J. Physiol. (1952) ii8, 46I-478

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1 461 J. Physiol. (1952) ii8, 46I-478 A COMPARISON OF THE EFFECTS OF ANAPHYLACTIC SHOCK AND OF CHEMICAL HISTAMINE RELEASERS BY J. L. MONGAR AND H. 0. SCIJILD From the Department qf Pharmacology, University College, London (Received 26 May 1952) One of the characteristic features of histamine release in anaphylaxis is that the quantity of histamine released by the antigen frqm different tissues varies and bears no constant relation to the amount originally present in the tissue (Schild, 1939). Some organs of the guinea-pig, for example, intestine, release in anaphylaxis only a small fraction of their histamine content-of the order of 3 % or less, whilst other tissues such as the aorta and the uterus, release in anaphylaxis more than 30% of the histamine contained in the tissue. This is in contrast to the unspecific action of grinding the tissue with sand in saline, which causes practically all the histamine originally contained in the tissue to be freed. It was of interest to find out whether chemical histamine releasers would release histamine selectively as it is released in anaphylaxis, or unselectively as in mechanical disintegration of the tissue. In Part I of this paper we report experiments in which the quantity of histamine released from different organs of the guinea-pig in anaphylaxis is compared with that released by D-tubocurarine or the powerful histamine releaser 48/80 (Baltzly, Buck, de Beer & Webb, 1949; Paton, 1951). Our results show that under similar conditions there is a close correlation between the fraction of histamine released in sensitized tissues by the chemical releasers and by the specific antigen. In view of this close quantitative agreement between the two types of releaser it seemed possible that previous administration of a chemical releaser might prevent the anaphylactic release of histamine and perhaps also the anaphylactic muscular contraction. The experiments relating to this are reported in Part II. METHODS The methods used for detecting histamine release were similar to those previously used by Schild (1939) and Rocha e Silva & Schild (1949). They consisted in first washing thin pieces of tissue (weighing mg) taken from young sensitized guinea-pigs for 20 min in about 100 ml.

2 462 J. L. MONGAR AND H. 0. SCHILD Tyrode solution at 370 C. The tissues were then suspended in 1 ml. of Tyrode solution at 370 C, well stirred by bubbling oxygen. The quantity of histamine diffusing from the tissue into the surrounding fluid in the presence of either the specific antigen or a chemical histamine releaser was measured by assaying on guinea-pig intestine, using a four-point method and automatic apparatus (Mongar & Schild, 1950). For the purpose of comparing the releases by these two classes of substances, adjacent pieces of tissue of similar thickness and structure from the same animal were used. Before being assayed on the guinea-pig ileum the two solutions were made identical in composition by adding chemical releaser to one and antigen to the other in order to equalize any possible interference of these with the biological assay of histamine. To prevent bacterial action the solutions were boiled for 1 min. They were then diluted at least five times with Tyrode solution as high concentrations of both 48/80 and D-tubocurarine tend to decrease the sensitivity of the preparation. By interposing doses of histamine between those of the diffusates it was possible to obtain satisfactory assays with concentrations up to 0-2 mg/ml. 48/80 and 1 mg/ml. D-tubocurarine in the assay solutions. The spontaneous rate of release of histamine from the tissues suspended in Tyrode solution was measured and subtracted from the total release. It was usually less than 5% of the total activity. In referring to the gut-contracting substance as 'histamine' we are aware that a slow-reacting, gut-contracting substance is released under certain conditions by antigen and chemical releasers. In our experiments on isolated tissues, however, the active substance liberated in either case was completely antagonized by concentrations of mepyramine just sufficient to antagonize completely equiactive doses of histamine. Highly sensitized guinea-pigs were obtained by injecting 100 mg of commercial egg albumin (B.D.H. flake) intraperitoneally and 100 mg subcutaneously, 3-6 weeks before the experiments. D-Tubocurarine (B.P.) was used; the 48/80 was kindly supplied by Dr Kellaway of the Wellcome Laboratories as 'standard depressor substance 48-80a'. It has a formula of the type (Baltzly et al. 1949): CH2NR2 CH2NR2 CH2NR2 CH2 CH2 CH2 Z CH2 CH2 z OCH3 OCH3 OCH3 Where R is H or CH, and Z and Z' are H, CH2OH or CH2C1. tetramer. It also contains some dimer and RESULTS I. Comparison of chemical releasers and antigen It has previously been shown that when the isolated diaphragm of the rat is immersed in a solution of D-tubocurarine or when isolated tissues of a guineapig sensitized to egg albumen are immersed in a solution of egg albumen, histamine diffuses from the isolated tissue into the surrounding fluid (Rocha e Silva & Schild, 1949; Schild, 1939). We have now found that D-tubocurarine and compound 48/80 will release histamine from many isolated tissues of the guinea-pig, if these are immersed in Tyrode solution at 370 C containing the histamine liberators in sufficient concentration.

