JOURNAL

Vol.

OF

APPLIED

41, No. 1, July

PHYSKOLOGY

1976.

Printed

in U.S.A.

Comparison of drug-induced responses of rabbit trachea and bronchus JEROME H. FLEISCH AND PATRICIA J. CALKINS Division of Pharmacological Research, The Lilly Research Laboratories, Eli Lilly and Company, Irzdianupolis, Indiana 46206

was not blocked by pyrilamine, burimamide, metiamide, or propranolol. We conclude that the use of large airway smooth muscle in vitro is probably a good first step in determining the action of unknown compounds on the bronchial tree. However, results obtained by such analysis must be tempered by the knowledge that even two closely associated sections of respiratory smooth muscle can be pharmacologically distinct.

FLEISCH, JEROME H., AND PATRICIA J+ CALKINS. Comparison of drug-induced responses of rabbit trachea and bronchus. J. Appl. Physiol. 41( 1): 62-66. 1976. -The effect of various smooth muscle stimulants and relaxants was examined on isolated rabbit trachea and bronchus. Trachea contracted maximally in response to carbachol, slightly to KC1, and there was no response to serotonin or PGF2,U, Relaxation of carbachol-contracted trachea was elicited by papaverine, aminophylline, isoproterenol, bradykinin, or PC&. Histamine also relaxed the rabbit trachea. However, bronchi from the same animal contracted to this amine. As was the case with trachea, the bronchi contracted maximally to carbachol and slightly to KCl; serotonin and PG&,, were inactive. Unlike trachea, only papaverine or aminophylline completely relaxed the rabbit bronchus, The other relaxants tested produced smaller responses. Contractions induced by carbachol were blocked by atropine. Bronchial contractions caused by histamine were antagonized by pyrilamine. In contrast, relaxation of trachea caused by histamine was neither affected by pyrilamine nor burimamide, metiamide, or propranolol. We conclude that a lack of pharmacological uniformity exists in at least two smooth muscle subdivisions of a mammalian airway and that this must be taken into consideration when determining the action of drugs on the respiratory system,

METHODS

Male or female New Zealand rabbits (Sweetwater Farms Rabbitry, Hillsboro, Ohio) weighing 2-2.5 kg were killed by an air embolism. The lungs with the trachea attached were quickly excised and placed in Krebs-bicarbonate solution (pH: 7.4) of the following composition in millimoleslliter: KC1 4.6, CaCl, 92H,O 2.5, KH,PO, 1.2, MgSO,m7H,O 1.2, NaCl 118.2, NaHCO, 24.8, and dextrose 10.0. The trachea was separated at its juncture with the main bronchi cleaned, and helically cut according to the method of Patterson (15). The right and left bronchi were obtained by carefully teasing away the parenchyma with dissecting forceps followed by detachment of adhering bronchioles. A polyethylene tube (PE-50) was inserted into the main bronchus and advanced as far as possible. A 3-cm section of bronchus was excised from the distal end and helically cut in a fashion similar to the trachea. This tissue was devoid of cartilage rings, a characteristic which distinguished it from the upper portion of the main bronchus and the trachea (Fig. 1). The right and left bronchus reacted equally well to drugs. Each strip was suspended in a loml isolated tissue bath containing Krebs-bicarbon ate solution aerated with a mixture of 95% 0, and 5% CO,. Temperature was maintained at 37.5OC with a Haake constant-temperature circulating unit. Contractions were measured isometrically with a Grass FT 03 forcedisplacement transducer and recorded on a Grass polygraph as changes in grams of tension. Tracheal strips were subjected to an initial tension of 2 g, whereas strips of bronchus were kept at 1 g. The muscles were placed in tissue baths for l-2 h before drugs were tested. All drugs were dissolved in Krebs-bicarbonate solution with the exception of papaverine and aminophylline which were prepared in 0.9% physiological saline solution. Prostaglandins were stored in 95% ethanol; the final dilutions

drug receptors; histamine; carbachol; polypeptides; smooth muscle; prostaglandins; biogenic amines; bronchodilation

