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Inhibitory Effect of Tritoqualine (TRQ) on Histamine Release from Mast Cells
Inhibitory
Effect
of Tritoqualine
Release
from
(TRQ) on Histamine
Mast
Cells
Kohei UMEZU, Satoshi YUASA, Atsuko SUDOH, and Atsushi ICHIKAWA* Biosciences
Laboratory, 1000
*Department Kyoto
Research
Kamoshida, of
Health
Center,
Midori-ku, Chemistry,
University,
Faculty
Yoshida,
Accepted
Mitsubishi Yokohama of
Sakyo-ku. February
28,
Ryoji KIKUMOTO Chemical
227,
Pharmaceutical Kyoto
606,
Ind.
Ltd. ,
Japan Sciences, Japan
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 of which is shown in Fig. 1, has been clinically used on patients with allergic disorders such as pollinosis, asthma and urticaria (1-5) . Since Parrot (6, 7) and Carpi (8) first demonstrated the inhibitory action of TRQ on histidine decarboxylase (HDC) in the guinea pig kidney, the main mechanism of action of TRQ as an antiallergic drug has been proposed to involve the inhibition of histamine biosynthesis in mast cells. The inhibitory action of TRO on tissue histamine content has been shown in several experimental
inflammation models (9, 10). However, conflicting results have also been reported suggesting that the content of tissue hista mine was not changed after TRQ adminis tration (11, 12). In spite of the important role of mast cells in allergic histamine metabolism, there have been fewer investi gations about the effect of TRQ on the histidine decarboxylase of the mast cell type which is suggested to be specifically involved in histamine biosynthesis in mast cells (13) and histamine release by stimulants from 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
Fig. 1.
Chemical structure of tritoqualine
(TRQ).
and Methods
HDC from fetal rats and mastocytoma P cells and HDC assay: HDC was partially
815
purified from fetal rats according to Hakanson's method (14), and it was ob tained from mastocytoma P-815 cells ac cording to Wada's method (13). Briefly, 5 rat fetuses were homogenized in 200 ml of 0.1 M acetate buffer (pH 5.5), and the homogenate was then centrifuged. After the supernatant was treated with heat at 55'C for 5 min, the crude HDC was precipitated at 25-45% ammonium sulfate saturation. The precipitant was redissolved and dialyzed against 50 mM phosphate buffer (pH 7.0) and used as a crude HDC (final volume was 11.2 ml). Mastocytoma P-815 cells (90 ml packed cell volume) collected from the ascites of 200 mice were homogenized, centrifuged, and the crude HDC precipitated by 30-55% ammonium sulfate saturation of the super natant was successively chromatographed on columns of DEAE-Sephadex CL-6B, hy droxyapatite, Sephacryl S-300, carnosine sepharose 4B, and again on DEAE-Sepharose CL-6B. The whole procedure resulted in an overall 2,000-fold purification of the enzyme. The final enzyme preparation produced a single protein band on polyacrylamide gel electrophoresis. HDC activity was assayed using 14C-histidine by the procedure of Kobayashi (15) with some modification. Briefly, the assay mixture (500 III) contained 0.25 mmole of sodium potassium phosphate buffer (pH 6.9), 0.1 Pmole of pyridoxal-5 phosphate, 0.25 iimole of histidine containing 0.1 zeCi of 14C-histidine, the enzyme fraction and distilled water. The assay mixture was incubated at 37'C for 20-60 min, and the reaction was stopped by adding 0.5 ml of 2N HCI, followed by a 30 min incubation to absorb the CO2 on filter paper containing 0.2 ml of ethanolamine. Radioactivity was determined in a Beckman LS 100 scintillation counter after adding toluene-ethanol (7:3) with 0.4% 2,5-diphenyl oxazole (PPO) and 0.01% 1,4-bis-, 2-(4-methyl-5-phenyloxa zolyl) benzene (DM-POPOP). TRO was dissolved in 0.1 N HCI with 30 ppm t-butylated hydroxyanisole (BHA) at 0.3-5 mM to prevent the oxidation of TRQ or without BHA, and TRQ was added in the assay mixture at final concentrations of 3-50 /NM. Preparation of mast cells: Mast cells were
obtained from the peritoneal cavity fluid of male Wistar rats weighing 300-400 g, and these cells including other cells were used as they were or after purification by Ficoll density gradient centrifugation as described previously (16). Purity of mast cells was over 90%. After mast cells (0.5-2.O x 104 cells/tube) were preincubated for 10 min with TAO or vehicle, one of the histamine releasers was added to the reaction mixture, and 10 min later, the reaction was stopped by cooling the mixture in an ice-bath, followed by centrifugation at 500 g for 1 min. The histamine released in the supernatant fraction was measured by the methods of Shore (17) and Hakanson (18) with some modifications. Preparation of DNP-Ascaris and sensi tization: 2,4-Dinitrophenyl-coupled ascaris extract (DNP-Ascaris) and rat anti-DNP Ascaris antibody were prepared by the procedure of Azuma et al. with some modifications (19). Rat peritoneal mast cells were sensitized with rat anti-DNP-Ascaris antibody by two methods, in vivo and in vitro. Sensitization in vitro was done by mixing mast cells (8 X 106) with 4 ml of rat anti-DNP-Ascaris antibody (titer 1/256) and incubating it at 37°C for 60 min. After washing, cells were preincubated with phos phatidylserine (10 W) for 5 min; then in the presence of TRQ (10 W), then cells were incubated 10 min more, and DNP-Ascaris was added as an antigen. After 20 min, histamine released from mast cells was measured as described above. In vivo sensitization was conducted by intraperi toneally injecting 0.6 ml of rat anti-DNP Ascaris antibody (titer 1/256) to male Wistar rats weighing 200-300 g. Sensitized mast cells obtained from the peritoneal cavity fluid after 45 hr of the antibody injection were treated by the same procedure described above and were used for histamine release by antigen challenge. Histamine release from mastocytoma P 815 cells by compound 48/80: Mouse mastocytoma P-815 cells (2-4 x 105 cells/ ml in 500 ml flask) were maintained in suspension culture at 37'C in Fischer's medium supplemented with 5% fetal calf serum (20). Incubation with or without TRQ was continued by exchanging half of the
