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The effect of three novel thromboxane A2 receptor antagonists (S-1452, AA-2414 and ONO-3708) on the increase in pulmonary pressure caused by Forssman anaphylaxis in guinea-pigs
The Effect of Three Novel Throm~xane AZ Receptor Antagonists (S-1452, AA-2414 and ONO-3708) on the Increase in Pulmonary Pressure Caused by Forssman Anaphylaxis in Guinea-pigs H. Nagai, M. Kunimoto,
K. Yoshitake,
T. Iwama, A. Arimura” and A. Koda
Department of Pharmacology, Gifu Pharmaceutical University, S-6-1 Mitahora higashi, Gifu, 502, J~pu~ and ~Sh~o~~~~research ~u~~rutor~es, 5-12-4 Sag&u, Fuk~h~rn~-K~, Osaka, 5.53, J~pff~ &prim requests to HN) ABSTRACT. TBw&f&s were studied of three novel throm~xane AZ (TXA*) receptor ~~~~~ {S-1452, AA-2414 and ~NU-~~~~ on the increase in pufmonary pressure caused by Forssman a~~yla~s in guineapigs, Three TXAZ an~go~is~ at doses of between 1 and 10 m#kg ad~n~ter~ orally 1 h before the chsllenge clearly inhibited the puimonary pressure increase. At a dose of 10 mfig, all three an~gonists inhibited the pulmonary pressure increase caused by leukotriene Dd (LTDd) and U-46619, but not that caused by histamine, The decrease in peripheral platelet counts caused by Forssman anaphylaxis was also clear& inhibits by the three TXAz antagonists. However, the decreased peripheral leukocyte counts were unaffected by the three agents. The decrease in serum complement activity (CHw) was inhibited by S-1452 and AA-2414 at a dose of 10 mg/kg. In bronchoalveolar lavage fluid (BALF), significant increases in eosinophils and neutrophils were observed after Forssman anaphylaxis. Three TXA2 antagonists at a dose of 10 nqq’icg (except for AA2414 on ~in~ils~ did not affect the changes of leukocyte counts in BALF. Moreover, increases in the TXS, and fi-keto-PGFl, leveis of the BALF brought about by Forssman anaphylaxis were m&&e&d by the three TXA2 receptor antagonists. Histamine and LTDd were not changed in the BALF after Forssman ~~~hyI~is. These results indicate the ef#icaey of TXAz receptor antagonists on the increase in pulmonary pressure caused by Forssman anaphylaxis in ~inea-pin by direct antagonism to released TXA2.
INTRODUCTION Forssman (heterophiie) shock in the guinea-pig is an important model of immunological cytotoxic injury (l-3). A lethal response can be provoked by an intravenous injection of rabbit antiserum to sheep erythrocyte stroma. Postmortem examination reveals marked oedematous and massive haemorrhaging in the lungs, as well as a frothy blood-stained exudate which fiffs the trachea and exudes from the nose. Previously, we have reported the role of thromboxane A2 (TXA2) in Forssman shock, and the efficacy of OKY-046, a novel TXAz synthetase inhibitor, on Forssman fethal shack (4). Simultaneously, we reported an effect of OKY-046 on the Forssman antibody-induced airway resistance increase. When Forssman antibody was injected into guineapigs at a sublethal dose, a diphasic increase in &he p~Imona~ pressure was observed. Phase I occurs
immediate@ after the injection of antiserum, probably due to the release of serotonin and other mediators. Phase II pulmonary pressure increase QCcurs immediately thereafter, because of pulmonary oedema and obstruction caused by endothelial damage and ~xtravasation of haemorrhagic exudates into the respiratory tract. In this study, we investigated the effect of three TXA2 receptor antagonists on Forssman antibodyinduced airway resistance increase in guinea-pigs.
MATERIALS AND METHODS Animals Hartley male guinea-pigs, weighing 300-400 g, were used, Animals were purchased from Shizuaka Laboratory Animal Center. Materials
Date received 8 My
Calcium ~(Z~-lR,ZS,3S,~7-~3-phen~~ su~fony~ami~ bicycio [Z+21 hept-2-ye-~-hept~noate hydrate (S-1452,
1981
Date accepted 311September19% 233
234
Prostagiandins Leukotrienes
and Essential Fatty Acids
Shionogi Pharmaceutical Co Ltd, Osaka, Japan), (~)-7-(3,5,6-Trimethyl-l,4-benzoquinon-2-yl)-7-phenyl heptanoic acid (AA-2414, Takeda Pharmaceutical Co Ltd, Osaka, Japan) and (9,11), (11,12)-Dideoxa9cu,l lo-dimethylmetha~o-1 l,lZ-methano-I3,14-dihy~o-13-aza-14-o~o-15-~yc1~~~~1-16,17,1g,19,20pentanon-Ifi-epithromboxane AZ (ONO-3708, Ono Pharmaceutical Co Ltd, Osaka, Japan) were kindly donated by each company. Each drug was suspended in saline containing 0.5% carboxy methylcellulose (CMC). Hemolysin (Kyokuto Pharmaceutical Co Ltd, Tokyo, Japan), 9,lI~id~xy-9~,ll~~methanoe~xyprostaglandin Fz~ (U-46619, Funakoshi Co Ltd, Tokyo, Japan), leukotriene D4 (LTD4, Wako Pure Chemical Co Ltd, Osaka, Japan) and histamine (Wako Chemicals) were purchased.
M~u~ment
of pu~m~ary pressure
The increase in pulmonary pressure was measured
by the overflow method, as described by KonzettRossler and ourselves (5, 6). The guinea-pigs were initially anaesthetised with pentobarbital Na at a dose of 50 m&kg. The trachea was cannulated, and the animal artificially ventilated by a positive pressure pump at 60 stroke~min with 5 ml/stroke. Changes in pressure were measured by connecting a side arm from the tracheal cannula to an arterial pressure transducer, coupled to a strip chart recorder (Ugo Basil, Milano, Italy).
