Metaproterenol Responsiveness After Methacholine- and Histamine-Induced Bronchoconstriction

After Metaproterenol Responsiveness Methacholineand Histamine-lnduced

Bronchoconstriction*

Serge Elsasser, MD; Elio Donna, MD, FCCP; Cuneyt M. Demirozu, MD; Ignacio Danta, BS; and Adam Wanner, MD, FCCP We investigated whether the bronchodilator response to a p-adrenergic agonist is influenced by the

mechanism of induced bronchoconstriction. Normal subjects and asymptomatic asthmatics inhaled a dry aerosol (mass median aerodynamic diameter, 1.5 pm) with increasing concentrations of methacholine or histamine to produce a 35% decrease in specific airway conductance (SGaw), fol¬ lowed by a single inhalation of a metaproterenol aerosol. By studying normal subjects and asthmat¬ ics, we were able to compare metaproterenol responsiveness after widely divergent doses of the bronchoprovocative agents but the same degree of bronchoconstriction. Airway deposition of methacholine, histamine, and metaproterenol was measured using a quinine fluorescence tech¬ nique. Mean baseline SGaw, metaproterenol responsiveness, and metaproterenol mass deposited were similar in normal subjects and asthmatics. Likewise, mean SGaw after completion of metha¬ choline and histamine challenge, and the subsequently deposited metaproterenol mass were sim¬ ilar in the two groups. After methacholine challenge (mean±SD provocative drug mass causing a 35% decrease in SGaw, PM35: 8.94 ±5.96 pmol in normal subjects and 0.30±0.29 pmol in asthmat¬ ics), metaproterenol increased mean SGaw by 89±33% in normal subjects and by 190±55% in asthmatics (p<0.05, two-way analysis of variance). After histamine challenge (PM35, 2.92±2.49 pmol in normal subjects and 0.17±0.29 pmol in asthmatics), metaproterenol increased mean SGaw by 111±38% in normal subjects and 113±69% in asthmatics (p=not significant). Thus, for the same degree of bronchoconstriction, metaproterenol responsiveness was influenced by the dose of methacholine but not the dose of histamine. The differential metaproterenol response could be re¬ lated to a functional antagonism between muscarinic and P-adrenergic agonists.

(CHEST 1996; 110:617-23)

Key words: airway hyperresponsiveness; functional antagonism; histamine; methacholine; sympathomimetic agents Abbreviations: cAMP=cyclic adenosine monophosphate; MDI=metered-dose inhaler; PM35=deposited mass of metha¬ choline or histamine required for a 35% decrease in SGaw; SGaw=specific airway conductance

P-Adrenergic

agonists are the mainstay of broncho¬ dilator therapy in bronchial asthma. They seem to exert their effects on airway smooth muscle by increas¬ ing intracellular cyclic adenosine monophosphate (cAMP).1 An interaction between muscarinic cholin¬ ergic bronchoconstrictors and P-adrenergic agonists was shown in 1973 by Van den Brink2 who found that, at a high concentration of a muscarinic agonist, there was no response to isoproterenol in vitro. This phe¬ nomenon is referred to as functional antagonism. *From the Department of Medicine, University of Basel, Switzer¬ land (Dr. Elsasser); and the Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Miami School of Medicine at Mount Sinai Medical Center, Miami Beach, Fla (Drs. Donna, Demirozu, Danta, and Wanner). Supported by a grant from Stiftung fur Pneumologie, Switzerland, and Boehringer Ingelheim, Switzerland. Manuscript received November 6,1995; revision accepted April 18, 1996.

and Critical Reprint requests: Dr. Wanner, Division of Care Medicine, University of Miami SchoolPulmonary 4300 Al¬ Medicine, of ton Road, Miami FL 33140

Reach,

The relaxant effect of P-adrenergic agonists on air¬ way smooth muscle in vitro has subsequently been shown to depend on the mechanism of precontraction. Russell3 compared the relaxation response to isopro¬ terenol of canine airways precontracted with hista¬ mine, 5-hydroxytryptamine, or acetylcholine. He found that isoproterenol reversed contractions induced by histamine much more effectively than those induced by acetylcholine. Torphy et al4 extended this observa¬ tion by showing that higher concentrations of metha¬ choline reduced the maximal relaxation induced by prostaglandin E2 and isoproterenol, and that this was paralleled by a reduction in drug-stimulated cAMPdependent protein kinase activity. Furthermore, an inverse relationship has been shown in canine trachealis smooth muscle between methacholine sensitivity and either cAMP or cAMP-dependent protein kinase activity.5 Madison and Brown6 found that, in canine tracheal smooth muscle, increased cAMP is associated CHEST /110 / 3 / SEPTEMBER, 1996

