3. Smooth Muscle Pharmacology of Airway Smooth Muscle in Chronic Obstructive Pulmonary Disease in Asthma'? JUDITH L. BLACK Introduction The purpose of this review is to outline the similarities and differences in the in vitro pharmacologic behavior of human airway smooth muscle derived from subjects with welldefined asthma or chronic obstructive pulmonary disease (COPD). The airway hyperresponsiveness that characterizes asthma is manifested as airway narrowing to a variety of possibly unrelated stimuli. It is therefore reasonable to assume that the basic abnormality underlying this disease is in the airway smooth muscle. If this assumption were true, then airway smooth muscle isolated from asthmatic subjects would exhibit different pharmacologic behavior from that of nonasthmatic subjects. This could take the form of an increase in contractility or a decrease in relaxation responses. If, as has been suggested, COPD and asthma form part of a spectrum of respiratory disease (1), then given the above assumptions, there should be a gradation in in vitro responsiveness between asthma and COPD. If asthma is an airway smooth muscle abnormality, but unrelated to COPD, then a clear separation of their pharmacologic responsiveness would exist. Moreover, similarities and differences in pharmacologic behavior should hold true for all agonists. There is another possibility. If the basic abnormality in asthma does not lie solely in the smooth muscle, then there will be no clear differences in the in vitro responses of muscle from patients with asthma or COPD and muscle from those free of respiratory disease. In vitro pharmacologic behavior can be assessed in terms of, first, functional response and, second, receptor number and affinity as assessed in radioligand binding studies. Functional Studies The EC so (the concentration of an agonist that produces 50070 of the maximal response) or the pD 2 (its negative logarithm) is an estimate of agonist potency. Given that asthmatic subjects are more sensitive to various inhaled agonists, it would seem to be a valid method of comparing patient groups. It is extremely difficult to make comparisons of maximal relaxation or contractile responses between patient groups, and this is due to problems with expression of the data. Attempts to compare tissues from different patient groups by normalizing responses in terms of smooth muscle volume, tissue weight, and protein content have largely proved unsatisfactory (2). AM REV RESPIR DIS 1991; 143:1177-1181
Although, as stated above, changes in responsiveness should be consistent for all agonists, it is convenient to review available data in terms of the agonists studied.
Contraction Histamine The EC so values for tissue from asthmatic patients and from those with COPD are shown in figure 1 (3-15). It is apparent that there is good agreement between values obtained for asthmatic subjects in different studies (4-7,9-11, 13, 14). This is in spite of the fact that there are marked differences in patient characteristics. Those studied by Goldie and coworkers (5) died in status asthmaticus, whereas the asthma of the patients in the study by Whicker and colleagues (11) was sufficiently mild as to permit lung resection. Moreover, both these investigators noted that, compared with patients free of airway obstruction, histamine sensitivity in asthmatic subjects was decreased. de Jongste and coworkers (6) and Schellenberg and Foster (7) also observed no increase in sensitivity compared with that in normal subjects. In contrast to these four studies, there is one report in which an increase in sensitivity was found. Cerrina and coworkers (14) noted an increase in sensitivity to histamine but not to acetylcholine, prostaglandin E 2 (PGE 2 ) , or F 2U (PGF2U) ' Unlike tissue from asthmatic patients, there is a striking difference in the sensitivity of tissue from patients with COPD. The values obtained by Vincenc and coworkers (8) and in one of the studies of Armour and colleagues (3) are much lower (and therefore indicate greater histamine potency) than those from all other laboratories. Within individual studies, no significant differences exist between patients with COPD and normal subjects, and, overall, there is clearly no distinct separation between patients with COPD and those with asthma. Methacholine/Acetylcholine/Carbachol As for histamine, there is no clear separation of sensitivity to cholinergic stimulation (figure 2) between tissue from asthmatics or from subjects with COPD. Whicker and colleagues (11) and Goldie and coworkers (5) again reported that asthmatic tissue was less sensitive than normal tissue. It is again interesting to note the apparent increase in sensitivity of patients with COPD compared with other groups in the study by Armour and colleagues
(3). These patients were a carefully defined group, all with documented evidence of airway obstruction, half of whom had in vivo airway hyperresponsiveness when tested preoperatively, and half of whom were nonresponsive to inhaled methacholine. It is interesting to speculate as to whether these Australian patients with COPD are different in some way from those studied in other laboratories. Nevertheless, when all studies are considered, there are no differences in sensitivity in tissues from patients with COPD (3, 4, 12, 16-18) and asthmatics (4-7, 11).
Leukotrienes/Prostaglandins Dahlen and coworkers found that tissue from asthmatic subjects (10) was as sensitive to leukotrienes as that from nonasthmatics (19). Moreover, sensitivity to leukotrienes was not greater in those patients with COPD (12, 20) than those without (21). That differences do not exist between COPD (12) and asthma (6, 7, 10) with respect to leukotriene sensitivity is indicated in figure 3, and it is possible to infer from comparisons within individual studies that these patients in turn do not differ from those free of respiratory disease. Likewise, Cerrina and coworkers (14) could not differentiate between COPD and asthma when responses to PGE 2 and PGF2u were studied. Relaxation Isoproterenol and Other {3-Adrenoceptor Agonists The available information on sensitivity to isoproterenol in tissue from subjects with asthma (4-6,9,11) and those with COPD (4, 16, 22) is shown in figure 4. Although in individual studies there may be differences in l3-adrenoceptor function between the patient groups, when all studies are considered, differences between asthmatics, patients with COPD, and normal subjects are difficult to detect. Moreover, if sensitivity to other l3-adrenoceptor agonists such as salbutamol is compared in asthma (5) and COPD (17), the former patient group appears to be more 1 From the Department of Pharmacology, University of Sydney,New South Wales, Australia. 2 Supported by the National Health and Medical Research Council of Australia. 3 Correspondence and requests for reprints should be addressed to Judith L. Black, Department of Pharmacology, University of Sydney,New South Wales 2006, Australia.
1177
JUDITH L. BLACK
1178
•
Armour
..
•
.1 0
Cerrina Goldie
0 0 0
de Jongste
0
Dahlen
•
Whicker de Jongste
•
o•
Cerrlna
0
1 \,....
-8
Armour
••
Vincenc'••: -9
Fig. 1. ECso values in human isolated bronchus from patients with COPO (closed circles) or with asthma (open circles). Single symbols in an individual study indicate either that only one patient was studied or that only a mean value is available.
Roberts
o~~\ Schellenberg
-7
sensitive to this agonist. Thus, although there is in vitro evidence for a 13-adrenoceptor abnormality in asthma (5), it is difficult to understand why this would vary between 13-adrenoceptor agonists.