3 ANAPHYLACTIC. SHOCK AND HISTAMINE RELEASERS 463 We tried to find out whether the quantities of histamine thus released are related to the histamine content of tissues or unrelated as is the case in anaphylaxis, and whether there is parallelism between histamine released in anaphylaxis and by synthetic releasers. Before describing these experiments the time course of histamine release and the effect of concentration of releaser were investigated in order to choose optimal conditions for a comparison. (a) (b) 48/80 (1 mg/mi.) Egg albumin (1 mg/ml.) * Uterus X Lung A Aorta -o x Diffusion time (min) Diffusion time (min) Fig. 1. Comparison of time course of histamine release from various guinea-pig tissues by 48/80 and antigen. Time course of histamine release. Typical release experiments by 48/80 are illustrated in Fig. 1 a. These may be compared with histamine release by antigen from sensitized tissues, as illustrated in Fig. l b. It will be seen that irrespective of the tissue and releaser used, about three-quarters of the total release occurs in the first 10 min. It is difficult to determine with certainty when the release induced by a specific releaser has finished, since as a background to any specific release there is a slow spontaneous loss of histamine from all tissues even after they have been well soaked in Tyrode to wash out any histamine from damaged cells. We have therefore chosen a 10 min period as the standard time allowed for histamine release and diffusion out of the tissue into the Tyrode solution. Effect of concentration of releaser. In previous work on histamine release by D-tubocurarine from rat's diaphragm the quantity released depended critically on concentration: 1 mg/ml. D-tubocurarine produced a maximal release,

4 464 J. L. MONGAR AND H. 0. SCHILD whereas 0.1 mg/ml. was practically ineffective. A similar critical dependence on concentration was recently described by McIntire, Roth & Sproull (1951) in a study of histamine release from rabbits' blood by certain long-chain compounds. In the present experiments histamine release also increased markedly with concentration as shown in Fig. 2, but in contrast to the previous 50 A Egg albumin to 4 _ 48/80 / v 3~~~~~~~~~ C E S2 0 4 OA X A~~~~ t Fig io lo- 104 lo- 1o2 Concentration of releaser Effect of concentration of antigen and 48/80 on histamine release from lung of sensitized guinea-pigs. experiments in the rat's diaphragm it was not possible to reach a concentration giving a maximal rate of release. Even at the highest practicable concentrations the curves show no evidence of flattening out. It is interesting that histamine release increases with concentration not only with chemical releasers but also when the specific antigen is used. In order to compare chemical releasers and the anaphylactic reaction in different tissues the following procedure was adopted. In a preliminary series, the concentrations of 48/80, D-tubocurarine and egg albumin were determined which are equiactive when applied to the isolated diaphragm of a sensitized guinea-pig. The following concentrations produced approximately the same quantity of histamine from guinea-pig diaphragm: 48/80, 1 mg/ml.; D-tubocurarine, 6 mg/ml.; antigen (egg albumin), 1 mg/ml. These same concentrations were then tested on thirteen different tissues of the guinea-pig. Histamine release from thirteen different tissues by antigen and chemical releasers. In each case adjacent similar pieces of tissue from a sensitized guinea-pig were kept for 10 min in solutions of the chemical releasers or antigen, and the quantity of histamine diffusing into the surrounding fluid as well as that remaining in the tissue at the end of the experiment was determined, the latter after grinding the tissue with sand and boiling. The results are

