Study of the mechanism of inhibitory action of tranilast on chemical mediator release.

We investigated the mechanism of inhibitory action of tranilast on chemical mediator release by antigen-antibody reactions. Tranilast (10(-5)-10(-3) M) inhibited antigen (DNP-Ascaris)-induced histamine release from sensitized purified rat mast cells (PMC), but did not show an obvious influence on intracellular cyclic AMP. 45Ca uptake into PMC induced by antigen (300 micrograms/ml) was obviously suppressed by tranilast (10(-6)-10(-3) M). Tranilast (10(-4) M) inhibited antigen-induced histamine release from and 45Ca uptake into PMC independently of the presence or absence of glucose in the medium. On the other hand, 2-deoxyglucose (10(-2) M) markedly inhibited both responses in the absence but not in the presence of glucose. Tranilast slightly inhibited Ca-induced contraction of guinea pig taenia coli, but had no influence on aggregation of rabbit platelets. Verapamil (10(-6)-10(-4) M) had no effect on antigen-induced histamine release, but it markedly suppressed Ca-induced contraction and platelet aggregation. From these results, we suggest that the mechanism of inhibitory action of tranilast on the release of antigen-induced chemical mediator from mast cells involves the suppression of Ca uptake, but that its mode of action is apparently different from those of 2-deoxyglucose and verapamil.

N-(3, 4-dimethoxycinnamoyl) anthranilic acid (tranilast), one of the anti-allergic drugs, has been reported to inhibit the release of chemical mediators such as histamine and slow reacting substance of anaphylaxis (SRS A) by antigen-antibody reactions (1-9). Therefore, the clinical efficacies of tranilast on bronchial asthma (10), allergic dermatitis (11) and allergic rhinitis (12) are believed to be due to the inhibition of release of chemical mediators.
Although the effect of tranilast is distin guished from the drugs which increase in tracellular cyclic AMP (cAMP), disodium cromoglycate (DSCG) which increases in tracellular cAMP and inhibits the Ca uptake into cells and papaverine which acts on an energy-requiring process in the histamine release (5-7), the mechanisms of the inhi bitory action of tranilast on the release of chemical mediators induced by the antigen antibody reactions are not clarified yet. The concentrations at which tranilast inhibits the release of chemical mediators are approx. 10-5 to 10-3 M in various in vitro studies (3-9). Accordingly, the mechanisms of the inhi bitory action of tranilast may involve the sup pression of the same pathway involved in the release of chemical mediators.
It is generally known that mast cells bear IgE receptors and cross-linking of cell-bound IgE antibody molecules by multivalent ligands triggers the release of a variety of chemical mediators. The biochemical studies of the mechanisms of mast cell activation revealed that bridging of IgE receptors in duces phospholipid methylation, a transient increase in cAMP, mobilization of intracel lular Ca and Ca influx and enhancement of phosphatidylinositol turnover (13-15). Therefore, we investigated the effect of tranilast on the antigen-induced changes in intracellular cAM P level and Ca movement using rat purified mast cells (PMC).