culture medium containing the growing cells with the same amount of fresh medium including TRQ (10 /CM) or vehicle every 24 hr for 4 days. Mastocytoma P-815 cells (2-2.5 x107 cells) were harvested by centrifuging 50 ml of the suspension and washing the cells three times with phosphate buffered saline (PBS) at the indicated time. Mastocytoma cells (4-6X106 cells) in 1 ml of PBS were incubated for 10 min after treatment with 50 gig of compound 48/80, and released histamine was measured by the same procedure described above. The cell number was counted by a Coulter model Z counter (Coulter Electronics, Hialeah, FL, U.S.A.) (21). Elimination of TRQ from histamine: TRQ showed strong fluorescence in the emission and excitation wavelengths used for the fluorometric assay of histamine. Diaion HP-20 (Mitsubishi Chemical Ind., Ltd.) was used to separate TRQ from histamine, as TRQ was bound on the resin completely and the 0 phthalaldehyde complex of histamine passed through this resin column. Recovery of histamine was over 95%. Chemicals: TRQ was purchased from Table P-815
1. cells
Inhibitory
effect
of TRQ
on
histidine
Medichemie AG (Ettingen, Switzerland). Ficoll, DEAE-Sepharose CL-6B, Sephacryl S 300 and carnosine-Sepharose 4B were purchased from Pharmacia (Uppsala, Sweden). Histidine, L-(carboxy-14C), 30 mCi/mmol was purchased from New England Nuclear. MI-063 (3,5 dichlor-2,4 dihydroxy benzanilide) was synthesized by Mitsubishi Chemical Ind., Ltd., according to Umezawa's method (22). 7-Amino, 3 hydroxy, 4,5,6-triethoxyphthalide (HOEAP) was also synthesized by Mitsubishi Chemical Ind., Ltd. All other chemicals were analytical grade preparations obtained from commercial sources. Results Inhibitory effect of TRQ on HDC: As shown in Table 1, TRQ did not show any significant inhibitory action on HDC activity prepared from fetal rats at concentrations in the range of 10 to 50 ,FPM.No inhibitory effect was found when crude and completely purified HDC from mastocytoma cells was used. Compound MI-063, which has been proven to be a strong inhibitor of HDC (22), inhibited HDC activity almost 100% at
decarboxylase
prepared
from
fetal
rats
and
mastocytoma
from rat peritoneal mast cells induced by compound 48/80 (Fig. 2A), ATP (Fig. 2B) and DNP-Ascaris (Fig. 3). IC50 values of histamine release by compound 48/80 and ATP are 10 tiM and 13 tiM, respectively. As shown in Fig. 2A, cell injury by TRQ itself was not observed at concentrations of 1-33 ,uM. On the histamine release by Ca ionophore A23187, TRQ did not show any inhibitory effect (data not shown). There was no
0.15 W. As TRO was partially decomposed to cotarnine and HOEAP by oxygen dissolved in water, the possibility of these products inhibiting HDC activity was examined. None of them had any inhibitory action (data not shown). Effect of TRQ on histamine release from rat peritoneal mast cells: Results presented in Figs. 2 and 3 show that TRQ had dose related inhibitory activity on histamine release
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
Mast cells sensitization
effect
of TRQ
on histamine
release
by DNP-Ascaris
antigen
from
mast cells
of rats.
were sensitized according to the procedure described in Methods. A represents in vitro and B represents in vivo sensitization. The concentration of TRO was 10 tM in each
experiment.
White
phosphatidyl
serine.
and dotted Significantly
columns
represent
different
control
from each
and TRO treatment,
control,
*P<0.5,
respectively.
**P<0.01.
PS means
inhibitory action of TRQ metabolites, cotarnine or HOEAP, in the same system in vitro (data not shown). Effect of TRQ on histamine release from cultured mastocytoma P-81 5 cells stimulated by compound 48/80: Mouse mastocytoma P 815 cells in the exponential growth phase, as compared with rat peritoneal mast cells, contain a low level of histamine and are much less likely to release histamine by the stimulation of compound 48/80. To obtain the reproducible release of histamine (about 20% release of total histamine), at least 50 iig of compound 48/80 was required (Fig. 4). Though at 2 hr there was no difference between control and TRQ-treated masto cytoma P-81 5 cells with respect to histamine release by compound 48/80, at the 4th day,
Fig. 4. Time course change of response of mas tocytoma P-81 5 ce!ls against compound 48/80. Each value represents the mean and S.E. of 4 samples. Ordinate: relative % of histamine released from mastocytoma cells. Abscissa: time course. Insertion represents the time course change of the cell size and generation time of mastocytoma P-81 5 cells. (0): control, (0) : TRQ treated. *,**: statistically significant from the control at P<0.05 and 0.01 , respectively. Table
2.
