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Airway responses to histamine, acetylcholine, and propranolol in anaphylactic hypersensitivity in guinea pigs
Airway responses to histamine, acetylcholine, and propranolol in anaphylactic hypersensitivity in guinea Valentin
Popa,
James
S. Douglas,
and
Arend
Bouhuys
New Haven, Corm
Using changes in tidal volwm8 and dynamic lzlng compliance, airway responses to h&amine, acetylcholine, and propranolol were investigated in diferent groups of guinea pigs before and after active or passive sensitization. The threshold doses to inhaled histamine, acetylcholine, and propranolol were identical before and after sensitization. Similarly, the slope of dose response curves to these drugs and the time course of recovery from airway constriction were comparable in control and sensitized animals. The slopes of dose response curves to histamine and aoetylcholine were parallel. Threshold doses to acetylcholine (TdcB) and histamine (T,) were proportional. The average ratio T,,, /T, was 7.7. There was no correlation between threshold doses and slopes of dose response curves for propranolol and those for histamine or acetyloholine. Antigen challenge resulted in decreases of dynamic lung compliance and in increases of airway rssistake and frequency, which were maximal 60 to lb0 seconds after ohallenge. When functional parameters after antigen challenge had returned to normal, bronchial responses to histamine, aoetylcholine, and propranolol were exaggerated. These exaggerated respomes were repro&We, transient, variable from animal to animal, and related to the antigen dose. Guinea pig awaphylaxis does not lead to the prolonged hypersensitivity to chemical mediators, which characterizes human reaginio asthma, but to a temporary enhancement of responses to acetylcholine, histamine, and propranolol.
In patients with bronchial asthma, the bronchi respond to smaller doses of acetylcholine,l histamine, and beta adrenergic blocking drugs2 than bronchi of persons without asthma. This “hyperreactivity” could be due to effects of circulating reaginic antibodies or to alterations of the muscular mechanisms that produce airway constriction, or to both. There are similarities between circulating antibodies in anaphylactic and in reaginic bronchial asthma.3 These led us to study airway constriction, induced by drugs, in guinea pigs rendered anaphylactic by active or passive sensitization, We used two different challenge From the Yale University Lung Research Center and the John B. Pierce Foundation Laboratory. Supported in part by USPHS grants HL-14534 and HL-14179. Presented in part at the meeting of the American Physiological Society, Chicago, Ill., April 12-16, 1971. Received for publication Sept. 20, 1972. Reprint requests to: Dr. A. Bouhuys, Yale University Lung Research Center, 333 Cedar St., New Haven, Conn. 06510. Vol. 51, No. 6, pp. 344-356
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FIG. 1A. Tidal volume recording before and after a subthreshold (0.35 mg. per milliliter) dose of histamine (AVT,-0 per cent) (top). Dynamic lung compliance [solid line) and resistance (dashed line) before and after challenge (bottom).
conditions : (1) during a period in which circulating antibody is present but t‘here is no contact with antigen and (2) during a period immediately following antigen challenge. As the experimental animal, we used the unanesthetized spontaneously breathing guinea pig,4 and we measured airway responses with methods previously developed in this laboratory.5F 6 Thus, we used an animal whose airway responses to drugs and antigen are similar to those of man, and we measured its responses to drugs under physiological conditions. METHODS Female albino guinea pigs, Hartley strain (200 to 500 Gm.) were used. Some animals were actively sensitized by a single intramuscular injection of 5 per cent w/v egg albumin s.olution (0.7 ml.) as described by Herxheimer. 7 Other animals were passively sensitized by a single intraperitioneal injection (0.22 mg. antibody nitrogen) of rabbit antiegg albumin precipitating serum” (quantitatively determined as in Reference 8). Histamine, t acetylcholine,$ propranolol,$ and egg albumin/l were administered as aerosols generated in a PetersenRooth generator. Doses were expressed u per cent w/v antigen or milligrams per milliliter histamine dihydrochloride, milligrams per milliliter acetylcholine hydrochloride, or milligrams per milliliter propranolol hydrochloride in the fluid aerosolized. Airway responses were initially determined using a simple noninvasive technique suitable *Rabbit antichieken egg albumin antisera, lyophilized (Nutritional Biochemicals Corp., Cleveland, Ohio). tHistamine dihydrochloride, Calbiochem, Los Angeles, Calif. $Acetylcholine chloride, Calbiochem, Los Angeles, Calif. $Propranolol hydrochloride, Ayerst Labs., New York, N. Y. IlAlbumin egg, 5x crystallized, Nutritional Biochemicals Corp., Cleveland, Ohio.
346
Popa,
Douglas,
FIG. 1B. Measurable compliance (ACodyn, milliliter).
and
Bouhuys
J. ALLERGY CLIN.
changes of tidal volume (AVT, -14 per cent) and -25 per cent) after a threshold dose of histamine
IMMUNOL. JUNE 1973
dynamic lung (0.5 mg. per
for repeated bronchial testing. Animals were placed in a body plethysmograph, and tidal volume, flow, and frequency were recorded.5 Previous data as well as experiments in the present animals have demonstrated that tidal volume closely follows changes in dynamic lung comp1iance.s The volume changes are smaller than compliance changes and are not linearly related to drug doses. A ohange in tidal volume of greater than 10% was considered to be a positive inhalation test to the nebulized agent when tidal volume was stable (< 6 per cent variation in the control condition and when an inhalation test with 0.9 per cent w/v saline caused no change of tidal volume). Animals not meeting these criteria were rejected. The first dose of drug that decreased tidal volume > 10 per cent was considered the threshold dose if the doses immediately higher and lower gave positive and negative tests, respectively. Dose steps varied according to the responses obtained, usually 0.25 mg. per milliliter per dose. Threshold doses were determined for each drug at least 5 times over a period of 3 days. Antigen inhalation tests were immediately preceded by a challenge with 0.9 per cent w/v saline. In unsensitized animals the highest concentration of antigen used caused no change in tidal volume or dynamic lung compliance or both (Table I). A more accurate determination of threshold doses to histamine, aoetylcholine, propranolol, and antigen was made using dynamic lung compliance.5 This requires the recording of tidal volume, flow, and intrapleural pressure, with an invasive technique suitable for terminal experiments of about 12 hours in duration. A decrease of compliance of at least 15 per cent was considered a positive test; this was about twice the spontaneous variation of compliance over a 3 to 5 minute period. Criteria for threshold doses were similar to those cited previously. With this technique dose response curves could be produced and their slopes determined. The recovery of dynamic lung compliance to control levels could be monitored breath by breath. The experimental protocols are summarized in Table I.