AGENTS can exert their effects locally on the smooth muscle of the terminal bronchioles and alveolar ducts as well as reflexly on the trachea (3). Nevertheless, most studies involving the action of drugs on respiratory smooth muscle utilize the isolated mammalian trachea as the test organ (6). Thus, unless large and small airways of the tracheobronchial tree are pharmacologically identical, results obtained with this tissue might be misleading. In the present study, rabbit trachea and the terminal end of the main bronchus were shown to contract maximally to carbachol and slightly to KCl. Histamine contracted the bronchus but relaxed trachea previously contracted with carbachol. The trachea was also relaxed by papaverine, aminophylline, isoproterenol, bradykinin, or PGE,. In contrast, the rabbit bronchus only relaxed completely with papaverine and aminophylline. The other relaxants produced lesser responses. Histamine-induced contraction of the bronchus was antagonized by pyrilamine, whereas relaxation of the trachea BRONCHOACTIVE

62

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DRUGS

ON

TRACHEA

AND

63

BRONCHUS TABLE 1. KB and PA, values for atropine in rabbit and rat trachea Rabbit

KB -r- SE* PA2 Slope5

Trachea

4.92 x 10-l” M -c 0.68t 9.28 -1.04

Rat Trachea

7.32

x IO-lo M -t 1 03t 9.18 . -1.01

* K, = [antagonist]/(dose ratio - 1). t Mean ments each of which comprised the average of 3 Ke with different concentrations of the antagonist. 12 determinations. 5 Slope of the plots log (dose [log antagonist].

FIG. 1. Rabbit trachea, bronchus and bronchioles devoid of parenchyma. A refers to trachea; B refers to terminal end of the main bronchus. Parenchyma must be carefully removed to avoid bronchiolar damage. In this specimen, a branch of the right main bronchus was torn off during preparation. [Photographed by Dean E. Timmerman. I

were made in Krebs-bicarbonate solution. All agonists, except KCl, papaverine, and aminophylline were kept on ice throughout the experiment and added to the baths in cumulative doses until a complete dose-response curve was obtained. The bath fluid was exchanged several times after each dose-response curve. The antagonists were kept in contact with the tissues for at least 1 h before agonist-induced responses were obtained. To eliminate the action of isoproterenol on the scant number of.alpha receptors located in mammalian trachea (6), phentolamine (3 x lo-” M), an alpha receptor blocking agent, was incorporated into the bath fluid. No more than three agonists were investigated on each tissue. Drug-induced relaxations were obtained in tissues previously contracted with a concentration of carbachol which produced an ED,,,. This amount was determined for each individual tissue. Values for Kg, the dissociation constant of the receptor-inhibitor complex, were calculated by the method of Furchgott (10) using the following equation: KB = [BY (dose ratio - 1). Dose ratio refers to the concentration of agonist required to elicit 50% of maximal response (ED,,,) in the presence of a concentration, [B], of antagonist divided by the ED,, in the absence of antagonist. This formula can only be used when the agonists and antagonists are competitive with one another. The K, values were independent of the concentration of the

-C SE of values t Mean ratio -

5 experiobtained -C SE of 1) vs. -

antagonist which is one indication that such a relationship exists. Also, the slopes of the plots of log (dose ratio - 1) vs. - log [antagonist] were close to the theoretical value of -1 (Table 1) (1). These values and their corresponding pA, values (-log concentration of the antagonist necessary to produce a twofold increase of the ED& were determined by regression analysis using a PDP-10 computer. Dose-response curves were determined on four individual tissues. Three of the preparations were subjected to different concentrations of the antagonist; the fourth was a control and served as an indicator of tissue sensitivity during the course of the experiment. If such changes were noted, dose ratios were corrected accordingly. The following drugs were used: carbamylcholine chloride (carbachol), serotonin creatinine sulfate (Bhydroxytryptamine), histamine dihydrochloride, bradykinin, and atropine sulfate (Sigma Chemical Co.); PGFza and PGE, (Analabs, Inc.); papaverine HCl and potassium chloride (Mallinckrodt); aminophylline (Merck and Co.,); Z-isoproterenol d-bitartrate (Winthrop Laboratories); nitroglycerin (Eli Lilly and Co.); pyrilamine maleate (K & K Laboratories); phentolamine mesylate (gift of Ciba-Geigy Corp.); burimamide HCl and metiamide HCl (gifts of Smith Kline & French); and propranolol HCl (gift of Ayerst Laboratories). RESULTS

