Nimesulide decreases superoxide production by inhibiting phosphodiesterase type IV

European Journal of Pharmacology Molecular Pharmacology Section 268 (1994) 415-423

ELSEVIER

molecularpharmacology

Nimesulide decreases superoxide production by inhibiting phosphodiesterase type IV Maurizio Bevilacqua ay*,Tarcisio Vago a, Gabriella Baldi a, Elio Renesto a, Franc0 Dallegri b, Guido Norbiato a aServizio di Endocrinologia, Ospedale Luigi Sacco (Vialba), via GB Grassi 74, 20157 Milano, Italy b Clinica Medica I, Dipartimento di Medic&a Interna, Kale Benedetto Xv, 6, I-16132 Genoua, Italy Received 21 April 1994; accepted 20 May 1994

Abstract Nimesulide, the prototype of a new class of anti-inflammatory drugs, dose-dependently decreases the production of the superoxide anion (0;) in N-formyl-methionyl-leucyl-phenylalanine (fMLP)-and in phorbol myristate acetate (PMA)-stimulated polymorphonuclear leukocytes. The inhibition of 0; is possibly related to its inhibitory effect on polymorphonuclear leukocyte cytosolic phosphodiesterase type IV (IC,, = 39 f 2 PM), to the related increase in CAMP (P < 0.01 at 1 PM) and the subsequent

increase in protein kinase A activity. In fact H-89, a specific protein kinase A inhibitor, counteracts the inhibitory effect of nimesulide on 0; production by fMLP and PMA. The activation of protein kinase A may prompt the phosphorylation of a number of substrates, thus inhibiting the assembly of NADPH-oxidase in the plasma membrane. Accordingly, nimesulide decreases PMA-induced assembly of NADPH-oxidase in polymorphonuclear leukocytes plasma membranes by about 35%. Protein kinase A activation may also interfere with chemotaxis. Nimesulide inhibits stimulated chemotaxis and the effect is decreased by H-89. Inhibition of phosphodiesterase type IV may explain many of nimesulide’s effects. Key words: Nimesulide;

Polymorphonuclear

leukocyte; Phosphodiesterase

1. Introduction The increase in CAMP in polymorphonuclear leukocytes inhibits chemotaxis (Harvath et al., 1991; Rivkin et al., 1975), degranulation and 0; release (Becker et al., 1987; Bourne et al., 1971; Cox and Karnovsky, 1973; Marone et al., 1980; Noursharg and Hoult, 1986; Zurier et al., 1973; Furui et al., 1989; Busse and Sosman, 1984; Wong and Freund, 1981). It also promotes the collapse of microfilaments, antagonizing actin organization induced by cytokines (Nathan and Sanchez, 1990). Other physiological responses inhibited by elevated CAMP in polymorphonuclear leukocytes include calcium mobilization (Tecoma et al., 1986; Takenawa et al., 1986; Kato et al., 19861, inositol phospholipid metabolism (Takenawa et al., 1986; Kato

* Corresponding

author.

0922-4106/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved %?DI0922-4106(94)00083-B

type IV; Superoxide anion; CAMP; Chemotaxis

et al., 1986) and arachidonic acid release (Takenawa et al., 1986). CAMP levels in polymorphonuclear leukocytes are controlled by adenylate cyclase (Lad et al., 1984) and phosphodiesterase type IV, the main CAMP degrading enzyme present in these cells (Nielson et al., 1990, Schudt et al., 1991). Nimesulide, a prototype of a new class of anti-inflammatory agents (Ward and Brogden, 1988; Zimmerli et al., 19911, is a potent drug which has been shown to decrease fMLP (Capsoni et al., 1987) and PMA-stimulated 0; production in polymorphonuclear leukocytes (Bevilacqua et al., 1988; Zimmerli et al., 1991) and to protect the alpha-1-proteinase inhibitor from oxidative inactivation by a scavenging effect on hypochlorous acid (Dallegri et al., 1992). The mechanism by which nimesulide decreases 0; production is unknown. As an increase in intracellular CAMP can affect 0; production in polymorphonuclear leukocytes, we evaluated the effect of nimesulide on CAMP

416

M. Bet~ilacqua et al. / European Journal of Pharmacology

generation in polymorphonuclear leukocytes and on the biochemical pathways involved in cAMP metabolism; i.e. degradation by phosphodiesterase type IV (Nielson et al., 1990; Schudt et al., 1991), production by adenylate cyclase (Lad et al., 1984) and by cAMP increasing fMLP related pathways (Snyderman et al.; 1990). Finally, since cAMP levels are involved in the chemotaxis control (Harvath et al., 1991), we also evaluated the effect of nimesulide on fMLP-stimulated chemotaxis.