VOLUME 51 NUMBER 6
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347
lung compliance FIG. 1C. Responses of tidal volume (&VT, -36 per cent) and dynamic - 38 per cent) to a higher than threshold dose of histamine (0.75 mg. per Wodyn, milliliter). Histamine doses administered for 30 seconds (solid block below Codyn and RI records) as an aerosol to the same animal.
RESULTS Threshold doses of histamine, acetylcholine, after active or passive sensitization
and propranolol
before
and
Typical responses to different doses of histamine are shown in Figs. 1A to lC, which indicate changes of tidal volume (Vr), dynamic lung compliance (Codyn), and airway resistance (RI) to increasing doses of histamine aerosols. In each experiment control data are on the left (A, B, C), and data after challenge are on the right (A’, B’, C’). With the techniques used, threshold doses of histamine, acetylcholine, and propranolol aerosols were comparable in the different groups of animals and varied from 0.25 to 2.5 mg. per milliliter f’or histamine, 2.5 to 12.5 mg. per milliliter for acetylcholine, and 5 to 75 mg. per milliliter for propranolol. Over the time course of the study, the average body weight of the animals increased 3.3 times, and average tidal and minute volumes increased six-fold. These changes with growth did not affect the threshold doses to the drugs during a 60 day period. Thus, threshold doseswere characteristic of the animal and were independent of body weight, tidal volume, or minute volume. Threshold doses were not altered by placement of a catheter for pleural pressure measurements as required for compliance and resistance determinations. .Active or passive sensitization did not alter the threshold doses to histamine, acetylcholine, or propranolol. There was a significant correlation (r = 0.86 ; p < 0.001) between the threshold doses of histamine and acetylcholine (Fig. 2)) ‘but no correlation between these and the threshold doses of propranolol.
348
Popa,
Douglas,
TABLE 1. Materials,
Type of sensitization Control
Active
methods,
No. of animals in group 7
13
8
Active
6
7
Bouhuys
J. ALLERGY CLIN.
Procedures
Days of challenge
to antigen
58 60* 60' 60*
0
21
0
35
21
Fig.2 Fig. 5
60% 60* Fig. 2 60' 60"
21
~-I
H = Histamine dihydrochloride. AcCh = Acetylcholine chloride. P = Propranolol hydrochloride. Ag = Antigen (egg albumin). T.D. = Threshold dose. D.R = Dose response. ( ) = Number of animals from group. *Airway responses measured by changes of dynamic
Responses
IMMUNOL. JUNE 1973
and procedures
T.D. of H, A&h, P D.R. curves to H, A&h, P Recovery from responses to H, AcCh, P (5) Control for inhaled Ag (6) T.D. of H, AcCh, P before and after sensitization D.R. curves to H, A&h (4) H, A&h, P responses immediately after Ag challenge (10) 50y0 T.D. of H immediately after small antigen doses 50% T.D. of H immediately after large Ag doses D.R. curves to H, AcCh, P Recovery from responses to H, A&h, P T.D. of H, AcCh, P before and after sensitization 50% T.D. of H immediately after large Ag doses D.R. curves to H, A&h, P Recovery from responses to H, A&h, P \
Active
Passive
and
24
Fig. 5 Table II Table II
21*
Table II
21* 21*
Fig. 5
2*
Fig. 2
2*
Table II
lung compliance.
challenge
Challenge of sensitized animals with aerosolized egg albumin resulted in decreased tidal volumes and dynamic lung compliance, and increases in frequency and airway resistance .g A typical response to a low antigen dose is shown in Fig. 3. The duration of these changes was increased with the dose of antigen used in the challenge. Responses
to histamine,
acetylcholine,
and propranolol
after
antigen
challenge
Animals were challenged with varying amounts of antigen, and once tidal volume or dynamic lung compliance or both had returned to normal, responses to subthreshold doses of drugs were examined. Actively and passively sensitized animals (Table I) were found to react to subthreshold doses of histamine at varying times after antigen challenge (Fig. 4). These exaggerated responses were characteristic and reproducible in individual animals (Table II) and were dependent upon the dose of antigen and the magnitude of the response the antigen challenge produced (Table II,
Airway
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349
. I 0
0.5
1.0
Histamine
1
1.5 Threshold (mg/ml)
2.0
2.5
Dose
FIG. 2. Relationship between threshold doses of histamine and threshold doses of acetylcholine in control and sensitized animals. Acetylcholine threshold dose (milligrams per milliliter) is plotted against histamine threshold dose (milligrams per milliliter) from sensitized, or A, passively sensitized guinea the same animal in 0, control, 0, actively pigs. There is a highly significant correlation (r = + 0.86) between the threshold doses for the two drugs, and the closeness of the two regression lines shown (x on y and y on x) is an indication of the goodness of fit. The ratio of threshold doses of acetylcholine/doses of histamine is 7.74.
IFig. 5). They lasted up to 180 minutes after antigen challenge, but in no animal was the enhanced bronchial reactivity permanent. After antigen challenge there were also enhanced responses to acetylcholine or propranolol (Table II). Dose response
curves
to histamine,
acetylcholine,
and propranolol
Increasing doses of aerosolized histamine, acetylcholine, or propranolol produce dose-related decreases of dynamic lung compliance (Fig. 6). Dose response curves to histamine and acetylcholine were parallel in control animals as well as sensitized ones. Slopes of dose response curves with propranolol always differed from those for acetylcholine and histamine in the same animal (Fig. 6). The slopes of dose response curves to any of the drugs used did not appear to be altered by active or passive sensitization, and the distribution of these slopes in the control and sensitized groups of animals was not significantly different. Similarly, the time for recovery from bronchoconstriction induced by either drug was not different in the groups examined (Fig. 7). DISCUSSION
Individual airway responses to histamine, acetyleholine, and propranolol vary among individual guinea pigs. Far histamine, there is a log normal distribution of sensitivity in commercially bred and randomly selected animals.6 Genetic differences in sensitivity to antigen and histamine occur in specially bred strains.10 The responses to the drugs were reproducible in animals that were studied for up to 60 days and were independent of tidal volume, minute volume, or body weight. The surface area where aerosol particles are deposited
350
Popa,
Douglas,
and Bouhuys
J. ALLERGY CLIN.
IMMUNOL. JUNE 1973
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352
J. ALLERGY CLIN.
Popa, Douglas, and Bouhuys
ot-----@ 30
Duration
60
90
of enhanced
120 responses
150
IMMUNOL. JUNE 1973
160
(mm)
FIG. 5. Typical periods of enhanced responses to histamine after challenge with various concentrations of antigen. Animals were challenged with various doses of antigen to produce either a 20 to 30 per cent decrease of dynamic lung compliance (Codyn) (curves 1, 2, and 3) or a 40 to 50 per cent decrease of Codyn (curves 4, 5, and 6), or a 67 to 80 per cent decrease of Codyn (curves 7, 8, and 9). After challenge, Codyn values were allowed to return to their control values, and the animals were then challenged with a subthreshold dose (t/z threshold dose) of histamine. These, doses would produce no response if there was no enhancement of bronchial reactivity. Responses to histamine aerosols (ordinate) are per cent of control Codyn before challenge, at various times [minutes, abscissa) after Codyn values had returned to their control values after antigen challenge. Dashed lines are responses from passively sensitized animals, solid lines from actively sensitized animals. The greater the response to antigen, the greater the responses to histamine and the longer their duration. 10XX are two guinea pigs who showed no exaggerated responses to t/z TD at 30 minutes but gave exaggerated responses to the TD (-A Codyn > 40 per cent) 15 minutes later.
