Inflammation, Vol. 16, No. 6, 1992
E F F E C T S OF N I F L U M I C A C I D O N POLYPHOSPH(3INOSITIDE METABOLISM
IN P O L Y M O R P H O N U C L E A R
FROM HEALTHY AND
M. TISSOT, ~ M. RO ~H-ARVEILLER, J. FONTAGNE, 2 and J. P. GIROUD I UDOpartement de Pharmacologie, URA CNRS 595 ZAssociation Claude Bernard HOpital Cochin 27 r~ e du Faubourg Saint-Jacques 75014 Paris, France
Abstract--Thermal injury in iats leads to an impairment of polymorphonuclear leukocyte (PMN) functions, pa-ticularly oxidative metabolism and phosphoinositide turnover. As prostaglandin E2, which has immunosuppressive properties, is released in high levels after bum trauma, we investigated the in vitro and in vivo effects of a nonstemidal antiinflammatory drag, niflumic acid, on oxidative and phosphoinositide metabolism in PMNs from healthy and burned rats. Given the role of fluoride ions on PMN, the influence of nif umic acid was compared with that of sodium fluoride (NaF) at equivalent doses of ]7-. In vitro, niflumic acid and sodium fluoride had no effect on oxidative metabolisra in stimulated by formyl methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ) or nonstimulated PMNs from healthy and burned rats. Niflumic acid sli?~htly increased the production of inositol phosphate by nonstimulated PMNs from healthy and burned rats. Niflumic acid and NaF partly restored the stimulating effect of FMLP on inositol phosphate production by PMNs from burned rats. In vivo treatment with niflumic acid and NaF increased the oxidative metabolism of PMNs from burned rats but not healthy rats. Niflumic acid, more than NaF, restored the activity of both stimulants on phosphoinositide metabolism in PMNs from burned rats. In conclusion, at non-antiinflammatory doses, while inhibiting cyclooxygenase activity, niflumic acid exerts a complex effect on the burn~ induced depression of PMN functions. The fluoride anion induces similar but generally weaker effects and see-as to be involved in the restoring effects of niflumic acid on PMN functions in bu~nned rats.
0360 3997/92/1200-0645506.50/0 9 1992 PlenumPublishingCorporation
Tissot et al.
INTRODUCTION Severe thermal injury leads to alterations of various components of host defenses, including decreased humoral and cellular immunity (1) and inhibition of polymorphonuclear leukocyte (PMN) functions such as chemotaxis, phagocytosis, and bacterial killing (2). A temporal association between the depression of PMN bactericidal activity, complement consumption, and production of arachidonic acid metabolites via the cyclooxygenase pathway occurs in thermally injured animals (3). Elevated levels of E-series prostaglandins (PGE) are released from macrophages/monocytes and skin cells after burn injury (3). These PGEs inhibit various PMN functions including chemotaxis, aggregation, superoxide production, and lysosomal enzyme release (4). It has been suggested that defective PMN bactericidal activity caused by experimental thermal injury is related to the elevation of cAMP, and that PGEI plays an important role in this phenomenon (4). PGE also modulates humoral and cellular immune functions, acting as a feedback inhibitor for T-cell proliferation, lymphokine production, cytotoxicity, and other functions (5-7). Nonsteroidal antiinflammatory drags (NSAIDs) were thus tested for their ability to counteract some detrimental effects of bum injury (8). Inhibition of prostaglandin (PG) production by NSAIDs generally results in enhanced immunological functions, as demonstrated in several in vivo and in vitro studies (7, 9). One such drug, niflumic acid, shows immunostimulating properties in mice (10). We have previously shown, in a rat model of thermal injury, alterations of PMN responses to classical stimulants such as FMLP and OZ three days after injury. The oxidative burst was diminished (11), possibly due to alterations in signaling systems and particularly in phosphoinositide (PI) breakdown. The stimulating effects of FMLP and OZ on PI metabolism are reduced or abolished after burn injury (12). In the present study, we investigated the effects of niflumic acid, a NSAID inhibitor of cyclooxygenase, on the oxidative burst and PI breakdown in PMNs from healthy and thermally injured rats both in vitro and in vivo. As the F - ion may interfere with G-proteins involved in stimulus-coupling mechanisms (13, 14), we investigated its relative importance in the activity of niflumic acid by comparing the effects of NaF at equivalent doses.