2. Materials and methods

2.1. Materials The following chemicals were used: fMLP, PMA, phenylmethylsulfonyl fluoride, dimethyl sulfoxide (DMSO), superoxide dismutase, isobutyl-methylxanthine (IBMX), histopaque 1077, GTP, cAMP, ATP, forskolin, bovine serum albumin (all from Sigma Chemical, St. Louis, MO); Hanks' balanced salt solution, calcium and magnesium free HBSS, RPMI 1640 medium, fetal calf serum and Dulbecco's phosphate buffered saline (PBS) (from Gibco, Paisley, UK), H-89, N-J2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide 2 HCI (from Biomol Research Lab., PA), RO 20-1724 (from Research Biochemicals, Natick, MA). Milrinone was kindly donated by Boots, Nottingham, UK. Pentoxyfylline was donated by Hoechst AG, Italy. Nimesulide was donated by LPB Istituto Farmaceutico, Cinisello Balsamo, Milan, Italy. Stock solutions of PMA and nimesulide (all 10 raM) in DMSO were prepared and stored at -20°C until required. The other substances were dissolved just prior to use. Buffers were adjusted to pH 7.4 and 285 mosmol/kg H2o (Fiske Osmometer).

2.2. Isolation of polymorphonuclear leukocytes Polymorphonuclear leukocytes were isolated at 4°C by using pre-cooled buffers and refrigerated centrifuges. Buffy coats were obtained from healthy blood donors and cells were isolated using a modified Boyum's procedure (1968). Briefly, the buffy coat layers were transferred to 50 ml polypropylene tubes and 5 ml of 0.6% Dextran T500 in saline were added to sediment erythrocytes. After 30 min, the leukocyte-rich supernatant was pelletted at 275 x g for 5 min, resuspended in PBS, underlayered with Histopaque 1077 and centrifuged at 400 x g for 40 min. The resulting pellet, consisting mainly of polymorphonuclear leukocytes, was washed at 275 x g for 5 rain; contaminating erythrocytes were then removed by hypotonic lysis (exposure for 10 s to 0.2% sodium chloride followed by a return to isotonicity by the addition of 1.6% sodium

Molecular Pharmacology Section 268 (1994) 415-423

chloride, repeated twice). Polymorphonuclear leukocytes obtained in this way were 95% pure and 94% viable (Dallegri et al., 1987). They were counted and used immediately, according to the procedures described below.

2.3. 0 5 production by polymorphonuclear leukocytes O j produced by polymorphonuclear leukocytes was evaluated by the superoxide dismutase-inhibited reduction of cytochrome C, as previously reported (Dallegri et al., 1987). Briefly, a suspension of polymorphonuclear leukocytes (2 X 10 6 cells in 2 ml final volume) in HBSS with added calcium and magnesium was pre-incubated at 37°C for 10 rain in polystyrene cuvettes in the presence of 0.08 mmol/1 ferricytochrome C and a range of concentrations of nimesulide or RO 20-1724. H-89 was added at the final concentration of 1 /xM. The reaction was started by adding fMLP (0.1 ~M final concentration) or PMA (10 nM final concentration); the reduction of ferricytochrome C was monitored at 550 nM in a Beckman Du-50 spectrophotometer (equipped with thermostatic cuvette holder) at 10-s intervals for 10 rain. A molar extinction coefficient of 21 m o l . c m - I 1-1 was used to determine the O F produced. Superoxide dismutase (50 rag/I) was added to the incubation mixture in the reference cuvettes.

2.4. Measurement of cAMP accumulation Polymorphonuclear leukocytes were suspended in complete HBSS medium (pH 7.4) containing 0.2% BSA at a concentration of 2 X 10 6 cells/ml. Polymorphonuclear leukocytes were incubated with or without increasing concentrations of nimesulide, RO 20-1724 a n d / o r H-89 for 1, 3 and 30 rain at 37°C. In experiments involving fMLP, the chemoattractans (0.1 /xM) was added after a pre-icubation of cells with the drugs for 10 rain. The reactions were stopped after 1, 3 and 30 min by boiling the samples for 2 rain. After centrifugation at 600 x g for 5 min, cAMP contained in the supernatant was determined by a radioimmunoassay, according to the manufacturer's protocol (Cyclic AMP [12sI] RIA Kit, Dupont NEN Research Products, Boston, MA).