617

with inhibition of phosphoinositide hydrolysis in re¬ sponse to histamine, but not methacholine. These studies suggest the presence of a functional antagonism between muscarinic and P-adrenergic agonists in canine airway smooth muscle. Such an antagonism has also been observed in isolated human bronchial mus¬ cle preparations.' However, in an in vivo dog model, Jenne et al8 found equal isoproterenol protection against methacholine- and histamine-induced bron¬ choconstriction. This may be explained by the fact that at least part of histamine-induced airway constriction is vagally mediated in several animal species, including the dog.9 The purpose of the present study was to determine whether such a differential metaproterenol respon¬ siveness is demonstrable in intact humans. We there¬ fore compared the bronchodilator effect of a standard¬ ized dose of metaproterenol on methacholine- or histamine-induced bronchoconstriction. By studying asymptomatic asthmatics with nonspecific airway hyperresponsiveness and normal subjects without airway we were able to use two widely hyperresponsiveness, different dosages of the bronchoconstricting agents to induce the same degree of bronchoconstriction. Since total aerosol deposition in the airways may vary considerablymaneuver among subjects even if a controlled is used, we used a quinine breathing fluorescence technique to determine the actual mass of methacholine, histamine, and metaproterenol de¬ in the airways.9"11 We found that higher doses posited of methacholine but not histamine produce metapro¬ terenol resistance. Materials Patients Fourteen normal and 14

and

Methods

asymptomatic asthmatic subjects par¬

ticipated in the study. The protocols were approved by the institu¬ tional review board and informed consent was obtained from all was based on a history of episodic subjects. The diagnosis of asthma confirmed previously by histamine or wheezing and had been methacholine challenges revealing airway hyperresponsiveness. Exclusion criteria included a history of recent (<6 weeks) respira¬ tory tract infections, current cigarette smoking, an airway resistance of more than 250% of predicted, or maintenance therapy with oral bronchodilators, glucocorticosteroids, or antiallergic drugs. Treat¬ ment with inhaled (3-adrenergic agonists (used on demand by some of the subjects) was withheld for at least 12 h prior to testing.

Experimental Techniques

index of airway caliber, airway resistance was measured in volume body plethysmograph (Collins; Braintree, Mass). The reciprocal of airway resistance was divided by thoracic gas volume to obtain specific airway conductance (SGaw). Total aerosol deposition in the airways was determined with a of phosphate-buff¬ previously described technique.1112 A solution ered physiologic saline solution containing 300 ug/mL quinine base (Sigma Chemicals Co; St. Louis) was mixed in a fixed ratio of 1:1 with buffered saline solution containing varying concentrations of methacholine sulfate or histamine base (Sigma Chemicals Co) or As

an

a constant

618

5% metaproterenol HCI. Quinine was used as a fluorescent marker calculate airway deposition. The solutions were nebulized by a nebulizer (Puritan-Bennett model "rain drop;" Puritan-Bennett Co; Kansas City, Kan) using compressed air at a flow rate of 5 L/min. The initial aerosol was mixed with a large amount of dry air, and the resulting dry aerosol was led into a 30-L anesthesia bag, which was placed in a sealed box connected directly to a dry seal spirometer. The mass median aerodynamic diameter of the dry aerosol of buffered saline solution, measured by a 7-stage cascade impactor (Anderson), was 1.5 um (geometric SD, 2.1). The aerosol concen¬ tration in the bag was first determined by sampling approximately 3 L of the bag volume through a 0.5-um pore membrane filter and then extracting the collected sample into 10 mL of buffered saline solution and measuring the fluorescence in the extracted solution by a UV fluorometer (model 112; Sequoia-Tumer Corp; Mountain to

View, Calif).