Reagents. Niflumicacid (UPSA Laboratories, Agen, France) was dissolved in phosphate buffer (Na2HPO4, 2H20 11.21 g, KH2PO40.49 g, NaC1 3.00 g, H20 qsp 1000 ml). Cytochrome c, FMLP, cytochalasinB, OZ, luminol, bovine serum albumin (BSA), and sodiumfluoride(NaF) were obtained from Sigma (St. Louis, Missouri), Dowex AG1-X8 ion-exchangeresin (200-400
Effects of Niflumic Acid
mesh, formate form) from BioF,ad (Richmond, California), and myo-[2-3H] inositol from Amersham (Les Ulis, France). Animals. Male Sprague-Dawley rats (Drprr, St. Doulchard, France) weighing 180-200 g were used. Under ether anesth~sia, third-degree thermal injury of 20-25% of the body surface area was induced according to the method of Walker and Mason (15). After recovery from anesthesia, the animals were housed two per cage and given water and food ad libitum for four days. In in vivo experiments, six groups were treated per os (0.5 ml) daily for four days, two (healthy and burned rats) receiving niflumk acid (0.1-10 mg/kg), two (healthy and bumed rats) receiving NaF (0.05-5mg/kg) and two (healthy and burned rats) receiving water. The first administration was given 2-3 h after injury. In in vitro experiments, PMNs from untreated healthy and burned animals were incubated with buffer (control~) and with various concentrations of the drugs. PMN Collection. On the fourth day after injury, PMNs were elicited by an intrapleural injection of 1 ml of isologous decomplemented rat serum (16). Four hours later, PMNs ( > 9 5 % , purity) were collected in phosphate-buffered saline (PBS). A portion of the PMNs collected from each rat was kept for 0 2 generation studies and the chemiluminescence assay, while the rest was pooled (four to six animals per group) and labeled. PMNLabeling. As previously described by Tissot et al. (12), PMNs were centrifuged and resuspended in Hanks' balanced salt solution (HBSS) containing 20 mM HEPES (pH 7.4) supplemented with 0.025% bovine serum albumin (BSA). Cells were adjusted to a concentration of 10s/ 10 ml/tube, labeled with 200 ~Ci of myo-[2-3H]inositol and incubated for 16-20 h at 37~ with gentle shaking. At the end of the incubation period, the ceils were washed in PBS containing 20 m M HEPES, 0.025% BSA, and 10 mM LiCI (to inhibit inositol phosphate-dephosphorylating enzymes). After washing, cells were pooled, resuspended in 20 mM HEPES-HBSS supplemented with 0.1% BSA and 10 mM LiC1 (prewarmed to 37~ and oxygenated with 95 % 02/5 % CO2) and the concentration was adjusted to 107 cells/600-/~l aliquot. Aliquots were dispensed into Eppendorf microtubes (in duplicate or triplicate) and incubated for 10 rain at 37~ The test reagents were then added at various concentrations and for various contact times. Labeled PMN Stimulation In Vitro. Stimulating agents were applied to PMNs from untreated healthy and burned rats as follows; (1) PBS for 15 sec (controls), or (2) niflumic acid (10-9-10 -5 M) for 10 min, or (3) NaF (3 x 10-9-3 • 10 -5 M) for 10 min (equivalent doses of F - to those of niflumic acid). In some cases complementary incubations were carried out as follows, with: (4) FMLP (10 -6 M) for 15 sec, added after .