Inhibitory Effect of Tritoqualine (TRQ) on Histamine Release from Mast Cells

Kohei UMEZU, Satoshi YUASA, Atsuko SUDOH, Ryoji KIKUMOTO and Atsushi ICHIKAWA*

Biosciences Laboratory, Research Center, Mitsubishi Chemical Ind. Ltd. , 1000 Kamoshida, Midori-ku, Yokohama 227, Japan *Department of Health Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Yoshida, Sakyo-ku. Kyoto 606, Japan

Accepted February 28, 1985

Abstract-Tritoqualine (TRO), used clinically as an antiallergic drug, did not inhibit histidine decarboxylase activity (HDC, EC. 4.1.1.22.) partially purified from fetal rats and the enzymes prepared from mastocytoma P-815 cells. However, TRQ inhibited the histamine release from rat peritoneal mast cells induced by compound 48/80 and ATP. TRQ was also effective in inhibiting antigen-induced histamine release in rat mast cells sensitized actively or passively by the homologous anti DNP-Ascaris antibody. Preincubation of cultured mastocytoma P-815 cells in a medium including TRQ inhibited non-cytotoxically the histamine release of mas tocytoma cells induced by compound 48/80, and the effect of TRQ became more marked with lengthening of the culture period in the presence of TRQ. It was concluded from these results that one of the main actions of TRO as an antiallergic drug was not the inhibitory action on HDC, but might be ascribed to its inhibitory effect on histamine release from mast cells.

Tritoqualine (TRQ), the chemical structure inflammation models (9, 10). However, of which is shown in Fig. 1, has been clinically conflicting results have also been reported used on patients with allergic disorders such suggesting that the content of tissue hista as pollinosis, asthma and urticaria (1-5) . mine was not changed after TRQ adminis Since Parrot (6, 7) and Carpi (8) first tration (11, 12). In spite of the important demonstrated the inhibitory action of TRQ role of mast cells in allergic histamine on histidine decarboxylase (HDC) in the metabolism, there have been fewer investi guinea pig kidney, the main mechanism of gations about the effect of TRQ on the action of TRQ as an antiallergic drug has been histidine decarboxylase of the mast cell type proposed to involve the inhibition of histamine which is suggested to be specifically involved biosynthesis in mast cells. The inhibitory in histamine biosynthesis in mast cells (13) action of TRO on tissue histamine content and histamine release by stimulants from has been shown in several experimental mast cells. The present study was carried out in order to clarify these two questionable points. The present results showed that TRQ inhibited the release mechanism of histamine prom mast cells, but not HDC activities prepared from rat fetal tissues and mouse mastocytoma cells.