5 ANAPHYLACTIC SHOCK AND HISTAMINE RELEASERS 465 summarized in Fig. 3 and shown in detail in Tables 1 and 2. In Fig. 3 histamine release is expressed as a percentage of the initial histamine content calculated as the sum of histamine released and histamine extracted at the end of the experiment. 0-5 r WI -'5 '_ v o F 0X2 I F 48/80 (1 mg/ml.) * Egg albumin (1 mg/ml.) E D-Tubocurarine (6 mg/ml.) 0-1 I 0 E a- 0 Fig. 3. II0 ] "0 W- -~0. C '4 0 3 so c -I E 0 U, V 0 J L Ov.c " v -,c 0. Parallelism of histamine release by antigen and chemical releasers from thirteen guinea-pig tissues. The following main conclusions may be drawn from these results: (1) The percentage of histamine released by either antigen or synthetic releasers varies markedly in different tissues. It is smallest in the intestine and largest in the diaphragm, aorta and uterus. These differences cannot be explained in terms of length of diffusion path; for example uterus and small intestine, both of which are thin hollow organs, are at the two ends of the scale. The absolute quantities of histamine released also vary markedly as shown in Table 3, but in this case the order is somewhat different: the lungs move to the top of the scale in view of their high histamine content and the diaphragm towards the bottom in view of its low histamine content. Even so the gastrointestinal tissues form the most inactive group. (2) There is a remarkable overall parallelism between percentage histamine released in the anaphylactic reaction and by the two chemical releasers. to~

6 466 J. L. MONGAR AND H. 0. SCHILD TABLE 1. Comparison of histamine release by egg albumin (1 mg/mi.) and 48/80 (1 mg/mi.) from sensitized guinea-pig tissues Tissue Diaphragm Aorta Uterus Heart Bladder Skin Trachea Oesophagus Lung Stomach Spleen Large intestine Small intestine Histamine release (.tg/g) Egg albumin 48/ Histamine remaining (pg/g) Egg albumin 48/ ' Particularly noticeable is the small release of histamine by the intestine in anaphylaxis as well as with 48/80 or D-tubocurarine (it will be shown later, however, that a different result is obtained when chemical releasers and antigen are combined), whereas diaphragm, aorta and uterus release a large proportion of their histamine content with either type of releaser. The

7 ANAPHYLACTIC SHOCK AND HISTAMINE RELEASERS 467 TABLE 2. Comparison of histamine release by egg albumin (1 mg/ml.) and D-tubocurarine (6 mg/ml.) from sensitized guinea-pig tissues Histamine release Histamine remaining Gtg/g) ('4g/g) D-Tubo- D-Tubo- Tissue Antigen curarine Antigen curarine Diaphragm Aorta Uterus Heart Bladder Skin Trachea Oesophagus (9-9) Lung Stomach Spleen Large intestine Small intestine TABLE 3. Histamine content of guinea-pig tissues and average histamine release by antigen or chemical releasers Histamine Histamine content release Fraction Tissue (pg/g) (.tg/g) released Aorta (10) Lung (11) Uterus (9) Trachea (4) Oesophagus (8) Heart (7) Spleen (6) Bladder (9) Skin (9) Diaphragm (10) Small intestine (10) Large intestine (7) Stomach (7) The figures quoted are the geometric means of individual results. The number of experiments on each tissue is given in brackets. parallelism is not complete, for example, in the aorta and heart, 48/80 releases appreciably more histamine than antigen. This may be due to the fact that not all individual cells have become sensitized, hence the antigen interacts with fewer cells than the chemical releaser. It is also noticeable that 6 mg/ml. D-tubocurarine is generally rather less active than 1 mg/ml. 48/80 except in the skin where curare seems to be particularly active. PH. CXVIII. 30