Changes
in histamine
content
of
histamine release from mastocytoma P-81 5 cells was inhibited about 90% compared with the vehicle control. Under this culturing condition with TRQ, doubling time and cell size of mastocytoma P-81 5 cells were not influenced by treatment of TRO (Fig. 4). The histamine content in mastocytoma P 815 cells maintained 24 hr longer in the culture was 1.8-2.2 /cg/108 cells, and TRQ treated cells had almost the same histamine content (2.1-2.2 /cg/108 cells). No sig nificant difference was observed (Table 2). Discussion Since it has been reported that TRQ inhibited HDC in vitro (6, 7), some researchers have identified this compound from their 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 L-amino acid decarboxylase. The specific decarboxylase has been purified from fetal rat tissues and studied in detail by Hakanson (14) and Watanabe et al. (13). The non specific enzyme has been purified from guinea pig kidney and studied by Lovenberg et al. (23). It was concluded that of these enzymes, only the specific enzyme functioned significantly in vivo to form histamine and
mastocytoma
P-815
cells
incubated
for
4 days
that the aromatic amino acid decarboxylase was not so important because of its low V,,,,,, and very low affinity for histidine (24). There are high HDC activities in the stomach, the whole fetus and the brain as well as mast cells in rats. Watanabe et al. reported that there are at least two isozymes of HDC, HDC in the fetus which belongs to the mast cell type and HDC in the brain which belongs to the non-mast cell type (13). Since it is known that it is very difficult to purify HDC from mast cells because HDC becomes labile once cellular integrity is lost (25), we used HDC purified from fetal rats and mastocytoma cells for estimating the inhibitory effect of TRQ on HDC activity. As shown in Table 1, TRQ did not inhibit HDC activity when prepared from fetal rats in concentrations from 10 to 50 1nM. Cotarnine and HOEAP which are decomposition pro ducts of TRQ did not show any inhibitory effect in this assay system. The same result was obtained using the crude and purified HDC from mastocytoma cells. MI-063 used as a reference drug showed a very strong inhibition in both HDC tests, showing that our assay systems were working well. These results were inconsistent with those of previous reports (6, 7). Parrot used the guinea pig kidney as an enzyme source for histamine synthesis and assayed the HDC activity by means of measuring the amount of histamine produced in the incubation mixture with a bioassay using an ileum preparation. As already mentioned, the main enzyme involved in decarboxylating histidine and forming histamine in the kidney might be the I-aromatic amino acid decarboxylase, and not HDC. The difference between our result and that of Parrot may be derived from the different source of the enzymes. In addition, TRQ has a nonspecific inhibitory action on the smooth muscle contraction induced by acetylcholine, serotonin and histamine at concentrations above 20 /iM (data not shown). Therefore, the possibility that con taminating TRQ in the assay medium inhibits the ileum contraction is not completely ruled out. From our results, it was concluded that TRQ does not inhibit HDC. TRQ inhibited the histamine release from mast cells by various stimulants, not only
that by the antigen DNP-Ascaris, but those by other chemical substances such as com pound 48/80 and ATP. The IC50 values of TRQ for the histamine release induced by compound 48/80 and ATP were 10 /cM and 13 W, respectively (Figs. 2 and 3). In addition, we observed the actions of TRQ to inhibit the Ca influx into mast cells and to inhibit the phospholipid turn over in the membrane of mast cells by measuring 32P or 14C-arachidonic acid metabolisms (to be reported elsewhere). To investigate the contact effect of TRQ with cell membranes, mastocytoma P-81 5 cells were cultured with TRQ during the period from 2 hr to 6 days, and it was observed that the histamine release response of mastocytoma P-81 5 cells by compound 48/80 decreased, depending on the incubation time or contact time with TRQ. Under this condition, no change was observed in the doubling time of cells, the cell sizes and the histamine content between mastocytoma P-81 5 cells incubated in the presence of TRO and the nontreated cells (Fig. 4 and Table 2), indicating that there was no direct cell toxicity by TRQ. The result that the histamine content did not change in the TRQ treated cells indicated that the histamine biosynthesis was not inhibited with the treatment of TRQ. Decrease in the mastocytoma P-81 5 cells against compound 48/80 might be derived from the increment of accumulated TRQ in the cell membrane. As TRQ is a hydrophobic compound, it may be accumulated in the membrane with the increase of contact time with cells. However, further investigation is needed to prove whether the action of TRQ is restricted to mastocytoma cells only or is observed commonly in other cells. Thus, it seems reasonable to postulate from these results that the action mechanisms of TRQ as an antiallergic drug might be the inhibitory effect on histamine release from mast cells, rather than the inhibition on HDC activity. Some researchers reported the other effects of TRQ except the antiallergic action, for instance, the application of TRO for rat hepatoma induced by dimethylamino azobenzene (26) and clinical application of TRQ for chronic hepatitis (27). Though they