Bal
0
•
-4
-5
-6
Theophylline and Forskolin As can be seen in figure 5, little information is available on sensitivity to these agonists in asthma (6,9, 11)or in COPD (16, 17). What is striking is the lack of variability between patients with COPD in in vitro responsiveness to forskolin (16, 17).It has been suggested that the abnormality in COPD lies in the coupling mechanism distal to the 13-adrenoceptor (22). If this were so, it would be expected that agents that stimulate adenylate cyclasedirectly and bypass the 13-adrenoceptor would differentiate between patient groups. This appears not to be the case with forskolin. No studies have been performed on theophylline sensitivity in patients with COPD. Of the three in asthmatic tissue, two have reported no difference from normal subjects (5, 11), but Bai (9) did observe a decreased sensitivity in postmortem trachea.
log histamine (M)
•• • •••
•
•
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Armour Cerrina
...
.,.
•••• .....
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o
Goldie de Jongste
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Radioligand Binding Studies Radioligand binding studies provide a direct assessment of receptor number and receptor affinity. The possibility exists that in asthma and/or COPD, the fundamental abnormality lies in an alteration of these parameters. Thus, a change in the characteristics of those receptors, stimulation of which results in alteration of airway smooth muscle tone, may produce airway hyperresponsiveness. Studies of changes in receptor characteristics are beset with multiple problems and this is especially relevant when human airway tissue is under investigation. First, those receptors of interest are likely to be those situated on airway smooth muscle. The percentage of muscle in airway preparations available for in vitro study, whether they are obtained from surgical resection or post-mortem, is very low (l-161t7o) (3~ 20). This means that in order to obtain sufficient material (as estimates of receptor numbers are usually expressed in terms of milligram of protein), tissues from several patients have to be pooled. Alternatively, in order to obtain sufficient material for study of individual patients, many investigators have used samples of peripheral lung only. These studies therefore provide us with
0
I
i
I
i
i
-9
-8
-7
-6
-5
I
-10
Fig. 2. ECso values for acetylcholine, methacholine, or carbachol in human isolated bronchus from patients with COPO (closed circles) or with asthma (open circles). Single symbols in an individual study indicate either that only one patient was studied or that only a mean value is available.
• Taylor
log cholinergic a'ionist (M)
t.. ...
Fig. 3. ECso values in human isolated bronchus for leukotrienes from patients with COPO (closed triangles) or with asthma (open triangles). Also shown are EC so values for prostaglandin F2a (PGF 2a) in patients with COPO (closed squares) or with asthma (open squares) and, correspondingly, for prostaglandin E2 (PGE~ (closed and open circles). Single symbols in an individual study indicate either that only one patient was studied or that only a mean value is available.
Roberts
.. deJongsfe
leukotrienes
A
deJongsfe
A A
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2
. Carrino
i i i
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-8
-7
-6
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•
von Koppen.
• •• • de Jongste • •• • • • • • •• • • •• Whicker ••
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•
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•••
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0
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:
.~
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forskolin
-6
Taylor de Jongste
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.5
de Jongste Sai
log isoprenaline (M)
Fig. 4. ECso values in human isolated bronchus from patients with COPO (closed circles) or with asthma (open circles). Single symbols in an individual study indicate either that only one patient was studied or that only a mean value is available.
j
j
-8
-7
, -6
0
Whicker Goldie 0
0
theophylline
I
j
i
-5
-4
-3
log agonist (M)
Fig. 5. EC so values for forskolin in human isolated bronchus from patients with COPO (closed squares) or with asthma (open squares). Also shown are values for theophylline from asthmatic subjects (open circles). Single symbols in an individual study indicate either that only one patient was studied or that only a mean value is available.
1179
PHARMACOLOGY OF AIRWAY SMOOTH MUSCLE IN COPO AND ASTHMA
information about characteristics of receptors only on very small airways. In addition, those receptors situated on other structures such as mucous glands and blood vessels would be included. The physiologic relevance of these findings may therefore be questioned. Second, wide variabilities (> 1 log unit) in the sensitivity of some receptor populations (23) have been recorded within a particular patient group, and this means that comparisons between different patient groups such as asthmatic and COPD groups need to be performed on very large numbers of subjects. Third, the relevance and interpretation of information on receptor characteristics is questionable. Increasingly, it is apparent that there is little relationship between receptor number and pharmacologic response (24). From this it is possible to infer that not all receptors are functional. Some receptors may be "spare," i.e., not directly linked to a physiologic response and these could be "recruited" under a variety of experimental and pathologic conditions. Lastly, the influence of preoperative or antemortem conditions such as medication and tissue storage also may constitute confounding variables, as these may cause upregulation or downregulation of receptor populations. There are few reports of ligand binding studies in human airway tissue and even fewer in which a comparison can bedrawn between patients with CO PD and those with asthma. Those receptor populations that have received most attention are muscarinic and a- and ~-adrenoceptors.
TABLE 1 BINDING PARAMETERS OF ~-ADRENOCEPTOR LIGANDS IN HUMAN LUNG TISSUE*
Ligand
Patient Characteristics
n
Bmax (fmo/fmg protein)
Barnes (25)
[3H1DHA
COPD No obstructive lung disease
7 1
467 ± 76 309
van Koppen (22)
[1 25 11CYP
COPD No obstructive lung disease
8 (Bronchus) 9 (Bronchus)
0.372 ± 0.069 0.376 ± 0.064
Szentivanyi (27)
PH1DHA
Asthma COPD No obstructive lung disease
12 18 49
160 ± 9.4 194 ± 21.6 212 ± 20.2
Raaijmakers (26)
[3H1DHA
COPD No obstructive lung disease
12 24
511 ± 47 396 ± 33
Source
Definitionof abbreviations: DHA = dihydroalprenolol; CYP = cyahopindolol; Bmax dissociation constant. • Studies were performed on lung parenchymal tissue unless otherwise indicated.
0.82 ± 2 0.70 11.34 ± 0.05 11.22 ± 0.05
2.3 ± 0.5 1.7 + 0.2
= maximal number of binding
sites; Kd
=
TABLE 2 BINDING CHARACTERISTICS OF ll-ADRENOCEPTOR LIGANDS IN HUMAN LUNG TISSUE*
Ligand
Patient Characteristics
n
Bmax (fmo/fmg protein)
Kd (nmo/fL)
Barnes (25)
[3Hlprazosin
COPD No obstructive lung disease
7 1
600 62
1.75 1.1
Raaijmakers (26)
[3Hlprazosin
COPD No obstructive lung disease
12 24
2,202 ± 220 491 ± 52
10.7 ± 2.01 4.8 ± 1.8
Szentivanyi (27)
[3H1DHE
Asthma COPD No obstructive lung disease
12 18 49
Source
172 ± 8.8 62 ± 21.8 38 ± 11.8
= dihydroergotamine; Bmax = maximum number of binding sites; Kd • These studies were carried out on lung parenchymal tissue.