8 468 J. L. MONGAR AND H. 0. SCHILD (3) There is no correlation between histamine content of tissues and histamine release. Histamine releasefrom unsensitized tissues. All comparisons were made with sensitized tissues. With normal tissues there is, of course, no release by the antigen but 48/80 releases amounts comparable to those with sensitized tissues. Table 4 compares the results of tests on normal tissues with those obtained on sensitized ones. TABLE 4. Comparison of histamine release by 48/80 from normal and sensitized guinea-pig tissues Geometric means (,ug/g) Normal Sensitized Aorta 6 (2) 10 (4) Lung 6 (3) 5 (4) Skin 0 3 (3) 0 6 (4) Oesophagus 0.6 (1) 1-2 (3) Small intestine 0-5 (3) 0-4 (4) Diaphragm 1-2 (2) 0'6 (4) The figures in brackets represent the number of separate determinations. II. Interaction of chemical releasers and antigen In all the tissues examined, only a fraction of the total histamine contained in the tissue was released by chemical releaser or antigen. Since for each tissue the fractions released were similar, it seemed possible that the previous administration of a chemical releaser might by exhausting the available histamine prevent a subsequent histamine release and muscular contraction in anaphylaxis. The interactions of chemical releasers and antigen on (a) histamine release, (b) muscular contraction, are described in the following two sections. (a) Histamine release The results of these interaction studies proved to be complex. It was not simply a case of exhaustion of available histamine by one releaser leaving the tissue refractory to the action of the other. Depending on the tissue used, there was a variety of interactions ranging from simple depletion or 'desensitization' of the tissue, to the reverse process, '.sensitization' of one releaser by the other. For example, in the intestine previous contact with 48/80 or D-tubocurarine produces a greatly increased histamine release by the antigen. The technique generally adopted in these experiments was as follows: the tissue was placed in solutions of one releaser until the rate of release fell to a steady low value. It was then placed in solutions of a second releaser and the change in rate of histamine release was measured. Interaction of chemical releasers and antigen in isolated aorta and lung. In the sensitized lung and aorta 48/80 (1 mg/ml.) is able to release most of the

9 ANAPHYLACTIC SHOCK AND HISTAMINE RELEASERS 469 available histamine, so that when the antigen is added there is no appreciable increase in the rate of release (Fig. 4). 12 c 1-0 E >! 0-8 c 0-6 I-n..v4 W-r. 40% Aorta 15% * Egg albumin (1 mg/ml.) El 48/80 (1 mg/ml.) Lung 17% 5% U I I -I -I Fig. 4. Release of histamine from aorta and lung of sensitized guinea-pigs; 48/80 followed by egg albumin. (The percentage figures are in terms of the total histamine content of the tissue.) 1-2 c 1.0 S -8 }0X8.,a 00-6 E0-4._ I: n.2 - L 21% Aorta Egg albumin (1 mg/ml.) 0l] 48/80 (1 mg/ml.) 12% Lung 8% 0s A1 30 Fig. 5. Release of histamine from aorta and lung of sensitized guinea-pigs; egg albumin followed by 48/80. If the experiment is reversed, the antigen being added before the chemical releaser the results are not the same. Fig. 5 shows that 48/80 is still able to release further histamine from a tissue previously treated with antigen. Attempts were made to abolish histamine release by antigen with lower concentrations of the chemical releasers which, though not releasing much 30-2