Me~u~ment
of blood parameters
The numbers of platelets and leukocytes were counted by a Coulter counter (MEK-3100, Nihon Koden, Tokyo, Japan). Haemolytic complement activity was measured by the CHSe method described by Kabat-Mayer (7).
M~u~ment of TXBz, B-keto-PGF,,, LTD4 and histamine in the BALF
In order to determine the amounts of TXAz and PGI2, their stable breakdown products - TXB;? and 6-keto-PGFi, - were measured. The BALF was obtained by injecting saline containing indomethacin at a concentration of 10s7 M. The fluid was centrifuged at 5000 rpm for 30 min at o”C, and then applied to a C-18 column. The column was washed with 4% acetic acid and distilled water. TXB:! and 6-keto-PGFi, were eluted with lc#l% methanol. Then, the effluent was evaporated and dissolved in assay buffer (phosphate buffer saline containing gelatin and thimerosal). The amounts of TXBz and 6-keto-PGF,, were measured using a radioimmunoassay kit (Amersham Japan, Tokyo, Japan). The recovery rates of TXB2 and 6-ketoPGF1, were 75 and lOO%, respectively. In order to measure the amount of LTD, in the BALF, BALF was obtained by injecting saline containing AA-861 (a potent 5-lipoxygenase inhibitor) at a concentration of lo-? M. The fluid was centrifuged at 5000 rpm for 30 min at 0°C. Three milliliters of supernatant were mixed with 12 ml ethanol. These samples were centrifuged at 10 000 rpm for 10 min at 4°C. The supernatant was evaporated below 40°C. Samples were applied to a C-18 column, after adjusting to pH 5.1 with 1 N HCl and washing with distilled water (20 ml) and petroleum ether (20 ml). Following this procedure, LTD4 was eiuted with 20 ml methanol. Then, the effluent was evaporated and the amount of LTD4 in the residual solution measured using an Amersham radioimmunoassay kit. To determine the amount of histamine, BALF was obtained by injecting saline. The BALF was centrifuged at 5000 rpm for 10 min at 4”C, and the amount of histamine assayed by the fluorescence method of May et al (8). Statistics
Measurement of Ieukocyte counts in the b~ch~lv~1~ lavage fluid (BALF)
In order to obtain the BALF, the animals were sacrificed by the intraperitoneal injection of pentobarbital (250 mg’kg), 30 min after antiserum injection. The BALF was collected by the slow injection of 10 ml sterile saline into the trachea through a cannula, whereafter the fluid was withdrawn. It was injected into the lungs again, and the procedure repeated 10 times. The BALF was centrifuged at 1000 rpm for 10 mm at 4°C. The number of nucleated cells in the Iavage fluid was counted in a Biirker chamber, and a differential count was made on a smear prepared with a c~ocent~fuge and day-Giemsa stained.
Data were expressed as a mean -t- SEM. Results for increased pulmonary pressure were evaluated by Dunnet’s multiple comparison (9). The other results were evaluated by Student’s t-test. RESULTS Effect of the three TXAz receptor uncool on pulmonary pressure changes caused by Forssman anaphylaxis and various mediators
The three TXA2 receptor antaganists were administered p.o, 1 h before the injection of antiserum or mediators. At doses between 1 and 10 mg/kg, the three antagonists inhibited increased pulmona~ pressure Forssman anaphylaxis (Fig. 1).
TXA, Receptar Aatapnists
and Pulmonary Pressure
2%
CCI ONE-3708
Fig= I Effect of S-1452, AA-2414 and 0NO-3708 on the increase in pufmonary pressure caused by Farssman anaphylaxis in guinea-pigs. Each drug was ~dmj~istered p.a. I h priar to the injectian of Fotssman antibady. Each paint represents the mean f SEM of B-24 animals. [Af 0: control l : S-1452, 1 m&g A: S-1452, IO m& D: S-1452, 3 mg’kg [S] 0: control e: AA-2414, 1 mx g A: AA-2414, 3 mg!kg A: AA-2414, 10 mg/kg 0: ONW708, 1 m&g [C] A: control A: ONO-3708, 10 mg/kg * ; p < O.QS, I ; p > 0.01 (from control)
Add~t~o~~~~y,30 m~kg doses of all three TXAz receptor ~~t~~o~jsts inhabited increased p~~rno~~~ pressure due to LTD4 and U-46619 (Fig. 2). The increase of pulmonary pressure caused by histamine was inhibited only by S-14.52. Effect of the three receptor ~~~n~s~ on Mod parameter changes caused by Forssman WlpItj&ST&?! The injection of Forssman antibody resulted in a
clear decrease in the ~ripb~ra~ platelet and leukocyte counts, as weH as the ha~rn~~i~ complement activity (Fig. 3). S-1452 at a dose of 10 mg/kg inhibited the reduction of these parameters. AA-2414 at doses of 3 and 10 mdkg inhibited the decrease in platelet count and hemolytic complement activity, but did not affect the leukocyte count. ONO-3708 at a dose of 10 m&g also inhibited the decrease in platelet count, but did not affect the other parameters.
236
Prosta~andi~s Leukotrienes and EssentialFatty Acids
100, Histamine
[AI
[ B 3 LTD4
C C 1 U-46619
0 .