and the mass of effector tissue are probably changed approximately in the same proportion during growth. Thus the amount of drug deposited from a given aerosol will increase, but deposition per unit surface area is probably relatively constant. The finding of a constant threshold dose is in agreement with recent concepts of lung growthI and with studies with histamine and acetylcholine.7, lo Threshold doses of histamine, acetylcholine, and propranolol were not changed by active or passive sensitization. Our data confirm results of Takino, Sugahara, and Horino,lO who found comparable acetylcholine “preconvulsion times” before and after sensitization of guinea pigs, and our previous results in which active sensitization did not affect responses to intravenous histamine in the guinea pig.5 The lack of effect might be due to insufficient antibody formation. However, Hicks and Okpako12 have demonstrated that small doses of egg albumin (1 mg. intraperitoneally) gave a higher level of hypersensitization than large doses (50 or 100 mg. intraperitoneally). No large differences were seen in hypersensitivity on the twenty-first and thirty-sixth days ; antibody titers peaked at the thirty-sixth day and remained at this level until the sixteenth
Airway
VOLUME 51 NUMBER 6
\\ LO- \ Q ! ‘y+t 2 k
8 40 Fig. 6 0 2
in guinea
353
pigs
8 2@loot_
0’
(60
responses
g 80\
\
0 ‘4
Q g 60
8
P
! /
.
.
F
2 40h
20 -
R
s L 4.0 2.5 1.5 1.0 0.5 mg/ml hlstamme dlhydrochloride IO I5 25 40 5.0 mg /ml acetylcholine chloride and propronolol hydrochloride
20-
a\ 30
60
90 150 Seconds
300
600
, 900
0, acetylcholine, 0, and propranolol, A, in a FIG. 6. Dose response curves to histamine, guinea pig 60 days after active sensitization. Dynamic lung compliance (per cent of control) is plotted against dose of drug [expressed as milligrams per milliliter salt in nebulizer) administered as an aerosol. Note that the histamine and acetylcholine dose response curves have similar slopes.
FIG. 7. Recovery
periods after histamine challenge in sensitized and unsensitized animals. Two actively sensitized animals (0, A) and two unsensitized animals (0, A.) were challenged with doses of histamine aerosol to produce an 80 per cent decrease of dynamic lung compliance. Values of Codyn expressed as per cent of the control immediately before challenge (ordinate) are plotted at various time intervals (seconds, abscissa) after histamine challenge. Recovery periods are of similar duration in either group of animals.
week after sensitization.12 Thus we used an immunization schedule shown to be effective, and we measured responses at the peak level of hypersensitization. In passively sensitized animals, Kabat and BoldP obtained fatal anaphylactic reactions after challenge with only 25 per cent of the antibody dose we gave. Thus, the presence of anaphylactic antibody in the guinea pig does not appear to cause a long-term decrease in the threshold to histamine, acetylcholine, and propranolol. Enhanced responses to histamine, acetylcholine, and propranolol have been observed within 2 hours after antigen challenge, Enhancement of response was short-lived (15 to 30 minutes) after small antigen doses but lasted 30 to 120 minutes after large antigen doses (Fig. 4). Secondly, its duration following the same change in lung compliance varied from animal to animal, For instance, a 40 per cent change in compliance was followed by potentiated responses lasting, in different animals, 90, 120, or even 150 minutes (Fig. 4). Thirdly, exaggerated responses were transient ; after a variable interval, threshold responses to histamine, acetylcholine, or propranolol returned to their prechallenge level (Fig. 4, Table II). The reason for these temporarily enhanced responses to drugs after antigen responses is not clear. They occurred at a time when all functional parameters had returned to control levels. Hence, altered initial conditions (i.e., a minimal degree of airway constriction) are unlikely. It is perhaps more likely that some
354
Popa,
Douglas,
TABLE II. Enhanced
type of sensitization
and Bouhuys-
responses
Day of challenge
J. ALLERGY CLIN.
to subthreshold
No. of animals
Active
of
histamine
Antigen dose x threshold
21 60*+ 21 2
::
2-5 2-5 20-100 20-100 10-20 20
after
varying
Response -w
%*
amounts
of
to antigen 1I -hCodvn%
20-30 20-30 100 80-100 50-65 45-63
t
-4 80-100 Active 6 60-80 Passive 5 60-70 Animals were challenged with varying doses of antigen (column 4); responses to these antigen column 6). When compliance returned to base line conditions, responses to subthreshold histamine before *-AV,% = Decrease of tidal volume expressed as per cent of control value immediately t-ACodyno/, = Decrease of dynamic lung compliance expressed as per cent of control value STime in minutes. $- = Test not performed. ((0 = Responses similar to threshold dose. 8Same animals as used in experiments on line 1, tested on day 24 after active sensitization. #Animals also showed enhanced responses to acetylcholine. *‘Same animals as used in experiments on line 3, tested on day 60 after active sensitization. t tAnimals also showed enhanced responses to propranolol. t$Two animals did not react to subthreshold dose but gave exaggerated responses to threshold doses Active
8 8
doses
IMMUNOL. JUNE 1973
of the chemical mediators of the antigen response’* are still present in smooth muscle in subthreshold levels. Although released histamine is rapidly metabolized in tissues, other mediators such as SRS-A persist longer. By definition, subthreshold doses of mediators may be present in tissues at a point in time when responses are no longer measurable. The exogenously administered drugs may augment the effects of these mediator substances and thus cause enhanced responses. For instance, the effect of released histamine can be potentiated by propranolol,6 and released SRS-A may potentiate contractions induced by exogenous histamine.15 The gradual return to normal responses would agree with the gradual decrease of the levels of the humoral mediators after the antigen response. Thus, the temporary increases of bronchial reactivity are probably due to subtle changes in initial conditions that are not reflected in changes of dynamic lung compliance. There is a clear relationship between the threshold doses (Fig. 2) and slopes of dose response curves (Fig. 6) of acetylcholine and histamine in guinea pigs, with a mean ratio of threshold doses of 7.74. The interaction between histamine and eholinergic mechanisms is discussed in detail in another paper from our laboratory.6 In our guinea pigs, threshold doses to propranolol were considerably higher than those of histamine or acetylcholine and were not related to them. Similarly, in man there is a close relationship between threshold doses of aerosolized acetylcholine and histamine with a ratio of between 5 and 10.ls Threshold doses to inhaled propranolol are higher than those of histamine or acetylcholine.17 Thus, it appears that airway responses to 3 bronchoconstrictor drugs are similar in man and in guinea pigs. In human bronchial asthma, one finds long-term hyperreactivity to inhaled histamine, acetylcholine, and pro-
Airway
VOLUME 51 NUMBER 6
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pigs
355
antigen
No. 30
86 6#
-ii-
-
5
66
51t
of animals I
60
v 4 3
responding I
90
to subthreshold I
1 2
I
;# Btt 1
2
doses were measured using tidal volume (V,, doses were evaluated. challenge with antigen. immediately before challenge with antigen.