Drug-induced contraction of rabbit trachea and bronchus. Previous studies from various laboratories have indicated that acetylcholine and its congeners contract all sections of airway smooth muscle. This was indeed the case in’our study. Carbachol contracted both trachea and bronchus; the ED50)s being nearly identical, 5.4 x 10m7M in the trachea and 7.0 x lop7 M in the bronchus (Fig. 2). Histamine produced a dose-related contraction of the bronchus with an ED,, of 7.6 x lo-” M. In contrast, the trachea did not contract in response to histamine, it relaxed (see below). Maximal concentrations of histamine contracted the rabbit bronchus to approximately 70% of the tension produced by a maximal concentration of carbachol (carbachol 1.6 + 0.2 g,’ n = 15; histamine 1.1 rt 0.1 g,’ n = 12). Since this was not a great difference, we elected to express the results for these two agonists in percent of their maximal response (Fig. 2). On the other hand, KC1 which contracted both trachea and bronchus, caused a maximal response 1 Mean

of the number

of experiments

indicated

1?1SE.

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64

J. I-I. FLEISCH RABBIT

AND

P. J.

CALKINS

BRONCHUS

/ z

20ii

rn

go m

,,

/ /

10”

A

10-6

l@

:

HISTAMINE

10M4

10-3

10-1

MOLAR

: 9

z z

x10&-

RABBIT

TRACHEA * O

00-

:=:

CONTROL

0 -=--a

AFTER l&h ATROPI NE

A-A

(6) (6)

60-

A-A o-o

PGFzo WI

1

10-8

lO-7

10-6

CARBACHOL,

MOLAR

CONCENTRATION

OF

AGONISTS

2. Contractile responses of rabbit bronchus (upper) and rabbit trachea (lower) to various agonists. Responses to KC1 were plotted as a percent of the carbachol maximum. Maximal response to histamine was equivalent, to 70% of that for carbachol but has been expressed in terms of its maximal response. Each point is the mean of the number of experiments indicated in the legend * SE. FIG.

equivalent to only 30% of the carbachol maximum and therefore was expressed as such. Serotonin and PGF,, were devoid of activity on these preparations (Fig. 2,. Angiotensin (n = 3), vasopressin (n = Z), and nicotine (n = 1) had littl e or no effect on either rabbit trachea or bronchus. Effect of antagonists on contractile responses to carbachol and histamine. The influence of atropine, a muscarinic cholinergic receptor blocking agent, and pyrilamine, an H, receptor antagonist, were examined on the contractile responses to carbachol and histamine, respectively. Pyrilamine was a potent antagonist of histamine on the rabbit bronchus (Fig. 3, upper panel). The ED,, after 1 x 10S7 M pyrilamine was 59 times greater than its corresponding control. This produced an uncorrected K, value of 1.7 x 1O-g M for this single concentration of antagonist which is similar to that previously reported for the rabbit and guinea pig aorta (7, 8). Since K, values are one criterion for characterizing pharmacological receptors, this result indicates that histamine receptors in rabbit aorta, rabbit bronchus, and guinea pig aorta are similar in nature. We then determined the effect of 1 x lows M atropine on responses of rabbit trachea and bronchus to carbachol (Fig. 3, lower panel). This concentration produced a muscarinic receptor blockade sufficient to in-

10-5

1o-4

10-3

MOLAR

FIG. 3. Influence of pyrilamine on responses of the rabbit bronchus to histamine (upper) and the effect of atropine on responses of trachea ( -----) and bronchus (- -> to carbachol (Lower). Each point is the mean of the number of experiments in parentheses + SE.