I
0
1
2
3
4
k
5 Minutes
I
10
c
’
0
1
I
3
2
I
t
4 5 Minutes
I
10
L
I
0
1
3
I
I
I
I
t
2
4 5 Minutes
10
Fig. 2 Effect of S-1452, AA-2414 and ONO-3708 on the increase in pulmonary pressure caused by histamine, LTD, and U-46619 in guinea-pigs. Each drug at a dose of 10 m&g was administered p.o. 1 h prior to the injection of each mediator. Each point represents the mean + SEM of 5 animals. 0: control, e: S-1452, A: AA-2414, A: ONO-3708 * ; p < 0.05, +; p > 0.01 (from control)
Platelet 0
20
40
(106 cells/ml) 60
Platelet
120 140
0 20 -m
-iI
40
Platelet
(106 cells/ml) 60
80 120
0 20 40 -I+
140
N
N
c
c
s-1 s-3 s-10
A-l A-3 A-10
t 0 r
Leukocyte 0.1
I
(lo7 cells/ml) 0.2 0.3 t
Leukocyte
cells/ml)
Comp:;ment
I
c S-l s-3 s-10
Leuko;y;e (10: ;ellse;f
. I
I
i
N C A-l A-3 A-10
I
t
N C o-1 O-10
0 t
0
I
A-l A-3 A-10
s-1 s-3 s-10
N
(lo7
N c
:
120 140
o-1 O-10
0 e
1
I
(106 cells/ml) 60
Comp;emen;OO(CHsol
(CH ) 100 #o
200
I
t
0
I N C o-1 O-10
I
I
150
200
I
I
t
counts Fig. 3 Effect of S-1452, AA-2414 and ONO-3708 on the changes of platelet counts, . . .leukocyte . _. and serum complement actrvity (CH,,) caused by Forssman anaphylaxis in guinea-pigs. Each drug was admmrstereo p.o. 1 h prior to the injection of Forssman antibody. Each column represents the mean ? SEM of 6-11 animals. q: N; Normal (before the injection of Forssman antibody) &$ C; Control (after the injection of Forssman antibody) S-3 (S-1452, 3 mg/kg) 0: S-l (S-1452, 1 m&g) A-l (AA-2414, 1 m&g) S-10 (S-1452, 10 m&g) A-10 (AA-2414, 10 m& ) A-3 (AA-2414, 3 m&g) O-10 (ONO-3708, 10 m PB;g) O-l (ON?-3708, 1 m&g) * ; p < 0.05, ; p > 0.01 (from control)
Effect of the three TXA, receptor an~gonis~ on the leukocyte count and the levels of TXB2, 6-keto-PGF,, LTD4 and histamine in the BALF Figure
4 shows the changes
of leukocyte
count
in the BALF. The numbers of total cells, eosinophils and neutrophits increased after Forssman anaphylaxis. AA-2414 at a dose of 10 mg/kg inhibited the increase in eosinophils but other changes were unaffected by the three antagonists. Atthough
TX& Receptor Antagonists Nafrophages f% to total cells) 0 SO
The number af cells (X IO5 cells/ml)
I
t
100
1
Noraal Control S-1452 AA-2414 ONO-3708 n
Eosinophifes [# to total cells) 0 20
I
237
40
4
l!!z!z
~ Neutroohiles (% to :btal cetls)
and Pulmonary Pressure
Lymphocytes 3
Norma 1
Control S-1452 AA-2414 ONO-3708 Fig. 4 Effect of S-1452, AA-2414 and ON03708 on the changes of number of total leukocytes, macrophages, eosinophils, neutrophiles and iymph~ytes in bronchoalv~Iar lavage fluid (BALF) caused by Forssman anaphylaxis in guinea-pegs. Each drug at a dose of 10 mg&g was administered p.o. I h prior to the injection of Forssman antibody. Each column represents the mean + SEM of 5-9 animals. 8: Normal (before the injection of Forssman antibody) @: Control (after the injection of Forssman antibody) * ; p < o.f)5, : p > 0.01 (from control)
Table Effects of three TXA2 receptor antagonists on the changes of levels of histamine, LTD4, TXBz and 6-keto-PGFtb, in BALF
Normal Control S-X4.52 AA-2414 ONO-3708
Histamine (q/ml)
LTD, (&ml)
TXB:
1.0 1.2 1.4 1.0 1.1
0.0 0.0 0.0 0.0 0.0
1.6 2.7 3.3 3.6 2.5
+ 0.5 +. 0.5 * 0.6 It 0.5 f 0.5
+ + + * +
0.0 0.0 0.0 0.0 0.0
+ + + + +
6-keto-PC& 0.3* 0.3 1.0 1.1 0.5
41 87 76 70 80
(pg/ml)
rt 2* It 9 rf: 8 Yk5 -c 4
Each value represents the mean + SEM of 5-9 animals. Each drugs at a dose of 10 m&g was administered pa_ 1 h prior to the injection of Fcrrssman antibody. * ; p < 0.05 ffron contml).
the amounts of TXB, and 6-keto-PGF,, in the BALF increased significantly, the amounts of LTD4 and histamine were unchanged by Forssman anaphyiaxis (Table). The three TXA2 receptor antagonists, however, did not affect the increased levels of TXB2 and 6-keto-PGFi, in the BALF.