120
2 column
histamine 150
I
1 1 0
5)
and dynamic
doses 180
at I
210
I
240
--0
---
0 -0
-
-
-
lung
compliance
(Codyn,
pranolol and prolonged recovery periods after airway conatriction.ls~ *’ In our actively sensitized guinea pigs, such an alteration of responses to constrictor dl*ugs, in the long term, did not occur. This difference between asthmatic man and anaphylaetic guinea pig may be related to a difference in the nature of the antibodies involved. However, it has been suggested that the circulating antibodies in anaphylactic and reaginic bronchial asthma are of the same type.3 It. stems to us more likely that the hyperreactivity of airway smooth muscle in asthma is dependent on events that are unrelated to the formation of circulating antibodies, since the induction of homocytotrophic antibody formation in guinea pigs dots not result in hypcrreactivity of airway smooth muscle to histamine, acetylcholine, and propranolol. REFERENCES Comparative action of aeetyl beta-methylcholine and histamine on the in normals, patients with hay fever and subjects with bronchial asthma, J. Clin. Invest. 26: 430, 1947. McNeil], R. S.: Effect of a beta adrenergic-blocking agent, propranolol, on asthmatics, T,ancet 2: 1101, 1964. Becker, E. I,., and Austen, F. K.: In Miescher, P. A., and Miiller-Eberhard, H. J., editors: Text-Ilook of immunopathology, New York, 1968, Grune & Stratton, Inc. Armitage, P., Herxheimer, H., and Rosa, L.: The protective action of antihistamines in tlrc mraphylnctic microshock of the guinea-pig, Br. J. Pharmncol. 7: 625, 1952. dilatation and conl)ouglas, .J., Dennis, M., Ridgway, P., and Bouhuys, A.: Airway striction in spontaneously breathing guinea pigs, J. Pharmacol. Exp. Ther. 180: 98,
1 Curry,
J. J.:
respiratory tract
2 3 4 5
19i2.
6 Douglas, J. S., Dennis, M. W., Ridgway, P., and Bouhuys, A.: Airway constriction in guinea pigs: Interaction of histamine and autonomic drugs, J. Pharmxcol. Exp. Ther. 184: 169, 1973. 7 Herxheimer, H.: Repeatable “microshocks” of constant strength in guinea pig anaphylaxis, .T. Phgsiol. 117: 251, 1952. 8 Gitlin, I).: Use of ultraviolet absorption spectroscopy in the quantitative precipitation reaction, J. Immunol. 62: 437, 1949. C.: The mechanical properties 9 Stein, M., Schiavi, R. C., Ottenberg, P., and Hamilton, of the lungs in experimental asthma in the guinea pig, J. ALLERGY 32: 8-16, 1961. and non:LO Takino, Y., Sugahara, K., and Horino, I.: Two lines of guinea pigs sensitive sensitive to chemical mediators and anaphylaxis, J. ALLERGY 47: 247, 1971.
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11 Zapletal, A., Motoyama, E. K., van de Woestijne, K. P., Hunt, V. R., and Bouhuys, A.: Maximum expiratory flow volume curves and airway conductance in children and adolescents, J. Appl. Physiol. 26: 308316, 1969. 12 Hicks, R., and Okpako, D. T.: The influence of the size of the sensitizing dose of antigen on the development and duration of anaphylactic hypersensitivity in the guinea pig, Int. Arch. Allergy Appl. Immunol. 33: 131, 1968. 13 Kabat, E. A., and Boldt, M. H.: A quantitative study of passive anaphylaxis in the guinea pig, J. Immunol. 48: 181, 1944. 14 Okpako, D. T.: The effect of graded doses of antigen on bronchoconstrictor and mediator release responses in anaphylaxis of the isolated perfused guinea pig lung, Int. Arch. Allergy Appl. Immunol. 40: 620-630, 1971. 15 Chakravarty, hr.: A method for the assay of slow reacting substance, Acta Physiol. Rcand. 46: 298313, 1959. R.: Hypersensibilite cholinergohistaminique pulmonaire de l’asthmatique, 16 Tiffeneau, Acta Allergol. (Suppl.) 5: 187-221, 1958. I.: Untersuehungen iiber die bronchokonstriktorische 17 Herxheimer, H., and Langer, Wirkung des beta Rezeptorblockers Propranolol bei Meerschweinchen und Patienten mit Asthma Bronchiale, Klin. Wochenschr. 45: 1149-1153, 1967. 18 Bouhuys, A., and finsson, R. Lichtneckert, S., Lindell, S. E., Lundgren, C., Lundin, G., and Ringquist, T. R.: Effects of histamine on pulmonary ventilation in man, Clin. Sci. 19: 79-94, 1960. J., Parrel, L., and Lava& P.: Etude spirographique comparative 19 Duron, B., Humbert, des crises d’asthme spontanees et artificiellement provoquees, J. Fr. Med. Chir. Thorac. 22: 5-24, 1968.
Popa,
James
S. Douglas,
and
Arend
Bouhuys
New Haven, Corm
Using changes in tidal volwm8 and dynamic lzlng compliance, airway responses to h&amine, acetylcholine, and propranolol were investigated in diferent groups of guinea pigs before and after active or passive sensitization. The threshold doses to inhaled histamine, acetylcholine, and propranolol were identical before and after sensitization. Similarly, the slope of dose response curves to these drugs and the time course of recovery from airway constriction were comparable in control and sensitized animals. The slopes of dose response curves to histamine and aoetylcholine were parallel. Threshold doses to acetylcholine (TdcB) and histamine (T,) were proportional. The average ratio T,,, /T, was 7.7. There was no correlation between threshold doses and slopes of dose response curves for propranolol and those for histamine or acetyloholine. Antigen challenge resulted in decreases of dynamic lung compliance and in increases of airway rssistake and frequency, which were maximal 60 to lb0 seconds after ohallenge. When functional parameters after antigen challenge had returned to normal, bronchial responses to histamine, aoetylcholine, and propranolol were exaggerated. These exaggerated respomes were repro&We, transient, variable from animal to animal, and related to the antigen dose. Guinea pig awaphylaxis does not lead to the prolonged hypersensitivity to chemical mediators, which characterizes human reaginio asthma, but to a temporary enhancement of responses to acetylcholine, histamine, and propranolol.