The subjects took a single breath of the aerosol via a 4-way-valve from residual volume position to total lung capacity position at an inspiratory flow rate of 0.5 L/s controlled by visual feedback using a large electrical flow indicator. Without breath-holding, the sub¬ jects then exhaled passively to residual volume position through an exhaust filter identical to the presample filter, at a flow rate of 0.5 L/s controlled by a downstream vacuum source and critical flow orifice. From the fluorescence eluted from the exhaust filter, the concentration, and the inspired volume, the inspired ofaerosol amount aerosol deposited in the airway was calculated according to the following formula: MD=(Fi-IV-A-10/PS)-(0.9.Fe-10.A), where Mo=drug mass deposited, F^fluorescence of presample aerosol, Fe=fluorescence of exhaled aerosol, IV=inspired volume, A=slope of concentration (metaproterenol, methacholine, or histamine) vs fluorescence by linear regression (calibration), PS=volume of pre¬ sample, 10=mL of dilution for filter paper, 0.9=conversion factor BTPS-ATPS.

Experimental Protocol The study consisted of three parts. In the first experiment, the bronchodilator effect of metaproterenol on basal airway tone was determined in eight asthmatic subjects and eight normal control subjects. After measuring baseline SGaw, the subjects took 1 inha¬ lation of the 5% metaproterenol/quinine solution described above.

After 10 min, SGaw was determined again and the percent increase in SGaw was calculated. In the second experiment, seven asthmatics (including one from the first experiment) and seven normal control subjects (including one from the first experiment) performed a methacholine chal¬ lenge. After determination of baseline SGaw, the subjects inhaled one breath of a solution containing quinine in buffered saline solu¬ tion. If the SGaw did not differ more than 10% from baseline, methacholine (for asthmatics, 0.078 mg/mL; for normal control subjects, 1 mg/mL) with a fixed quinine concentration was inhaled and SGaw was measured 10 min later. The procedure was then repeated with a doubling of the methacholine concentration after each SGaw measurement until a 35% decrease from saline solution/quinine was reached. The subjects then took 1 inhalation of the 5% metaproterenol/quinine solution and SGaw was measured again after 10 min. A least square fit was used to obtain a linear re¬ gression of SGaw on methacholine mass deposited, and the prov¬ ocation dose required to decrease SGaw by 35% was calculated

(PM35).

In the third set of experiments, the

same

7 asthmatics and the

subjects performed a dose-response curve of with doubling doses histamine (initial concentration for asth¬ matics was 0.0625 mg/mL and for normal control subjects it was 1 mg/mL) in the same manner as described for methacholine. Again, after a decrease of SGaw by 35%, 1 inhalation of the 5% metaprosame

7 normal control

Clinical Investigations

Table

1.Anthropometric Data and Pretest Baseline SGaw of the Study Population* Weight, kg

Age>

Height,

Experiment 1: baseline metaproterenol responsiveness 77.6±10 175±10 29.9±5.1 Normal subjects (n=8) 36.9±9.3 70.8±16 170±10 Asthmatics (n=8) 2 3: after methacholine and (M) and histamine (H) metaproterenol responsiveness Experiments 74.9±20 172±11 29.1+4.2 Normal subjects (n=7) Asthmatics (n=7)

78.0±19.2

26.'

SGaw,

cm-1H2Q-s-1

Male/Female

cm

178±11

6/2 3/5

0.19d 0.05 0.15:! 0.05

5/2

0.15±0.03 0.16±0.04 0.15±0.05 0.14±0.06

3/4

(M) (H) (M) (H)

*Mean±SD.

terenol/quinine solution was given and the SGaw measurement was repeated after 10 min. were not conducted in random

The second and third experiments order and the investigators were not blinded to the bronchoconstricting aerosol. The experiments were separated by at least 48 h.

Statistical Analysis A two-way unbalanced analysis of variance was carried out on the change in SGaw by subject (normal subjects, asthmatics) and drug

(methacholine, histamine). Dose-response

curves were

analyzed

after log transformation of the deposited metaproterenol mass with linear regression. When normal distribution was not present, a Spearman rank correlation was used. Significance was accepted at

p<0.05.

Results

anthropometric characteristics and baseline SGaw of the test populations are summarized in Table 1. There were no systematic differences among the groups except for mean baseline SGaw that tended to be lower in asthmatics. There was a significant nega¬ tive correlation between the percentage of deposition and the preceding SGaw for all inhalations performed The

(r=0.31; p<0.001; n=44).