These results indicate the efficacy of TXA2 receptor antagonists on increased pulmanary pressure caused by Forssman anaphylaxis in guinea-pigs. Forssman anaphylaxis is considered to be an important experimental model for pu~rno~a~ thromboembolism (11). At present, specific drugs are not available for the treatment of pulmonary thromboembolism. Non-specific antiinflammatory drugs and, more oceasionafly, jmmunosuppressive drugs such as aspirin
and glucocorticoids, have been used, individuahy and combined. Other results suggest a possible use of TXAz receptor antagonists as a remedy for pulmonary thromboembolism. As for the mediator of the Forssman antibodyinduced pulmonary pressure increase in guinea-pigs, three major factors have been proposed (4, 12-20). Firstly, serotonin is a primary mediator, causing increased pulmonary pressure in phase I. Some investigators have reported the release of serotonin during shock and the efficacy of anti-serotonergic agents on increased pufmonary pressure in phase I. Secondly, superoxide radicals (Oz-) may be one of the mediators for pulmonary pressure increase in phase IX, because of the selective efficacy of superoxide dismutase in phase IL Thirdly, our previous and present results indicate that TXAz seems to be a crucial mediator in the increase of pulmonary pressure during phases I and ff. The previous study
238
Frosta~andins
Leukotrienes
and Essential Fattv Acids
indicated the efficacy of OKY-046, a selective TXA2 synthetase inhibitor, on Forssman antibody-induced pulmonary pressure increase and the increment of TXBz in the -plasma after Forssman anaphylaxis. Our present results support the previous findings on the use of TXAz receptor antagonists and BALF. The three TXA, receptor antagonists clearly inhibited increased pulmonary pressure caused by the Forssman antibody. Moreover, the amount of TXBz in the BALF was increased after the injection of antibody. These data confirm the role of TXAz in the increase of pulmonary pressure during Forssman anaphylaxis in guinea-pigs. The mode of action of TXA:! on pulmonary pressure increase is, however, still obscure. Two possible mechanisms are postulated for the role of TXA2 in increased pulmonary pressure. One is the direct action of TXAz on airway smooth muscle: TXA2 may cause a potent contraction of airway smooth muscle. The other mechanism may be related to the vasoconstriction of pulmonary blood vessels, resulting in decreased pulmona~ circulation and may cause an increase in pulmonary pressure during Forssman anaphylaxis. These two mechanisms may contribute to increased pulmonary pressure. We need to carry out further experiments to study the effect of Forssman anaphylaxis on the pulmonary circulation of guineapigs. In conclusion, these results indicate the efficacy of three novel TXAZ receptor antagonists (S-1452, AA-2414 and ONO-3708) on increased pulmonary pressure caused Forssman anaphylaxis in guineapigs.
References Redfern, W. W.: A study of the primary toxicity of heterophile immune rabbit serum for guinea pigs and its apparent relaxation to the phenomenon of anaphyla&. Am. J. Hygiene 6: 276-310 (1926) Glovsky, M. M., Ward, P. A., Becher, E. L. and Halbrook, N. J.: Role of fumaropimaric acid in guinea pig complement dependent and non-complement dependent biological reaction. J. Immunol. 102, l-11 (1969) Nagai, H., Kurimoto, Y. and Koda, A.: im~unopha~acological approach to Forssman shock. Microbial. Immunol. 24. 649-655 (1980) Nagai, H., Yakuo, I., Inagaki,‘N., Koda; A., ’ Hamano, S., Ujiie, A. and Nakazawa, M.: Role of thromboxane A, (TXA j in guinea pig Forssman shock and the effect of OKY-046, TXA, synthetase
inhibitor. Prostaglandins Leukotrienes and Medicine 26, 133-141 (1987) 5. Konzett, H. and Rossler, R.: Versuchanordnung zu Untersuchengen an der Bronchialmuskulater. Arch. Exp. Path. Pharmak. 195, 71-74 (1940) 6. Nagai, H., Suda, H., Kitagaki, K., Goto, S., Muira, T. and Koda, A.: Anti-allergic effects of Ketanserin on animal models of allergic reactions. Archieves internationales de pharmacodynamie et de Therapie 307, 172-182 (1990) 7. Kabat, E. A. and Mayer, M. M.: Experimantal lmmunochemist~, p. 135, Charles, C. Thomas, Publisher, Springfield, Illinors (1964) 8. May, C. D., Lyman, M., Alberta, R. and Cheng, J.: Procedures for immunochemical study of histamine release from leukocytes with small volume of blood. J. Allergy 46, 12-20 (1970) 9. Dunnett, C. W.: A multiple comparison procedure for comparing several treatment with a control. J. Am. Stat. Assuc. 50. 1096-1121 (1950) IO. Pelczarska, A. B. and Roszkowski, A: P.: Inhibitors of Forssman guinea pig anaphylaxis. J. Pharmacol. EXD. Ther. 185. 116-126 (I973) 11. Becher, E. L. and Austen K. F.: Anaphylaxis in Textbook of Immunopathology, vol. 1. (ed., by Miesher, P. A, and Miiller-Ebrhard, H. J.) Grune and Stratton, New York, 117-135 (1976) 12. Hitomi, Y. and Fujii, S.: Inhibition of various immunologic reaction in vivo, by a new synthetic complement inhibitor. Int. Arch. Allergy appl. Immunol. 69.262-270 (1982) 13. Butler, K. D. and Smith, J. R.: Mechanism of Forssman induced bronchospasms and their inhibition. Br. J. Pharmacol. 73, 25-32 (1981) 14. Newman, S.. Glovsky, M. M., Ghekiere, L. and Alenty, A.: Quantitative requirements for C3 to induce Forssman systemic shock and cutaneous hemorrhagic vasculitis in guinea pigs. J. Allergy Clin. lmmunol. 59. 327-333 (1977) 15. Davies, G. E.: Inhibition of guinea pig complement in vitro and in vivo by carrageenin. Immunology 6, 561-568 (1963) 16. Spear, G: S. and Kihara, I.: Complement and heterouhile shock. Johns Hookins Med. J. 126. I 2f0-2i6 (1970) 17. Tsai. C. C., Taichman, N. S., Pulver. W. H. and Schonbaum E.: Heterophile antibodies and tissue injury III. A role for platelets in the development of lethal vascular injury during Forssman shock in guinea pigs. Am. J.-P&ho]. 72, 179-196 (1973) 18. Taichman. N. S.. Creiehton. M.. Steohenson. A. and Tsai, C. C.: Heterophil antibodies and tissue injury, 1. Ultrastructure of pulmonary vascular lesions produced by Forssman antiserum in guinea pigs. Immunology 22, 93-102 (1972) 19. Nakagami, K., Kobayashi, T., Takayanagi, N., Yano, J. and Miura, M.: Increase in pulmonary resistance induced by Forssman antiserum in guinea pigs. Folia, pharmacol. japon 82, 429-441 (1983) (in Japanese) 20. Nakagami, K., Inamura, M., Nakano, Y. and Kobayashi, Y.: Effect of liposome-encapsulated recombinant human superoxide dismutase (liposome-r-h-SOD) on the lung injury induced by Forssman antiserum in guinea pigs. Folia pharmacol. japon 97, 41-49 (1991)