In patients with bronchial asthma, the bronchi respond to smaller doses of acetylcholine,l histamine, and beta adrenergic blocking drugs2 than bronchi of persons without asthma. This “hyperreactivity” could be due to effects of circulating reaginic antibodies or to alterations of the muscular mechanisms that produce airway constriction, or to both. There are similarities between circulating antibodies in anaphylactic and in reaginic bronchial asthma.3 These led us to study airway constriction, induced by drugs, in guinea pigs rendered anaphylactic by active or passive sensitization, We used two different challenge From the Yale University Lung Research Center and the John B. Pierce Foundation Laboratory. Supported in part by USPHS grants HL-14534 and HL-14179. Presented in part at the meeting of the American Physiological Society, Chicago, Ill., April 12-16, 1971. Received for publication Sept. 20, 1972. Reprint requests to: Dr. A. Bouhuys, Yale University Lung Research Center, 333 Cedar St., New Haven, Conn. 06510. Vol. 51, No. 6, pp. 344-356
VOLUME 51 NUMBER 6
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345
FIG. 1A. Tidal volume recording before and after a subthreshold (0.35 mg. per milliliter) dose of histamine (AVT,-0 per cent) (top). Dynamic lung compliance [solid line) and resistance (dashed line) before and after challenge (bottom).
conditions : (1) during a period in which circulating antibody is present but t‘here is no contact with antigen and (2) during a period immediately following antigen challenge. As the experimental animal, we used the unanesthetized spontaneously breathing guinea pig,4 and we measured airway responses with methods previously developed in this laboratory.5F 6 Thus, we used an animal whose airway responses to drugs and antigen are similar to those of man, and we measured its responses to drugs under physiological conditions. METHODS Female albino guinea pigs, Hartley strain (200 to 500 Gm.) were used. Some animals were actively sensitized by a single intramuscular injection of 5 per cent w/v egg albumin s.olution (0.7 ml.) as described by Herxheimer. 7 Other animals were passively sensitized by a single intraperitioneal injection (0.22 mg. antibody nitrogen) of rabbit antiegg albumin precipitating serum” (quantitatively determined as in Reference 8). Histamine, t acetylcholine,$ propranolol,$ and egg albumin/l were administered as aerosols generated in a PetersenRooth generator. Doses were expressed u per cent w/v antigen or milligrams per milliliter histamine dihydrochloride, milligrams per milliliter acetylcholine hydrochloride, or milligrams per milliliter propranolol hydrochloride in the fluid aerosolized. Airway responses were initially determined using a simple noninvasive technique suitable *Rabbit antichieken egg albumin antisera, lyophilized (Nutritional Biochemicals Corp., Cleveland, Ohio). tHistamine dihydrochloride, Calbiochem, Los Angeles, Calif. $Acetylcholine chloride, Calbiochem, Los Angeles, Calif. $Propranolol hydrochloride, Ayerst Labs., New York, N. Y. IlAlbumin egg, 5x crystallized, Nutritional Biochemicals Corp., Cleveland, Ohio.
346
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FIG. 1B. Measurable compliance (ACodyn, milliliter).
and
Bouhuys
J. ALLERGY CLIN.
changes of tidal volume (AVT, -14 per cent) and -25 per cent) after a threshold dose of histamine
IMMUNOL. JUNE 1973
dynamic lung (0.5 mg. per
for repeated bronchial testing. Animals were placed in a body plethysmograph, and tidal volume, flow, and frequency were recorded.5 Previous data as well as experiments in the present animals have demonstrated that tidal volume closely follows changes in dynamic lung comp1iance.s The volume changes are smaller than compliance changes and are not linearly related to drug doses. A ohange in tidal volume of greater than 10% was considered to be a positive inhalation test to the nebulized agent when tidal volume was stable (< 6 per cent variation in the control condition and when an inhalation test with 0.9 per cent w/v saline caused no change of tidal volume). Animals not meeting these criteria were rejected. The first dose of drug that decreased tidal volume > 10 per cent was considered the threshold dose if the doses immediately higher and lower gave positive and negative tests, respectively. Dose steps varied according to the responses obtained, usually 0.25 mg. per milliliter per dose. Threshold doses were determined for each drug at least 5 times over a period of 3 days. Antigen inhalation tests were immediately preceded by a challenge with 0.9 per cent w/v saline. In unsensitized animals the highest concentration of antigen used caused no change in tidal volume or dynamic lung compliance or both (Table I). A more accurate determination of threshold doses to histamine, aoetylcholine, propranolol, and antigen was made using dynamic lung compliance.5 This requires the recording of tidal volume, flow, and intrapleural pressure, with an invasive technique suitable for terminal experiments of about 12 hours in duration. A decrease of compliance of at least 15 per cent was considered a positive test; this was about twice the spontaneous variation of compliance over a 3 to 5 minute period. Criteria for threshold doses were similar to those cited previously. With this technique dose response curves could be produced and their slopes determined. The recovery of dynamic lung compliance to control levels could be monitored breath by breath. The experimental protocols are summarized in Table I.
VOLUME 51 NUMBER 6
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347
lung compliance FIG. 1C. Responses of tidal volume (&VT, -36 per cent) and dynamic - 38 per cent) to a higher than threshold dose of histamine (0.75 mg. per Wodyn, milliliter). Histamine doses administered for 30 seconds (solid block below Codyn and RI records) as an aerosol to the same animal.