Materials and Methods
Preparation of the suspension of PMC and peritoneal exudate cells (PEC) of rats a) Rat PMC: Murine hybridoma which secretes monoclonal IgE antibodies of anti 2, 4-dinitrophenyl (DNP) specificity was obtained by a fusion of X653Ag8 tumor cells and spleen cells from DNP-ascaris-hyperim munized BALB/c mice. The supernatants of murine hybridoma give a threshold reaction of the passive cutaneous anaphylaxis (PCA) in the rat at a dilution of 1/2000. The super natant was injected into the pleural and peri toneal cavities of male Wistar rats. After 16 hr, animals were exsanguinated by decapitation, and the exudate cells were harvested from the pleural and peritoneal cavities with a medium of the following composition: 150 mM NaCI, 3.7 mM KCI, 3.0 mM Na2HPO4-12H20, 3.5 mM KH2PO4, 0.9 MM CaC12.2H2O, 5.6 mM glucose, 0.1% bovine serum albumin (BSA, Sigma, ST. Louis, MO, U.S.A.) and 5 units/ml heparin, pH 6.9. The cell suspension was centrifuged at 35 x g for 10 min at O 'C and layered over 5 ml of 38% BSA solution as described by Sullivan et al. (16). The cells were allowed to settle for 20 min and centri fuged at 460xg for 20 min. Subsequently, mast cells with a purity approx. 90% were obtained from the BSA layer. b) Rat PEC: Male Wistar rats were sacri ficed by a blow on the head and exsanguina tion by decapitation. Rat PEC suspension was prepared as described by Nakazawa et al. (5) using a phosphate-buffered salt solution (PBS) containing 137 mM NaCI, 2.7 mM KCI, 1.8 mM CaC12, 1.0 MM MgC12.6H2O, 5.6 mM glucose, 5 units/ml heparin and 5% (v/v) sodium-potassium phosphate buffer (0.1 M, pH 7.2). Assay of histamine and cAM P The suspension of sensitized rat PMC was incubated for 5 min at 37°C, 200 or 300icg/ ml of DNP-Ascaris was added as an antigen, and then the cells were incubated for a further 30 min. The reaction mixture was cooled in an ice bath and then centrifuged. the separated supernatant was mixed with 4% HCIO4 and boiled for 3 min. After cen trifugation, the histamine content of the supernatant was measured by the method described by Shore et al. (17). The cell pellet was frozen in liquid nitrogen; the freezing and thawing procedure was repeated 3 times, and finally, the material was lyophilized. The lyophilizate was dissolved in distilled water. The cAM P level was measured using a cAM P Assay Kit (Yamasa, Choshi, Japan). Assay of 45Ca uptake Rat PMC was suspended in the Tyrode's solution containing 5.0 mM 4-(2-hydroxy ethyl)-1-piperazineethane sulfonic acid (HEPES, Sigma, St. Louis, MO, U.S.A.), 5.0 mM 2-(N-morpholino)-ethanesulfonic acid (MES, Sigma), 0.1% BSA and 5 units/ml heparin, pH 6.9. The suspension of rat PMC was set on silicon oil (d=1.05, Aldrich, Milwaukee, WI, U.S.A.) for 5 min at 37°C as described by Ishizaka et al. (13). Three ,uCi/ ml 45CaC12 (Amersham, Buchinghamshire, England) and 300 or +00 ug/ml of DNP Ascaris was simultaneously added to the cell suspension, and the reaction mixture was further incubated for 5 min at 37°C. Tris-A EDTA buffer, pH 7.6 (0.025 M Tris, 0.12 M NaCI, 5 mM KCI, 0.01 M EDTA and 0.02% human serum albumin (Miles, Elkhart, IN, U.S.A.)), was added to the mixture and then it was centrifuged to remove the supernatant. The radioactivities of 45Ca in the cell pellets were measured by a scintillation counter (Tri Carb 4640, Packard, Downers Grove, IL, U.S.A.). Ca-induced contraction of guinea pig taenia coli The taenia coli was isolated from a male guinea pig. The preparation was mounted at a load of 1 g in the Krebs' solution not con taining Ca (Ca(-) Krebs) and incubated at 28'C under aeration. After incubating for more than 1 hr, 1 mM of CaCl2 was added to the incubation medium. The contraction was isometrically recorded using force-displace ment transducer (Model SB-1T, Nihon Kohden, Tokyo, Japan). Aggregation of rabbit platelets Rabbit blood was collected from the auricular artery and mixed with one-tenth volume of 3.8% sodium citrate. From each blood sample, platelet-rich plasma (PRP) and platelet poor plasma (PPP) were prepared by centrifugation. Collagen (Horm, Munchen, W. Germany) as coagulant was added to PR P (0.45 ml). Platelet aggregation was moni tored using a platelet aggregation tracer (Model PAT-4A, Nikokizai, Tokyo, Japan). Test drugs Tranilast (Kissei, Matsumoto, Japan), verapamil (Sigma, St. Louis, MO, U.S.A.), DSCG (Fujisawa, Osaka, Japan), 2 deoxyglucose (Nakarai, Kyoto, Japan) and antimycin A (Boehringer Mannheim, Mannheim, W. Germany). Tranilast was dis solved in 1% NaHCO3 and other drugs were dissolved in the incubation medium or 0.9% saline. All test drugs were diluted to the desired concentration by the incubation medium or 0.9% saline. Drugs were added 1 min before the addition of antigen when testing for the effect on cAM P level, 45Ca uptake and histamine release, and they were added also 1 min before the addition of col lagen when testing for the effect on platelet aggregation. When testing for the effect on Ca-induced contraction of guinea pig ileum, drugs were added 5 min before the addition of CaCl2.

