contribute to tissue damage by the cellular component of inflammatory processes, such as degranulation, and was less effective in suppressing the TPA-stimulated oxidative burst.

Variabilin has inhibited neutrophil responses triggered by structurally divergent agonists that induce neutrophil responsiveness through different pathways, which suggests that this marine product, apart from a possible influence on ligand-receptor interactions, may inhibit intracellular signal transduction pathways. In this respect, inhibition of sPLA₂ activity has been related to neutrophil exocytosis, because released arachidonic acid or lysophospholipids could act as fusogens (Barnette et al., 1994), as well as to superoxide generation by human eosinophils (White et al., 1993). The inhibition of myeloperoxidase activity could also participate in the control of neutrophil-mediated tissue injury by this marine compound.

LTB₄ biosynthesis by human neutrophils in vitro and PGE₂ and LTB₄ generation in vivo in the mouse air pouch were inhibited by variabilin. Because the measured endpoint of our assays was the generation of eicosanoids, compounds able to inhibit cyclo-oxygenase or 5-lipoxygenase enzymes could appear as PLA₂ inhibitors. In our experiments, variabilin had no effect on arachidonic acid metabolism directly in cell-free assays for 5-lipoxygenase, cyclo-oxygenase 1 or cyclo-oxygenase 2 activities. In addition, the data presented here demonstrate that variabilin does not inhibit in vitro prostaglandin generation in the digestive tract. In contrast, known inhibitors of sPLA₂ such as scalaradial (Marshall et al., 1994b) are in vitro inhibitors of 5-lipoxygenase, which explains the high potency of this compound on LTB₄ generation by human neutrophils.

Furthermore, variabilin failed to inhibit the in vivo generation of eicosanoids in the presence of an excess of substrate (arachidonic acid-induced edema) but decreased with the same potency LTB₄ and PGE₂ levels generated from endogenous substrate in the mouse air pouch. This result indicates that variabilin affected arachidonate availability rather than metabolism. It is interesting to note that group II sPLA₂ has been related to inflammatory responses to TPA or carrageenan (Miyake et al., 1993; Tramposch et al., 1994), which have been inhibited by variabilin. Thus our results support an inhibitory action of variabilin on PLA₂ in human intact cells as well as in experimental models in mice.

Our data indicate that variabilin is a dual inhibitor of human sPLA₂ and cPLA₂ that is able to control the production of arachidonic acid metabolites in vitro and in vivo. This marine compound exerts anti-inflammatory effects after topical or p.o. administration to laboratory animals, probably because the reduction in arachidonic acid availability leads to inhibition of the biosynthesis of inflammatory mediators, with the partial contribution of inhibitory actions on neutrophil degranulation or lysosomal enzymes.