RESULTS Threshold doses of histamine, acetylcholine, after active or passive sensitization
and propranolol
before
and
Typical responses to different doses of histamine are shown in Figs. 1A to lC, which indicate changes of tidal volume (Vr), dynamic lung compliance (Codyn), and airway resistance (RI) to increasing doses of histamine aerosols. In each experiment control data are on the left (A, B, C), and data after challenge are on the right (A’, B’, C’). With the techniques used, threshold doses of histamine, acetylcholine, and propranolol aerosols were comparable in the different groups of animals and varied from 0.25 to 2.5 mg. per milliliter f’or histamine, 2.5 to 12.5 mg. per milliliter for acetylcholine, and 5 to 75 mg. per milliliter for propranolol. Over the time course of the study, the average body weight of the animals increased 3.3 times, and average tidal and minute volumes increased six-fold. These changes with growth did not affect the threshold doses to the drugs during a 60 day period. Thus, threshold doseswere characteristic of the animal and were independent of body weight, tidal volume, or minute volume. Threshold doses were not altered by placement of a catheter for pleural pressure measurements as required for compliance and resistance determinations. .Active or passive sensitization did not alter the threshold doses to histamine, acetylcholine, or propranolol. There was a significant correlation (r = 0.86 ; p < 0.001) between the threshold doses of histamine and acetylcholine (Fig. 2)) ‘but no correlation between these and the threshold doses of propranolol.
348
Popa,
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TABLE 1. Materials,
Type of sensitization Control
Active
methods,
No. of animals in group 7
13
8
Active
6
7
Bouhuys
J. ALLERGY CLIN.
Procedures
Days of challenge
to antigen
58 60* 60' 60*
0
21
0
35
21
Fig.2 Fig. 5
60% 60* Fig. 2 60' 60"
21
~-I
H = Histamine dihydrochloride. AcCh = Acetylcholine chloride. P = Propranolol hydrochloride. Ag = Antigen (egg albumin). T.D. = Threshold dose. D.R = Dose response. ( ) = Number of animals from group. *Airway responses measured by changes of dynamic
Responses
IMMUNOL. JUNE 1973
and procedures
T.D. of H, A&h, P D.R. curves to H, A&h, P Recovery from responses to H, AcCh, P (5) Control for inhaled Ag (6) T.D. of H, AcCh, P before and after sensitization D.R. curves to H, A&h (4) H, A&h, P responses immediately after Ag challenge (10) 50y0 T.D. of H immediately after small antigen doses 50% T.D. of H immediately after large Ag doses D.R. curves to H, AcCh, P Recovery from responses to H, A&h, P T.D. of H, AcCh, P before and after sensitization 50% T.D. of H immediately after large Ag doses D.R. curves to H, A&h, P Recovery from responses to H, A&h, P \
Active
Passive
and
24
Fig. 5 Table II Table II
21*
Table II
21* 21*
Fig. 5
2*
Fig. 2
2*
Table II
lung compliance.
challenge
Challenge of sensitized animals with aerosolized egg albumin resulted in decreased tidal volumes and dynamic lung compliance, and increases in frequency and airway resistance .g A typical response to a low antigen dose is shown in Fig. 3. The duration of these changes was increased with the dose of antigen used in the challenge. Responses
to histamine,
acetylcholine,
and propranolol
after
antigen
challenge
Animals were challenged with varying amounts of antigen, and once tidal volume or dynamic lung compliance or both had returned to normal, responses to subthreshold doses of drugs were examined. Actively and passively sensitized animals (Table I) were found to react to subthreshold doses of histamine at varying times after antigen challenge (Fig. 4). These exaggerated responses were characteristic and reproducible in individual animals (Table II) and were dependent upon the dose of antigen and the magnitude of the response the antigen challenge produced (Table II,
Airway
VOLUME 51 NUMBER 6
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pigs
349
. I 0
0.5
1.0
Histamine
1
1.5 Threshold (mg/ml)
2.0
2.5
Dose
FIG. 2. Relationship between threshold doses of histamine and threshold doses of acetylcholine in control and sensitized animals. Acetylcholine threshold dose (milligrams per milliliter) is plotted against histamine threshold dose (milligrams per milliliter) from sensitized, or A, passively sensitized guinea the same animal in 0, control, 0, actively pigs. There is a highly significant correlation (r = + 0.86) between the threshold doses for the two drugs, and the closeness of the two regression lines shown (x on y and y on x) is an indication of the goodness of fit. The ratio of threshold doses of acetylcholine/doses of histamine is 7.74.
IFig. 5). They lasted up to 180 minutes after antigen challenge, but in no animal was the enhanced bronchial reactivity permanent. After antigen challenge there were also enhanced responses to acetylcholine or propranolol (Table II). Dose response
curves
to histamine,
acetylcholine,
and propranolol
Increasing doses of aerosolized histamine, acetylcholine, or propranolol produce dose-related decreases of dynamic lung compliance (Fig. 6). Dose response curves to histamine and acetylcholine were parallel in control animals as well as sensitized ones. Slopes of dose response curves with propranolol always differed from those for acetylcholine and histamine in the same animal (Fig. 6). The slopes of dose response curves to any of the drugs used did not appear to be altered by active or passive sensitization, and the distribution of these slopes in the control and sensitized groups of animals was not significantly different. Similarly, the time for recovery from bronchoconstriction induced by either drug was not different in the groups examined (Fig. 7). DISCUSSION
Individual airway responses to histamine, acetyleholine, and propranolol vary among individual guinea pigs. Far histamine, there is a log normal distribution of sensitivity in commercially bred and randomly selected animals.6 Genetic differences in sensitivity to antigen and histamine occur in specially bred strains.10 The responses to the drugs were reproducible in animals that were studied for up to 60 days and were independent of tidal volume, minute volume, or body weight. The surface area where aerosol particles are deposited
350
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and Bouhuys
J. ALLERGY CLIN.
IMMUNOL. JUNE 1973
‘(‘1301q y*lmas) aeuai au!wo$s!y Ja+!l!ll!w Jad ‘6~ yz o+ sasuodsaa ‘(yzolq yylj) axo!ldwo:, fiun( ~!wouxp ~0 -~txp ua6!gur~ Jay0 sJnol( p ~DWJOU 04 pauJn@J IOso~an ap!JOlLpoJph#p au!wD@y Ja+!l!ll!w Jad -6UJ SL’() aLU!g s!yi 4y (xtxIallDy3 Ua6!&iD Jags pJo3aJ IA asoaJ3ap +uaI Jad 9p D ah06 6un) 3!w~uAp ‘awnloA ~~p!r $0 uo!gtxq!lix u! a6utx.p aloN) ‘(y301q y+~no~) (DWJOU 04 pawnia~ p13y mms!saJ ADMJ!D purr ‘axxxgdwo> ‘ppolq pJ!L#) a6ualltxp u!cunqlD 66a @a~ Jad 01 D ~a+j~ sJnoy OM~ ‘(ysolq puo3as) @Ia:, Jad SE pasDaJ?ap axm!ldwo3 6unl 3!umUAp ~‘2 40 asop KI Jayt/ .(wo&(oq) (au!1 paqsap) axm+s!saJ pm (au!1 p!los) ammldwo~ 6unl aU!WD+S!y Ja@l!ll!UJ Jad .6w oau!nG paz!g!suas Alamym UD u! awnloA IDP!~ jdo+) a6uallmp uaB!gm myo au!w~cs!y o+ asuodom pa~~x4~3 ‘p 'gj