crease the ED,, of carbachol in the bronchus by approximately 7 and in the trachea by 9. The uncorrected K, values for atropine were 1.8 x lo-” M in the bronchus and 1.2 x lop9 M in the trachea suggesting that the cholinergic receptors in these two segments of airway are most likely identical. However, they were significantly higher than those reported for the rat trachea (9). This indicated a possible difference in tracheal cholinergic receptors from rabbits and rats. It then became necessary to critically determine K, and pA2 values (correcting for changes in tissue sensitivity, etc.) for atropine in rat and rabbit trachea using carbachol as the agonist. Table 1 shows that the K, and pA2 values for atropine obtained in such a manner were for all intents and purposes identical. Drug-induced relaxations of rabbit trachea and bronchus. Rabbit trachea and bronchus responded differently to smooth muscle relaxants (Fig. 4). With the exception of nitroglycerin (up to 3 x lo-” M due to solubility characteristics), the trachea relaxed well to all drugs tested. Only papaverine or aminophylline were able to relax the bronchus completely; there were varying degrees of smaller responses to isoproterenol, bradykinin, PGE2, and nitroglycerin. PGE, might have caused complete relaxation of the tissue if concentrations above 3 x lo+ M were used. However, the high cost of the drug coupled with the physiological irrelevance of these concentrations precluded such experiments. Nevertheless, it is obvious from Fig. 4 that even if complete relaxation to PGE, were obtained on the

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DRUGS

ON

TRACHEA

AND

65

BRONCHUS

RABBIT

BRONCHUS

RABBIT

TRACHEA 1 min

PAPAVERINE

A-A

d -0 om-m

BRADYKININ PGE, NITROGLYCERIN 01

h t 10 -6

40-

HISTAMINE

1

IO-'

1O-s

10 -6

1O-3

1O-4

i

(M)

‘:[ J 4 4 t 10-6

HISTAMINE

RABBIT

ii

t 10-5

t 3x1o’6

10

tM)

3x10-6

-5

t 10-4

t 3x10-5

10+-4

t

3x1o-5

TRACHEA

z %oc rr

-

9

Bo-

0

SRADYKININ

4 -7

cd;

4 t 3x10-7

IO

-6

t 3x10-6

FIG. 5. Relaxation of carbachol-contracted tamine (upper and middZe) and bradykinin shows a smooth dose-response relationship obtained on occasion. Middle tracing shows more frequently. These rhythmic responses difficult to quantitate. 10-7

10-4

MOLAR

1O-s

CONCENTRATION

IO

OF

-4

+s t 3X10-

rabbit trachea by his(Lower). Upper tracing to histamine that was the type of response seen were unpredictable and

lo4

AGONISTS

4. Relaxant responses of carbachol-contracted chus (upper) and rabbit trachea (Lower) to various point is the mean of the number of experiments legend * SE. FIG.

10

rabbit agonists. indicated

bronEach in the

bronchus, the ED,,, would have been at least 150 times greater than that seen in the trachea. Interestingly, the ED& for papaverine and aminophylline were 2-3 times smaller in bronchus than in trachea. The lower tracing in Fig. 5 shows an experiment in which 1 x 10p7 M bradykinin produced a substantial relaxation of the carbachol-contracted trachea. Higher concentrations were without effect. The magnitude of such responses to bradykinin were unpredictable. Relaxation of rabbit trachea by histamine. The upper and middle panels of Fig. 5 demonstrate that histamine is capable of relaxing rabbit trachea. The middle tracing illustrates the type of response seen most frequently to histamine whereas the upper tracing was obtained on occasion. Due to the histamine-induced rhythmic activity, this response was difficult to quantitate. That this action of histamine was not blocked by 1 x IO-’ M pyrilamine indicated that an H, receptor mechanism was not involved. In fact, most of our experiments were run with Krebs-bicarbonate solution containing pyrilamine. We then attempted to block the histamine-induced relaxation of rabbit trachea with burimamide or metiamide, H, receptor blocking agents. Burimamide, up to 3 x 1O-4M (rz = 5), or metiamide, up to 3 x lo-*? M (n = 3), had no discernible effect on the response to histamine. As indicated above, the data generated in response to histamine were not definitive, and thus it is

possible that a small depression of the response caused by the H, antagonists could have gone unnoticed. Maengwyn-Davies (14) showed that histamine relaxed the cat tracheal chain; this response was partially antagonized by pronethalol, a beta receptor blocking agent. To ascertain whether or not a similar situation exists in rabbit trachea, we examined the effect of propranolol, another beta receptor antagonist on the relaxation caused by histamine. Propranolol (1 x lo-” M, IZ = 2; 1 x lo+ M, n = 1) did not, alter the response of rabbit trachea to histamine. DISCUSSION