Effect
of tranilast on the release of histamine: As shown in Fig. 1, the histamine release from sensitized rat PMC by 300 fig/ml of DNP-Ascaris was 40%. Tranilast at 10-5 to 10-3 M suppressed the histamine release in a dose-dependent manner. The IC50 of tranilast is approx. 10-4 M. DSCG (10-3 M) inhibited the release of histamine by about 55%. Effect of tranilast on the release of hista mine and cAMP content in rat PMC: Figure  2 shows the relationship between the release of histamine and the intracellular cAMP level in sensitized rat PMC. When 300 fig/ml of DNP-Ascaris was added to rat PMC, the histamine release was induced. The release reaction was rapid until 1 min after the an tigen addition, and then it proceeded slowly. On the other hand, the intracellular cAMP decreased immediately after the antigen ad dition, then increased to a peak at 15 sec. The cAMP level restored to the initial control value 30 sec after and remained at this level afterwards. Although 10-5 to 10-3 M of tranilast markedly inhibited the release of histamine from rat in a dose-dependent manner, it had no apparent influence on the change in the intracellular cAMP.
Effect of tranilast and antimycin A on 45Ca uptake into rat PMC: Figure 3 shows the effect of tranilast and antimycin A on the 45Ca uptake into rat PMC by 300 ug/ml of DNP-Ascaris. The 45Ca uptake into the cells increased 5 min after the addition of antigen to about 2 times the value in the absence of antigen. Tranilast at concentrations of 10-6 to 10-3 M markedly suppressed the 45Ca uptake in a dose-dependent manner, but did not influence the spontaneous 45 Ca uptake (Fig. 4). Antimycin A at a concentration of 10-5 M obviously decreased the antigen induced 45Ca uptake into the cells. The degree of antigen-induced histamine release was similar regardless of the presence or absence of glucose in the medium (data not shown). The antigen induced histamine release from and 45Ca uptake into rat and PEC occurred in spite of the absence of glucose in the medium. As shown in Fig.S, 10-4M of tranilast suppressed histamine release and 45Ca uptake by 70 to 80% independently of the presence or absence of glucose. On the other hand, whereas 2 deoxyglucose, one of the metabolic inhibitors, slightly suppressed by 15 to 35% the hista mine release and 45Ca uptake in the presence of glucose, it markedly inhibited both re sponses in the absence of glucose (Fig. 5) Effect of tranilast and verapamil on the histamine release, Ca-induced contraction and platelet aggregation: Figure 6A shows the effect of tranilast and verapamil on the histamine release induced by 200 itg/ml of DNP-Ascaris from sensitized rat PEC. Tranilast (10-4 to 10-3 M) markedly inhibited the histamine release, but 10-6 to 10-4 M of verapamil had no influence on the histamine release.    CaCI2 (1 mM) caused a marked contrac tion of the guinea pig taenia coli immersed in the Ca(-) Krebs' solution (data not shown). As shown in Fig. 6B, tranilast at 10-4 M did not influence the Ca-induced contraction. However, at a concentration of 3x10-4 M or higher, it slightly inhibited the contraction. On the other hand, 10-8 to 10-5 M of verapamil markedly reduced the contraction in a dose-dependent manner. The aggregation of platelets in rabbit PRP induced by 10 to 20 ug/ml of collagen was hardly inhibited by tranilast even at 10-3 M. On the other hand, verapamil markedly sup pressed it at a concentration of 10-4 M (Fig.  6C).