~ min pretreatment with 10 mM cytochalasin B, or (5) FMLP + cytochalasin B as above, after 10 min incubation with niflumic acid (10 -7 and 10 -6 M), or (6) FMLP + cytochalasin B, after 10 min incubation with NaF (3 • 10 -7 and 3 x 10 -6 M). Stimulating agents were applied as follows to PMNs from healthy and burned rats treated in vivo with niflumic acid, NaF (at equivalent doses of F-), or the buffer: (1) PBS for 15 sec, or (2) FMLP (10 -6 M) for 15 sec, added after 5 min pretreatment with 10 m M cytochalasin B, or (3) OZ (100 mg/106 cells) for 90 sec. The reactions were stopped by the addition of 100 t~l of 40 % perchloric acid (PCA) to obtain a final PCA concentration of z.-5 %, followed by three cycles of freezing-thawing. [3tl} Inositol Phosphate and [3H]Inositol Lipid Analysis. Analyses were performed as previously described (12). After centrifugation, the hydrosoluble perchloric acid supematants, containing inositol phosphates (InsP), were diluted and neutralized. The inositol lipids were extracted from the PCA-insoluble pellets and deacylated. The [3H]inositol phosphate and [3H]glycerophosphoryl esters were separated by means of anion-exchange chromatography on Dowex AG1-X8 columns using the buffer system described by Downes and Michell (17), Berridge et al. (18), and Creba et al. (19). 02 Generation. Superoxide O~-) generation was measured in terms of ferricytochrome c reduction (horse heart, type IlI), as described by Johnston et al. (20). PMNs (2 x 106 cells/ml)
Tissot et al.
and 150 ,ul/ml of a 0.4 mM solution of ferricytochrome c were incubated for 15 min at 37~ with or without the drags in the presence of OZ (500/zg/10 6 cells) for 15 min, or FMLP (10 -7 M) for 5 min. The final volume of the reaction mixture was adjusted to 1 ml. The reaction was stopped by placing the tubes in an ice-water bath, followed by centrifugation at 800g for 10 min at 4~ The amount of O~- produced was calculated from the difference in the absorbance of the samples before and after incubation, using an extinction coefficient of 20 mM ~cm -~ at 550 nm. The results are expressed in nanomoles of Of released per minute per 10 6 cells and the percentage response was calculated relative to the appropriate control. The specificity of the reaction was checked by addition of superoxide dismutase, which inhibited at least 90% of OZ stimulation. ChemiluminescenceAssay. The PMN chemiluminescent response (CL) was measured using a Packard Picolite luminometer (21). CL was measured after placing 100/zl of cell suspension (5 x 106 cells/ml) in 6 x 50-mm borosilicate tissue-culture tubes in the apparatus for 5 min in the dark at 37~ After equilibration, 20/~1 of luminol solution was added at a final concentration of 4 x 10 -5 M. When background light emission became constant, 150 ttl of the suspension of OZ (1500 #g/106 cells) or FMLP (10 -v M) was added and light emission was recorded for 20 min. Data Analysis. Since tritiated inositol incorporation into phosphoinositides varies from one pool of cells to another [in particular, PMNs from burned rats are much more strongly labeled than those from healthy rats (12)], results are expressed as percentages of controls (aliquots incubated with PBS), in mean +SEM. The statistical significance of differences between means was estimated using the Mann-Whitney U test for percentages and Student's paired t test on real values for O~generation and chemiluminescence.