Effect of Metaproterenol on Baseline Airway Tone In normal subjects, metaproterenol (mean±SD deposited mass [95±52 pg]) increased SGaw from

o

o to LJ

cm^HfeO.s-1 (p<0.002). In asthmatics, metaproterenol responsiveness was not different: for a mean deposited metaproterenol mass of 104±91 pg, mean SGaw rose from 0.16±0.06 to 0.20±0.07 cm-1H20.s~1 (p<0.005) (Fig 1). There was no significant relationship between the increase in SGaw and the deposited mass of metaproterenol in normal subjects or asthmatics.

0.19±0.05 to 0.23±0.06

Effect of Metaproterenol After Methacholine and

Histamine Challenge Mean SGaw after methacholine and histamine was comparable in normal subjects challenges asthmatics

(Table 2).

The mean provocative mass of methacholine to in¬ duce a 35% decrease in SGaw was 29 times greater in normal subjects than in asthmatics (Table 2). For comparable mean masses of metaproterenol (102 ±41 pg in normal subjects; 98±40 pg in asthmatics), the absolute and percent increase in mean SGaw after methacholine-induced bronchoconstriction was greater in asthmatics than in normal subjects (Figs 2 and 3). There was no relationship between deposited mass and increase in SGaw (Spearman metaproterenol rank correlation, p>0.05).

301

1.50

20 4

1.00

o: i

< UJ

2

O z

O

and

104

0.50

o

x

x

0.00

CZ3 NORMALS

C3D ASTHMATICS

Figure 1. Percent increase in SGaw (left panel) and percent increase in SGaw corrected for deposited

dose (right panel) after a single inhalation of metaproterenol in normal control subjects metaproterenol and asthmatics

(n=8)

(n=8).

CHEST /110 / 3 / SEPTEMBER, 1996

619

Table 2.Methacholine- and Histamine-induced Bronchoconstriction * SGaw at PM35,

PM35,

cm-1H20.s-1

pmol

Methacholine Control subjects (n=7) Asthmatics (n=7)

0.07±0.03

0.09±0.02

8.94±5.96 0.30±0.29

Control subjects (n=7) Asthmatics (n=7)

0.09+0.02 0.08±0.04

2.92±2.49 0.17±0.29

Histamine

*Mean±SD.

The mean histamine mass to induce a 35% decrease

subjects subjects; proterenol asthmatics) given after histamine challenge led to a increase in mean SGaw in normal subjects comparable and asthmatics (Figs 2 and 3). A borderline significant correlation between the deposited mass of metapro¬ terenol and the increase in SGaw was found for the whole group (Spearman rank correlation; p=0.05). The difference between the responses of normal subjects and asthmatics to metaproterenol was not the same for each drug level. To examine the interaction, normal and asthmatics were compared at each drug subjects level separately, using simple effects analysis. The dif¬ ference between normal subjects and asthmatics for methacholine was significant (p<0.05), whereas for histamine the difference was not significant. than in SGaw was 17 times greater in normal in asthmatics. identical mean masses of meta¬ 98 ±50 pg in (98 ±40 pg in normal

Nearly

Discussion

We conclude the following from these observations:

(1) inhaled metaproterenol is equally effective in reversing histamine-induced bronchoconstriction in normal subjects and asthmatics; (2) metaproterenol is more effective in reversing methacholine-induced bronchoconstriction in asthmatics than in normal (3) the relative metaproterenol resistance in subjects; normal subjects is dependent on the methacholine dose and not the magnitude of bronchoconstriction; and (4) the methacholine-associated metaproterenol resistance is not related to baseline

metaproterenol in the

responsiveness or metaproterenol deposition airway. Airway Deposition of Delivered Drugs