K. Yoshitake,
T. Iwama, A. Arimura” and A. Koda
Department of Pharmacology, Gifu Pharmaceutical University, S-6-1 Mitahora higashi, Gifu, 502, J~pu~ and ~Sh~o~~~~research ~u~~rutor~es, 5-12-4 Sag&u, Fuk~h~rn~-K~, Osaka, 5.53, J~pff~ &prim requests to HN) ABSTRACT. TBw&f&s were studied of three novel throm~xane AZ (TXA*) receptor ~~~~~ {S-1452, AA-2414 and ~NU-~~~~ on the increase in pufmonary pressure caused by Forssman a~~yla~s in guineapigs, Three TXAZ an~go~is~ at doses of between 1 and 10 m#kg ad~n~ter~ orally 1 h before the chsllenge clearly inhibited the puimonary pressure increase. At a dose of 10 mfig, all three an~gonists inhibited the pulmonary pressure increase caused by leukotriene Dd (LTDd) and U-46619, but not that caused by histamine, The decrease in peripheral platelet counts caused by Forssman anaphylaxis was also clear& inhibits by the three TXAz antagonists. However, the decreased peripheral leukocyte counts were unaffected by the three agents. The decrease in serum complement activity (CHw) was inhibited by S-1452 and AA-2414 at a dose of 10 mg/kg. In bronchoalveolar lavage fluid (BALF), significant increases in eosinophils and neutrophils were observed after Forssman anaphylaxis. Three TXA2 antagonists at a dose of 10 nqq’icg (except for AA2414 on ~in~ils~ did not affect the changes of leukocyte counts in BALF. Moreover, increases in the TXS, and fi-keto-PGFl, leveis of the BALF brought about by Forssman anaphylaxis were m&&e&d by the three TXA2 receptor antagonists. Histamine and LTDd were not changed in the BALF after Forssman ~~~hyI~is. These results indicate the ef#icaey of TXAz receptor antagonists on the increase in pulmonary pressure caused by Forssman anaphylaxis in ~inea-pin by direct antagonism to released TXA2.
INTRODUCTION Forssman (heterophiie) shock in the guinea-pig is an important model of immunological cytotoxic injury (l-3). A lethal response can be provoked by an intravenous injection of rabbit antiserum to sheep erythrocyte stroma. Postmortem examination reveals marked oedematous and massive haemorrhaging in the lungs, as well as a frothy blood-stained exudate which fiffs the trachea and exudes from the nose. Previously, we have reported the role of thromboxane A2 (TXA2) in Forssman shock, and the efficacy of OKY-046, a novel TXAz synthetase inhibitor, on Forssman fethal shack (4). Simultaneously, we reported an effect of OKY-046 on the Forssman antibody-induced airway resistance increase. When Forssman antibody was injected into guineapigs at a sublethal dose, a diphasic increase in &he p~Imona~ pressure was observed. Phase I occurs
immediate@ after the injection of antiserum, probably due to the release of serotonin and other mediators. Phase II pulmonary pressure increase QCcurs immediately thereafter, because of pulmonary oedema and obstruction caused by endothelial damage and ~xtravasation of haemorrhagic exudates into the respiratory tract. In this study, we investigated the effect of three TXA2 receptor antagonists on Forssman antibodyinduced airway resistance increase in guinea-pigs.
MATERIALS AND METHODS Animals Hartley male guinea-pigs, weighing 300-400 g, were used, Animals were purchased from Shizuaka Laboratory Animal Center. Materials
Date received 8 My
Calcium ~(Z~-lR,ZS,3S,~7-~3-phen~~ su~fony~ami~ bicycio [Z+21 hept-2-ye-~-hept~noate hydrate (S-1452,
1981
Date accepted 311September19% 233
234
Prostagiandins Leukotrienes
and Essential Fatty Acids
Shionogi Pharmaceutical Co Ltd, Osaka, Japan), (~)-7-(3,5,6-Trimethyl-l,4-benzoquinon-2-yl)-7-phenyl heptanoic acid (AA-2414, Takeda Pharmaceutical Co Ltd, Osaka, Japan) and (9,11), (11,12)-Dideoxa9cu,l lo-dimethylmetha~o-1 l,lZ-methano-I3,14-dihy~o-13-aza-14-o~o-15-~yc1~~~~1-16,17,1g,19,20pentanon-Ifi-epithromboxane AZ (ONO-3708, Ono Pharmaceutical Co Ltd, Osaka, Japan) were kindly donated by each company. Each drug was suspended in saline containing 0.5% carboxy methylcellulose (CMC). Hemolysin (Kyokuto Pharmaceutical Co Ltd, Tokyo, Japan), 9,lI~id~xy-9~,ll~~methanoe~xyprostaglandin Fz~ (U-46619, Funakoshi Co Ltd, Tokyo, Japan), leukotriene D4 (LTD4, Wako Pure Chemical Co Ltd, Osaka, Japan) and histamine (Wako Chemicals) were purchased.
M~u~ment
of pu~m~ary pressure
The increase in pulmonary pressure was measured
by the overflow method, as described by KonzettRossler and ourselves (5, 6). The guinea-pigs were initially anaesthetised with pentobarbital Na at a dose of 50 m&kg. The trachea was cannulated, and the animal artificially ventilated by a positive pressure pump at 60 stroke~min with 5 ml/stroke. Changes in pressure were measured by connecting a side arm from the tracheal cannula to an arterial pressure transducer, coupled to a strip chart recorder (Ugo Basil, Milano, Italy).
Me~u~ment
of blood parameters
The numbers of platelets and leukocytes were counted by a Coulter counter (MEK-3100, Nihon Koden, Tokyo, Japan). Haemolytic complement activity was measured by the CHSe method described by Kabat-Mayer (7).
