In Vitro Anaphylaxis in Guinea-Pig Skin: Amplification by Burimamide

IN VITRO ANAPHYLAXIS IN GUINEA-PIG SKIN: AMPLIFICATION BY BURIMAMIDE

SHOSO YAMAMOTO, M.D., DAVID FRANCIS, B.Se., AND MALCOLM W. GREAVES, M.D., PH.D., F.R.C.P. Institute of Dermatology, London, England

The effects of burimamide, an H.-antihistamine, on the anaphylactic reaction in the skin of ovalbumin-sensitized guinea pigs were studied in vitro. Burimamide enhanced the concentration of histamine in the supernatant fraction of antigen-challenged sensitized guinea-pig skin in a dose-related way, but did not alter the concentration of residual histamine in the skin after antigen challenge. The enhanced histamine concentration in the supernatant was not due to in creased histamine synthesis by the target cells during the reaction because the increase was not inhibited by a histidine decarboxylase inhibitor, Brocresine. In further experiments it was shown that guinea-pig skin possesses potent histamine degrading enzyme activity which is inhibited by burimamide. We suggest that inhibition of these degrading enzymes leads to the increase in histamine concentration in the presence of burimam ide.

Some responses to histamine, including gastric mg wet weight approximately), the number per tube acid secretion, increase in heart rate, and inhibition being constant in anyone experim ent. T he sli ces we re of myometrium, cannot be antagonized by 'c onven washed 3 times in cold Tyrode solution prior to incuba tional antihistamines. Ash and Schild [1] proposed tion. two classes of histamine receptors, H. and H •. Skin an.aphylaxis. Triplicate samples were used in all Black and his colleagues [2] described a specific experiments. Anaphylaxis was obtained by incubating skin sli ces with ovalbumin antigen (5 x crystallized, competitive H2 antagonist, burimamide, and sug Koch-Light Laboratories Ltd.) in concentrations of 10 gested that burimamide together with a conven /L g/ml for 15 min at 37°C. The reaction volume was 2 ml. tional (H I antagonist) antihistamine might prove a The reaction was term in ated by decantation of the more effective combination in the management of supernatant Tyrode solution. Supernatant and residual whealing skin reactions due to histamine than histamine in each sample were expressed in absolu te either class of antihistamine alone. However the amounts. All histamine resul ts were expressed after effect of H2 antagonists on anaphylaxis in sk in is subtraction of t he corresponding negative co ntrol valu es. unknown. We therefore report the effects of buri Under these conditions histamine release in positive mamide on anaphylaxis in guinea-pig skin in vitro. control samples was approximately 50% of total hista mine in most experim ents. The effect ofburimamide was MATERIALS AND METHODS studied by preincubating burimamide with t he skin sli ces for 5 m in at 37°C prior to challenge by antigen (prein The effect of burim amide on anaphylax is was studied cubation volume 1 ml), burimamide control samples usin g skin of ovalbumin-sensitized guinea pigs by the being similarl y treated. method of Yeoh, Tay, and Greaves [3 ). Histamine assay. Histamine was measured by bioas Materials. Burimamide was a gift of Dr. Brimblecome say using the atropinized guinea-pig il eum preparation (Smith, Kline and French Laboratories Ltd.). Brocresine and an automatic bioassay apparatus. Histamine activ (NSD 1055) and egg albumin (for sensitization) were ity was estimated by comparison with contractile re obtained from Smith and Nephew Ltd., and British Drug sponses to several concentrations of standard hi stamine Houses, respectively. solu tions. That the smooth muscle contracting activity Male guinea pigs weighing 200 to 300 gm Sensitization. released from the skin was entirely due to histamine was were sensitized by two subcutaneous injections of 0.5 ml coni'irmed using the specific antagonist mepyramine egg albumin solu tion (1 %). T he sensitized guin ea pigs (10 - 6 M). Neither burimamide nor Brocresine interfered