Materials and Methods HDC from fetal rats and mastocytoma P Fig. 1. Chemical structure of tritoqualine (TRQ). 815 cells and HDC assay: HDC was partially purified from fetal rats according to obtained from the peritoneal cavity fluid of Hakanson's method (14), and it was ob male Wistar rats weighing 300-400 g, and tained from mastocytoma P-815 cells ac these cells including other cells were used as cording to Wada's method (13). Briefly, 5 rat they were or after purification by Ficoll fetuses were homogenized in 200 ml of 0.1 M density gradient centrifugation as described acetate buffer (pH 5.5), and the homogenate previously (16). Purity of mast cells was was then centrifuged. After the supernatant over 90%. After mast cells (0.5-2.O x 104 was treated with heat at 55'C for 5 min, the cells/tube) were preincubated for 10 min crude HDC was precipitated at 25-45% with TAO or vehicle, one of the histamine ammonium sulfate saturation. The precipitant releasers was added to the reaction mixture, was redissolved and dialyzed against 50 mM and 10 min later, the reaction was stopped by phosphate buffer (pH 7.0) and used as a cooling the mixture in an ice-bath, followed crude HDC (final volume was 11.2 ml). by centrifugation at 500 g for 1 min. The Mastocytoma P-815 cells (90 ml packed histamine released in the supernatant fraction cell volume) collected from the ascites of was measured by the methods of Shore (17) 200 mice were homogenized, centrifuged, and Hakanson (18) with some modifications. and the crude HDC precipitated by 30-55% Preparation of DNP-Ascaris and sensi ammonium sulfate saturation of the super tization: 2,4-Dinitrophenyl-coupled ascaris natant was successively chromatographed on extract (DNP-Ascaris) and rat anti-DNP columns of DEAE-Sephadex CL-6B, hy Ascaris antibody were prepared by the droxyapatite, Sephacryl S-300, carnosine procedure of Azuma et al. with some sepharose 4B, and again on DEAE-Sepharose modifications (19). Rat peritoneal mast cells CL-6B. The whole procedure resulted in an were sensitized with rat anti-DNP-Ascaris overall 2,000-fold purification of the enzyme. antibody by two methods, in vivo and in The final enzyme preparation produced a vitro. Sensitization in vitro was done by single protein band on polyacrylamide gel mixing mast cells (8 X 106) with 4 ml of rat electrophoresis. HDC activity was assayed anti-DNP-Ascaris antibody (titer 1/256) and using 14C-histidine by the procedure of incubating it at 37°C for 60 min. After Kobayashi (15) with some modification. washing, cells were preincubated with phos Briefly, the assay mixture (500 III) contained phatidylserine (10 W) for 5 min; then in the 0.25 mmole of sodium potassium phosphate presence of TRQ (10 W), then cells were buffer (pH 6.9), 0.1 Pmole of pyridoxal-5 incubated 10 min more, and DNP-Ascaris phosphate, 0.25 iimole of histidine containing was added as an antigen. After 20 min, 0.1 zeCi of 14C-histidine, the enzyme fraction histamine released from mast cells was and distilled water. The assay mixture was measured as described above. In vivo incubated at 37'C for 20-60 min, and the sensitization was conducted by intraperi reaction was stopped by adding 0.5 ml of toneally injecting 0.6 ml of rat anti-DNP 2N HCI, followed by a 30 min incubation to Ascaris antibody (titer 1/256) to male Wistar absorb the CO2 on filter paper containing rats weighing 200-300 g. Sensitized mast 0.2 ml of ethanolamine. Radioactivity was cells obtained from the peritoneal cavity determined in a Beckman LS 100 scintillation fluid after 45 hr of the antibody injection counter after adding toluene-ethanol (7:3) were treated by the same procedure described with 0.4% 2,5-diphenyl oxazole (PPO) and above and were used for histamine release by 0.01% 1,4-bis-, 2-(4-methyl-5-phenyloxa antigen challenge. zolyl) benzene (DM-POPOP). TRO was Histamine release from mastocytoma P dissolved in 0.1 N HCI with 30 ppm 815 cells by compound 48/80: Mouse t-butylated hydroxyanisole (BHA) at 0.3-5 mastocytoma P-815 cells (2-4 x 105 cells/ mM to prevent the oxidation of TRQ or ml in 500 ml flask) were maintained in without BHA, and TRQ was added in the suspension culture at 37'C in Fischer's assay mixture at final concentrations of medium supplemented with 5% fetal calf 3-50 /NM. serum (20). Incubation with or without TRQ Preparation of mast cells: Mast cells were was continued by exchanging half of the culture medium containing the growing cells Medichemie AG (Ettingen, Switzerland). with the same amount of fresh medium Ficoll, DEAE-Sepharose CL-6B, Sephacryl S including TRQ (10 /CM) or vehicle every 24 300 and carnosine-Sepharose 4B were hr for 4 days. Mastocytoma P-815 cells purchased from Pharmacia (Uppsala, (2-2.5 x107 cells) were harvested by Sweden). Histidine, L-(carboxy-14C), 30 centrifuging 50 ml of the suspension and mCi/mmol was purchased from New washing the cells three times with phosphate England Nuclear. MI-063 (3,5 dichlor-2,4 buffered saline (PBS) at the indicated time. dihydroxy benzanilide) was synthesized by Mastocytoma cells (4-6X106 cells) in 1 ml Mitsubishi Chemical Ind., Ltd., according to of PBS were incubated for 10 min after Umezawa's method (22). 7-Amino, 3 treatment with 50 gig of compound 48/80, hydroxy, 4,5,6-triethoxyphthalide (HOEAP) and released histamine was measured by the was also synthesized by Mitsubishi Chemical same procedure described above. The cell Ind., Ltd. All other chemicals were analytical number was counted by a Coulter model Z grade preparations obtained from commercial counter (Coulter Electronics, Hialeah, FL, sources. U.S.A.) (21). Elimination of TRQ from histamine: TRQ Results showed strong fluorescence in the emission Inhibitory effect of TRQ on HDC: As and excitation wavelengths used for the shown in Table 1, TRQ did not show any fluorometric assay of histamine. Diaion HP-20 significant inhibitory action on HDC activity (Mitsubishi Chemical Ind., Ltd.) was used to prepared from fetal rats at concentrations in separate TRQ from histamine, as TRQ was the range of 10 to 50 ,FPM.No inhibitory effect bound on the resin completely and the 0 was found when crude and completely phthalaldehyde complex of histamine passed purified HDC from mastocytoma cells was through this resin column. Recovery of used. Compound MI-063, which has been histamine was over 95%. proven to be a strong inhibitor of HDC (22), Chemicals: TRQ was purchased from inhibited HDC activity almost 100% at