‘ap!JO]tpOJpkj!p z+.um~A~ .B!d
352
J. ALLERGY CLIN.
Popa, Douglas, and Bouhuys
ot-----@ 30
Duration
60
90
of enhanced
120 responses
150
IMMUNOL. JUNE 1973
160
(mm)
FIG. 5. Typical periods of enhanced responses to histamine after challenge with various concentrations of antigen. Animals were challenged with various doses of antigen to produce either a 20 to 30 per cent decrease of dynamic lung compliance (Codyn) (curves 1, 2, and 3) or a 40 to 50 per cent decrease of Codyn (curves 4, 5, and 6), or a 67 to 80 per cent decrease of Codyn (curves 7, 8, and 9). After challenge, Codyn values were allowed to return to their control values, and the animals were then challenged with a subthreshold dose (t/z threshold dose) of histamine. These, doses would produce no response if there was no enhancement of bronchial reactivity. Responses to histamine aerosols (ordinate) are per cent of control Codyn before challenge, at various times [minutes, abscissa) after Codyn values had returned to their control values after antigen challenge. Dashed lines are responses from passively sensitized animals, solid lines from actively sensitized animals. The greater the response to antigen, the greater the responses to histamine and the longer their duration. 10XX are two guinea pigs who showed no exaggerated responses to t/z TD at 30 minutes but gave exaggerated responses to the TD (-A Codyn > 40 per cent) 15 minutes later.
and the mass of effector tissue are probably changed approximately in the same proportion during growth. Thus the amount of drug deposited from a given aerosol will increase, but deposition per unit surface area is probably relatively constant. The finding of a constant threshold dose is in agreement with recent concepts of lung growthI and with studies with histamine and acetylcholine.7, lo Threshold doses of histamine, acetylcholine, and propranolol were not changed by active or passive sensitization. Our data confirm results of Takino, Sugahara, and Horino,lO who found comparable acetylcholine “preconvulsion times” before and after sensitization of guinea pigs, and our previous results in which active sensitization did not affect responses to intravenous histamine in the guinea pig.5 The lack of effect might be due to insufficient antibody formation. However, Hicks and Okpako12 have demonstrated that small doses of egg albumin (1 mg. intraperitoneally) gave a higher level of hypersensitization than large doses (50 or 100 mg. intraperitoneally). No large differences were seen in hypersensitivity on the twenty-first and thirty-sixth days ; antibody titers peaked at the thirty-sixth day and remained at this level until the sixteenth
Airway
VOLUME 51 NUMBER 6
\\ LO- \ Q ! ‘y+t 2 k
8 40 Fig. 6 0 2
in guinea
353
pigs
8 2@loot_
0’
(60
responses
g 80\
\
0 ‘4
Q g 60
8
P
! /
.
.
F
2 40h
20 -
R
s L 4.0 2.5 1.5 1.0 0.5 mg/ml hlstamme dlhydrochloride IO I5 25 40 5.0 mg /ml acetylcholine chloride and propronolol hydrochloride
20-
a\ 30
60
90 150 Seconds
300
600
, 900
0, acetylcholine, 0, and propranolol, A, in a FIG. 6. Dose response curves to histamine, guinea pig 60 days after active sensitization. Dynamic lung compliance (per cent of control) is plotted against dose of drug [expressed as milligrams per milliliter salt in nebulizer) administered as an aerosol. Note that the histamine and acetylcholine dose response curves have similar slopes.
FIG. 7. Recovery
periods after histamine challenge in sensitized and unsensitized animals. Two actively sensitized animals (0, A) and two unsensitized animals (0, A.) were challenged with doses of histamine aerosol to produce an 80 per cent decrease of dynamic lung compliance. Values of Codyn expressed as per cent of the control immediately before challenge (ordinate) are plotted at various time intervals (seconds, abscissa) after histamine challenge. Recovery periods are of similar duration in either group of animals.
week after sensitization.12 Thus we used an immunization schedule shown to be effective, and we measured responses at the peak level of hypersensitization. In passively sensitized animals, Kabat and BoldP obtained fatal anaphylactic reactions after challenge with only 25 per cent of the antibody dose we gave. Thus, the presence of anaphylactic antibody in the guinea pig does not appear to cause a long-term decrease in the threshold to histamine, acetylcholine, and propranolol. Enhanced responses to histamine, acetylcholine, and propranolol have been observed within 2 hours after antigen challenge, Enhancement of response was short-lived (15 to 30 minutes) after small antigen doses but lasted 30 to 120 minutes after large antigen doses (Fig. 4). Secondly, its duration following the same change in lung compliance varied from animal to animal, For instance, a 40 per cent change in compliance was followed by potentiated responses lasting, in different animals, 90, 120, or even 150 minutes (Fig. 4). Thirdly, exaggerated responses were transient ; after a variable interval, threshold responses to histamine, acetylcholine, or propranolol returned to their prechallenge level (Fig. 4, Table II). The reason for these temporarily enhanced responses to drugs after antigen responses is not clear. They occurred at a time when all functional parameters had returned to control levels. Hence, altered initial conditions (i.e., a minimal degree of airway constriction) are unlikely. It is perhaps more likely that some