ascribed the effect of TRIO to its HDC inhibitory action, it will be necessary to reconsider the mechanisms of TRIO, judging from our results. References 1 Von Andres, H.U. and Krebs, A.: Behandelung allergischer Hautkrankheiten mit Hypostamine (Tritoqualine) unter besonderer Berucksichtig ung des Pruritus. Praxis 57, 536-539 (1968) 2 Hohlbrugger, H.: Erfahrungen mit Hypostamine beim heuschnupfen and anderen allergischen Erkrankungen der oberen Luftwege. Therapie woche 18, 1357-1365 (1968) 3 Probst, G.: Klinische Erfahrungen bei der Behandlung des Pruritus mit einem Histidin Dekarboxylase-Hemmer (Hypostamine). Theraa piewoche 20, 1380-1384 (1970) 4 Francois. R. and Lamit, J.: Therapeutic trial of hypostamine in various allergic manifestation in the child. Pediatrie 19, 760-762 (1964) 5 Humbert, G., Carron, R. and Jenne, M.: Clinical study of the effect of a histidine decarboxylase inhibitor in allergic diseases of the child. Pediatrie 19, 827-833 (1964) 6 Kosmicki, B., Mordelet-Dambrine, M., Lallonette, P. and Parrot, J.-L.: Action of tritoqualine on the activity of non specific histidine decarboxylase after injection of 5-hydroxytryptamine. J. Phar macol. 5, 331-342 (1974) 7 Parrot, J.-L.: Recherches sur le mode d'action d'une nouvelle serie de derives utilisables dans la therapeuytique de I'allergiei. J. Physiol. (Paris) 47, 263 (1955) 8 Carpi, C. and Maggi, G.C.: Azione antiistidina decarbossilasica in vivo della tritoqualine. Boll. Soc. Ital. Biol. Sper. 44, 543-546 (1968) 9 Stresseman, E.: The effect of tritoqualine, a tetrahydroisoquinoline derivative, on the ana phylactic microshock in guinea pigs. Int. Arch. Allergy 30, 382-384 (1966) 10 Von Hahn, F., Teschendorf, H.J., Kretzschmar, R., Gossow, U., Glanjamann, Ch., Filipowski, P. and Somorjai, K.: Zur frage der antiallergischen Wirkung von Tritoqualine. Arzneimittelforsch. 20, 1490-1496 (1970) 11 Koda, A., Nagai, H., Watanabe, S., Dansako, H., Inoue, Y., Sakamoto, K. and Nakagami, K.: Antiallergic action of tritoqualine. Japan. J. Allergol. 22, 640-648 (1973) 12 Schayer, R.W. and Reilly, M.A.: Effect of histidine decarboxylase inhibitors and other drugs on histamine formation in vivo. Agents Actions 4, 133-138 (1974) 13 Watanabe, T., Yamada, M., Taguchi, Y., Kubota,
14
15
16
17
18
19
20
21
22 23
24
25
26
H., Maeyama, K., Yamatodani, A., Fukui, H., Shiosaka, S., Tohyama, M. and Wada, H.: Puri fication and properties of histidine decarboxylase isozymes and their pharmacological significance. Advances in the Biosciences, Edited by UvnJs, B. and Tasaka, K., Vol. 33, p. 93-106, Pergamon Press, Oxford and New York (1982) Hakanson, R.: Histidine decarboxylase in the fetal rat. Biochem. Pharmacol. 12, 1289-1296 (1963) Kobayashi, Y.: Determination of histidine decar boxylase activity by liquid scintillation counting of CO2. Anal. Biochem. 5, 284-290 (1962) Hayashi, H.. Ichikawa, A., Saito, T. and Tomita, K.: Inhibitory role of cyclic adenosine 3',5' monophosphate in histamine release from- rat peritoneal mast cells in vitro. Biochem. Phar macol. 25, 1907-1913 (1976) Shore, P.A., Burkhalter, A. and Cohn, V.H.: A method for the fluorometric assay of histamine in tissues. J. Pharmacol. Exp. Ther. 127, 182-186 (1959) Hakanson, R., Ronnberg, A.-L. and Sjolund, K.: Fluorometric determination of histamine with OPT. Anal. Biochem. 47, 356-370 (1972) Azuma, H., Banno, K. and Yoshimura, T.: Pharmacological properties of N-(3',4'dimethoxycinnamoyl)anthranilic-acid (N-5'), a new anti-atopic agent. Br. J. Pharmacol. 58, 483-488 (1976) Negishi, M., Ichikawa, A. and Tomita, K.: Calcium uptake in mastocytoma P-81 5 cells. Biochim. Biophys. Acta 687, 179-188 (1982) Negishi, M., Ichikawa, A., Oshio, N., Yatsunami, K. and Tomita, K.: Cell cycle specific fluctuations of adenosine 3',5'-monophosphate and pro staglandin binding in synchronized mastocytoma P-815 cells. Biochem. Pharmacol. 31, 173-179 (1982) Umezawa, H.: Patent Japan. Kokai Tokkyo Knho 51, 146432 (1976) Lovenberg, W., Weissbach, H. and Udenfriend, S.: Aromatic L-amino acid decarboxylase. J. Biol. Chem. 237, 89-93 (1962) Levine, R.J. and Noll, W.W.: Histidine decarboxy lase and its inhibition. Ann. N.Y. Acad. Sci. 166, 246-256 (1969) Beaven, M.A., Roderick, N.B., Shaffe, R.E. and Soll, A.H.: Histamine synthesis in intact and disrupted rat mast cells. Biochem. Pharmacol. 31, 1189-1195 (1982) Bini, A., Frontini, E., Nicolin, A. and Olivani, P.: Histamine in DAB induced hepatoma and effects of tritoqualine and coumpound 48/80 on the incidence and development of the tumor.
Arch. Int. Pharmacodyn. Ther. 196, 291-292 (1972) 27 Ishii, K., Suzuki, O., Maruyama, K., Nagata, H., Kiryu, Y. and Tsuchiya, M.: Therapeutic effect of
histidine
decarboxylase
active hepatitis. 110 (1978)
inhibitor
Gastroenterol.