Table 1. Inhibitory effect of TRQ on histidine decarboxylase prepared from fetal rats and mastocytoma P-815 cells 0.15 W. As TRO was partially decomposed from rat peritoneal mast cells induced by to cotarnine and HOEAP by oxygen dissolved compound 48/80 (Fig. 2A), ATP (Fig. 2B) in water, the possibility of these products and DNP-Ascaris (Fig. 3). IC50 values of inhibiting HDC activity was examined. None histamine release by compound 48/80 and of them had any inhibitory action (data not ATP are 10 tiM and 13 tiM, respectively. As shown). shown in Fig. 2A, cell injury by TRQ itself Effect of TRQ on histamine release from was not observed at concentrations of 1-33 rat peritoneal mast cells: Results presented in ,uM. On the histamine release by Ca ionophore Figs. 2 and 3 show that TRQ had dose A23187, TRQ did not show any inhibitory related inhibitory activity on histamine release effect (data not shown). There was no

Fig. 2. Effect of TRQ on histamine release by stimulants. A: Inhibitory effect of TRQ on histamine release by compound 48/80 from rat peritoneal mast cells. Ordinate: relative histamine released against total histamine contained in mast cells. Abscissa: concentration of TRQ (<): The change of histamine release after compound 48/80 was added (0.1 ,ug/ml). (s): The change of histamine release under the condition of no compound 48/80. B: Inhibitory effect of TRO on histamine release by ATP (0.3 mM) from mast cells of rats.

Fig. 3. Inhibitory effect of TRQ on histamine release by DNP-Ascaris antigen from mast cells of rats. Mast cells were sensitized according to the procedure described in Methods. A represents in vitro sensitization and B represents in vivo sensitization. The concentration of TRO was 10 tM in each experiment. White and dotted columns represent control and TRO treatment, respectively. PS means phosphatidyl serine. Significantly different from each control, *P<0.5, **P<0.01. inhibitory action of TRQ metabolites, histamine release from mastocytoma P-81 5 cotarnine or HOEAP, in the same system in cells was inhibited about 90% compared vitro (data not shown). with the vehicle control. Under this culturing Effect of TRQ on histamine release from condition with TRQ, doubling time and cell cultured mastocytoma P-81 5 cells stimulated size of mastocytoma P-81 5 cells were not by compound 48/80: Mouse mastocytoma P influenced by treatment of TRO (Fig. 4). 815 cells in the exponential growth phase, as The histamine content in mastocytoma P compared with rat peritoneal mast cells, 815 cells maintained 24 hr longer in the contain a low level of histamine and are culture was 1.8-2.2 /cg/108 cells, and TRQ much less likely to release histamine by the treated cells had almost the same histamine stimulation of compound 48/80. To obtain content (2.1-2.2 /cg/108 cells). No sig the reproducible release of histamine (about nificant difference was observed (Table 2). 20% release of total histamine), at least 50 iig of compound 48/80 was required (Fig. 4). Discussion Though at 2 hr there was no difference Since it has been reported that TRQ between control and TRQ-treated masto inhibited HDC in vitro (6, 7), some researchers cytoma P-81 5 cells with respect to histamine have identified this compound from their release by compound 48/80, at the 4th day, in vivo and in vitro experiments as a phar macological tool to decrease histamine con tent in tissues (9, 10). On the contrary, Schayer and Reilly reported that TRQ was ineffective in altering endogenous histamine formation in the stomachs of mice and rats or rather caused a significant increase in histamine formation in the skin of mice after treatment of TRO (12). The inconsistent findings prompted us to carry out the present study with the aim of understanding the pharmacological mechanism of TRQ as an antiallergic drug. The synthesis of histamine from L histidine can be catalyzed in vitro by either of the two enzymes, the specific histidine decarboxylase and the nonspecific aromatic Fig. 4. Time course change of response of mas L-amino acid decarboxylase. The specific tocytoma P-81 5 ce!ls against compound 48/80. decarboxylase has been purified from fetal Each value represents the mean and S.E. of 4 samples. rat tissues and studied in detail by Hakanson Ordinate: relative % of histamine released from (14) and Watanabe et al. (13). The non mastocytoma cells. Abscissa: time course. Insertion represents the time course change of the cell size specific enzyme has been purified from and generation time of mastocytoma P-81 5 cells. guinea pig kidney and studied by Lovenberg (0): control, (0) : TRQ treated. *,**: statistically et al. (23). It was concluded that of these significant from the control at P<0.05 and 0.01 , enzymes, only the specific enzyme functioned respectively. significantly in vivo to form histamine and