In Vitro Effect of Niflumic Acid and NaF on Phosphoinositide Metabolism in PMNs from Healthy and Burned Rats (Figures 1 and 2). N i f l u m i c acid o n l y s i g n i f i c a n t l y i n c r e a s e d p r o d u c t i o n o f i n o s i t o l p h o s p h a t e s [inositol 1 - p h o s p h a t e (IP), i n o s i t o l 1 , 4 - b i s p h o s p h a t e (IP2) a n d inositol 1 , 4 , 5 - t r i s p h o s p h a t e (IP3)] b y P M N s f r o m h e a l t h y a n d b u r n e d rats at 10 - 7 M (IP3 is s h o w n b y w a y o f a n e x a m p l e in F i g u r e 1; P < 0 . 0 2 to P < 0.01 c o m p a r e d to t h e totally i n a c t i v e d o s e s o f 10 - 9 a n d 10 - 5 M ) . W e c h e c k e d t h a t the b u f f e r u s e d to d i s s o l v e n i f l u m i c a c i d h a d n o effect o n t h e v a r i o u s p a r a m e t e r s studied. N a F f r o m 3 x 10 - 9 to 3 x 10 -5 M h a d n o s i g n i f i c a n t effect o n I n s P m e t a b o l i s m in P M N f r o m h e a l t h y o r b u r n e d rats. P r e i n c u b a t i o n w i t h n i f l u m i c acid at 10 - 7 M o r N a F at 3 • 10 - 7 M did n o t m o d i f y t h e s t i m u l a t i n g effect o f F M L P o n I n s P p r o d u c t i o n in P M N s f r o m h e a l t h y rats, b u t it partly r e s t o r e d t h e effect o f F M L P in P M N f r o m b u r n e d rats. N o s i g n i f i c a n t v a r i a t i o n w a s o b s e r v e d i n i n o s i t o l lipid c o n t e n t s o f P M N s f r o m h e a l t h y o r b u r n e d rats a f t e r s t i m u l a t i o n b y n i f l u m i c acid o r N a F , p a r t i c u l a d y in p h o s p h a t i d y l inositol 4 , 5 - b i s p h o s p h a t e (PIP2).
In Vivo Effect of Niflumic Acid and NaF on Phosphoinositide Metabolism in PMNs from Healthy and Burned Rats (Figure 3). I n h e a l t h y rat P M N s , d a i l y t r e a t m e n t f o r f o u r d a y s w i t h n i f l u m i c acid at 10 m g / k g o r N a F at 5 m g / k g d i d n o t m o d i f y t h e s t i m u l a t i n g effect o f F M L P o r O Z .
Effects of Niflumic Acid
"6 g a.
=* o= Q. O
g =. 50
Fig. 1, Effect on IP3 release of in vitro incubation of PMNs from healthy (a) and burned rats (b) with various concentrations of niflumic acid (Nit) or equivalent concentrations of sodium fluoride (NaF). Cellular IP 3 is expressed as the ratio (% of control) of cpm recovered from stimulated cells relative to unstimulated cells (100%). The data are the mean _ SEM of four to six independent experiments. (a) Only 10 7 IV[ Nif significantly increased IP3; 10 -9 and 10 5 M Nif and equivalent concentrations of NaF were ineffective (P < 0.05 to P < 0.01). (b) 10 7 M Nif induced a significantly higher release of IP3 than other concentrations of Nif or NaF (P < 0,05 to P < 0.01).