2.5. Isolation of polymorphonuclear leukocytes plasma membranes and measurement of NADPH-oxidase The isolation of cytosol and plasma membranes was performed according to Gabig et al. (1987). Cells were suspended in 0.13 M NaC1, 0.34 M sucrose, 10 mM potassium phosphate, 1 mM EGTA, at pH 7.0 containing 500 /xM phenylmethylsulfonyl fluoride and ruptured, using three 15-second bursts of 30% power from the microtip of an Artek System sonic dismernbrator,

M. Bevilacqua et al. / European Journal of Pharmacology - Molecular Pharmacology Section 268 (1994) 415-423

model 300 (PBI, Milano, Italy). The unbroken cells and debris were pelletted by centrifugation (800 x g for 15 rain) and the supernatant was underlayered with a 40% w / v sucrose cushion made in the sonication buffer and centrifuged at 100 000 g for 30 min at 4°C in a Beckman L3-50 ultracentrifuge (Beckman Instruments, Fullerton, CA). After centrifugation the membranebound NADPH-oxidase, layered at the interface between the top phase and the sucrose cushion, was recovered, washed and the protein content was evaluated with Bradford's method (1976). In some experiments, polymorphonuclear leukocytes suspended in 0.13 M NaCI, 10 mM potassium phosphate, 1 mM EGTA, 1 mM CaCI 2, 1 mM MgSO4, pH 7.0 and warmed at 37°C, were stimulated with PMA (1/zg/ml) to promote the migration of NADPH-oxidase from cytosol to plasma membrane and superoxide anion production. These experiments were performed either in the presence or absence of nimesulide. Twenty-five minutes after the PMA addition, the cells were washed and ruptured as before. The membranes were used to measure NADPH-oxidase activity (McPhail et al., 1984; McPhail and Snyderman, 1983). In some experiments, nimesulide was added to samples containing plasma membranes isolated from polymorphonuclear leukocytes pretreated with PMA to evaluate the effect of the drug on NADPH-oxidase. 2.6. Polymorphonuclear leukocytes and platelet isolation and cytosol extraction

Polymorphonuclear leukocytes and platelets were isolated as described previously (Vago et al., 1985), resuspended in Tris-HC1 50 mM, MgCI 2 5 mM, phenylmethylsulfonyl fluoride 2 mM, pH 7.4 and sonicated at 4°C (3 x 15 s at 30% power). The sonicated material was centrifuged (400 x g for 10 rain, at 4°C), the supernatant subjected to ultracentrifugation (100000 x g for 30 min) and the cytosol used as a source of phosphodiesterase activity. 2. 7. Phosphodiesterase activity

Phosphodiesterase activity of the cytosols obtained from polymorphonuclear leukocytes or platelets was evaluated in presence or absence of the various drugs. Cytosol proteins (20/xg) were preincubated at 37°C for 10 min in a final volume of 200/xl of 50 mM Tris-HCl buffer (pH 7.4) containing 5 mM MgC12, 2 mM phenylmethylsulfonyl fluoride and the appropriate concentrations of the drugs. The reaction was started by adding 0.2 nmol of [3H]cAMP (about 2 x 10 6 cpm) and was stopped after 10 min by placing the tubes in boiling water for 1 min and then adding 25 ixl of a 0.1 M cAMP solution, containing about 100000 cpm of [14C]5'AMP as a recovery marker. The [3H]5' AMP

417

produced was recovered by gel chromatography using Davis and Daly's method (1979); quantified by a [3H]/[14C] beta count and corrected for [14C]5' AMP recovery. 2.8. Adenylate cyclase

Adenylate cyclase in polymorphonuclear leukocytes plasma membranes was assessed as the [32p]cAMP generation from radiolabeled substrate. Briefly, polymorphonuclear leukocytes plasma membranes (50 /xg of protein) were incubated in assay buffer containing 40 mM Tris HCI (pH 7.4), 15 mM MgC12, 0.1 mM EGTA, 1 mM IBMX, 50 /xM GTP, 0.5 mM [ce32p]adenosine triphosphate (ATP, New England Nuclear; about 4 X 106 cpm/tube), 20 U / m l of creatine phosphokinase, 20 mM phosphocreatine, 1 mM cAMP and various concentrations of drugs in a final reaction volume of 200 /xl. After 10 min at 37°C, the reaction was interrupted by adding 150/zl of a solution containing 6% sodium dodecyl sulfate, 90 mM ATP, 3 mM cAMP. Approximately 10 000 cpm [3H]cAMP were then added to monitor the recovery of cAMP from the subsequent separation, and the tubes were boiled for 5 min. The amount of [32p]cAMP generated was evaluated by a liquid scintillation count after separation by column chromatography (Salomon et al., 1974; Bevilacqua et al., 1991). Assay blanks yielded about 10% of basal activity for all cases; column recovery, evaluated by [3H]cAMP, ranged from 58% to 75%. 2.9. Chemotaxis