Definitionof abbreviations: DHE
{3-Adrenoceptors The proposal that a deficit in ~-adrenoceptor function may contribute to airway hyperresponsiveness has prompted several investigators to compare adrenoceptor characteristics in airway tissue from patients with and without respiratory disease (table 1). Barnes and coworkers (25) found that neither ~-adrenoceptor density nor affinity differed in patients with COPD from that in tissue from one subject free of respiratory disease. This was confirmed in larger numbers of subjects by Raaijmakers and coworkers (26) who, however, reported a decrease in the ratio of ~2/~1 receptors in patients with COPO. An extensive study combining both functional and binding techniques by van Koppen and colleagues (22) detected no difference in ~-receptor characteristics in either tracheal or bronchial smooth muscle from patients with COPD. The only study in which asthmatic tissue has been investigated is that of Szentivanyi and coworkers (27) who studied three groups of patients: those with (1) no respiratory disease, (2) nonreversible airway obstruction, and (3) asthma, and found that total ~-receptor density decreased progressively in these groups.
that ~-receptor populations are not altered by respiratory disease, three studies have reported concomitant changes in c-adrenoceptor number (table 2). In the same study in which Barnes and coworkers (25) reported no change in ~-adrenoceptor density, there was, in contrast, a to-fold increase in n-adrenoceptor density in tissue from patients with COPD. Both a decrease in affinity and a 5-fold increase in density were reported by Raaijmakers and coworkers (26), and these changes were reversed by the presence of disodium cromoglycate. In addition, Szentivanyi and coworkers (27) observed a progressive increase in binding to dihydroergotamine ([3H]DHE) across their patient groups (see above). These findings of increased o-adrenoceptor number need further verification since ligands such as [3H]DHE may not be specific for c-adrenoceptors. In addition, vascular tissue in the lung parenchyma used in these studies contains c-adrenoceptors. If COPD and asthma were associated with an increase in vascularity, then an increase in o-adrenoceptor number could be explained on this basis. The relevance of changes in a-adrenoceptor characteristics to the in vivo situation is not clear. Although asthmatic subjects exhibit bronchoconstriction to inhaled n-adrenoceptor agonists (28, 29), this is not true of patients with COPD (30).
a-Adrenoceptors Although the majority of studies has reported
Cholinergic Receptors The concept that a cholinergic receptor dys-
Adrenoceptors
Kd (nmo/fL)
= dissociation constant.
function may occur in COPD and/or asthma has been addressed by a number of groups. The issue has been complicated by recent evidence that there are subtypes of cholinergic receptors that are labeled by selective ligands in human airways such as pirenzepine (PZ) (31). The possibility exists that a decrease in the number of inhibitory muscarinic receptors or an increase in those that are excitatory could contribute to the abnormalities in these respiratory disorders (32). No radioligand binding studies have as yet been performed on tissue from asthmatic subjects. Raaijmakers and colleagues (33) reported a decrease in receptor density in peripheral lung tissue from patients with COPD - a surprising finding in view of the fact that these patients are more responsive than normal subjects to cholinergic agents. These findings were not confirmed, however, in a subsequent extensive study by load and Casale (23) who found that cholinergic receptor characteristics were not altered in patients with COPD. Thus, insufficient data exist at present to compare cholinergic receptor characteristics in asthma and COPD, although it seems likely (table 3) that the latter group do not differ from normal subjects. Summary Only a small number of studies investigating the in vitro pharmacologic properties of airway smooth muscle in asthma and wellcharacterized COPD have been performed.
1180
JUDITH L. BLACK
TABLE 3 BINDING PARAMETERS OF MUSCARINIC LIGANDS IN HUMAN LUNG TISSUE·
Source
Patient Characteristics
Ligand
n
Bmax (fmo/fmg protein)
Kd (nmo/fL)
Raaijmakers (33)
[3H)QNB
COPD No obstructive lung disease
4 8
25.6 ± 11.2 85 ± 14.5
0.12 ± 0.02 0.085 ± 0.009
Joad (23)
[3H)QNB
COPD No obstructive lung disease
19 6
29 ± 4 21 ± 5
0.07 ± 0.01 0.06 ± 0.01
van Koppen (24)
[3H)QNB
No respiratory disease (trachea)
14
123 ± 16
0.047
Bloom (31)
[3H)QNB
No respiratory disease
6
55.6 ± 12
0.0143
Definition of abbreviations: ONS
= (- )-quinuclidinylbenzilate;
Smax
= maximum number
of binding sites; Kd
= dissociation
constant. * Studies were performed on lung parenchymal tissue unless otherwise stated.
Further detailed studies on well-defined patient groups are required. The majority of available evidence would suggest that once airway smooth muscle is removed from its in vivo milieu, it loses the characteristics of hyperresponsiveness. This would explain why there are no clear differences in the pharmacologic responsiveness of tissue from patients with asthma or COPD and those with no obstructive disease. Future in vitro studies should be directed towards reproducing the in vivo environment. This would entail the establishment of a chronic inflammatory condition created by the continuous presence of neural and humoral factors.
Acknowledgment The writer thanks Dr. Carol Armour and Miss Susan Whicker for advice on the content of the manuscript and Miss Ann McGregor for its careful preparation.