Japon.
on
chronic 13, 105 -
Effect
of Tritoqualine
Release
from
(TRQ) on Histamine
Mast
Cells
Kohei UMEZU, Satoshi YUASA, Atsuko SUDOH, and Atsushi ICHIKAWA* Biosciences
Laboratory, 1000
*Department Kyoto
Research
Kamoshida, of
Health
Center,
Midori-ku, Chemistry,
University,
Faculty
Yoshida,
Accepted
Mitsubishi Yokohama of
Sakyo-ku. February
28,
Ryoji KIKUMOTO Chemical
227,
Pharmaceutical Kyoto
606,
Ind.
Ltd. ,
Japan Sciences, Japan
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 of which is shown in Fig. 1, has been clinically used on patients with allergic disorders such as pollinosis, asthma and urticaria (1-5) . Since Parrot (6, 7) and Carpi (8) first demonstrated the inhibitory action of TRQ on histidine decarboxylase (HDC) in the guinea pig kidney, the main mechanism of action of TRQ as an antiallergic drug has been proposed to involve the inhibition of histamine biosynthesis in mast cells. The inhibitory action of TRO on tissue histamine content has been shown in several experimental
inflammation models (9, 10). However, conflicting results have also been reported suggesting that the content of tissue hista mine was not changed after TRQ adminis tration (11, 12). In spite of the important role of mast cells in allergic histamine metabolism, there have been fewer investi gations about the effect of TRQ on the histidine decarboxylase of the mast cell type which is suggested to be specifically involved in histamine biosynthesis in mast cells (13) and histamine release by stimulants from 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
Fig. 1.
Chemical structure of tritoqualine
(TRQ).
and Methods
HDC from fetal rats and mastocytoma P cells and HDC assay: HDC was partially
815
purified from fetal rats according to Hakanson's method (14), and it was ob tained from mastocytoma P-815 cells ac cording to Wada's method (13). Briefly, 5 rat fetuses were homogenized in 200 ml of 0.1 M acetate buffer (pH 5.5), and the homogenate was then centrifuged. After the supernatant was treated with heat at 55'C for 5 min, the crude HDC was precipitated at 25-45% ammonium sulfate saturation. The precipitant was redissolved and dialyzed against 50 mM phosphate buffer (pH 7.0) and used as a crude HDC (final volume was 11.2 ml). Mastocytoma P-815 cells (90 ml packed cell volume) collected from the ascites of 200 mice were homogenized, centrifuged, and the crude HDC precipitated by 30-55% ammonium sulfate saturation of the super natant was successively chromatographed on columns of DEAE-Sephadex CL-6B, hy droxyapatite, Sephacryl S-300, carnosine sepharose 4B, and again on DEAE-Sepharose CL-6B. The whole procedure resulted in an overall 2,000-fold purification of the enzyme. The final enzyme preparation produced a single protein band on polyacrylamide gel electrophoresis. HDC activity was assayed using 14C-histidine by the procedure of Kobayashi (15) with some modification. Briefly, the assay mixture (500 III) contained 0.25 mmole of sodium potassium phosphate buffer (pH 6.9), 0.1 Pmole of pyridoxal-5 phosphate, 0.25 iimole of histidine containing 0.1 zeCi of 14C-histidine, the enzyme fraction and distilled water. The assay mixture was incubated at 37'C for 20-60 min, and the reaction was stopped by adding 0.5 ml of 2N HCI, followed by a 30 min incubation to absorb the CO2 on filter paper containing 0.2 ml of ethanolamine. Radioactivity was determined in a Beckman LS 100 scintillation counter after adding toluene-ethanol (7:3) with 0.4% 2,5-diphenyl oxazole (PPO) and 0.01% 1,4-bis-, 2-(4-methyl-5-phenyloxa zolyl) benzene (DM-POPOP). TRO was dissolved in 0.1 N HCI with 30 ppm t-butylated hydroxyanisole (BHA) at 0.3-5 mM to prevent the oxidation of TRQ or without BHA, and TRQ was added in the assay mixture at final concentrations of 3-50 /NM. Preparation of mast cells: Mast cells were
obtained from the peritoneal cavity fluid of male Wistar rats weighing 300-400 g, and these cells including other cells were used as they were or after purification by Ficoll density gradient centrifugation as described previously (16). Purity of mast cells was over 90%. After mast cells (0.5-2.O x 104 cells/tube) were preincubated for 10 min with TAO or vehicle, one of the histamine releasers was added to the reaction mixture, and 10 min later, the reaction was stopped by cooling the mixture in an ice-bath, followed by centrifugation at 500 g for 1 min. The histamine released in the supernatant fraction was measured by the methods of Shore (17) and Hakanson (18) with some modifications. Preparation of DNP-Ascaris and sensi tization: 2,4-Dinitrophenyl-coupled ascaris extract (DNP-Ascaris) and rat anti-DNP Ascaris antibody were prepared by the procedure of Azuma et al. with some modifications (19). Rat peritoneal mast cells were sensitized with rat anti-DNP-Ascaris antibody by two methods, in vivo and in vitro. Sensitization in vitro was done by mixing mast cells (8 X 106) with 4 ml of rat anti-DNP-Ascaris antibody (titer 1/256) and incubating it at 37°C for 60 min. After washing, cells were preincubated with phos phatidylserine (10 W) for 5 min; then in the presence of TRQ (10 W), then cells were incubated 10 min more, and DNP-Ascaris was added as an antigen. After 20 min, histamine released from mast cells was measured as described above. In vivo sensitization was conducted by intraperi toneally injecting 0.6 ml of rat anti-DNP Ascaris antibody (titer 1/256) to male Wistar rats weighing 200-300 g. Sensitized mast cells obtained from the peritoneal cavity fluid after 45 hr of the antibody injection were treated by the same procedure described above and were used for histamine release by antigen challenge. Histamine release from mastocytoma P 815 cells by compound 48/80: Mouse mastocytoma P-815 cells (2-4 x 105 cells/ ml in 500 ml flask) were maintained in suspension culture at 37'C in Fischer's medium supplemented with 5% fetal calf serum (20). Incubation with or without TRQ was continued by exchanging half of the