As early as 1913, Golla and Symes (11) stated that “the tracheal muscle differs in its reactions from that of the bronchioles.” In 1958, Brocklehurst (2) showed that trachea and bronchioles from the same animal have surprisingly different reactions to drugs. Eyre (4, 5) then observed that histamine contracts trachea and major bronchi of sheep but relaxes lesser bronchi and bronchioles. Somlyo and Somlyo (16) noted that rabbit bronchus is invariably contracted by histamine whereas histamine may either relax or contract rabbit tracheal smooth muscle. Although these studies suggested that the smooth muscle of the respiratory tree is not pharmacologically uniform, this fact does not appear lo be widely appreciated. -The present study was designed to determine in a more definitive manner whether two sections of airway smooth muscle from a commonly used laboratory animal would react similarly to a variety of drugs. We chose the rabbit because of the relatively large size of the main bronchus and its divisions. Preliminary exper-

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66 iments demonstrated that the terminal end of the main bronchus could be secured easily and prepared for isolated tissue recordings. Responses of this muscle were compared to responses of trachea obtained from the same animal. The results clearly demonstrated that these two tissues are not pharmacologically identical. As was expected, carbamylcholine (carbachol) contracted the rabbit trachea and bronchus. The carbachol ED,& and the K, values for atropine were taken as evidence that the muscarinic cholinergic receptors in the rabbit trachea and bronchus are similar. Responses of the tissues to KC1 were also alike, but rather small. Of the relaxants tested, only papaverine or aminophylline produced analogous responses in trachea and bronchus. Nitroglycerin, serotonin, PGFzcy, angiotensin, and vasopressin were ineffective on these tissues. The pharmacol ogic heterogenei ty of bronchus and trachea became evid .ent by their action in response to histamine, PGEz, isoproteienol, and bradykinin. Histamine contracted the bronchus bu .t produced dose-dependent relaxations of the trachea. The histamine-induced contraction of the rabbit bronchus is mediated via H, receptors as evidenced by its antagonism by pyrilamine. Relaxation of carbachol-contracted trachea by histamine was complicated by the simultaneous production of rhythmi c activity. Such a response pattern is unusual and we can &er no explanation as tO its cause. Histamine-induced relaxation of the trachea was not blocked by pyrilamine, burimamide, metiamide, or propranolol. This intriguing finding permits a number of interpretations. The first is that there are H, receptors in the rabbit trachea that are not blocked by burimamide or metiamide. Another possibility might be that histamine releases a nonadrenergic mediator which causes relaxation; this substance would not be blocked by propranolol. A third suggests that the rabbit

J. H.

FLEISCH

AND

P. J. CALKINS

trachea contains a class of histamine receptors, the significance of which has not heretofore been appreciated (H,). PGE, and isoproterenol relaxed rabbit trachea and bronchus. Both agonists showed greater activity on trachea than bronchus. Recently Joiner et al. (13) concluded that PGE, relaxed canine bronchi previously contracted with carbachol whereas PGF2, had no effect or occasionally elicited some contractions. In the present study, we found that PGFza was devoid of activity on the trachea as well as the bronchus. Interestingly, Sweatman and Collier (17) described a dual action of the prostaglandins on human bronchial smooth muscle; PGF2, contracted this tissue and PGE, and PGE2 caused a relaxation. Bradykinin has been shown to either contract or relax guinea pig tracheal muscle depending on tissue tone (12). We observed a small relaxation of the rabbit bronchus. Large relaxations could be produced in strips of rabbit trachea but these responses were often unpredictable and therefore could not be evaluated. This study, coupled with previous observations on the lack of pharmacological uniformity in the airways, indicates that the use of single isolated sections of airway smooth muscle (e.g., trachea, bronchus) for the evaluation of new compounds could yield misleading results. For example, a drug may appear to be a bronchodilator in one section but constrict all other divisions. Furthermore, these results suggest that circulating hormones or mediators released by antigen-antibody interactions may differentially affect the various airway subdivisions; their total action on the smooth muscle of the lung, therefore, must result from their individual action on each of the component parts. Received

for publication

October

1975.