In b u r n e d rat P M N s , t r e a t m e n t w i t h n i f l u m i c acid c o m p l e t e l y (10 m g / k g ) o r p a r t i a l l y (1 m g / k g ) r e s t o r e d t h e s t i m u l a t i n g effect o f F M L P a n d O Z ( P < 0.01 a n d P < 0 . 0 5 , r e s p e c t i v e l y , v e r s u s u n t r e a t e d c o n t r o l s ) , a l t h o u g h t h e effect o f t h e h i g h e r d o s e (10 nag/kg) w a s n o t s i g n i f i c a n t l y different f r o m t h a t o f t h e l o w e r d o s e . T r e a t m e n t w i t h N a F at 5 m g / k g s i g n i f i c a n t l y i n c r e a s e d IP3 p r o d u c -
Tissot et al. 200 r 0 =. m 0 r
Fig. 2. Effect on IP 3 release of incubation of PMNs from burned rats with FMLP (10 -6 M), Nif (10 -7 M) and NaF (3 • 10 -7 M) and of FMLP after preincubation with Nif or NaF. Each value is the mean of duplicate determinations. Data are representativeof those obtained in three separate experiments. tion relative to untreated rats (P < 0.05), while 0.5 mg/kg had no effect. The effect of treatment with 10 mg/kg niflumic acid was significantly greater (P < 0.05) than that of 5 mg/kg NaF (F- equivalent doses). Smaller and larger doses of niflumic acid (0.1, 20, and 50 mg/kg) had no effect (data not shown). PIP 2 contents were significantly decreased in PMNs stimulated by FMLP after niflumic acid treatment (P < 0.05, versus untreated controls). The effects of the two doses, 1 or 10 mg/kg, were not significantly different. Effect of Niflumic Acid and NaF on PMN Oxidative Metabolism. PMN oxidative metabolism was decreased by approximately 20% by bum injury. Thus, in order to compare the action of the drugs on PMNs collected from healthy and burned rats, results were expressed as a percentage of untreated, healthy control values. Figure 4 shows that niflumic acid, at doses below 10 mg/kg, did not significantly modify O~- production by OZ-stimulated PMNs from healthy rats, whereas higher doses, which are antiinflammatory, inhibited this parameter (data not shown). In contrast, doses from 0.1 to 10 mg/kg significantly enhanced 0 2 generation by PMNs from burned rats (P < 0.05 to P < 0.01). Higher doses were inhibitory (data not shown). The effect was not dose-related. Similar results were obtained with NaF from 0.05 to 5 mg/kg (P < 0.01 with 0.05 and 0.5 mg/kg; Figure 5). Higher doses were ineffective. Figure 6 shows the results obtained in a series of experiments (N --- 6) comparing the effects of the lowest doses of niflumic acid (0.1 mg/kg) and NaF (0.05 mg/kg). O~- generation by PMNs from burned untreated rats was significantly depressed; neither drug modulated the response of PMNs from healthy rats but totally restored that of PMNs from burned rats (P < 0.01). Similar results were obtained in the chemiluminescence assay whatever the stimulant used (data not shown). In contrast, in vitro experiments showed no
Effects o f Niflumic A c i d
e~ lOO. -6
Fig. 3. Effect on PMN IP3 and PIP 2 variations of treatment of healthy and burned rats with Nif or NaF. (a) Incubation of PMNs from healthy rats with FMLP or OZ with or without (controls) Nif or NaF. Mean _+ SEM of th~ee independent experiments. Treatment had no effect. (b) Incubation of PMNs from burned rats with FMLP or OZ with or without Nif or NaF. Mean +_ SEM of five independent experiments. IP:~: significant differences were observed with both stimulants between untreated and treated rats with 10 mg/kg (P < 0.01) or 1 mg/kg (P < 0.05) of Nif; between treated rats with 10 mg/kg of Nil arLd 5 mg/kg of NaF (P < 0.05); and between treated (5 mg/kg NaF) and untreated rats with OZ as PMN stimulus. PIPz: significant differences were observed with FMLP between untreated and treated rats with 10 mg/kg or 1 mg/kg of Nif (P < 0.05).
s i g n i f i c a n t a c t i v i t y o f n i f l u m i c acid ( 1 0 - 9 - 1 0 -5 M ) o r s o d i u m fluoride (3 • 10 - 9 , 3 • 10 -5 a n d 1.5 • 10 - 4 M ) . H i g h e r c o n c e n t r a t i o n s o f n i f l u m i c acid w e r e i n h i b i t o r y (data n o t s h o w n ) .