culture medium containing the growing cells with the same amount of fresh medium including TRQ (10 /CM) or vehicle every 24 hr for 4 days. Mastocytoma P-815 cells (2-2.5 x107 cells) were harvested by centrifuging 50 ml of the suspension and washing the cells three times with phosphate buffered saline (PBS) at the indicated time. Mastocytoma cells (4-6X106 cells) in 1 ml of PBS were incubated for 10 min after treatment with 50 gig of compound 48/80, and released histamine was measured by the same procedure described above. The cell number was counted by a Coulter model Z counter (Coulter Electronics, Hialeah, FL, U.S.A.) (21). Elimination of TRQ from histamine: TRQ showed strong fluorescence in the emission and excitation wavelengths used for the fluorometric assay of histamine. Diaion HP-20 (Mitsubishi Chemical Ind., Ltd.) was used to separate TRQ from histamine, as TRQ was bound on the resin completely and the 0 phthalaldehyde complex of histamine passed through this resin column. Recovery of histamine was over 95%. Chemicals: TRQ was purchased from Table P-815
1. cells
Inhibitory
effect
of TRQ
on
histidine
Medichemie AG (Ettingen, Switzerland). Ficoll, DEAE-Sepharose CL-6B, Sephacryl S 300 and carnosine-Sepharose 4B were purchased from Pharmacia (Uppsala, Sweden). Histidine, L-(carboxy-14C), 30 mCi/mmol was purchased from New England Nuclear. MI-063 (3,5 dichlor-2,4 dihydroxy benzanilide) was synthesized by Mitsubishi Chemical Ind., Ltd., according to Umezawa's method (22). 7-Amino, 3 hydroxy, 4,5,6-triethoxyphthalide (HOEAP) was also synthesized by Mitsubishi Chemical Ind., Ltd. All other chemicals were analytical grade preparations obtained from commercial sources. Results Inhibitory effect of TRQ on HDC: As shown in Table 1, TRQ did not show any significant inhibitory action on HDC activity prepared from fetal rats at concentrations in the range of 10 to 50 ,FPM.No inhibitory effect was found when crude and completely purified HDC from mastocytoma cells was used. Compound MI-063, which has been proven to be a strong inhibitor of HDC (22), inhibited HDC activity almost 100% at
decarboxylase
prepared
from
fetal
rats
and
mastocytoma
from rat peritoneal mast cells induced by compound 48/80 (Fig. 2A), ATP (Fig. 2B) and DNP-Ascaris (Fig. 3). IC50 values of histamine release by compound 48/80 and ATP are 10 tiM and 13 tiM, respectively. As shown in Fig. 2A, cell injury by TRQ itself was not observed at concentrations of 1-33 ,uM. On the histamine release by Ca ionophore A23187, TRQ did not show any inhibitory effect (data not shown). There was no
0.15 W. As TRO was partially decomposed to cotarnine and HOEAP by oxygen dissolved in water, the possibility of these products inhibiting HDC activity was examined. None of them had any inhibitory action (data not shown). Effect of TRQ on histamine release from rat peritoneal mast cells: Results presented in Figs. 2 and 3 show that TRQ had dose related inhibitory activity on histamine release
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
Mast cells sensitization
effect
of TRQ
on histamine
release
by DNP-Ascaris
antigen
from
mast cells
of rats.
were sensitized according to the procedure described in Methods. A represents in vitro and B represents in vivo sensitization. The concentration of TRO was 10 tM in each
experiment.
White
phosphatidyl
serine.
and dotted Significantly
columns
represent
different
control
from each
and TRO treatment,
control,
*P<0.5,
respectively.
**P<0.01.
PS means
inhibitory action of TRQ metabolites, cotarnine or HOEAP, in the same system in vitro (data not shown). Effect of TRQ on histamine release from cultured mastocytoma P-81 5 cells stimulated by compound 48/80: Mouse mastocytoma P 815 cells in the exponential growth phase, as compared with rat peritoneal mast cells, contain a low level of histamine and are much less likely to release histamine by the stimulation of compound 48/80. To obtain the reproducible release of histamine (about 20% release of total histamine), at least 50 iig of compound 48/80 was required (Fig. 4). Though at 2 hr there was no difference between control and TRQ-treated masto cytoma P-81 5 cells with respect to histamine release by compound 48/80, at the 4th day,
Fig. 4. Time course change of response of mas tocytoma P-81 5 ce!ls against compound 48/80. Each value represents the mean and S.E. of 4 samples. Ordinate: relative % of histamine released from mastocytoma cells. Abscissa: time course. Insertion represents the time course change of the cell size and generation time of mastocytoma P-81 5 cells. (0): control, (0) : TRQ treated. *,**: statistically significant from the control at P<0.05 and 0.01 , respectively. Table
2.