354
Popa,
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TABLE II. Enhanced
type of sensitization
and Bouhuys-
responses
Day of challenge
J. ALLERGY CLIN.
to subthreshold
No. of animals
Active
of
histamine
Antigen dose x threshold
21 60*+ 21 2
::
2-5 2-5 20-100 20-100 10-20 20
after
varying
Response -w
%*
amounts
of
to antigen 1I -hCodvn%
20-30 20-30 100 80-100 50-65 45-63
t
-4 80-100 Active 6 60-80 Passive 5 60-70 Animals were challenged with varying doses of antigen (column 4); responses to these antigen column 6). When compliance returned to base line conditions, responses to subthreshold histamine before *-AV,% = Decrease of tidal volume expressed as per cent of control value immediately t-ACodyno/, = Decrease of dynamic lung compliance expressed as per cent of control value STime in minutes. $- = Test not performed. ((0 = Responses similar to threshold dose. 8Same animals as used in experiments on line 1, tested on day 24 after active sensitization. #Animals also showed enhanced responses to acetylcholine. *‘Same animals as used in experiments on line 3, tested on day 60 after active sensitization. t tAnimals also showed enhanced responses to propranolol. t$Two animals did not react to subthreshold dose but gave exaggerated responses to threshold doses Active
8 8
doses
IMMUNOL. JUNE 1973
of the chemical mediators of the antigen response’* are still present in smooth muscle in subthreshold levels. Although released histamine is rapidly metabolized in tissues, other mediators such as SRS-A persist longer. By definition, subthreshold doses of mediators may be present in tissues at a point in time when responses are no longer measurable. The exogenously administered drugs may augment the effects of these mediator substances and thus cause enhanced responses. For instance, the effect of released histamine can be potentiated by propranolol,6 and released SRS-A may potentiate contractions induced by exogenous histamine.15 The gradual return to normal responses would agree with the gradual decrease of the levels of the humoral mediators after the antigen response. Thus, the temporary increases of bronchial reactivity are probably due to subtle changes in initial conditions that are not reflected in changes of dynamic lung compliance. There is a clear relationship between the threshold doses (Fig. 2) and slopes of dose response curves (Fig. 6) of acetylcholine and histamine in guinea pigs, with a mean ratio of threshold doses of 7.74. The interaction between histamine and eholinergic mechanisms is discussed in detail in another paper from our laboratory.6 In our guinea pigs, threshold doses to propranolol were considerably higher than those of histamine or acetylcholine and were not related to them. Similarly, in man there is a close relationship between threshold doses of aerosolized acetylcholine and histamine with a ratio of between 5 and 10.ls Threshold doses to inhaled propranolol are higher than those of histamine or acetylcholine.17 Thus, it appears that airway responses to 3 bronchoconstrictor drugs are similar in man and in guinea pigs. In human bronchial asthma, one finds long-term hyperreactivity to inhaled histamine, acetylcholine, and pro-
Airway
VOLUME 51 NUMBER 6
responses
in guinea
pigs
355
antigen
No. 30
86 6#
-ii-
-
5
66
51t
of animals I
60
v 4 3
responding I
90
to subthreshold I
1 2
I
;# Btt 1
2
doses were measured using tidal volume (V,, doses were evaluated. challenge with antigen. immediately before challenge with antigen.
120
2 column
histamine 150
I
1 1 0
5)
and dynamic
doses 180
at I
210
I
240
--0
---
0 -0
-
-
-
lung
compliance
(Codyn,
pranolol and prolonged recovery periods after airway conatriction.ls~ *’ In our actively sensitized guinea pigs, such an alteration of responses to constrictor dl*ugs, in the long term, did not occur. This difference between asthmatic man and anaphylaetic guinea pig may be related to a difference in the nature of the antibodies involved. However, it has been suggested that the circulating antibodies in anaphylactic and reaginic bronchial asthma are of the same type.3 It. stems to us more likely that the hyperreactivity of airway smooth muscle in asthma is dependent on events that are unrelated to the formation of circulating antibodies, since the induction of homocytotrophic antibody formation in guinea pigs dots not result in hypcrreactivity of airway smooth muscle to histamine, acetylcholine, and propranolol. REFERENCES Comparative action of aeetyl beta-methylcholine and histamine on the in normals, patients with hay fever and subjects with bronchial asthma, J. Clin. Invest. 26: 430, 1947. McNeil], R. S.: Effect of a beta adrenergic-blocking agent, propranolol, on asthmatics, T,ancet 2: 1101, 1964. Becker, E. I,., and Austen, F. K.: In Miescher, P. A., and Miiller-Eberhard, H. J., editors: Text-Ilook of immunopathology, New York, 1968, Grune & Stratton, Inc. Armitage, P., Herxheimer, H., and Rosa, L.: The protective action of antihistamines in tlrc mraphylnctic microshock of the guinea-pig, Br. J. Pharmncol. 7: 625, 1952. dilatation and conl)ouglas, .J., Dennis, M., Ridgway, P., and Bouhuys, A.: Airway striction in spontaneously breathing guinea pigs, J. Pharmacol. Exp. Ther. 180: 98,
1 Curry,
J. J.:
respiratory tract
2 3 4 5
19i2.
6 Douglas, J. S., Dennis, M. W., Ridgway, P., and Bouhuys, A.: Airway constriction in guinea pigs: Interaction of histamine and autonomic drugs, J. Pharmxcol. Exp. Ther. 184: 169, 1973. 7 Herxheimer, H.: Repeatable “microshocks” of constant strength in guinea pig anaphylaxis, .T. Phgsiol. 117: 251, 1952. 8 Gitlin, I).: Use of ultraviolet absorption spectroscopy in the quantitative precipitation reaction, J. Immunol. 62: 437, 1949. C.: The mechanical properties 9 Stein, M., Schiavi, R. C., Ottenberg, P., and Hamilton, of the lungs in experimental asthma in the guinea pig, J. ALLERGY 32: 8-16, 1961. and non:LO Takino, Y., Sugahara, K., and Horino, I.: Two lines of guinea pigs sensitive sensitive to chemical mediators and anaphylaxis, J. ALLERGY 47: 247, 1971.
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and
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11 Zapletal, A., Motoyama, E. K., van de Woestijne, K. P., Hunt, V. R., and Bouhuys, A.: Maximum expiratory flow volume curves and airway conductance in children and adolescents, J. Appl. Physiol. 26: 308316, 1969. 12 Hicks, R., and Okpako, D. T.: The influence of the size of the sensitizing dose of antigen on the development and duration of anaphylactic hypersensitivity in the guinea pig, Int. Arch. Allergy Appl. Immunol. 33: 131, 1968. 13 Kabat, E. A., and Boldt, M. H.: A quantitative study of passive anaphylaxis in the guinea pig, J. Immunol. 48: 181, 1944. 14 Okpako, D. T.: The effect of graded doses of antigen on bronchoconstrictor and mediator release responses in anaphylaxis of the isolated perfused guinea pig lung, Int. Arch. Allergy Appl. Immunol. 40: 620-630, 1971. 15 Chakravarty, hr.: A method for the assay of slow reacting substance, Acta Physiol. Rcand. 46: 298313, 1959. R.: Hypersensibilite cholinergohistaminique pulmonaire de l’asthmatique, 16 Tiffeneau, Acta Allergol. (Suppl.) 5: 187-221, 1958. I.: Untersuehungen iiber die bronchokonstriktorische 17 Herxheimer, H., and Langer, Wirkung des beta Rezeptorblockers Propranolol bei Meerschweinchen und Patienten mit Asthma Bronchiale, Klin. Wochenschr. 45: 1149-1153, 1967. 18 Bouhuys, A., and finsson, R. Lichtneckert, S., Lindell, S. E., Lundgren, C., Lundin, G., and Ringquist, T. R.: Effects of histamine on pulmonary ventilation in man, Clin. Sci. 19: 79-94, 1960. J., Parrel, L., and Lava& P.: Etude spirographique comparative 19 Duron, B., Humbert, des crises d’asthme spontanees et artificiellement provoquees, J. Fr. Med. Chir. Thorac. 22: 5-24, 1968.