M~u~ment of TXBz, B-keto-PGF,,, LTD4 and histamine in the BALF
In order to determine the amounts of TXAz and PGI2, their stable breakdown products - TXB;? and 6-keto-PGFi, - were measured. The BALF was obtained by injecting saline containing indomethacin at a concentration of 10s7 M. The fluid was centrifuged at 5000 rpm for 30 min at o”C, and then applied to a C-18 column. The column was washed with 4% acetic acid and distilled water. TXB:! and 6-keto-PGFi, were eluted with lc#l% methanol. Then, the effluent was evaporated and dissolved in assay buffer (phosphate buffer saline containing gelatin and thimerosal). The amounts of TXBz and 6-keto-PGF,, were measured using a radioimmunoassay kit (Amersham Japan, Tokyo, Japan). The recovery rates of TXB2 and 6-ketoPGF1, were 75 and lOO%, respectively. In order to measure the amount of LTD, in the BALF, BALF was obtained by injecting saline containing AA-861 (a potent 5-lipoxygenase inhibitor) at a concentration of lo-? M. The fluid was centrifuged at 5000 rpm for 30 min at 0°C. Three milliliters of supernatant were mixed with 12 ml ethanol. These samples were centrifuged at 10 000 rpm for 10 min at 4°C. The supernatant was evaporated below 40°C. Samples were applied to a C-18 column, after adjusting to pH 5.1 with 1 N HCl and washing with distilled water (20 ml) and petroleum ether (20 ml). Following this procedure, LTD4 was eiuted with 20 ml methanol. Then, the effluent was evaporated and the amount of LTD4 in the residual solution measured using an Amersham radioimmunoassay kit. To determine the amount of histamine, BALF was obtained by injecting saline. The BALF was centrifuged at 5000 rpm for 10 min at 4”C, and the amount of histamine assayed by the fluorescence method of May et al (8). Statistics
Measurement of Ieukocyte counts in the b~ch~lv~1~ lavage fluid (BALF)
In order to obtain the BALF, the animals were sacrificed by the intraperitoneal injection of pentobarbital (250 mg’kg), 30 min after antiserum injection. The BALF was collected by the slow injection of 10 ml sterile saline into the trachea through a cannula, whereafter the fluid was withdrawn. It was injected into the lungs again, and the procedure repeated 10 times. The BALF was centrifuged at 1000 rpm for 10 mm at 4°C. The number of nucleated cells in the Iavage fluid was counted in a Biirker chamber, and a differential count was made on a smear prepared with a c~ocent~fuge and day-Giemsa stained.
Data were expressed as a mean -t- SEM. Results for increased pulmonary pressure were evaluated by Dunnet’s multiple comparison (9). The other results were evaluated by Student’s t-test. RESULTS Effect of the three TXAz receptor uncool on pulmonary pressure changes caused by Forssman anaphylaxis and various mediators
The three TXA2 receptor antaganists were administered p.o, 1 h before the injection of antiserum or mediators. At doses between 1 and 10 mg/kg, the three antagonists inhibited increased pulmona~ pressure Forssman anaphylaxis (Fig. 1).
TXA, Receptar Aatapnists
and Pulmonary Pressure
2%
CCI ONE-3708
Fig= I Effect of S-1452, AA-2414 and 0NO-3708 on the increase in pufmonary pressure caused by Farssman anaphylaxis in guinea-pigs. Each drug was ~dmj~istered p.a. I h priar to the injectian of Fotssman antibady. Each paint represents the mean f SEM of B-24 animals. [Af 0: control l : S-1452, 1 m&g A: S-1452, IO m& D: S-1452, 3 mg’kg [S] 0: control e: AA-2414, 1 mx g A: AA-2414, 3 mg!kg A: AA-2414, 10 mg/kg 0: ONW708, 1 m&g [C] A: control A: ONO-3708, 10 mg/kg * ; p < O.QS, I ; p > 0.01 (from control)
Add~t~o~~~~y,30 m~kg doses of all three TXAz receptor ~~t~~o~jsts inhabited increased p~~rno~~~ pressure due to LTD4 and U-46619 (Fig. 2). The increase of pulmonary pressure caused by histamine was inhibited only by S-14.52. Effect of the three receptor ~~~n~s~ on Mod parameter changes caused by Forssman WlpItj&ST&?! The injection of Forssman antibody resulted in a
clear decrease in the ~ripb~ra~ platelet and leukocyte counts, as weH as the ha~rn~~i~ complement activity (Fig. 3). S-1452 at a dose of 10 mg/kg inhibited the reduction of these parameters. AA-2414 at doses of 3 and 10 mdkg inhibited the decrease in platelet count and hemolytic complement activity, but did not affect the leukocyte count. ONO-3708 at a dose of 10 m&g also inhibited the decrease in platelet count, but did not affect the other parameters.
236
Prosta~andi~s Leukotrienes and EssentialFatty Acids
100, Histamine
[AI
[ B 3 LTD4
C C 1 U-46619
0 .