were used between 3 and 8 weeks later. wit h the responses of t he gu inea-pig ileum to histamine in Animals were killed by stunning followed by Skin. the concentrations used. However, as an additional exsanguination. Shaved abdominal skin was sli ced into precaution, equal concentrations of the drugs were added 500-/Lm-thick sli ces using a hand microtome. The sli ces to the standard histamine solu tions during bioassay. were separated into a li quots of 4 or I) per tube (totaling 70 RESULTS Manuscript received March 12, 1976; accepted for publication July 20, 1976. Effect of Burimamide on Co ncentration of This work was supported by grants from t he Nuffield Histamine Trust and the Medical Research Council. Reprint requests to: Dr. M. W. Greaves, Institute of The concentration of histamine in the super Dermatology, Homerton Grove, London E9 6BX, natants of antigen-treated skin samples was higher England. in the presence of burimamide. In 8 experiments 696 Dec. 1976 SKIN ANAPHYLAXIS AND BURIMAMIDE 697 burimamide in concentrations of 10-Histidine Decarboxylase Inhibitor Hi stamine The possibility that the higher histamine concen tration of the supernatants with burimamide was due to increased biosynthesis of histamine was considered. We therefore examined the effect of Concentration of Burimamide (M I Brocresine on the increased histamine concentra FIG. 1. Effect of burimamide on concentration of his tion in sa mples containing burimamide. Brocresine tamine in supernatants of ant igen-chall enged samples. n (4-bromo-3-hydroxybenzyloxyamine, NSD 1055) is ; 8, mean ± SEM. an effective histidine decarboxylase inhibitor in vitro [4,5 ]. Brocresine at a concentration of 10 - ' 100 M (4 experiments) and 10 - 6 M (2 experiments) failed to prevent the increase in histamine caused by burimamide. These results do not support the pos Hi stam ine sibility that the increase in the histamine in the activi ty presence of antigen and burimamide is due to ~, increased histamine synthesis. 50 Enzymic Histamine Degradation: Inhibiton by Burimamide The alternative possibility was considered that guinea-pig skin contains histamine-degrading enzymes which normally cause decay of superna tant histamine during the period of incubation, and 10 20 30 40 50 60 that burimamide inhibits this process. T i me ( mins . Skin slices (4 per tube) were incubated in the presence of 80 ng histamine (concentration 160 FIG. 2. T im e-dependent hi stamine degradation dur ing incubation of histamine with guinea pig skin in ng/mi) for periods ranging up to 60 min. The Tyrode solut ion at 37°C. Ordinate ; % reduction of results of one experiment are shown in Figure 2. initial histam ine concentration (160 ng/ ml) in the pres ence of 4 slices of gu in ea-pig abdominal skin (wet we ight 70 mg approxim ately). No significant degradation TABLE. Amount of histamine released by antigen in occurred at 2°C. su.pernatant in the presence and absence of burimamide The histamine activity of the supernatant Tyrode Each value represents the mean of triplicate samples. solution declined progressively during incubation, Mean residual histamine ; 42 .4 ng ± 7.6 SEM (no 15 % of activity being lost at 15 min and 70 % at 60 burimamide), 47.5 ng ± 9.3 SEM (10 - ' M burimam ide). min. The reduced activity was not due to absorp Burim8micie (M) tion or uptake by the skin slices because t here was ExpL no increase in the histamine content of the skin 0 10 - ' slices (determined by boiling for 10 min and bioassay after cooling). 27.7 65.6 1 That the observed decay in histamine activity 2 40.0 57.7 was enzym ic was supported by the temperature 46.3 77.0 3 sensitivity of the reaction. In two experiments, 4 4 27.3 44.6 skin sli ces were incubated with 80 ng histamine 5 97.7 148.0 (concentration 160 ng/ml) for 60 min at 37°C and 67.4 70.0 6 2°C. At 37°C histamine activity decayed by 71% 7 53.3 143.7 but there was no significant reduction in histamine 8 20.6 37 .7 activity after in cubation at 2°C. 698 YAMAMOTO, FRANCIS, AND GREAVES Vol. 67, No . 6