Table 2. Changes in histamine content of mastocytoma P-815 cells incubated for 4 days that the aromatic amino acid decarboxylase that by the antigen DNP-Ascaris, but those was not so important because of its low V,,,,,, by other chemical substances such as com and very low affinity for histidine (24). pound 48/80 and ATP. The IC50 values of There are high HDC activities in the TRQ for the histamine release induced by stomach, the whole fetus and the brain as compound 48/80 and ATP were 10 /cM and well as mast cells in rats. Watanabe et al. 13 W, respectively (Figs. 2 and 3). In reported that there are at least two isozymes addition, we observed the actions of TRQ to of HDC, HDC in the fetus which belongs to inhibit the Ca influx into mast cells and to the mast cell type and HDC in the brain which inhibit the phospholipid turn over in the belongs to the non-mast cell type (13). membrane of mast cells by measuring 32P Since it is known that it is very difficult to or 14C-arachidonic acid metabolisms (to be purify HDC from mast cells because HDC reported elsewhere). To investigate the becomes labile once cellular integrity is contact effect of TRQ with cell membranes, lost (25), we used HDC purified from fetal mastocytoma P-81 5 cells were cultured with rats and mastocytoma cells for estimating the TRQ during the period from 2 hr to 6 days, inhibitory effect of TRQ on HDC activity. As and it was observed that the histamine shown in Table 1, TRQ did not inhibit HDC release response of mastocytoma P-81 5 activity when prepared from fetal rats in cells by compound 48/80 decreased, concentrations from 10 to 50 1nM. Cotarnine depending on the incubation time or contact and HOEAP which are decomposition pro time with TRQ. Under this condition, no ducts of TRQ did not show any inhibitory change was observed in the doubling time effect in this assay system. The same result of cells, the cell sizes and the histamine was obtained using the crude and purified content between mastocytoma P-81 5 cells HDC from mastocytoma cells. MI-063 used incubated in the presence of TRO and the as a reference drug showed a very strong nontreated cells (Fig. 4 and Table 2), inhibition in both HDC tests, showing that indicating that there was no direct cell our assay systems were working well. toxicity by TRQ. The result that the histamine These results were inconsistent with those of content did not change in the TRQ treated previous reports (6, 7). Parrot used the cells indicated that the histamine biosynthesis guinea pig kidney as an enzyme source for was not inhibited with the treatment of TRQ. histamine synthesis and assayed the HDC Decrease in the mastocytoma P-81 5 cells activity by means of measuring the amount against compound 48/80 might be derived of histamine produced in the incubation from the increment of accumulated TRQ in mixture with a bioassay using an ileum the cell membrane. As TRQ is a hydrophobic preparation. As already mentioned, the main compound, it may be accumulated in the enzyme involved in decarboxylating histidine membrane with the increase of contact time and forming histamine in the kidney might be with cells. However, further investigation is the I-aromatic amino acid decarboxylase, and needed to prove whether the action of TRQ not HDC. The difference between our result is restricted to mastocytoma cells only or is and that of Parrot may be derived from the observed commonly in other cells. different source of the enzymes. In addition, Thus, it seems reasonable to postulate TRQ has a nonspecific inhibitory action on from these results that the action mechanisms the smooth muscle contraction induced by of TRQ as an antiallergic drug might be the acetylcholine, serotonin and histamine at inhibitory effect on histamine release from concentrations above 20 /iM (data not mast cells, rather than the inhibition on HDC shown). Therefore, the possibility that con activity. taminating TRQ in the assay medium inhibits Some researchers reported the other effects the ileum contraction is not completely of TRQ except the antiallergic action, for ruled out. 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