I
0
1
2
3
4
k
5 Minutes
I
10
c
’
0
1
I
3
2
I
t
4 5 Minutes
I
10
L
I
0
1
3
I
I
I
I
t
2
4 5 Minutes
10
Fig. 2 Effect of S-1452, AA-2414 and ONO-3708 on the increase in pulmonary pressure caused by histamine, LTD, and U-46619 in guinea-pigs. Each drug at a dose of 10 m&g was administered p.o. 1 h prior to the injection of each mediator. Each point represents the mean + SEM of 5 animals. 0: control, e: S-1452, A: AA-2414, A: ONO-3708 * ; p < 0.05, +; p > 0.01 (from control)
Platelet 0
20
40
(106 cells/ml) 60
Platelet
120 140
0 20 -m
-iI
40
Platelet
(106 cells/ml) 60
80 120
0 20 40 -I+
140
N
N
c
c
s-1 s-3 s-10
A-l A-3 A-10
t 0 r
Leukocyte 0.1
I
(lo7 cells/ml) 0.2 0.3 t
Leukocyte
cells/ml)
Comp:;ment
I
c S-l s-3 s-10
Leuko;y;e (10: ;ellse;f
. I
I
i
N C A-l A-3 A-10
I
t
N C o-1 O-10
0 t
0
I
A-l A-3 A-10
s-1 s-3 s-10
N
(lo7
N c
:
120 140
o-1 O-10
0 e
1
I
(106 cells/ml) 60
Comp;emen;OO(CHsol
(CH ) 100 #o
200
I
t
0
I N C o-1 O-10
I
I
150
200
I
I
t
counts Fig. 3 Effect of S-1452, AA-2414 and ONO-3708 on the changes of platelet counts, . . .leukocyte . _. and serum complement actrvity (CH,,) caused by Forssman anaphylaxis in guinea-pigs. Each drug was admmrstereo p.o. 1 h prior to the injection of Forssman antibody. Each column represents the mean ? SEM of 6-11 animals. q: N; Normal (before the injection of Forssman antibody) &$ C; Control (after the injection of Forssman antibody) S-3 (S-1452, 3 mg/kg) 0: S-l (S-1452, 1 m&g) A-l (AA-2414, 1 m&g) S-10 (S-1452, 10 m&g) A-10 (AA-2414, 10 m& ) A-3 (AA-2414, 3 m&g) O-10 (ONO-3708, 10 m PB;g) O-l (ON?-3708, 1 m&g) * ; p < 0.05, ; p > 0.01 (from control)
Effect of the three TXA, receptor an~gonis~ on the leukocyte count and the levels of TXB2, 6-keto-PGF,, LTD4 and histamine in the BALF Figure
4 shows the changes
of leukocyte
count
in the BALF. The numbers of total cells, eosinophils and neutrophits increased after Forssman anaphylaxis. AA-2414 at a dose of 10 mg/kg inhibited the increase in eosinophils but other changes were unaffected by the three antagonists. Atthough
TX& Receptor Antagonists Nafrophages f% to total cells) 0 SO
The number af cells (X IO5 cells/ml)
I
t
100
1
Noraal Control S-1452 AA-2414 ONO-3708 n
Eosinophifes [# to total cells) 0 20
I
237
40
4
l!!z!z
~ Neutroohiles (% to :btal cetls)
and Pulmonary Pressure
Lymphocytes 3
Norma 1
Control S-1452 AA-2414 ONO-3708 Fig. 4 Effect of S-1452, AA-2414 and ON03708 on the changes of number of total leukocytes, macrophages, eosinophils, neutrophiles and iymph~ytes in bronchoalv~Iar lavage fluid (BALF) caused by Forssman anaphylaxis in guinea-pegs. Each drug at a dose of 10 mg&g was administered p.o. I h prior to the injection of Forssman antibody. Each column represents the mean + SEM of 5-9 animals. 8: Normal (before the injection of Forssman antibody) @: Control (after the injection of Forssman antibody) * ; p < o.f)5, : p > 0.01 (from control)
Table Effects of three TXA2 receptor antagonists on the changes of levels of histamine, LTD4, TXBz and 6-keto-PGFtb, in BALF
Normal Control S-X4.52 AA-2414 ONO-3708
Histamine (q/ml)
LTD, (&ml)
TXB:
1.0 1.2 1.4 1.0 1.1
0.0 0.0 0.0 0.0 0.0
1.6 2.7 3.3 3.6 2.5
+ 0.5 +. 0.5 * 0.6 It 0.5 f 0.5
+ + + * +
0.0 0.0 0.0 0.0 0.0
+ + + + +
6-keto-PC& 0.3* 0.3 1.0 1.1 0.5
41 87 76 70 80
(pg/ml)
rt 2* It 9 rf: 8 Yk5 -c 4
Each value represents the mean + SEM of 5-9 animals. Each drugs at a dose of 10 m&g was administered pa_ 1 h prior to the injection of Fcrrssman antibody. * ; p < 0.05 ffron contml).
the amounts of TXB, and 6-keto-PGF,, in the BALF increased significantly, the amounts of LTD4 and histamine were unchanged by Forssman anaphyiaxis (Table). The three TXA2 receptor antagonists, however, did not affect the increased levels of TXB2 and 6-keto-PGFi, in the BALF.
These results indicate the efficacy of TXA2 receptor antagonists on increased pulmanary pressure caused by Forssman anaphylaxis in guinea-pigs. Forssman anaphylaxis is considered to be an important experimental model for pu~rno~a~ thromboembolism (11). At present, specific drugs are not available for the treatment of pulmonary thromboembolism. Non-specific antiinflammatory drugs and, more oceasionafly, jmmunosuppressive drugs such as aspirin
and glucocorticoids, have been used, individuahy and combined. Other results suggest a possible use of TXAz receptor antagonists as a remedy for pulmonary thromboembolism. As for the mediator of the Forssman antibodyinduced pulmonary pressure increase in guinea-pigs, three major factors have been proposed (4, 12-20). Firstly, serotonin is a primary mediator, causing increased pulmonary pressure in phase I. Some investigators have reported the release of serotonin during shock and the efficacy of anti-serotonergic agents on increased pufmonary pressure in phase I. Secondly, superoxide radicals (Oz-) may be one of the mediators for pulmonary pressure increase in phase IX, because of the selective efficacy of superoxide dismutase in phase IL Thirdly, our previous and present results indicate that TXAz seems to be a crucial mediator in the increase of pulmonary pressure during phases I and ff. The previous study
238
Frosta~andins
Leukotrienes
and Essential Fattv Acids
indicated the efficacy of OKY-046, a selective TXA2 synthetase inhibitor, on Forssman antibody-induced pulmonary pressure increase and the increment of TXBz in the -plasma after Forssman anaphylaxis. Our present results support the previous findings on the use of TXAz receptor antagonists and BALF. The three TXA, receptor antagonists clearly inhibited increased pulmonary pressure caused by the Forssman antibody. Moreover, the amount of TXBz in the BALF was increased after the injection of antibody. These data confirm the role of TXAz in the increase of pulmonary pressure during Forssman anaphylaxis in guinea-pigs. The mode of action of TXA:! on pulmonary pressure increase is, however, still obscure. Two possible mechanisms are postulated for the role of TXA2 in increased pulmonary pressure. One is the direct action of TXAz on airway smooth muscle: TXA2 may cause a potent contraction of airway smooth muscle. The other mechanism may be related to the vasoconstriction of pulmonary blood vessels, resulting in decreased pulmona~ circulation and may cause an increase in pulmonary pressure during Forssman anaphylaxis. These two mechanisms may contribute to increased pulmonary pressure. We need to carry out further experiments to study the effect of Forssman anaphylaxis on the pulmonary circulation of guineapigs. In conclusion, these results indicate the efficacy of three novel TXAZ receptor antagonists (S-1452, AA-2414 and ONO-3708) on increased pulmonary pressure caused Forssman anaphylaxis in guineapigs.