10 470 J. L. MONGAR AND H. 0. SCHILD histamine themselves, might interfere with the antigen-antibody reaction. No such effect was observed. Fig. 6 shows that immersion of aorta in 0 01 mg/ml. 48/80 for 15 min does not interfere with subsequent histamine release by E bo ~,0-4 C E t~0-2- X ~~5% * Egg albumin (1 mg/ml.) [j 48/80 (0-01 mg/ml.) 35% Fig. 6. Release of histamine from aorta of sensitized guinea-pig. Dilute 48/80 (0-01 mg/ml.) followed by egg albumin. antigen. A similar lack of interference is shown when antigen and 48/80 act together, as shown in Table 5; 0-01 and 0-1 mg/ml. 48/80 cause no reduction in the release by antigen. Conversely, the antigen causes no reduction in the release by 1 mg/ml. 48/80. TABLE 5. Histamine release by antigen and 48/80 acting together on isolated guinea-pig lung Concn. egg Conen. Histamine albumin 48/80 release (mg/ml.) (mg/ml.) (Pg/g) 1 Nil Nil In general, it appears that chemical releasers and specific antigen compete for the same releasable histamine and that prevention of histamine release in anaphylaxis by previous administration of 48/80 is due to exhaustion of available histamine rather than to interference with the antigen-antibody reaction. Interaction of chemical releaser and antigen in isolated uterus. The uterus shows a behaviour that is in principle similar to aorta and lungs except that much higher concentrations of 48/80 are required to abolish histamine release by antigen. With 1 mg/ml. 48/80 subsequent release by antigen is not prevented,

11 ANAPHYLACTIC SHOCK AND HISTAMINE RELEASERS 471 but after 10 mg/ml. 48/80 release of histamine in anaphylaxis is practically completely abolished (Fig. 7). Interaction of chemical releaser and antigen in small intestine. A different kind of interaction is encountered with small intestine; far from preventing (b) n- 49% 2-8 E 2-4,9 2-0 V * Egg albumin w4880 Tyrode S 6. 1 II 6! X a 0*1162 i 0. 1' S 0-4 Fig I kluaw - I, I., I Release of histamine from uterus of sensitized guinea-pig by 48/80 followed by egg albumin (1 mg/ml.). 48/80 concentration: (a) 1 mg/ml., (b) 10 mg/ml. the release in anaphylaxis, chemical releasers potentiate it. Small intestine contains large amounts of histamine (20-60 ug/g) of which only a few per cent can normally be released in anaphylaxis, or by chemical releasers. When, however, the tissue is placed in an antigen solution after having been in a solution of 48/80, a large release is obtained, many times that given by the control tissue which had only been treated with antigen (Fig. 8). There are some further points about this phenomenon which make it interesting. Experiment shows that in a piece of intestine that has been completely desensitized and has ceased to release histamine by antigen, the application of 48/80 produces some change which renders the tissue again highly sensitive to the antigen (Fig. 9). Fig. 9 shows that this resensitization occurs even when

12 472 J. L. MONGAR AND H. 0. SCHILD the chemical releaser has been thoroughly washed out in Tyrode solution for an hour; the change produced is a permanent one. This potentiation is not a specific action of 48/80; D-tubocurarine has the same action but not to the E _ 0 _ 1 0 (a) * Egg albumin (1 mg/ml.) /80 (10 mg/ml.) (b) _ E 'I % Fig. 8. Release of histamine from small intestine of sensitized guinea-pig. Potentiation of egg albumin release by 48/80. (a) Control with egg albumin alone, (b) 48/80 (10 mg/ml.) followed by egg albumin. 0.1 (a) * Egg albumin (1 mg/ml.) t O [L 48/80 (10 mg/ml.), 0@2 V IU Fig. 9. Release of histamine from small intestine of sensitized guinea-pig. 'Resensitization' to egg albumin by 48/80. (a) Control; desensitization to second dose of antigen, (b) 'resensitization' to antigen by 48/80. Between drug additions tissue was washed in Tyrode solution. same degree. Finally, it occurs only with concentrations of releaser that are high enough to release some histamine themselves. Fig. 10 shows four parallel experiments on the same piece of sensitized intestine in which increasing concentrations of 48/80 were applied before the antigen; 041 mg/ml.