The mean PM35 for methacholine of 8.94 pmol for normal control subjects and 0.30 pmol for asthmatics was in accordance with earlier reports. Donna et al12 a PM35 of 6.95 pmol in normal control sub¬ reported and jects 0.439 pmol in asthmatics. Gillett et al,13 us¬ ing 99mTc-marked methacholine, found a provocation dose (PD35) of 10.5 pmol for normal control subjects and 1.23 pmol for asthmatics. To our knowledge, there are no comparable data on the effective deposited dose 620

of metaproterenol in humans. However, Ahrens et al14 that 1.3 mg aerosolized metaproterenol was reported effective against methacholine-induced broncho¬ spasm. Given a mean lower airway deposition of 10% for wet aerosols generated by jet nebulizers and metered-dose inhalers (MDIs), the estimated effec¬ tive deposited dose of 130 pg in the study ofAhrens et al14 is consistent with our measured mean total airway deposition that ranged between 65 and 103 pg for a dry aerosol; aerosol deposition in the tubing and upper airway was probably small considering that the aerosol was inhaled from a large anesthesia bag. In an oropha¬ model, Kim et al16 found that at an inspiratory ryngeal flow rate of 0.33 L/s, 240 pg of a 650-pg dose of delivered by MDI reached the lower metaproterenol This airways. number, however, does not take into ac¬ count the exhaled portion of the delivered drug. The total airway deposition of metaproterenol was very similar in normal subjects and asthmatics and thus does not explain our observed difference in metapro¬ terenol responsiveness between normal subjects and asthmatics after methacholine challenge. The sites of aerosol deposition in the airways cannot be determined with the method used in this study.

Effect of Metaproterenol on Baseline Airway Tone Our finding of comparable increases in SGaw after a (3-adrenergic agonist in normal subjects and asth¬ matics confirms earlier studies. Sobol et al17 reported a 58% increase in SGaw following isoproterenol inha¬ lation in normal subjects compared to a 41% increase in asthmatics with normal baseline SGaw. Tattersfield et al18 found virtually identical dose-response curves for salbutamol in normal subjects and asymptomatic asthmatics. After 1.3 mg metaproterenol by MDI, Fairshter and Wilson19 reported a 21% increase in SGaw in healthy subjects. Barnes and Pride,20 how¬ ever, found that asthmatics needed a larger dose of salbutamol to achieve maximal bronchodilatation. However, the asthmatics studied exhibited spontane¬ ous bronchoconstriction, which may explain the di¬ minished response to (3-adrenergic agonists. Taken against a difference in together, these ofresults argue smooth muscle to (3-adren¬ responsiveness airway control and abetween normal subjects ergic agonist with normal baseline asthmatics lung symptomatic function. While maximum bronchodilation may have occurred at different times after metaproterenol in¬ halation in different subjects, we chose 10 min to standardize the protocol. Thus, baseline metaproterenol responsiveness was similar in the normal subjects and asthmatics and could not explain the difference in the metaproterenolinduced reversal of methacholine-induced broncho¬ constriction between the two groups. A difference in Clinical

Investigations

250

0.35

200

MP

PH

Figure 3. Percent increase in SGaw induced by metaproterenol inhalation after methacholine and histamine challenge, respectively (seven normal subjects; seven asthmatics). Asterisk=p<0.05 vs nor¬ mal subjects.

0.35 0.30

4

published potency comparisons,23 an apparent plateau of efficacy seems to be reached for SGaw at 300 pg metaproterenol24 in normal subjects. However, Barnes and Pride20 reported that maximal bronchodilatation is reached at a significantly higher dose in asthmatics than normal control subjects. It is therefore possible that in our normal subjects, maximal bronchodilatation would have been reached at a lower metaproterenol the dose

0.25 0.20 -J 0.15

g

POST HISTAMINE

POST METHACHOLINE

o.io

4

0.05

4

dose than

given.

Effect of Metaproterenol After Methacholine and Histamine Challenge By design, SGaw prior to metaproterenol inhalation was similar in normal subjects and asthmatics with ei¬

0.00

Figure 2. Effect of metaproterenol (MP) on SGaw posthistamine

(PH), top, and postmethacholine (PMCH), bottom, induced bronchoconstriction in seven normal (N) and seven asthmatic (A) subjects. Horizontal bars reflect the mean.