References Redfern, W. W.: A study of the primary toxicity of heterophile immune rabbit serum for guinea pigs and its apparent relaxation to the phenomenon of anaphyla&. Am. J. Hygiene 6: 276-310 (1926) Glovsky, M. M., Ward, P. A., Becher, E. L. and Halbrook, N. J.: Role of fumaropimaric acid in guinea pig complement dependent and non-complement dependent biological reaction. J. Immunol. 102, l-11 (1969) Nagai, H., Kurimoto, Y. and Koda, A.: im~unopha~acological approach to Forssman shock. Microbial. Immunol. 24. 649-655 (1980) Nagai, H., Yakuo, I., Inagaki,‘N., Koda; A., ’ Hamano, S., Ujiie, A. and Nakazawa, M.: Role of thromboxane A, (TXA j in guinea pig Forssman shock and the effect of OKY-046, TXA, synthetase
inhibitor. Prostaglandins Leukotrienes and Medicine 26, 133-141 (1987) 5. Konzett, H. and Rossler, R.: Versuchanordnung zu Untersuchengen an der Bronchialmuskulater. Arch. Exp. Path. Pharmak. 195, 71-74 (1940) 6. Nagai, H., Suda, H., Kitagaki, K., Goto, S., Muira, T. and Koda, A.: Anti-allergic effects of Ketanserin on animal models of allergic reactions. Archieves internationales de pharmacodynamie et de Therapie 307, 172-182 (1990) 7. Kabat, E. A. and Mayer, M. M.: Experimantal lmmunochemist~, p. 135, Charles, C. Thomas, Publisher, Springfield, Illinors (1964) 8. May, C. D., Lyman, M., Alberta, R. and Cheng, J.: Procedures for immunochemical study of histamine release from leukocytes with small volume of blood. J. Allergy 46, 12-20 (1970) 9. Dunnett, C. W.: A multiple comparison procedure for comparing several treatment with a control. J. Am. Stat. Assuc. 50. 1096-1121 (1950) IO. Pelczarska, A. B. and Roszkowski, A: P.: Inhibitors of Forssman guinea pig anaphylaxis. J. Pharmacol. EXD. Ther. 185. 116-126 (I973) 11. Becher, E. L. and Austen K. F.: Anaphylaxis in Textbook of Immunopathology, vol. 1. (ed., by Miesher, P. A, and Miiller-Ebrhard, H. J.) Grune and Stratton, New York, 117-135 (1976) 12. Hitomi, Y. and Fujii, S.: Inhibition of various immunologic reaction in vivo, by a new synthetic complement inhibitor. Int. Arch. Allergy appl. Immunol. 69.262-270 (1982) 13. Butler, K. D. and Smith, J. R.: Mechanism of Forssman induced bronchospasms and their inhibition. Br. J. Pharmacol. 73, 25-32 (1981) 14. Newman, S.. Glovsky, M. M., Ghekiere, L. and Alenty, A.: Quantitative requirements for C3 to induce Forssman systemic shock and cutaneous hemorrhagic vasculitis in guinea pigs. J. Allergy Clin. lmmunol. 59. 327-333 (1977) 15. Davies, G. E.: Inhibition of guinea pig complement in vitro and in vivo by carrageenin. Immunology 6, 561-568 (1963) 16. Spear, G: S. and Kihara, I.: Complement and heterouhile shock. Johns Hookins Med. J. 126. I 2f0-2i6 (1970) 17. Tsai. C. C., Taichman, N. S., Pulver. W. H. and Schonbaum E.: Heterophile antibodies and tissue injury III. A role for platelets in the development of lethal vascular injury during Forssman shock in guinea pigs. Am. J.-P&ho]. 72, 179-196 (1973) 18. Taichman. N. S.. Creiehton. M.. Steohenson. A. and Tsai, C. C.: Heterophil antibodies and tissue injury, 1. Ultrastructure of pulmonary vascular lesions produced by Forssman antiserum in guinea pigs. Immunology 22, 93-102 (1972) 19. Nakagami, K., Kobayashi, T., Takayanagi, N., Yano, J. and Miura, M.: Increase in pulmonary resistance induced by Forssman antiserum in guinea pigs. Folia, pharmacol. japon 82, 429-441 (1983) (in Japanese) 20. Nakagami, K., Inamura, M., Nakano, Y. and Kobayashi, Y.: Effect of liposome-encapsulated recombinant human superoxide dismutase (liposome-r-h-SOD) on the lung injury induced by Forssman antiserum in guinea pigs. Folia pharmacol. japon 97, 41-49 (1991)