References 1. Orie NGM, Sluiter HJ, de Vries K, Tammeling GJ, Witkop J. The host factor in bronchitis. In: Orie NGM, Sluiter HJ, eds. Bronchitis. Assen, The Netherlands: Royal Vangorcum, 1961; 43-59. 2. de Jongste JC, van Strik R, Bonta IL, Kerrebijn KF. Measurement of human small airway smooth muscle function in vitro with the bronchiolar strip preparation. J Pharmacol Methods 1985; 14:111-8. 3. Armour CL, Black JL, Berend N, Woolcock AJ: The relationship between bronchial hyperresponsivenessto methacholine and airway smooth muscle structure and reactivity. Respir Physiol1984; 58:223-33. 4. Cerrina J, Le RoyLadurie M, Labat C, Raffestin B, Bayol A, Brink C. Comparison of human bronchial muscle responsesto histamine in vivo with histamine and isoproterenol agonists in vitro. Am Rev Respir Dis 1986; 134:57-61. 5. Goldie RG, Spina D, Henry Pl, Lulich KM, Paterson JW. In vitro responsiveness of human asthmatic bronchus to carbachol, histamine, l3-adrenoceptor agonists and theophylline. Br J Clin Pharmacol 1986; 22:669-76. 6. de Jongste JC, Mons H, Bonta IL, Kerrebijn KF. Human asthmatic airway responses in vitro - a case report. Eur J Respir Dis 1987; 71:23-9. 7. Schellenberg RR, Foster A. In vitro responses of human asthmatic airway and pulmonary vascu1arsmooth muscle. Int Arch Allergy Appl Immunol 1984; 75:237-41. 8. Vincenc KS, Black JL, Yan K, Armour CL, Donnelly P, Woolcock AJ. Comparison of in vivo
and in vitro responses to histamine in human airways. Am Rev Respir Dis 1983; 128:875-9. 9. Bai TR. Abnormalities in airway smooth muscle in fatal asthma (abstract). Am Rev Respir Dis 1989; 139:A258. 10. Dahlen S-E, Hansson G, Hedqvist P, Bjorck T, Granstrom E, Dahlen B. Allergen challenge of lung tissue from asthmatics elicits bronchial contraction that correlates with the release of leukotrienes C 4 , D 4 , and E 4 • Proc Nat! Acad Sci USA 1983; 80:1712-6. 11. Whicker SD, Armour CL, Black JL. Responsiveness of bronchial smooth muscle from asthmatic patients to relaxant and contractile agonists. Pulmon Pharmacol 1988; 1:25-31. 12. de Jongste lC, Mons H, van Strik R, Bonta IL, Kerrebijn KF. Comparison of human bronchiolar smooth muscle responsiveness in vitro with histological signs of inflammation. Thorax 1987; 42:870-6. 13. Roberts JA, Rodger IW, Thomson NC. Airway responsiveness to histamine in man: effect of atropine on in vivo and in vitro comparison. Thorax 1985; 40:261-7. 14. Cerrina J, Labat C, Haye-Legrande I, Raffestin B, BenvenisteJ, Brink C. Human isolated bronchial muscle preparations from asthmatic patients: effects of indomethacin and contractile agonists. Prostaglandins 1989; 37:457-70. 15. Armour CL, Lazar NM, Schellenberg RR, et al. A comparison of in vivo and in vitro human airway reactivity to histamine. Am Rev Respir Dis 1984; 129:907-10. 16. de Jongste JC, Mons H, Bonta IL, Kerrebijn KF.Relaxation of human peripheral airwaysmooth muscle in vitro does not correlate with severity of chronic airflow limitation in vivo. Pulmon Pharmacol 1989; 2:75-9. 17. Taylor SM, Pare PD, Armour CL, Hogg JC, Schellenberg RR. Airway reactivity in chronic obstructive pulmonary disease. Am Rev Respir Dis 1985; 132:30-5. 18. Roberts JA, Raeburn D, Rodger IW, Thomson NC. Comparison of in vivo airway responsiveness and in vitro smooth muscle sensitivity to methacholine in man. Thorax 1984; 39:837-43. 19. Dahlen S-E, Hedqvist P, Hammarstrom S, Samuelsson B. Leukotrienes are potent constrictors of human bronchi. Nature 1980; 288:484-6. 20. Roberts JA, Rodger IW,Thomson NC. In vivo and in vitro human airway responsiveness to leukotriene D 4 in patients without asthma. J Allergy Clin Immunol 1987; 80:688-94. 21. Roberts JA, Giembycz MA, Raeburn D, Rodger IW, Thomson NC. In vitro and in vivo effect of verapamil on human airway responsiveness to leukotriene D4 • Thorax 1986; 41:12-6. 22. van Koppen CJ, Rodrigues de Miranda JF, Beld AJ, van Herwaarden CLA, Lammers J-WJ,
van Ginneken CAM. Beta adrenoceptor binding and induced relaxation in airway smooth muscle from patients with chronic airflow obstruction. Thorax 1989; 44:28-35. 23. Joad JP, Casale TB. [3H]quinuclidinyl benzilate binding to the human lung muscarinic receptor. Biochem Pharmacol 1988; 37:973-6. 24. van Koppen CJ, Rodrigues de Miranda JF, Beld AJ, Hermanussen MW, Lammers J-WJ, van Ginneken CAM. Characterization of the muscarinic receptorin human tracheal smooth muscle.NaunynSchmiedebergs Arch Pharmacol1985; 331:247-52. 25. Barnes PJ, Karliner JS, Dollery CT. Human lung adrenoceptors studied by radioligand binding. Clin Sci 1980; 58:457-61. 26. Raaijmakers JAM, Wassink GA, Kreukniet J, Terpstra GK. Adrenoceptors in lung tissue: characterization, modulation, and relations with pulmonary function. Eur J Respir Dis Suppl135 1984; 65:215-20. 27. Szentivanyi A, Heim 0, Schultze P. Changes in adrenoceptor densities in membranes of lung tissue and lymphocytes from patients with atopic disease. Ann N Y Acad Sci 1979; 332:295-8. 28. Black JL, Salome CM, YanK, Shaw J. Comparison between airways response to an n-adrenoceptor agonist and histamine in asthmatic and non-asthmatic subjects. Br J Clin Pharmacol1982; 14:464-6. 29. Black JL, Salome C, Yan K, Shaw J. The action of prazosin and propylene glycol on methoxamine-induced bronchoconstriction in asthmatic subjects. Br J Clin Pharmacol 1984; 18:349-53. 30. Du Toit 11, Woolcock AJ, Salome CM, Sundrum R, Black JL. Characteristics of bronchial hyperresponsiveness in smokers with chronic airflow limitation. Am Rev Respir Dis 1986; 134: 498-501. 31. Bloom JW, Halonen M, Yamamura HI. Characterization of muscarinic cholinergic receptor subtypes in human peripheral lung. J Pharmacol Exp Ther 1988; 244:625-32. 32. Barnes PJ, Minette P, Maclagen J. Muscarinic receptor subtypes in airways. Trends Pharmacol Sci 1988; 9:412-6. 33. Raaijmakers JAM, Terpstra GK, van Rozen AJ, Witter A, Kreukniet J. Muscarinic cholinergic receptors in peripheral lung tissue of normal subjects and of patients with chronic obstructive lung disease. Clin Sci 1983; 66:585-90. 34. Mitchell HW, Willet KE, Sparrow MI>. Perfused bronchial segment and bronchial strip: narrowing vs. isometric force by mediators. J Appl Physiol 1989: 66:2704-9.