The chemotaxis assay was performed using a modified Boyden chamber method (Harvath et al., 1980; Senior et al., 1989; Senior et al, 1992). The Histopaque/Dextran-separated polymorphonuclear leukocytes were suspended in RPMI 1640 and 1% fetal calf serum at a concentration of 1.5 X 106 cells/ml. A two-section chamber (Neuroprobe, CA) and 5/zm pore size polycarbonate PVP free filters (Nucleopore, Pleasanton, CA) were used. As negative controls the lower chamber wells contained 27/xl of RPMI 1640, 1% fetal calf serum, 0.1% DMSO; as positive controls they contained the same medium with 10 nM fMLP, with or without nimesulide and 1 IxM of H-89. To evaluate if H-89 is chemoattractans we placed in the upper chamber 75 000 polymorphonuclear leukocytes per well suspended in RPMI 1640, 1% fetal calf serum, 0.1% DMSO with H-89 (1 txM) in the lower chamber. Every assay was repeated in six wells. The chamber was incubated for 1 hour at 37°C; then the filter was removed, fixed with methanol and stained with eosin G and thiazine in phosphate buffer pH 6.6 (Diff QuikBaxter, Dade AG, Suisse), mounted on a glass slide and examined under light microscopy. Chemotaxis was

M. Betqlacqua et al./ European Journal of Pharmacology - Molecular Pharmacology Section 268 (1994) 415-423

418

evaluated by counting the number of cells migrating through the filter in five 1000 × fields (Harvath et al., 1980). 2.10. Statistical evaluation

Table 1 Effect of nimesulide (N) and R O 20-1724 on the maximum rate of

superoxide anion production/106 cells/rain in polymorphonuclear leukocytes stimulated with fMLP (100 nM) and P MA (10 nM) (mean ± SD of n experiments) in the absence or in the presence of the protein kinase A inhibitor, H-89 (1 #M); cells were pre-incubated 10 min with drugs before addition of fMLP or P M A

Data were analyzed with ANOVA.

3. Results 3.1. Effect of nimesulide on 0 2 production in polymorphonuclear leukocytes After oral administration plasma nimesulide reaches a concentration of 1-5 /xM (Castoldi et al, 1988). As shown in Table 1 and in the representative experiment in Fig. 1, nimesulide dose-dependently (1-100 /xM) inhibited Oj production stimulated by fMLP and by PMA in polymorphonuclear leukocytes. RO 20-1724, a specific phosphodiesterase type IV inhibitor, gave results similar to those obtained with nimesulide. 3.2. Effect of nimesulide on cAMP in polymorphonuclear

leukocytes Nimesulide and the prototype phosphodiesterase inhibitor RO 20-1724 dose-dependently increased cAMP levels in intact polymorphonuclear ieukocytes, the effect being significant at 1/xM for nimesulide (P < 0.01) and at 10 ~ M for RO 20-1724 (P < 0.01)7 after 30 min of incubation (Table 2). The transient increase (1 rain and 3 min after fMLP addition) of cAMP by fMLP was potentiated by the addition of nimesulide and RO 20-1724 (Table 2). PMA did not increase cAMP in polymorphonuclear leukocytes (data not shown). Since intracellular cAMP levels can be increased by activating adenylate cyclase or by inhibiting phospho-

fMLP f M L P + N 0.1/xM f M L P + N 1 /zM f M L P + N 10/ z M f M L P + N 100/zM f M L P + R O 20-1724 1 / x M f M L P + R O 20-1724 10/zM fMLP + R O 20-1724 100/zM fMLP + H-89 f M L P + H - 8 9 + N 10/ z M f M L P + H - 8 9 + N 100/xM PMA P M A + N 0.1 # M P M A + N 1/zM P M A + N 10/ z M P M A + N 100 # M P M A + R O 20-1724 1 tzM P MA + R O 20-1724 10/xM PMA + R O 20-1724 100 izM P MA + H-89 P M A + H - 8 9 + N 10 # M P M A + H - 8 9 + N 100/xM ap bp cp dp cp