I n v i t r o w e f o u n d a w e a k , b u t significant, e n h a n c e m e n t b y n i f l u m i c acid o f PI m e t a b o l i s m in P M N s f r o m h e a l t h y a n d b u r n e d rats o n l y at 10 -7 M . N a F , at F - e q u i v a l e n t d o s e s , h a d n o effect. P r e i n c u b a t i o n w i t h n i f l u m i c acid at this
Tissot et al. 20 O
Fig. 4. Comparison of the chemiluminescent response to OZ of PMNs from burned and healthy animals treated with niflumic acid (Nif 0.1, 1, and 10 mg/kg) with the response in untreated animals. The results are expressed as the percentage enhancement of O;- production relative to control values. The results obtained with PMNs from healthy animals were not significantly different from controls, whereas those obtained with PMNs from burned animals were significantly enhanced (*P < 0.05; **P < 0.01).
O .~ 40-
II s 30" o~
~j'~ 20" ItS
O' NaF -10 ' 0,05
Fig. 5. Same representation of the results obtained after treatment of the animals with NaF (0.05, 0.5, and 5 mg/kg). Only the lowest doses had a significant effect (**P < 0.01); doses higher than 5 mg/kg were ineffective (ns).
dose did not modify the stimulating action of FMLP on InsP liberation by PMNs from healthy rats, although preincubation with 10 - 7 M niflumic acid and 3 x 10 -7 M NaF partly restored the stimulating effect of FMLP following burn injury. In contrast, neither drug exerted any effect on PMN oxidative metabolism at low concentrations, whereas niflumic acid (at concentrations higher than 3 x 10 -5 M) inhibited the PMN response to OZ and FMLP, as is generally observed with antiinflammatory concentrations of NSAIDs (22). According to Abramson et al. (23), neutrophil functions would be inhibited by NSAIDs by a disruption of G-protein-dependent events.
Effects of Niflumic Acid
12 oa 41 l 0 ~t
g '~ 0
Nil 0,l mg/kg
Fig. 6. Superoxide generatio:a by PMNs from healthy and burned rats (N = 6) (mean _+ SEM) treated or untreated (C) with ~iflumic acid (0.1 mg/kg) or NaF (0.05 mg/kg). Ceils were stimulated with OZ. The difference between healthy and burned controls was significant (P < 0.01) but not after treatment with Nif or NaF. The difference between treated burned animals and their own controls was significant (P < 0.01).
In vivo, niflumic acid and NaF had no effect on PI breakdown or oxidative metabolism in PMNs :~rom healthy rats, but partially (NaF) or completely (niflumic acid) restored the stimulating effects of F M L P and OZ on PI breakdown following thermal injury. Both substances, at F - equivalent doses, restored oxidative metabolism in PMNs from burned rats. It is well known that F M L P and OZ induce, after activation of their specific membrane receptor, raFid hydrolysis of PIP 2 by phospholipase C (PLC), via the activation of a G-protein, into the two intracellular messengers IP 3 and diacyl glycerol (DAG), which in turn activate protein kinase C (PKC) (18, 24). Fluoride anions are known to stimulate superoxide release by PMNs (25) by direct activation of various G-proteins, including that involved in PLC activation (13, 14, 26-28). However, it cannot be excluded that fluoride ions act on a signaling element that is not a G-protein. NaF would also appear to stimulate cytosolic PLC, followed by association o f the enzyme with the membrane (29). According to English et al. (13, 26), the reaction sequence triggered by F - involves specific hydrolysis of PIP 2, influx of extracellular Ca 2+, and activation of the superoxidegenerating enzyme. Our data show that NaF and niflumic acid stimulate PI breakdown in rat PMNs in vitro. At the concentrations used in our experiments, niflumic acid inhibited PG release. It has been reported that F - stimulates both the release of arachidonic acid (30) and PGI2 synthesis (31). On the other hand, F M L P and fluoride can stimulate both G S and Gi proteins that are linked to adenylate cyclase. According to Bokoch and Gilman (32), there is a synergistic effect between NaF
Tissot et al.