Comments Dr. Holgate: Mitchell and Sparrow have developed an in vitro technique for investigating resistivechanges in airway function in perfused airways (34). They report that smaller degrees of smooth muscle contraction are required to produce large changes in resistance than are conventionally used in airway strips. Could it be that the in vitro preparations that are used to investigate hyperresponsiveness. are inappropriate? Dr. Black: This is certainly possible. These experiments are very interesting, and I think we will obtain most valuable information from these investigations. Perhaps in future the use of this type of preparation will allow
PHARMACOLOGY OF AIRWAY SMOOTH MUSCLE IN COPO AND ASTHMA
the demonstration of in vitro differences in pharmacologic responsiveness of muscle from different patients. Dr. O'Byrne: Did PGE 2 have any postsynaptic effects on human airway smooth muscle in vitro? Dr. Black: Yes, PGE 2 has a biphasic action on human airway smooth muscle, relaxation at low, contraction at high concentrations. Dr. Pare: (1) Why did you in your initial statements rule out the study of maximal tension generated by human smooth muscle preparations? (2) In vitro studies of asthmatic airway smooth muscle by de Jongste and Schellenberg showed an increased tension generation. Dr. Black: I did not rule out the importance of maximal tension. I said it was extremely difficult to normalize maximal tension values between different patient groups. It is a question of how you express the data. Several
groups have tried to normalize for wet weight, dry weight, mg of muscle or mg of protein, largely without success. There is little relationship between amount of smooth muscle and maximal responses. (2) These studies are on small numbers of subjects. de Jongste studied only one asthmatic subject and Schellenberg two. Their results may not necessarily be confirmed in larger numbers of patients. Dr. Corrigan: How far is the capacity of airway smooth muscle to cause bronchoconstriction affected by the elastic recoil pressure of the tissue in which the muscle is situated? Dr. Black: Certainly this is not affected to a great extent when we study airways in vitro. In vivo it is probably important. Dr. Bleecker: Most comparisons of in vivo hyperresponsiveness with in vitro smooth muscle function have been performed in specimens obtained from long-standing cigarette smokers who are having a thoracotomy for
1181
lung cancer. Are there still additional studies that should be performed or have you put this topic to rest? Specifically, should smooth muscle from normal lungs and from asthmatics be studied? Dr. Black: I believe we need more studies on well-characterized patient groups, especially asthmatics. Perhaps a good method for comparing maximal responses in tissues from different patients could be established, and this would be very valuable. Dr. Skoogh: How do we know that not all airway smooth muscle tissue is hyperresponsive in vitro? Dr. Black: Wedo not know whether all tissue is hyperresponsive. There is some evidence that transplanted lungs are more responsive in vivo, which would indicate that a centrally emanating inhibitory pathway has been severed or lost.
Although, as stated above, changes in responsiveness should be consistent for all agonists, it is convenient to review available data in terms of the agonists studied.
Contraction Histamine The EC so values for tissue from asthmatic patients and from those with COPD are shown in figure 1 (3-15). It is apparent that there is good agreement between values obtained for asthmatic subjects in different studies (4-7,9-11, 13, 14). This is in spite of the fact that there are marked differences in patient characteristics. Those studied by Goldie and coworkers (5) died in status asthmaticus, whereas the asthma of the patients in the study by Whicker and colleagues (11) was sufficiently mild as to permit lung resection. Moreover, both these investigators noted that, compared with patients free of airway obstruction, histamine sensitivity in asthmatic subjects was decreased. de Jongste and coworkers (6) and Schellenberg and Foster (7) also observed no increase in sensitivity compared with that in normal subjects. In contrast to these four studies, there is one report in which an increase in sensitivity was found. Cerrina and coworkers (14) noted an increase in sensitivity to histamine but not to acetylcholine, prostaglandin E 2 (PGE 2 ) , or F 2U (PGF2U) ' Unlike tissue from asthmatic patients, there is a striking difference in the sensitivity of tissue from patients with COPD. The values obtained by Vincenc and coworkers (8) and in one of the studies of Armour and colleagues (3) are much lower (and therefore indicate greater histamine potency) than those from all other laboratories. Within individual studies, no significant differences exist between patients with COPD and normal subjects, and, overall, there is clearly no distinct separation between patients with COPD and those with asthma. Methacholine/Acetylcholine/Carbachol As for histamine, there is no clear separation of sensitivity to cholinergic stimulation (figure 2) between tissue from asthmatics or from subjects with COPD. Whicker and colleagues (11) and Goldie and coworkers (5) again reported that asthmatic tissue was less sensitive than normal tissue. It is again interesting to note the apparent increase in sensitivity of patients with COPD compared with other groups in the study by Armour and colleagues
(3). These patients were a carefully defined group, all with documented evidence of airway obstruction, half of whom had in vivo airway hyperresponsiveness when tested preoperatively, and half of whom were nonresponsive to inhaled methacholine. It is interesting to speculate as to whether these Australian patients with COPD are different in some way from those studied in other laboratories. Nevertheless, when all studies are considered, there are no differences in sensitivity in tissues from patients with COPD (3, 4, 12, 16-18) and asthmatics (4-7, 11).
Leukotrienes/Prostaglandins Dahlen and coworkers found that tissue from asthmatic subjects (10) was as sensitive to leukotrienes as that from nonasthmatics (19). Moreover, sensitivity to leukotrienes was not greater in those patients with COPD (12, 20) than those without (21). That differences do not exist between COPD (12) and asthma (6, 7, 10) with respect to leukotriene sensitivity is indicated in figure 3, and it is possible to infer from comparisons within individual studies that these patients in turn do not differ from those free of respiratory disease. Likewise, Cerrina and coworkers (14) could not differentiate between COPD and asthma when responses to PGE 2 and PGF2u were studied. Relaxation Isoproterenol and Other {3-Adrenoceptor Agonists The available information on sensitivity to isoproterenol in tissue from subjects with asthma (4-6,9,11) and those with COPD (4, 16, 22) is shown in figure 4. Although in individual studies there may be differences in l3-adrenoceptor function between the patient groups, when all studies are considered, differences between asthmatics, patients with COPD, and normal subjects are difficult to detect. Moreover, if sensitivity to other l3-adrenoceptor agonists such as salbutamol is compared in asthma (5) and COPD (17), the former patient group appears to be more 1 From the Department of Pharmacology, University of Sydney,New South Wales, Australia. 2 Supported by the National Health and Medical Research Council of Australia. 3 Correspondence and requests for reprints should be addressed to Judith L. Black, Department of Pharmacology, University of Sydney,New South Wales 2006, Australia.
1177
JUDITH L. BLACK
1178
•
Armour
..
•
.1 0
Cerrina Goldie
0 0 0
de Jongste
0
Dahlen
•
Whicker de Jongste
•
o•
Cerrlna
0
1 \,....
-8
Armour
••
Vincenc'••: -9
Fig. 1. ECso values in human isolated bronchus from patients with COPO (closed circles) or with asthma (open circles). Single symbols in an individual study indicate either that only one patient was studied or that only a mean value is available.
Roberts
o~~\ Schellenberg
-7
sensitive to this agonist. Thus, although there is in vitro evidence for a 13-adrenoceptor abnormality in asthma (5), it is difficult to understand why this would vary between 13-adrenoceptor agonists.