13 ANAPHYLACTIC SHOCK AND HISTAMINE RELEASERS /80 caused no appreciable histamine release above the control values and produced no potentiation of the release by the antigen; 1 mg/ml. 48/80 releases about 2% of the histamine content and causes greatly increased histamine release by the antigen; 10 mg/ml. 48/80 produced by itself an unusually large histamine release but still caused potentiation of the anaphylactic release. * Egg albumin 1.0 _ 48/80 Tyrode 0-6 -~~~~~~~~ E i Fig. 10. Release of histamine from small intestine of sensitized guinea-pig. Effect of various concentrations of 48/80 on release by egg albumin. (b) Muscular contraction It has not been possible to abolish the muscular contraction in anaphylaxis by the previous administration of chemical releasers either in the uterus or ileum. Ileum. In the ileum it was unlikely a priori that histamine releasers would specifically prevent the muscular contraction in anaphylaxis since the corresponding histamine release is not prevented but on the contrary potentiated. Fig. 11 shows, indeed, that in the ileum the anaphylactic muscular contraction is not prevented by 48/80. Fig. 11 also shows that 48/80 itself contracts the isolated ileum. It was reported by Paton (1951) that 48/80 in concentrations up to 0-06 mg/ml. has no action on the intestine, but in our experience a first dose of 0-1 mg/ml. produces a contraction to which there is marked tachyphylaxis, and after two or three doses the muscle ceases to respond to 48/80. At this stage, however, the antigen still produces, as expected, a marked contraction. Uterus. In the uterus it seemed theoretically possible that the anaphylactic muscular contraction would be specifically prevented since a large concentration of 48/80 releases practically all available histamine from the uterus. Unfortunately this concentration (10 mg/ml.) also tends to damage irreversibly muscular contractility and make the muscle- insensitive to histamine. Never-

14 474 J. L. MONGAR AND H. 0. SCHILD theless, in two experiments in which the antigen was tested after this high concentration of 48/80, it still produced a definite contraction (Fig. 12). In one experiment the histamine released by the antigen into the bath fluid was Fig. 11. Contractions of small intestine of sensitized guinea-pig with 48/80 (0.1 mg/ml.) followed by egg albumin (1 mg/ml.). Two or three contractions with histamine (0-01,ug/ml.) have been produced between doses to test for changes in sensitivity. C, 48/80; E.A., egg albumin. 1' t ILt Hist /80 Hist. Egg albumin 0 1 /pg/mi. 1 mg/ml. 10 mg/ml. 0-1 pg/ml. 1 mg./ml. Fig. 12. Effect of massive dose of 48/80 on the histamine contraction and anaphylactic reaction of sensitized guinea-pig uterus. measured and found to be only slightly greater than the spontaneous release from the tissue (cf. Fig. 7). There is therefore some evidence that the anaphylactic muscular contraction of the uterus may persist even after histamine release has been practically abolished and the muscle has become insensitive to histamine. The latter is reminiscent of the production of an anaphylactic contraction on a histamine-poisoned uterus (Schild, 1936). Lower concentrations of 48/80 (0 5 mg/ml.) contract the uterus, and if sufficient time is allowed between injections the effect may be repeated without