< < < < <

0.01 0.01 0.05 0.01 0.01

vs vs vs vs vs

n

nmol O y. 106 PMN-l-min i

16 4 4 4 4 4 4 4 4 4 4 10 4 4 4 4 4 4 4 4 4 4

2.40 _-t-0.05 2.30 +_0.20 1.80 _+0.50 a 0.80 ± 0.20 a 0.45+0.17 ~ 1.75 ± 0.22 " 0.75 ± 0.25 ~ 0.50 ± 0.20 a 3.30 _+0.45 ~' 2.25 ± 0.35 c,d 2.13 ± 0.40 ~,d 7.40 + 1.05 7.25 ± 0.90 7.12±1.12 5.63 ± 0.80 b 4.05_+0.71 b 7.50 + 1.10 6.12±0.93 b 5.07 + 0.98 b 7.35 ± 0.95 6.89_+ 1.15 c 6.71 _+ 1.08 ~

fMLP alone P M A alone H-89 alone fMLP and nimesulide P M A and nimesulide

diesterase mediated cAMP degradation, we evaluated the effect of the drugs on these enzymes. Nimesulide (1, 10, 100/zM) and RO 20-1724 (1, 10, 100/xM) did not increase cAMP production by adenylate cyclase (basal 4 . 0 + 0 . 2 p m o l e s × m i n - I x m g protein -t"

Table 2

Per cent changes of cAMP accumulation in polymorphonuclear leukocytes

Nimesulide 1 p~M Nimesulide 10/xM Nimesulide 100/xM RO 20-1724 1 # M RO20-172410/xM RO 20-1724 100/xM fMLP0.1 /xM fMLP + Nimesulide 1 /~M fMLP + Nimesulide 10/zM fMLP + Nimesulide 100 txM fMLP + R O 20-1724 1 /.~M fMLP + R O 20-1724 10/zM fMLP + RO 20-1724 100/zM

1 min

3 min

30 min

95+ 7 117 + 11 134 + 12 87 ± 12 112_+11 131 + 12 256_+27 a 359 ± 51 a,b 398 ± 53 a,b 486 _+ 61 a.b 297 ± 38 a,b 415 _+ 72 a,b 309 ± 91 ~,b

1 1 9 + 12 143 + 21 226 ± 17 103 _+ 8 121± 9 198 ± 13 1 5 8 ± 17 340 ± 45 606 ± 107 715 ± 99 203 ± 26 419 ± 88 593 _+ 115

137± 6 160 _+ 15 264 ± 22 106 ± 11 1 3 2 ± 12 195 + 12 115±10 156 ± 13 295 ± 30 315 ± 27 160 _+ 15 186 ± 12 245 + 16

a

a a a,b a,b a,b a,b a,b a,b

a a a a a ~,b a,b a,b a.b a,b a.b

In experiments involving fMLP, the cells were pre-incubated with the drugs for 10 min. The incubations were stopped at 1, 3 and 30 min (mean + SD of 4 different experiments; a p < 0.01 vs basal; b p < 0.01 vs fMLP alone).

M. Bevilacqua et al. / European Journal of Pharmacology - MolecularPharmacologySection 268 (1994) 415-423 nimesulide 100/zM 4.2 + 0.2 and RO 20-1724 100 ~ M 3.5 + 0.5; not significant versus basal). Forskolin (100 /~M), a known stimulator of adenylate cyclase, increased the production of cAMP in polymorphonuclear leukocytes membranes (12.1 + 3.0 pmoles × min -1 x mg protein - 1., P < 0.01 vs basal). RO 20-1724 inhibited cAMP degradation (IC50 = 3 _+ 0.5 /~M), confirming that in human polymorphonuclear leukocytes the main cAMP degrading activity is due to phosphodiesterase type IV (Fig. 2). Nimesulide also dose-dependently inhibited cAMP degradation by phosphodiesterase in polymorphonuclear leukocytes cytosol (IC50 = 39 + 2/zM; Fig. 2). Nimesulide was less effective in inhibiting cAMP degradation in human platelet cytosol (IC50> 100 /zM) than in polymorphonuclear leukocytes (Fig. 2), whereas milrinone, a selective phosphodiesterase type III inhibitor, strongly decreased cAMP degradation in platelet cytosol (IC50 = 0.2 + 0.05 ~ M ) (Fig. 2).

PDE IV

419

PDE III

5,,

,~0( o 80

E •~ 60 '~ 4 0

20 s

4

-log[DRUG] (M)

Fig. 2. Effect of nimesulide (0), RO 20-1724 (a prototype phosphodiesterase type IV inhibitor) (e), milrinone (a prototype phosphodiesterase type III) (,x), IBMX ( I ) and pentoxifylline( • ) (both non-specific phosphodiesterase inhibitors) on cAMP degradation by phosphodiesterase in cytosol of human polymorphonuclear leukocytes (PDE IV) (ECs0 values are: nimesulide39_ 2 p.M, RO 20-1724 3+0.5 p~M, IBMX 8+1 /zM and pentoxyfylline > 100 /~M) or of human platelets (PDE III) (ECs0 values are: nimesulide > 100 #M; milrinone 0.2 _+0.02 ~M, IBMX 5 _+0.9 p.M).