Bal
0
•
-4
-5
-6
Theophylline and Forskolin As can be seen in figure 5, little information is available on sensitivity to these agonists in asthma (6,9, 11)or in COPD (16, 17). What is striking is the lack of variability between patients with COPD in in vitro responsiveness to forskolin (16, 17).It has been suggested that the abnormality in COPD lies in the coupling mechanism distal to the 13-adrenoceptor (22). If this were so, it would be expected that agents that stimulate adenylate cyclasedirectly and bypass the 13-adrenoceptor would differentiate between patient groups. This appears not to be the case with forskolin. No studies have been performed on theophylline sensitivity in patients with COPD. Of the three in asthmatic tissue, two have reported no difference from normal subjects (5, 11), but Bai (9) did observe a decreased sensitivity in postmortem trachea.
log histamine (M)
•• • •••
•
•
de Joncste e
Armour Cerrina
...
.,.
•••• .....
Whicker
0
o
Goldie de Jongste
• o
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.. Schellenberg
Roberts
Radioligand Binding Studies Radioligand binding studies provide a direct assessment of receptor number and receptor affinity. The possibility exists that in asthma and/or COPD, the fundamental abnormality lies in an alteration of these parameters. Thus, a change in the characteristics of those receptors, stimulation of which results in alteration of airway smooth muscle tone, may produce airway hyperresponsiveness. Studies of changes in receptor characteristics are beset with multiple problems and this is especially relevant when human airway tissue is under investigation. First, those receptors of interest are likely to be those situated on airway smooth muscle. The percentage of muscle in airway preparations available for in vitro study, whether they are obtained from surgical resection or post-mortem, is very low (l-161t7o) (3~ 20). This means that in order to obtain sufficient material (as estimates of receptor numbers are usually expressed in terms of milligram of protein), tissues from several patients have to be pooled. Alternatively, in order to obtain sufficient material for study of individual patients, many investigators have used samples of peripheral lung only. These studies therefore provide us with
0
I
i
I
i
i
-9
-8
-7
-6
-5
I
-10
Fig. 2. ECso values for acetylcholine, methacholine, or carbachol in human isolated bronchus from patients with COPO (closed circles) or with asthma (open circles). Single symbols in an individual study indicate either that only one patient was studied or that only a mean value is available.
• Taylor
log cholinergic a'ionist (M)
t.. ...
Fig. 3. ECso values in human isolated bronchus for leukotrienes from patients with COPO (closed triangles) or with asthma (open triangles). Also shown are EC so values for prostaglandin F2a (PGF 2a) in patients with COPO (closed squares) or with asthma (open squares) and, correspondingly, for prostaglandin E2 (PGE~ (closed and open circles). Single symbols in an individual study indicate either that only one patient was studied or that only a mean value is available.
Roberts
.. deJongsfe
leukotrienes
A
deJongsfe
A A
Schellenberg
Dahlen
O. PGF2 a o PGE
•
2
. Carrino
i i i
-9
-8
-7
-6
-5
log agonist (M)
•
von Koppen.
• •• • de Jongste • •• • • • • • •• • • •• Whicker ••
. . .. , .
•
••• • •••
0
•••
o Goldie deJongste
· ! · io
0
Cerrino
:
.~
D
forskolin
-6
Taylor de Jongste
••• ••• ••••
.5
de Jongste Sai
log isoprenaline (M)
Fig. 4. ECso values in human isolated bronchus from patients with COPO (closed circles) or with asthma (open circles). Single symbols in an individual study indicate either that only one patient was studied or that only a mean value is available.
j
j
-8
-7
, -6
0
Whicker Goldie 0
0
theophylline
I
j
i
-5
-4
-3
log agonist (M)
Fig. 5. EC so values for forskolin in human isolated bronchus from patients with COPO (closed squares) or with asthma (open squares). Also shown are values for theophylline from asthmatic subjects (open circles). Single symbols in an individual study indicate either that only one patient was studied or that only a mean value is available.
1179
PHARMACOLOGY OF AIRWAY SMOOTH MUSCLE IN COPO AND ASTHMA
information about characteristics of receptors only on very small airways. In addition, those receptors situated on other structures such as mucous glands and blood vessels would be included. The physiologic relevance of these findings may therefore be questioned. Second, wide variabilities (> 1 log unit) in the sensitivity of some receptor populations (23) have been recorded within a particular patient group, and this means that comparisons between different patient groups such as asthmatic and COPD groups need to be performed on very large numbers of subjects. Third, the relevance and interpretation of information on receptor characteristics is questionable. Increasingly, it is apparent that there is little relationship between receptor number and pharmacologic response (24). From this it is possible to infer that not all receptors are functional. Some receptors may be "spare," i.e., not directly linked to a physiologic response and these could be "recruited" under a variety of experimental and pathologic conditions. Lastly, the influence of preoperative or antemortem conditions such as medication and tissue storage also may constitute confounding variables, as these may cause upregulation or downregulation of receptor populations. There are few reports of ligand binding studies in human airway tissue and even fewer in which a comparison can bedrawn between patients with CO PD and those with asthma. Those receptor populations that have received most attention are muscarinic and a- and ~-adrenoceptors.
TABLE 1 BINDING PARAMETERS OF ~-ADRENOCEPTOR LIGANDS IN HUMAN LUNG TISSUE*
Ligand
Patient Characteristics
n
Bmax (fmo/fmg protein)
Barnes (25)
[3H1DHA
COPD No obstructive lung disease
7 1
467 ± 76 309
van Koppen (22)
[1 25 11CYP
COPD No obstructive lung disease
8 (Bronchus) 9 (Bronchus)
0.372 ± 0.069 0.376 ± 0.064
Szentivanyi (27)
PH1DHA
Asthma COPD No obstructive lung disease
12 18 49
160 ± 9.4 194 ± 21.6 212 ± 20.2
Raaijmakers (26)
[3H1DHA
COPD No obstructive lung disease
12 24
511 ± 47 396 ± 33
Source
Definitionof abbreviations: DHA = dihydroalprenolol; CYP = cyahopindolol; Bmax dissociation constant. • Studies were performed on lung parenchymal tissue unless otherwise indicated.