Discussion
In early studies, 9-stimulants and phospho diesterase inhibitors which increase intracel lular cAMP were shown to inhibit the ana phylactic release of histamine and SRS-A from mixed cell systems (18,19). Therefore, it was noted that intracellular cAMP plays an important role in release of chemical medi ators. Recent biochemical studies of the mechanisms of mast cell activation have sug gested that IgE-receptor bridging enhances membrane phospholipid methylation and subsequently activates adenylate cyclase, fol lowed by the increase in cAMP and activation of cAMP-dependent protein kinase and modulation of the releasing process of chemical mediators (20)(21)(22). However, mediator releases induced by immunological and non-immunological stimulations were observed to be either suppressed, unaffected, or enhanced by cAMP-elevating stimulus (23). The role of cAMP in chemical mediator release is not known yet. In our present study, tranilast had no significant effect on intracel lular cAMP in spite of the significant inhi bition of the antigen-induced histamine release. A reciprocal correlation between the intracellular cAMP level and histamine release in rat PEC as described by Kubota et al. (6) was not seen in the present study using rat PMC. The time course of the change in the intracellular cAMP, as shown in Fig. 2, did not coincide with that described by Ishizaka and Ishizaka (20,21). A role of cAMP in the antigen-induced release of chemical media tors from mast cells appears to be unclear in this study. However, our data support which described that the mechanism of inhibitory action of tranilast, unlike 9-stimulants and phosphodiesterase inhibitors, has no relation to changes in intracellular cAMP level occurr ing in the process of release of chemical medi ators (24).
Calcium ion is necessary in the process of anaphylactic histamine release (25, 26). The essential event which initiates the release of chemical mediators is an increase in the level of free calcium ions in the cytosol which may be derived from the extracellular environment or mobilized from intracellular Ca stores. Therefore, it is suggested that the Ca uptake across the plasma membrane acts as a trigger for the releasing process. The antigen antibody reaction-induced histamine release from mast cells was inhibited up to 90% by inhibitors of glycolysis and oxidative phos phorylation, whereas the calcium influx under similar conditions is inhibited only by approx. 30% (14, 25). A combination of antimycin A (inhibitor of oxidative phosphorylation) and lanthanum (a competitive antagonist for Ca) reduced Ca uptake by 60% (14, 25). It ap pears that metabolic inhibitors suppress the energy-requiring Ca uptake into mast cells for the granule extrusion, and the inhibition of histamine release by lanthanum is the results of a competition between external membrane-bound Ca and lanthanum at an extracellular site which is thought to be the Ca channel (27,28). In our study, effects of antimycin A on the antigen-induced histamine release (data not shown) from and Ca uptake into mast cells were similar to results de scribed above (14). On the other hand, tranilast suppressed the Ca uptake by about 90% (Fig. 3). Further, tranilast was apparently different from 2-deoxyglucose (an inhibitor of glycolysis) in the mode of inhibitory action on the antigen-induced histamine release and Ca uptake (Fig. 5). Therefore, we suggest that the inhibition of Ca uptake into mast cells by tranilast is different from that of metabolic inhibitors and lanthanum, and that tranilast suppresses the energy-requiring and cell membrane-bound Ca uptake into mast cells in antigen-antibody reactions.
The effect of verapamil, one of the voltage dependent Ca channel blockers, on the release of chemical mediators in immediate hypersensitive reactions is not clear yet (29). Indeed, verapamil did not suppress the IgE dependent histamine release in our study. Effects of verapamil on the histamine release, the Ca-induced contraction and the platelet aggregation are obviously different from those of tranilast. Therefore, the Ca permeability through the plasma membrane of mast cells seems to be different from that pertinent to smooth muscle contraction and platelet ag gregation, and the mechanism of Ca uptake in mast cells may not be related to the voltage dependent Ca channels.
It has been suggested that the intracellular Ca source is utilized in the histamine release from mast cells (30). Recently, it was demon strated that the dissociation of arachidonic acid from membrane phospholipids is induced by the activation of not only phospholipase A2 but also phospholipase C. The latter is one of the enzymes catalyzing the phosphatidyl inositol cycle and requires Ca, especially intracellular Ca (31). Therefore, to examine the mechanism of the inhibitory action of tranilast on the release of arachidonic acid and histamine, it will be necessary to in vestigate the effect of this drug on the mobilization of intracellular Ca in the future.