and the chemotactic peptide, and fluoride markedly stimulated both cAMP accumulation (by Gs) and arachidonate release (by Gi). The most prominent response to FMLP is G i activation, which in turn enhances the release of arachidonate. Fluoride can also activate phospholipase D (PLD) to release phosphatidic acid (PA) and DAG (33). Moreover, PKC and cAMP activation appear to inhibit inositol phospholipid turnover, at a step between G-protein and PLC (28, 34) or at the level of the receptor or receptor-G-protein complex, without inhibiting fluoride-G-protein and G-protein-PLC interactions (35). Brom et al. (29) found that prestimulation of PMNs by FMLP (10 -6 M) and subsequent addition of NaF (20 mM) led to a potentiation of the stimulating effect, whereas pretreatment with NaF inhibited the PMN response to FMLP. These results indicate a receptor down-regulation or an inhibition of the receptor-G-protein interaction. However, it must be noted that the concentrations used in the literature (5-20 mM, for 3-30 min) are far higher than those we used (10 -4- to 10-5-fold), and it is possible that our results are more the consequence of a "priming" effect than a direct activity on membrane or cytosolic elements (26, 28, 29, 36). In vivo, niflumic acid and NaF only had effects on PMNs from burned rats, restoring the stimulating effects of FMLP and OZ. Niflumic acid showed greater effects than NaF on PI metabolism but similar effects on the oxidative burst. Immunostimulating effects of NSAIDs have been described after burn injury (8) and Florentin et al. (10) have reported immunostimulating effects of niflumic acid in mice. Indomethacin, at appropriately weak doses, has been shown to stimulate FMLP-induced O~- generation (37), whereas antiinflammatory doses inhibit PMN functions. These effects have been attributed to the capacity of NSAIDs to inhibit cyclooxygenase and, thereby, the release of PG, which have immunosuppressive properties. In our experiments, the effective doses of niflumic acid were much lower than antiinflammatory doses but were sufficient to inhibit PG synthetases. Fluoride had similar effects on PI metabolism and O~- release but did not interfere with cyclooxygenase activity. Thus, inhibition of prostaglandin synthesis cannot alone explain the restoring effects of niflumic acid on PMN functions. These results are in accordance with those recently obtained in burned mice treated with indomethacin (38). The PMN defect acquired following burn injury would not appear to be due to decreased availability of chemoattractant receptors--increased FMLP binding is seen in thermally injured rabbit PMNs (39)--but rather to a malfunction in receptor processing. Some authors have described an antagonistic effect of NaF on certain actions of FMLP (40, 41), while others have reported a synergistic effect between NaF and FMLP (32, 42). It is possible that NaF acts on a regulatory factor that enhances the transformation of GDP into GTP (29). It is also possible that fluoride anions, which are able to activate G-proteins, could correct the uncou-
Effects of Niflumic Acid
p l i n g effects o f N S A I D s o n G - p r o t e i n - d e p e n d e n t e v e n t s . P a r t i c u l a r l y , a f t e r b u r n i n j u r y , f l u o r i d e ions c o u l d r e a c t i v a t e F M L P o r O Z m e m b r a n e r e c e p t o r s b y a " p r i m i n g " effect a n d t h e r e b y r e s t o r e t h e i r s t i m u l a t i n g effects. N i f l u m i c a c i d at n o n - a n t i i n t t a m m a t o r y d o s e s a n d N a F a p p e a r to h a v e c o m p a r a b l e effects o n P M N s t i m u l a t i n g - c o u p l i n g m e c h a n i s m s . H o w e v e r , n i f l u m i c acid is g e n e r a l l y m o r e p o t e n t t h a n N a F (at F - e q u i v a l e n t doses). F - c o u l d t h e r e f o r e b e i n v o l v e d in this a c t i v i t y , w h i c h is p r o b a b l y e n h a n c e d b y t h e rest o f the molecule. Acknowledgments--The authors wish to thank M. Lenoir, O. Muntaner, M. Semichon, and A. Thuret for their excellent technical assistance.
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Effects of Niflumic Acid
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