0.82 ± 2 0.70 11.34 ± 0.05 11.22 ± 0.05
2.3 ± 0.5 1.7 + 0.2
= maximal number of binding
sites; Kd
=
TABLE 2 BINDING CHARACTERISTICS OF ll-ADRENOCEPTOR LIGANDS IN HUMAN LUNG TISSUE*
Ligand
Patient Characteristics
n
Bmax (fmo/fmg protein)
Kd (nmo/fL)
Barnes (25)
[3Hlprazosin
COPD No obstructive lung disease
7 1
600 62
1.75 1.1
Raaijmakers (26)
[3Hlprazosin
COPD No obstructive lung disease
12 24
2,202 ± 220 491 ± 52
10.7 ± 2.01 4.8 ± 1.8
Szentivanyi (27)
[3H1DHE
Asthma COPD No obstructive lung disease
12 18 49
Source
172 ± 8.8 62 ± 21.8 38 ± 11.8
= dihydroergotamine; Bmax = maximum number of binding sites; Kd • These studies were carried out on lung parenchymal tissue.
Definitionof abbreviations: DHE
{3-Adrenoceptors The proposal that a deficit in ~-adrenoceptor function may contribute to airway hyperresponsiveness has prompted several investigators to compare adrenoceptor characteristics in airway tissue from patients with and without respiratory disease (table 1). Barnes and coworkers (25) found that neither ~-adrenoceptor density nor affinity differed in patients with COPD from that in tissue from one subject free of respiratory disease. This was confirmed in larger numbers of subjects by Raaijmakers and coworkers (26) who, however, reported a decrease in the ratio of ~2/~1 receptors in patients with COPO. An extensive study combining both functional and binding techniques by van Koppen and colleagues (22) detected no difference in ~-receptor characteristics in either tracheal or bronchial smooth muscle from patients with COPD. The only study in which asthmatic tissue has been investigated is that of Szentivanyi and coworkers (27) who studied three groups of patients: those with (1) no respiratory disease, (2) nonreversible airway obstruction, and (3) asthma, and found that total ~-receptor density decreased progressively in these groups.
that ~-receptor populations are not altered by respiratory disease, three studies have reported concomitant changes in c-adrenoceptor number (table 2). In the same study in which Barnes and coworkers (25) reported no change in ~-adrenoceptor density, there was, in contrast, a to-fold increase in n-adrenoceptor density in tissue from patients with COPD. Both a decrease in affinity and a 5-fold increase in density were reported by Raaijmakers and coworkers (26), and these changes were reversed by the presence of disodium cromoglycate. In addition, Szentivanyi and coworkers (27) observed a progressive increase in binding to dihydroergotamine ([3H]DHE) across their patient groups (see above). These findings of increased o-adrenoceptor number need further verification since ligands such as [3H]DHE may not be specific for c-adrenoceptors. In addition, vascular tissue in the lung parenchyma used in these studies contains c-adrenoceptors. If COPD and asthma were associated with an increase in vascularity, then an increase in o-adrenoceptor number could be explained on this basis. The relevance of changes in a-adrenoceptor characteristics to the in vivo situation is not clear. Although asthmatic subjects exhibit bronchoconstriction to inhaled n-adrenoceptor agonists (28, 29), this is not true of patients with COPD (30).
a-Adrenoceptors Although the majority of studies has reported
Cholinergic Receptors The concept that a cholinergic receptor dys-
Adrenoceptors
Kd (nmo/fL)
= dissociation constant.
function may occur in COPD and/or asthma has been addressed by a number of groups. The issue has been complicated by recent evidence that there are subtypes of cholinergic receptors that are labeled by selective ligands in human airways such as pirenzepine (PZ) (31). The possibility exists that a decrease in the number of inhibitory muscarinic receptors or an increase in those that are excitatory could contribute to the abnormalities in these respiratory disorders (32). No radioligand binding studies have as yet been performed on tissue from asthmatic subjects. Raaijmakers and colleagues (33) reported a decrease in receptor density in peripheral lung tissue from patients with COPD - a surprising finding in view of the fact that these patients are more responsive than normal subjects to cholinergic agents. These findings were not confirmed, however, in a subsequent extensive study by load and Casale (23) who found that cholinergic receptor characteristics were not altered in patients with COPD. Thus, insufficient data exist at present to compare cholinergic receptor characteristics in asthma and COPD, although it seems likely (table 3) that the latter group do not differ from normal subjects. Summary Only a small number of studies investigating the in vitro pharmacologic properties of airway smooth muscle in asthma and wellcharacterized COPD have been performed.
1180
JUDITH L. BLACK
TABLE 3 BINDING PARAMETERS OF MUSCARINIC LIGANDS IN HUMAN LUNG TISSUE·
Source
Patient Characteristics
Ligand
n
Bmax (fmo/fmg protein)
Kd (nmo/fL)
Raaijmakers (33)
[3H)QNB
COPD No obstructive lung disease
4 8
25.6 ± 11.2 85 ± 14.5
0.12 ± 0.02 0.085 ± 0.009
Joad (23)
[3H)QNB
COPD No obstructive lung disease
19 6
29 ± 4 21 ± 5
0.07 ± 0.01 0.06 ± 0.01
van Koppen (24)
[3H)QNB
No respiratory disease (trachea)
14
123 ± 16
0.047
Bloom (31)
[3H)QNB
No respiratory disease
6
55.6 ± 12
0.0143
Definition of abbreviations: ONS
= (- )-quinuclidinylbenzilate;
Smax
= maximum number
of binding sites; Kd
= dissociation
constant. * Studies were performed on lung parenchymal tissue unless otherwise stated.
Further detailed studies on well-defined patient groups are required. The majority of available evidence would suggest that once airway smooth muscle is removed from its in vivo milieu, it loses the characteristics of hyperresponsiveness. This would explain why there are no clear differences in the pharmacologic responsiveness of tissue from patients with asthma or COPD and those with no obstructive disease. Future in vitro studies should be directed towards reproducing the in vivo environment. This would entail the establishment of a chronic inflammatory condition created by the continuous presence of neural and humoral factors.
Acknowledgment The writer thanks Dr. Carol Armour and Miss Susan Whicker for advice on the content of the manuscript and Miss Ann McGregor for its careful preparation.