REFERENCES 1. ARUNLAKSHANA, O., AND II. 0. SCHILD. Some quantitative uses of drug antagonists. Brik J. Pharmacol. 14: 48-58, 1959. 2. BROCKLEHURST, W. The action of 5-hydroxytryptamine on smooth muscle. In: 5-Hydroxytryptamine, edited by G. P. Lewis. London: Pergamon, 1958, p. 172-176. 3. DE KOCK, M. A., J. A. NADEL, S. ZWI, H. J. H. COLEBATCH, AND C. R. OLSEN. New method for perfusing bronchial arteries: histamine bronchoconstriction and apnea. J. AppZ. Physiol. 21: 18% 194, 1966. 4. EYRE, P. The pharmacology of sheep tracheobronchial muscle: a relaxant effect of histamine on the isolated bronchi. Brit. J. PharmacoZ. 36: 409-417, 1969. 5. EYRE, P. Histamine HP-receptors in the sheep bronchus and cat trachea: The action of burimamide. Brit. J. Pharmacol. 48: 321323, 1973. 6. FLEISCH, J. H., K. M. KENT, AND T. COOPER. Drug receptors in smooth muscle. In: Asthma: PhysioZogy, ImmunopharmacoZogy, and Treatment, edited by K. F. Au&en and L. M. Lichtenstein. New York: Academic, 1973, p. 139-167. 7. FLEISCH, J. H., M. C. KRZAN, AND E. TITUS. Pharmacologic receptor activity of rabbit aorta: effect of dithiothreitol and Nethylmaleimide. Circulation Res. 33: 284-290, 1973. 8. FLEISCH, J. H., M. C. KRZAN, AND E. TITUS. Alterations in pharmacologic receptor activity by dithiothreitol. Am. J. Physiol. 227: 1243-1248, 1974.

9. FLEISCH, J. H., AND E. TITUS. Effect of local anesthetics on pharmacologic receptor systems of smooth muscle. J. Pharmacol. Exptl. Therap. 186: 44-51, 1973. 10. FURCHGOTT, R. F. Pharmacological differentiation of adrenergic receptors. Ann. N. Y. Acad. Sci. 139: 553-570, 1967. 11. GOLLA, F L., AND W. L. SYMES. The reversible action of adrenaline and some kindred drugs on the bronchioles. J. Pharmacol. ExptZ. Therap. 5: 87-103, 1913. 12. IORIO, L. C., AND J. W. CONSTANTINE. Bradykinin on isolated guinea pig tracheal muscle. J. Pharmacol. ExptZ. Therap. 169: 264-270, 1969. 13. JOINER, P. D., L. MINOR, L. B. DAVIS, P. J. KADOWITZ, AND A. L. HYMAN. Studies of pharmacology of canine isolated intrapulmonary bronchi (Abstract). Pharmacologist 17: 191, 1975. 14. MAENGWYN-DAVIES, G. D. The dual mode of action of histamine in the cat isolated tracheal chain. J. Pharm. Pharmacol. 20: 572573, 1968. 15. PATTERSON, R. The tracheal strip: observations on the response of tracheal muscle. J. Allergy 29: 165-172, 1958. 16. SOMLYO, A. P., AND A. V. SOMLYO. Biophysics of smooth muscle excitation and contraction. In: Airway Biodynamics: Physiology and Pharmacology, edited by A. Bouhuys. Springfield, Ill.: Thomas, 1970, p. 209-227. 17. SWEATMAN, W. J. F., AND H. 0. J. COLLIER. Effects of prostaglandins on human bronchial muscle. Nature 217: 69, 1968.

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Comparison of drug-induced responses of rabbit trachea and bronchus.

JOURNAL Vol. OF APPLIED 41, No. 1, July PHYSKOLOGY 1976. Printed in U.S.A. Comparison of drug-induced responses of rabbit trachea and bronchu...
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