Changes
in histamine
content
of
histamine release from mastocytoma P-81 5 cells was inhibited about 90% compared with the vehicle control. Under this culturing condition with TRQ, doubling time and cell size of mastocytoma P-81 5 cells were not influenced by treatment of TRO (Fig. 4). The histamine content in mastocytoma P 815 cells maintained 24 hr longer in the culture was 1.8-2.2 /cg/108 cells, and TRQ treated cells had almost the same histamine content (2.1-2.2 /cg/108 cells). No sig nificant difference was observed (Table 2). Discussion Since it has been reported that TRQ inhibited HDC in vitro (6, 7), some researchers have identified this compound from their 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 L-amino acid decarboxylase. The specific decarboxylase has been purified from fetal rat tissues and studied in detail by Hakanson (14) and Watanabe et al. (13). The non specific enzyme has been purified from guinea pig kidney and studied by Lovenberg et al. (23). It was concluded that of these enzymes, only the specific enzyme functioned significantly in vivo to form histamine and
mastocytoma
P-815
cells
incubated
for
4 days
that the aromatic amino acid decarboxylase was not so important because of its low V,,,,,, and very low affinity for histidine (24). There are high HDC activities in the stomach, the whole fetus and the brain as well as mast cells in rats. Watanabe et al. reported that there are at least two isozymes of HDC, HDC in the fetus which belongs to the mast cell type and HDC in the brain which belongs to the non-mast cell type (13). Since it is known that it is very difficult to purify HDC from mast cells because HDC becomes labile once cellular integrity is lost (25), we used HDC purified from fetal rats and mastocytoma cells for estimating the inhibitory effect of TRQ on HDC activity. As shown in Table 1, TRQ did not inhibit HDC activity when prepared from fetal rats in concentrations from 10 to 50 1nM. Cotarnine and HOEAP which are decomposition pro ducts of TRQ did not show any inhibitory effect in this assay system. The same result was obtained using the crude and purified HDC from mastocytoma cells. MI-063 used as a reference drug showed a very strong inhibition in both HDC tests, showing that our assay systems were working well. These results were inconsistent with those of previous reports (6, 7). Parrot used the guinea pig kidney as an enzyme source for histamine synthesis and assayed the HDC activity by means of measuring the amount of histamine produced in the incubation mixture with a bioassay using an ileum preparation. As already mentioned, the main enzyme involved in decarboxylating histidine and forming histamine in the kidney might be the I-aromatic amino acid decarboxylase, and not HDC. The difference between our result and that of Parrot may be derived from the different source of the enzymes. In addition, TRQ has a nonspecific inhibitory action on the smooth muscle contraction induced by acetylcholine, serotonin and histamine at concentrations above 20 /iM (data not shown). Therefore, the possibility that con taminating TRQ in the assay medium inhibits the ileum contraction is not completely ruled out. From our results, it was concluded that TRQ does not inhibit HDC. TRQ inhibited the histamine release from mast cells by various stimulants, not only
that by the antigen DNP-Ascaris, but those by other chemical substances such as com pound 48/80 and ATP. The IC50 values of TRQ for the histamine release induced by compound 48/80 and ATP were 10 /cM and 13 W, respectively (Figs. 2 and 3). In addition, we observed the actions of TRQ to inhibit the Ca influx into mast cells and to inhibit the phospholipid turn over in the membrane of mast cells by measuring 32P or 14C-arachidonic acid metabolisms (to be reported elsewhere). To investigate the contact effect of TRQ with cell membranes, mastocytoma P-81 5 cells were cultured with TRQ during the period from 2 hr to 6 days, and it was observed that the histamine release response of mastocytoma P-81 5 cells by compound 48/80 decreased, depending on the incubation time or contact time with TRQ. Under this condition, no change was observed in the doubling time of cells, the cell sizes and the histamine content between mastocytoma P-81 5 cells incubated in the presence of TRO and the nontreated cells (Fig. 4 and Table 2), indicating that there was no direct cell toxicity by TRQ. The result that the histamine content did not change in the TRQ treated cells indicated that the histamine biosynthesis was not inhibited with the treatment of TRQ. Decrease in the mastocytoma P-81 5 cells against compound 48/80 might be derived from the increment of accumulated TRQ in the cell membrane. As TRQ is a hydrophobic compound, it may be accumulated in the membrane with the increase of contact time with cells. However, further investigation is needed to prove whether the action of TRQ is restricted to mastocytoma cells only or is observed commonly in other cells. Thus, it seems reasonable to postulate from these results that the action mechanisms of TRQ as an antiallergic drug might be the inhibitory effect on histamine release from mast cells, rather than the inhibition on HDC activity. Some researchers reported the other effects of TRQ except the antiallergic action, for instance, the application of TRO for rat hepatoma induced by dimethylamino azobenzene (26) and clinical application of TRQ for chronic hepatitis (27). Though they