15 ANAPHYLACTIC SHOCK AND HISTAMINE RELEASERS 475 tachyphylaxis (Fig. 13). If the antigen is added after this concentration of 48/80 the anaphylactic reaction is in no way diminished as might be expected from the fact that histamine release in anaphylaxis is still pronounced after previous treatment with this concentration of 48/80. I C C C E.A. E.A. Fig. 13. Contractions of uterus of sensitized guinea-pig with 48/80 (0-5 mg/ml.) followed by egg albumin (1 mg/ml.). Administration of 48/80 does not prevent the anaphylactic reaction. C, 48/80; E.A., egg albumin. DISCUSSION The parallelism between histamine release by chemical releasers and antigen for thirteen isolated guinea-pig tissues is unlikely to be a fortuitous one; it may not, however, be universal. There are two factors which make the use of isolated guinea-pig tissues particularly suitable for showing up a parallelism of this kind. First, by using a diffusion method it is possible to be sure of the effective concentration of releaser used, in contrast to perfusion methods in which it is generally not possible to be sure of the concentration of injected substance at the site of action. Secondly, guinea-pig tissues are likely to be fully and uniformly sensitized, and moreover we have made a point of giving large sensitizing doses of egg albumin to ensure this. Although up to now it seemed that the gastro-intestinal tract was an exception to this uniform sensitization, since only a small fraction of its histamine content is released in anaphylaxis, our experiments suggest that the intestine is potentially fully sensitized but some additional factor such as pretreatment with a histamine releaser is required to bring this out. This parallelism may not apply to other species and it may not extend to other releasers; for example, we have recently found that cadaverine releases a large fraction of the histamine from the skin but not from the uterus. I

16 476 J. L. MONGAR AND H. 0. SCHILD It has been known for a long time that peptone shock resembles anaphylactic shock, and more recent work seems to suggest that the chemical histamine releasers have effects which are similar to those of peptones. Peptones are known to cause the release of histamine (Feldberg & O'Connor, 1937), an anti-coagulant substance (Rocha e Silva, Scroggie, Fidlar & Jaques, 1947), and a slow-reacting gut-contracting substance (Beraldo, 1950), and to produce a state of refractoriness after repeated injections. Similarly, chemical histamine releasers have been found to release besides histamine, heparin (MacIntosh & Paton, 1949), and a slow-reacting substance (Paton, 1951), and to cause a state of refractoriness after repeated injections (Paton & Schachter, 1951). A possible explanation of the parallelism between histamine release by chemical releasers and antigen would be the production of some degradation product of proteins following the antigen-antibody reaction, which acted as releasers much in the same way as 48/80 and D-tubocurarine. Quantitative examination of the anaphylactic reaction suggests that the amount of releaser thus formed could be very small. For example, from Fig. 2 it can be calculated that at a concentration of 0.1,ug/ml. egg albumin is capable of releasing approximately its own weight of histamine from lung tissue, i.e. one molecule of egg albumin is responsible for the release of some hundreds of molecules of histamine. The amount of 48/80 required for the same histamine release is about 1000 times greater. Therefore, if a releaser were produced by the antigen-antibody reaction it would probably be highly active-much more active than 48/80 or D-tubocurarine. A high activity implies a high affinity for the site where it acts so that it would not readily diffuse into the fluid. Hence even if a histamine releaser is produced as a result of the antigenantibody reaction it may not be easily detectable. The results of the interaction experiments on the intestine, however, suggest a rather more complex explanation. In this case only the successive application of chemical releaser and specific antigen produced a powerful release, neither agent was effective by itself, which suggests that they act presumably at different sites. Furthermore, whilst previous addition of the chemical releaser greatly potentiates the anaphylactic reaction, even when the chemical releaser used for 'sensitizing' has been washed out in Tyrode solution for 1 hr, the converse is not true; antigen does not potentiate the action of the chemical releasers which suggests that the effects of the latter are more far-reaching and permanent. Each type of releaser probably involves a loosening of the bonds between histamine and non-diffusible molecules in the cell to which the histamine is attached. In the aorta where the bonds appear to be rather weak either releaser is equally effective in liberating histamine. But in the case of intestine much firmer bindings seem to be involved necessitating the combined action of two releasers to obtain this substantial release.