3.3. Effect of H-89, a protein kinase A inhibitor, and nimesulide on f M L P and PMA stimulated Oy production To evaluate whether cAMP-dependent protein kinase (protein kinase A) plays a role in the action mechanism of nimesulide, we evaluated the effect of protein kinase A inhibitor H-89 (Snyder et al., 1992; Chijiwa et al., 1990; Ginty et al., 1991). H-89 is a recently synthesized isoquinolone-sulfonamide derivative with superior potency and selectivity for protein kinase A inhibition ( K i = 0.048 /zM) (Chijiwa et al., 1990). This class of compounds inhibits protein kinase activity (Chijiwa et al., 1990) by competing with ATP

for its binding sites to the catalytic unit of protein kinase A (Hidaka et al., 1984). The H-89 dose employed by Snyder et al. (1992) in isolated cells was 50 /~M. So in isolated cells H-89 was employed at doses greater than its K i on isolated enzyme, since to affect protein kinase A, H-89 had to be higher in the presence of elevated levels of intracellular cAMP and ATP and to obviate its low cell permeability. On this basis we employed H-89 at 1 /~M. As shown in Table 1 (a representative experiment in Fig. 1) H-89 on its own increased O 3 production in fMLP-treated polymorphonuclear leukocytes and decreased the inhibitory effect of nimesulide. In PMA-treated polymorphonuclear leukocytes, H-89 did not increase O 3 production on its own and decreased the nimesulide's inhibitory effect (Table 1). 3.4. Effect of nimesulide on NADPH-oxidase assembly in polymorphonuclear leukocytes plasma membranes

.E E ""4

~3 o

~2 --B

O

O

Time (mln)

Fig. 1. Representative experiment of the effect of 10/zM nimesulide on OF production in fMLP-stimulated human polymorphonuclear leukocytes in the absence or in the presence of H-89, a specific protein kinase A inhibitor. A = fMLP (100 nM); B = fMLP plus nimesulide 10 p.M; C= fMLP plus H-89 1 ~M; D = fMLP plus nimesulide plus H-89.

Isolated plasma membranes of polymorphonuclear leukocytes pre-treated with PMA produced O 3 on subsequent re-challenging with PMA (3.0 + 0.05 nmol O 2 × m i n - I X 100 /~g protein -~). When added to plasma membranes of PMA-pretreated polymorphonuclear leukocytes, nimesulide (10 /xM) did not reduce O~- release (2.9+0.05 nmol O 3 x m i n - 1 x 1 0 0 ~g p r o t e i n - l ) . However when nimesulide (10 g m ) was added to polymorphonuclear leukocytes with PMA before plasma membrane isolation, these membranes consistently produced less O 3 on a re-challenge with PMA (1.95 + 0.03 n moles O 3 x min -1 x 100/~g protein -1; ( p < 0.01).

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Table 3 Chemotaxis of neutrophils to 10 nM fMLP in the presence of increasing concentrations of nimesulide (N) with or without H-89; the data are reported as number of cells counted in 1000×fields, mean _+SD of n experiments

Control fMLP f M L P + N l /xM f M L P + N 10/zM f M L P + N 100 # M H-89 1 /xM fMLP + H-89 f M L P + H - 8 9 + N 1/~M f M L P + H - 8 9 + N 10/zM f M L P + H - 8 9 + N 100/xM

n

No. of PMN 1000 × field 1

8 4 4 4 4 4 4 4 4 4

23 + 4 68 + 7 " 58_+3 b 51 _+4 b 36_+5 b 19-+5 83 + 8 b 70-+5 67-+6 52+6 h

a p < 0.01 vs control; h p < 0.05 vs fMLP.

3.5. Effect of nimesulide on polymorphonuclear leukocytes chemotaxis With increasing concentrations of nimesulide, fMLP-stimulated chemotaxis of polymorphonuclear leukocytes was dose-dependently inhibited (Table 3). H-89 (1 /zM) completely reversed the effect of nimesulide (1-10 /zM) and partially reversed the effect of nimesulide (100 /xM) (Table 3). H-89 (1 #M) was devoid of chemotactic activity on its own but increased significantly fMLP-stimulated chemotaxis.