100 guinea pig, methylation and oxidation of hista £ mine occurred to approximately the same extent ::> (7). Brown and his colleagues compared hista U mine-N-methyl transferase activity in severallabo ""Cl.) c:: ratory species and found the highest level of E activity in guinea-pig skin (9). It has been reported ~ Vl that histamine methylation is inhibited by buri .= mamide in guinea-pig atrium (10) and in pig a gastric mucosa [ll). It is therefore possible t hat, in c:: 50 ...... = the skin, histamine released from target cells -="" during anaphylaxis might be inactivated by hista ~ mine methylation and that burimamide might ""c::r> -=Cl.) reverse this process. The molecular configuration c:: of burimamide resembles that of histamine. Both Cl.) u molecules are based on an imidazole ring, burima

~ Cl.) mide possessing a polymethyl- thiourea substituent CL group. It seems probable that the inhibitory action o~~------~------~- of burimamide on degradation of histamine is at o tributable to substrate compet it ion. The slower rate of degradation of added histamine by skin Concentrat ion of Bur imamide (M) (Fig. 2) compared with degradation of antige n FIG. 3. Effect of burimamide on degradation of hista evoked (endogenous) histamine (Fig. 1 and Tab.) mine by gu inea-pig skin, in two experiments. Ordinate = is explained by t he greater accessibility of endoge % degradation of histam ine activity in su pernatant. nous histamine substrate to the degrading enzyme during secretion of histamine within the tissue. The effect of burimamide on degradation of The inhibitory action of burimamide on histamine histamine by guinea- pig skin was studied in two degradation was not seen in control samples con experiments. Skin slices (4 per tube) were incu taining burimamide in the absence of antigen in bated with 80 ng histamine (concentration 160 which low concentrations of histamine were pres ng/rnl) at 37°C for 60 min in the presence of ent due to "spontaneous" release of histamine burirnamide (4 X 10 - 6 to 4 x 10 - 5 M) . The results during incubation. This can be explained by the given in Figure 3 show that burimamide caused a extremely low "spontaneous" release of histamine dose-related inhibition of histamine degradation. during the 15-min period of incubation . Burimamide may prove to have a suppressive DI SCUSSION effect on histamine-mediated inflammatory reac The present experiments have show n t hat buri tions in skin through an action on H 2 receptors of rnamide amplifies the anaphylactic reaction in skin blood vessels [1 2 ). Nevertheless our ev idence guinea-pig skin by reducing enzymic degradation raises t.he possibility that burimamide administra of released histamine in vitro, thus leading to tion in vivo may amplify the reaction through elevated concentrations of histamine following inhibition of histamine degradation in skin. Our challenge of sensitized skin by antigen. We were results also suggest t hat enzymic degradation of not able to detect evidence of enhancement of histamine may have an important role in regula antigen-stimulated histamine release by burima tion of skin anaphylaxis. rnide as suggested by Lichtenstein and Gillespie [6] a lthough the possibility of a small increase in REFERENCES release by burimamide cannot be excluded by our 1. Ash ASF, Schild HO: Receptors mediating some data. That the increase of histamine was not due to actions of histamine. Br J Pharmacol Chemother 27:427- 439, 1966 increased histamine formation was indicated by 2. Black JW, Duncan WAM, Durant CJ, Ganellin CR, the failure of Brocresine, a histidine decarboxylase Parsons EM: Definition and antagonism of hista inhibitor, to reverse the burimamide-induced in mine H, receptors. Nature (Lond) 236:385- 390, crease in histamine. Although the concentrations 1972 3. Yeo h TS, Tay CH, Greaves MW: An aphylactic we used are known to be effective in vitro (5 ), we release of histam ine from guinea pig skin in vitro. cannot exclude the possibility that higher concen lnt Arch Allergy Appl lmmunol 42:285- 490, 1972 trations might have produced inhibition. 4. Reid JD, Shepherd DM: Inhibit ion of histidine T h e observation that gu inea-pig skin contains decarboxylases. Life Sci (Oxford) 2:5- 8, 1963 5. Levine RJ , Sato TL, Sjoerdsma A: Inhibit ion of potent histamine-degrading enzyme activity was histamine synthesis in the rat by a-hydrazino not unexpected. At least two types of histamine analog of histidine and 4-bromo-3-hydroxybenzyl degrading enzymes have been identified. The prop oxya mine. Biochem PharmacoI14:139- 149, 1965 6. Lichtenstein LM, Gill espie E: The effects of the HI erties and distribution of diamine oxidase and and H , antihistamines on all ergic histamine release histamine-N-methyl transferase have long been and its inhibition by histamine. J Pharmacol Exp recognized [7- 9). It has been shown that, in the Ther 192:441- 450, 1975 Dec. 1976 SKIN ANAPHYLAXIS AND BURIMAMIDE 699

7. Schayer RW: Catabolism of physiological quantities mouse neoplastic mast cells in vitro. Br J Phar of histamine in vivo. Physiol Rev 39: 116- 126, 1959 macol 53:569-574, 1975 8. Zeller EA: Identity of histaminase and diamine oxi· 11. Barth H, Niemeyer I, Lorenz W: Studies on the mech dase. Fed Proc 24: 766- 768, 1965 anisms of inhibition of gastric histamine methyl 9. Brown DO, Tomchick R, Axel rod J: The distribution transferase by H 1- and H ,-receptor antagonists, and properties of a histamine· methylating enzyme. International Symposium on Histamine H , J BioI Chem 234:2948- 2950, 1959 Receptor Antagonists. Edited by CJ Wood, MA 10. Fantozzi R, Franconi F, Mannaioni PF, Masini E, Simkins. London, Deltakos, 1973, pp 11 5-126 Moroni F: Interaction of histamine H I - and H, 12. Wyllie JH, Hesselbo T , Black JW: Effects in man of receptor antagonists with histamine uptake and histamine H,-receptor blockade by burimamide. metabolism by guinea-pig isolated atrium and Lancet 2:1117- 1120, 1972