P-adrenergic receptor function, if present but not de¬ in normal subjects and asymptomatic our tected, have been expected to be reflected would asthmatics, a decreased by metaproterenol responsiveness in asth¬ matics. For example, a decrease in P-adrenoreceptor function has been described in those with severe asthma, but not in those with mild or asymptomatic asthma who do not use P-adrenergic agonists regular¬

ly.21'22 The fact that

standard

did not find a significant dosebetween the mass deposition of response relationship the and metaproterenol improvement in SGaw may be explained by overdosing of metaproterenol for this group with normal baseline airway caliber. Using we

ther methacholineconstriction.

or

histamine-induced broncho¬

Therefore, a difference in airway caliber

explain why the bronchodilator response to a standard metaproterenol dose was comparable in normal subjects and asthmatics after histamine chal¬ lenge, but smaller in normal subjects than asthmatics after methacholine challenge. Airway responses to methacholine and histamine differ in the predominant site of bronchoconstriction, the time course of induced bronchoconstriction, and the involvement of vagal pathways in bronchoconstric¬ tion. It is generally believed that methacholine acts on large airways, while histamine acts both on primarily and small airways.9'2^ P-Adrenergic agonists large dilate both large and small airways,26'2' whereas SGaw is influenced mainly by large airway caliber. We therefore cannot entirely exclude the possibility that we underestimated the effect of metaproterenol after histamine challenge and failed to detect a difference in cannot

CHEST /110 / 3 / SEPTEMBER, 1996

621

metaproterenol responsiveness between normal sub¬

jects and asthmatics.

Another difference between methacholine and his¬

tamine is the time course of the induced bronchocon¬ striction. Carrier et al28 found a mean duration of 34 to 230 min) bronchoconstriction of 130 min after methacholine, and of 42 min (range, 9 to 90 min) that this raises the after histamine.

(range,

possibility Although dose-response curve was not perfectly cumulative, it cannot be made responsible for the dif¬ ferent bronchodilator response to metaproterenol that was assessed within 15 min of the last methacholine or histamine dose. It should also be pointed out that the time required to reach the PM35 was comparable for both bronchoconstrictors in normal subjects and asth¬ matics because the starting doses for the dose-response curves were different between the two groups. We therefore believe that the metaproterenol re¬ sistance after methacholine challenge was not due to factors but to a functional antagonism experimental the two between agonists. While our results do not of functional antago¬ conclusivelycanprove the presence nism, they explain why the normal subjects who a much higher dose of methacholine to reach required the same degree of bronchoconstriction as the asth¬ matics exhibited significantly less bronchodilatation following metaproterenol than the asthmatics. Another hint for a functional antagonism was the lack of a dose-effect relationship for metaproterenol after meth¬ acholine challenge, whereas a borderline significant correlation between metaproterenol dose and im¬ provement in SGaw was found after histamine chal¬ of our results, Malo et al29 have re¬ lenge. Inthatsupport albuterol was more effective in inhaled ported improving FEVi during spontaneous airflow obstruc¬ bronchocon¬ the histamine

tion than after methacholine-induced striction in patients with asthma.

Different mechanisms involving receptor expres¬ sion, signal transduction, and second messenger sys¬ tems have been proposed for the functional antago¬ nism between P-adrenergic and muscarinic receptor activation in airway smooth muscle. Of special interest is the study of Grandordy et al30 who demonstrated that activation of protein kinase C via phosphoinositide a second messenger pathway involved in hydrolysis, muscarinic airway smooth muscle stimulation, reduces number and cAMP response to p-adrenergic receptor of those authors is in The observation isoproterenol. with the in vivo findings reported in this ar¬ keeping ticle. References 1 Nelson HS.

therapy of bronchial asthma. J Allergy Adrenergic 77:771-85

Clin Immunol 1986;

2 Van den Brink FG. The model of functional interaction: I. De¬

622

velopment and first check of a new model of functional synergism and antagonism. Eur J Pharmacol 1973; 22:270-78 3 Russell JA. Differential inhibitory effect of isoproterenol on con¬ tractions of canine airways. J Appl Physiol 1984; 57:801-07 4 Torphy TJ, Zheng C, Peterson SM, et al. Inhibitory effect of methacholine on drug-induced relaxation, cyclic AMP accumu¬ lation, and cyclic AMP-dependent protein kinase activation in canine tracheal smooth muscle.

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233:409-17

J Pharmacol Exp Ther 1985;

Jensen AD, Puckett AM, Rinard GA, et al. Methacholine sensi¬ tivity and cAMP protein kinase in tracheal smooth muscle. J Appl

Physiol 1986; 60:1043-53 6 Madison JM, Brown JK. Differential inhibitory effects of forskolin, isoproterenol, and dibutyril cyclic adenosine monophosphate

on

phosphoinositide hydrolysis in canine tracheal smooth muscle.

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15

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30

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623