4. Discussion

The main finding of this study was the inhibitory effect nimesulide had on cAMP phosphodiesterase in human polymorphonuclear leukocytes with the consequent increase in cAMP and the possible link, through protein kinase A, to the inhibition of NADPH-oxidase assembly and O 7 production. The inhibition was rather specific for phosphodiesterase type IV. In fact, the phosphodiesterase in polymorphonuclear leukocytes cytosol, whose cAMP degrading activity is almost entirely due to type IV (Nieison et al., 1990; Schudt et al., 1991) which is specifically inhibited by RO 20-1724, was decreased by nimesulide, whereas in platelet cytosol, where the cAMP degrading activity is due to phosphodiesterase type III and inhibited by milrinone, nimesulide was much less effective. The phosphodiesterase blockade was accompanied by an increase in basal and fMLP-stimulated cAMP levels in polymorphonuclear leukocytes. Nimesulide capacity for decreasing O2 production and increasing cAMP in intact cells was similar to that of RO 20-1724; its inhibitory effect on phosphodiesterase type IV in polymorphonuclear leukocytes cytosol was less than that displayed by RO 20-1724. The quantitative difference between

nimesulide and RO 20-1724 in inhibiting PDE type IV in polymorphonuclear leukocytes could be related to the better access nimesulide had to the intracellular compartment of intact cells. The discussion takes into consideration the biochemical links between cAMP, O j production by NADPH-oxidase and chemotaxis.

4.1. cAMP and O~production The role of cAMP in polymorphonuclear leukocytes is complex. In fact, polymorphonuclear leukocytes cAMP may be increased by agents that inhibit O~production, such as isoproterenol, PGE 2 and PDE IV inhibitors, and increased by agents that stimulate O 2 such as fMLP. Agents that inhibit O 7 production and increase cAMP have been reported to affect O2 induced by soluble stimuli whereas the respiratory burst elicited by non-soluble stimuli (PMA) has been reported to be unaffected (Sedgwick et al., 1985; Fujita et al., 1984) or only partially counteracted (Verghese et al., 1986) by these agents. Therefore, it has been suggested that regulation of polymorphonuclear leukocytes functions by Of-inhibiting and cAMP-elevating agents occurs at a step proximal to protein kinase C events (Kramer et al., 1988). However, in rat glomeruli (Miyanoshita et al., 1989) and in rat gastric parietal cells (Ostrowski and Bomsztyk, 1989) cAMP is clearly able to affect protein kinase C-related events also possibly due to the inhibition of protein kinase C translocation to membrane. In polymorphonuclear leukocytes, nimesulide was able to affect protein kinase C translocation (Bevilacqua et al., 1991) and PMA-induced superoxide generation (Bevilacqua et al., 1992; Zimmerli et al., 1992; this work). The fact that Fujita et al. (1984) and Sedgwick et al. (1985) did not observe inhibition of O j production by agents which increase cAMP in PMAstimulated polymorphonuclear leukocytes could be related to the different experimental settings: isolation of polymorphonuclear leukocytes by heparinized blood instead of acid citrate dextrose solution may increase cAMP levels thus blunting the effect of cAMP enhancing agents (see Discussion in Harvath et al., 1991) and the use of cytochalasin B may prolong and magnify the superoxide production and obscure the relatively minor inhibitory effect of cAMP-increasing agents. Interestingly, (see Discussion in Jesaitis et al., 1986), cytochalasin B disrupts plasma membrane order established by the membrane microfilament system: so in cytochalasin B-treated polymorphonuclear leukocytes (Fujita et al., 1984) the role of agents such cAMP which typically affect plasma membrane translocation may be underestimated. In fact cAMP-dependent kinase may phosphorylate a large series of proteins involved in polymorphonuclear leukocytes function control (Omann et al., 1987). For example, cAMP-depend-