ascribed the effect of TRIO to its HDC inhibitory action, it will be necessary to reconsider the mechanisms of TRIO, judging from our results. References 1 Von Andres, H.U. and Krebs, A.: Behandelung allergischer Hautkrankheiten mit Hypostamine (Tritoqualine) unter besonderer Berucksichtig ung des Pruritus. Praxis 57, 536-539 (1968) 2 Hohlbrugger, H.: Erfahrungen mit Hypostamine beim heuschnupfen and anderen allergischen Erkrankungen der oberen Luftwege. Therapie woche 18, 1357-1365 (1968) 3 Probst, G.: Klinische Erfahrungen bei der Behandlung des Pruritus mit einem Histidin Dekarboxylase-Hemmer (Hypostamine). Theraa piewoche 20, 1380-1384 (1970) 4 Francois. R. and Lamit, J.: Therapeutic trial of hypostamine in various allergic manifestation in the child. Pediatrie 19, 760-762 (1964) 5 Humbert, G., Carron, R. and Jenne, M.: Clinical study of the effect of a histidine decarboxylase inhibitor in allergic diseases of the child. Pediatrie 19, 827-833 (1964) 6 Kosmicki, B., Mordelet-Dambrine, M., Lallonette, P. and Parrot, J.-L.: Action of tritoqualine on the activity of non specific histidine decarboxylase after injection of 5-hydroxytryptamine. J. Phar macol. 5, 331-342 (1974) 7 Parrot, J.-L.: Recherches sur le mode d'action d'une nouvelle serie de derives utilisables dans la therapeuytique de I'allergiei. J. Physiol. (Paris) 47, 263 (1955) 8 Carpi, C. and Maggi, G.C.: Azione antiistidina decarbossilasica in vivo della tritoqualine. Boll. Soc. Ital. Biol. Sper. 44, 543-546 (1968) 9 Stresseman, E.: The effect of tritoqualine, a tetrahydroisoquinoline derivative, on the ana phylactic microshock in guinea pigs. Int. Arch. Allergy 30, 382-384 (1966) 10 Von Hahn, F., Teschendorf, H.J., Kretzschmar, R., Gossow, U., Glanjamann, Ch., Filipowski, P. and Somorjai, K.: Zur frage der antiallergischen Wirkung von Tritoqualine. Arzneimittelforsch. 20, 1490-1496 (1970) 11 Koda, A., Nagai, H., Watanabe, S., Dansako, H., Inoue, Y., Sakamoto, K. and Nakagami, K.: Antiallergic action of tritoqualine. Japan. J. Allergol. 22, 640-648 (1973) 12 Schayer, R.W. and Reilly, M.A.: Effect of histidine decarboxylase inhibitors and other drugs on histamine formation in vivo. Agents Actions 4, 133-138 (1974) 13 Watanabe, T., Yamada, M., Taguchi, Y., Kubota,
14
15
16
17
18
19
20
21
22 23
24
25
26
H., Maeyama, K., Yamatodani, A., Fukui, H., Shiosaka, S., Tohyama, M. and Wada, H.: Puri fication and properties of histidine decarboxylase isozymes and their pharmacological significance. Advances in the Biosciences, Edited by UvnJs, B. and Tasaka, K., Vol. 33, p. 93-106, Pergamon Press, Oxford and New York (1982) Hakanson, R.: Histidine decarboxylase in the fetal rat. Biochem. Pharmacol. 12, 1289-1296 (1963) Kobayashi, Y.: Determination of histidine decar boxylase activity by liquid scintillation counting of CO2. Anal. Biochem. 5, 284-290 (1962) Hayashi, H.. Ichikawa, A., Saito, T. and Tomita, K.: Inhibitory role of cyclic adenosine 3',5' monophosphate in histamine release from- rat peritoneal mast cells in vitro. Biochem. Phar macol. 25, 1907-1913 (1976) Shore, P.A., Burkhalter, A. and Cohn, V.H.: A method for the fluorometric assay of histamine in tissues. J. Pharmacol. Exp. Ther. 127, 182-186 (1959) Hakanson, R., Ronnberg, A.-L. and Sjolund, K.: Fluorometric determination of histamine with OPT. Anal. Biochem. 47, 356-370 (1972) Azuma, H., Banno, K. and Yoshimura, T.: Pharmacological properties of N-(3',4'dimethoxycinnamoyl)anthranilic-acid (N-5'), a new anti-atopic agent. Br. J. Pharmacol. 58, 483-488 (1976) Negishi, M., Ichikawa, A. and Tomita, K.: Calcium uptake in mastocytoma P-81 5 cells. Biochim. Biophys. Acta 687, 179-188 (1982) Negishi, M., Ichikawa, A., Oshio, N., Yatsunami, K. and Tomita, K.: Cell cycle specific fluctuations of adenosine 3',5'-monophosphate and pro staglandin binding in synchronized mastocytoma P-815 cells. Biochem. Pharmacol. 31, 173-179 (1982) Umezawa, H.: Patent Japan. Kokai Tokkyo Knho 51, 146432 (1976) Lovenberg, W., Weissbach, H. and Udenfriend, S.: Aromatic L-amino acid decarboxylase. J. Biol. Chem. 237, 89-93 (1962) Levine, R.J. and Noll, W.W.: Histidine decarboxy lase and its inhibition. Ann. N.Y. Acad. Sci. 166, 246-256 (1969) Beaven, M.A., Roderick, N.B., Shaffe, R.E. and Soll, A.H.: Histamine synthesis in intact and disrupted rat mast cells. Biochem. Pharmacol. 31, 1189-1195 (1982) Bini, A., Frontini, E., Nicolin, A. and Olivani, P.: Histamine in DAB induced hepatoma and effects of tritoqualine and coumpound 48/80 on the incidence and development of the tumor.
Arch. Int. Pharmacodyn. Ther. 196, 291-292 (1972) 27 Ishii, K., Suzuki, O., Maruyama, K., Nagata, H., Kiryu, Y. and Tsuchiya, M.: Therapeutic effect of
histidine
decarboxylase
active hepatitis. 110 (1978)
inhibitor
Gastroenterol.
Japon.
on
chronic 13, 105 -