17 ANAPHYLACTIC SHOCK AND HISTAMINE RELEASERS 477 Chemical releasers and anaphylactic muscular contraction One of our objects was to find out whether chemical releasers could be used to prevent the anaphylactic reaction of plain muscle either by interference with the antigen-antibody reaction or by exhaustion of histamine stores. There was no suggestion in our experiments ofinterference with antigen-antibody reaction, on the contrary after pretreatment with 48/80 the anaphylactic reaction in some cases was potentiated rather than diminished. Inhibition of anaphylaxis by exhaustion of histamine stores in the uterus also seemed impracticable, since the high concentrations of chemical releaser required damaged the muscle. In order to find out whether exhaustion of histamine stores will, indeed, abolish the anaphylactic contraction it would be necessary to use a releaser capable of releasing histamine from the tissue without damaging its contractility. SUMMARY 1. Histamine release in anaphylaxis and by chemical compounds was measured by immersing sensitized guinea-pig tissues in Tyrode solution containing known concentrations of the specific antigen or the chemical releaser. The histamine diffusing out was estimated biologically. 2. The fraction of the total histamine released in the course of 10 min by the synthetic polymer 48/80 (1 mg/ml.) and the naturally occurring D-tubocurarine (6 mg/ml.) varied from 2 to 40% according to the tissue used. The fraction released from each of thirteen tissues by the two chemicals was similar. Moreover, this parallelism extended to the anaphylactic release by egg albumin despite the apparently different nature of the two processes. 3. There was no correlation between histamine content and fraction released. 4. Chemical releasers had a similar activity on normal and sensitized tissues. 5. The effect of chemical releasers on histamine released by the antigen was studied. Addition of antigen after 1 mg/ml. 48/80 caused no appreciable further histamine release in the lung and aorta, and after 10 mg/ml. 48/80 in the uterus. The opposite effect was obtained with ileum; the release by antigen was greatly increased by pretreatment with 48/80 (1-10 mg/ml.) or D-tubocurarine. 6. The effect of chemical releasers on the anaphylactic contraction of isolated uterus and intestine was studied. It was found impossible to abolish muscular contraction by pretreatment with 48/ The mechanism of histamine release in anaphylactic shock is discussed in the light of these results.

18 478 J. L. MONGAR AND H. 0. SCHILD REFERENCES BALTZLY, R., BUCK, J. S., DE BEER, E. J. & WEBB, F. J. (1949). A family of long-acting depressors. J. Amer. chem. Soc. 71, BERALDO, W. T. (1950). Formation of bradykinin in anaphylactic and peptone shock. Amer. J. Phy8iol. 163, FELDBERG, W. & O'CONNOR, W. J. (1937). The liberation of histamine from the perfused lung by peptone. J. Phy8iol. 90, MCINTIRE, F. C., ROTH, L. W. & SPROULL, M. (1951). Histamine release in rabbit blood by simple molecules, inhibition and reaction rate studies. Amer. J. Phy8iol. 167, MACINTOSH, F. C. & PATON, W. D. M. (1949). The liberation of histamine by certain organic bases. J. Phy8iol. 109, MONGAR, J. L. & SCHILD, H. 0. (1950). Apparatus for pharmacological assay. J. Phy8iol. ii1, 47P. PATON, W. D. M. (1951). Compound 48/80: a potent histamine liberator. Brit. J. Pharmacol. 6, PATON, W. D. M. & SCHACHTER, M. (1951). The influence of an antihistamine drug on release of histamine in the unanaesthetized dog. Brit. J. Pharmacol. 6, RocHA E SILVA, M., SCROGGIE, E., FIDLAR, E. & JAQUES, L. B. (1947). Liberation of the histamine and heparin by peptone from the isolated dog's liver. Proc. Soc. exp. Biol., N. Y., 64, RocRA E SILVA, M. & SCHEILD, H. 0. (1949). The release of histamine by D-tubocurarine from the isolated diaphragm of the rat. J. Phy8iol. 109, SCHILD, H. 0. (1936). The reaction of the guinea-pig's uterus immersed in a histamine solution to histamine and anaphylaxis. J. Physiol. 86, 51 P-53P. SCHLD, H. 0. (1939). Histamine release in anaphylactic shock from various tissues of the guineapig. J. Phy8iol. 95,