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ent kinase may phosphorylate actin which may induce actin de-organization and decrease the cytosol recruitment of components involved in the activation of NADPH-oxidase at the plasma membrane level. We tested this possibility directly by measuring NADPHoxidase activity in plasma membranes isolated from polymorphonuclear leukocytes pre-treated with nimesulide or by adding nimesulide to plasma membranes of pre-stimulated neutrophils. Nimesulide did not affect NADPH-oxidase activity in isolated plasma membranes, ruling out the drug's direct effect on NADPHoxidase, however it inhibited NADPH-oxidase assembly in plasma membranes which would suggest an effect on recruitment of plasma membranes. Finally we isolated polymorphonuclear leukocytes with pre-cooled buffers in order to obviate membrane association of protein kinase C during the steps of cell preparation (Miloszewska et al., 1986). This might have enhanced the effect of agents which affect protein kinase C translocation (Bevilacqua et al., 1991). On the other hand, it is possible that the inhibitory effect of Nimesulide on PMA-stimulated O 3 might be due to other actions of the drug. The role of fMLP-induced increases in cAMP in the control of polymorphonuclear leukocytes function is well-known, fMLP induces a transient elevation in cAMP which peaked at 15-20 s, while the adenylate cyclase stimulating hormones, prostaglandin E1 and isoproterenol, produced a more prolonged increase in cAMP levels (see Discussion in Verghese et al., 1986). The biochemical mechanism employed by chemoattractans and Ca 2+ ionophore to increase cAMP in polymorphonuclear leukocytes is distinct from that utilized by PGE1 or isoproterenol. In fact chelation of extracellular Ca 2+ by EGTA or inhibition of intracellular Ca 2+ redistribution by TMB-8 (8-(N,N-diethylamine)-octyl3,4,5-trimethoxybenzoate) greatly reduced cAMP increases in response to fMLP or the Ca 2+ ionophore without affecting cAMP increases in response to PGE1 or isoproterenol. In the model proposed by Snyderman (Verghese et al., 1986) fMLP receptors are not coupled to adenylate cyclase and do not utilize either G i or G s to regulate the coupling to post-receptor events. Rather they utilize G protein in a transduction mechanism linked to the generation of Ca 2+ signal. Furthermore, as other Authors suggested (Snyderman et al., 1990, Didsbury et al., 1992) intracellular cAMP " . . . may provide an autoregulatory termination mechanism" that depresses the effect of chemoattractans. On this basis, the stimulatory effect that H-89 alone has on the superoxide anion production in fMLP-stimulated polymorphonuclear leukocytes (at doses which in vitro have proved to affect protein kinase A selectively) (Snyder et al., 1992, Chijiwa et al., 1990) suggests the cAMP has a tonic inhibitory effect on superoxide anion production in fMLP-stimulated polymorphonuclear leuko-

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cytes. Furthermore, the fact that H-89 does not stimulate superoxide production in PMA-stimulated polymorphonuclear leukocytes where cAMP is not involved, would suggest that cAMP plays this inhibitory role on fMLP-stimulated polymorphonuclear leukocytes only. Interestingly, the respiratory burst of fMLP-stimulated polymorphonuclear leukocytes stops on its own, whereas the burst stimulated by PMA continues indefinitely and stops only after substrate exhaustion. 4.2. c A M P a n d c h e m o t a x i s

The mechanism which links cAMP to chemotaxis is also unclear (Harvath et al., 1991). One hypothesis is that phosphorylation of actin by cAMP-protein kinase might result in actin de-organization and reduction of motility. Moreover, Tumour Necrosis Factor alpha, a potent cytokine, has been shown to activate polymorphonuclear leukocytes chemotaxis simply by decreasing cAMP levels (Nathan and Sanchez, 1990). Our data shows that nimesulide decreases chemotaxis in polymorphonuclear leukocytes; also, this effect is probably the consequence of phosphodiesterase type IV inhibition, the increase in cAMP and the activation of protein kinase A. H-89 (1/xM) increased fMLP-stimulated chemotaxis and reversed the inhibition of stimulated chemotaxis completely when nimesulide was used at 1 and 10 /xM, but only partially when nimesulide was used at 100 /xM. Therefore, it is possible that other effects of nimesulide are responsible for the inhibition of chemotaxis. Interestingly, as Harvath et al. (1991) pointed out, the elevation of cAMP affects chemotactic responsiveness differentially: in fact, isoproterenol strongly inhibited polymorphonuclear leukocytes chemotaxis and slightly enhanced cAMP levels, whereas forskolin strongly increased cAMP levels but affected chemotaxis only slightly. So it is possible that compartmentalization of cAMP might play a role in the effect on chemotaxis: on this basis it can be stated that the effect of nimesulide is similar to that of forskolin, i.e. strong effect on cAMP and relatively minor effect on chemotaxis. 4.3. Conclusion

In conclusion, nimesulide inhibits cAMP phosphodiesterase in human polymorphonuclear leukocytes. There is an increase in cytosolic cAMP that in turn activates protein kinase A which phosphorylates a number of substrates; this mechanism may account for the decrease in O 3 release and chemotaxis brought about by nimesulide. Since cAMP and phosphodiesterase are also important regulators of PAF production in polymorphonuclear leukocytes (Verhoeven et al., 1991) and of some basophil activities, this mech-

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anism may also explain other relevant activities of the drug with potential clinical interest, i.e. the inhibition of the release of histamine in human basophils (Patella et al., 1991; Casolaro et al., 1993; Verhoeven et al., 1993; Undem et aI., 1988) and of Platelet Activating Factor in polymorphonuclear leukocytes (Verhoeven et al., 1993).

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