References 1. Orie NGM, Sluiter HJ, de Vries K, Tammeling GJ, Witkop J. The host factor in bronchitis. In: Orie NGM, Sluiter HJ, eds. Bronchitis. Assen, The Netherlands: Royal Vangorcum, 1961; 43-59. 2. de Jongste JC, van Strik R, Bonta IL, Kerrebijn KF. Measurement of human small airway smooth muscle function in vitro with the bronchiolar strip preparation. J Pharmacol Methods 1985; 14:111-8. 3. Armour CL, Black JL, Berend N, Woolcock AJ: The relationship between bronchial hyperresponsivenessto methacholine and airway smooth muscle structure and reactivity. Respir Physiol1984; 58:223-33. 4. Cerrina J, Le RoyLadurie M, Labat C, Raffestin B, Bayol A, Brink C. Comparison of human bronchial muscle responsesto histamine in vivo with histamine and isoproterenol agonists in vitro. Am Rev Respir Dis 1986; 134:57-61. 5. Goldie RG, Spina D, Henry Pl, Lulich KM, Paterson JW. In vitro responsiveness of human asthmatic bronchus to carbachol, histamine, l3-adrenoceptor agonists and theophylline. Br J Clin Pharmacol 1986; 22:669-76. 6. de Jongste JC, Mons H, Bonta IL, Kerrebijn KF. Human asthmatic airway responses in vitro - a case report. Eur J Respir Dis 1987; 71:23-9. 7. Schellenberg RR, Foster A. In vitro responses of human asthmatic airway and pulmonary vascu1arsmooth muscle. Int Arch Allergy Appl Immunol 1984; 75:237-41. 8. Vincenc KS, Black JL, Yan K, Armour CL, Donnelly P, Woolcock AJ. Comparison of in vivo
and in vitro responses to histamine in human airways. Am Rev Respir Dis 1983; 128:875-9. 9. Bai TR. Abnormalities in airway smooth muscle in fatal asthma (abstract). Am Rev Respir Dis 1989; 139:A258. 10. Dahlen S-E, Hansson G, Hedqvist P, Bjorck T, Granstrom E, Dahlen B. Allergen challenge of lung tissue from asthmatics elicits bronchial contraction that correlates with the release of leukotrienes C 4 , D 4 , and E 4 • Proc Nat! Acad Sci USA 1983; 80:1712-6. 11. Whicker SD, Armour CL, Black JL. Responsiveness of bronchial smooth muscle from asthmatic patients to relaxant and contractile agonists. Pulmon Pharmacol 1988; 1:25-31. 12. de Jongste lC, Mons H, van Strik R, Bonta IL, Kerrebijn KF. Comparison of human bronchiolar smooth muscle responsiveness in vitro with histological signs of inflammation. Thorax 1987; 42:870-6. 13. Roberts JA, Rodger IW, Thomson NC. Airway responsiveness to histamine in man: effect of atropine on in vivo and in vitro comparison. Thorax 1985; 40:261-7. 14. Cerrina J, Labat C, Haye-Legrande I, Raffestin B, BenvenisteJ, Brink C. Human isolated bronchial muscle preparations from asthmatic patients: effects of indomethacin and contractile agonists. Prostaglandins 1989; 37:457-70. 15. Armour CL, Lazar NM, Schellenberg RR, et al. A comparison of in vivo and in vitro human airway reactivity to histamine. Am Rev Respir Dis 1984; 129:907-10. 16. de Jongste JC, Mons H, Bonta IL, Kerrebijn KF.Relaxation of human peripheral airwaysmooth muscle in vitro does not correlate with severity of chronic airflow limitation in vivo. Pulmon Pharmacol 1989; 2:75-9. 17. Taylor SM, Pare PD, Armour CL, Hogg JC, Schellenberg RR. Airway reactivity in chronic obstructive pulmonary disease. Am Rev Respir Dis 1985; 132:30-5. 18. Roberts JA, Raeburn D, Rodger IW, Thomson NC. Comparison of in vivo airway responsiveness and in vitro smooth muscle sensitivity to methacholine in man. Thorax 1984; 39:837-43. 19. Dahlen S-E, Hedqvist P, Hammarstrom S, Samuelsson B. Leukotrienes are potent constrictors of human bronchi. Nature 1980; 288:484-6. 20. Roberts JA, Rodger IW,Thomson NC. In vivo and in vitro human airway responsiveness to leukotriene D 4 in patients without asthma. J Allergy Clin Immunol 1987; 80:688-94. 21. Roberts JA, Giembycz MA, Raeburn D, Rodger IW, Thomson NC. In vitro and in vivo effect of verapamil on human airway responsiveness to leukotriene D4 • Thorax 1986; 41:12-6. 22. van Koppen CJ, Rodrigues de Miranda JF, Beld AJ, van Herwaarden CLA, Lammers J-WJ,
van Ginneken CAM. Beta adrenoceptor binding and induced relaxation in airway smooth muscle from patients with chronic airflow obstruction. Thorax 1989; 44:28-35. 23. Joad JP, Casale TB. [3H]quinuclidinyl benzilate binding to the human lung muscarinic receptor. Biochem Pharmacol 1988; 37:973-6. 24. van Koppen CJ, Rodrigues de Miranda JF, Beld AJ, Hermanussen MW, Lammers J-WJ, van Ginneken CAM. Characterization of the muscarinic receptorin human tracheal smooth muscle.NaunynSchmiedebergs Arch Pharmacol1985; 331:247-52. 25. Barnes PJ, Karliner JS, Dollery CT. Human lung adrenoceptors studied by radioligand binding. Clin Sci 1980; 58:457-61. 26. Raaijmakers JAM, Wassink GA, Kreukniet J, Terpstra GK. Adrenoceptors in lung tissue: characterization, modulation, and relations with pulmonary function. Eur J Respir Dis Suppl135 1984; 65:215-20. 27. Szentivanyi A, Heim 0, Schultze P. Changes in adrenoceptor densities in membranes of lung tissue and lymphocytes from patients with atopic disease. Ann N Y Acad Sci 1979; 332:295-8. 28. Black JL, Salome CM, YanK, Shaw J. Comparison between airways response to an n-adrenoceptor agonist and histamine in asthmatic and non-asthmatic subjects. Br J Clin Pharmacol1982; 14:464-6. 29. Black JL, Salome C, Yan K, Shaw J. The action of prazosin and propylene glycol on methoxamine-induced bronchoconstriction in asthmatic subjects. Br J Clin Pharmacol 1984; 18:349-53. 30. Du Toit 11, Woolcock AJ, Salome CM, Sundrum R, Black JL. Characteristics of bronchial hyperresponsiveness in smokers with chronic airflow limitation. Am Rev Respir Dis 1986; 134: 498-501. 31. Bloom JW, Halonen M, Yamamura HI. Characterization of muscarinic cholinergic receptor subtypes in human peripheral lung. J Pharmacol Exp Ther 1988; 244:625-32. 32. Barnes PJ, Minette P, Maclagen J. Muscarinic receptor subtypes in airways. Trends Pharmacol Sci 1988; 9:412-6. 33. Raaijmakers JAM, Terpstra GK, van Rozen AJ, Witter A, Kreukniet J. Muscarinic cholinergic receptors in peripheral lung tissue of normal subjects and of patients with chronic obstructive lung disease. Clin Sci 1983; 66:585-90. 34. Mitchell HW, Willet KE, Sparrow MI>. Perfused bronchial segment and bronchial strip: narrowing vs. isometric force by mediators. J Appl Physiol 1989: 66:2704-9.
Comments Dr. Holgate: Mitchell and Sparrow have developed an in vitro technique for investigating resistivechanges in airway function in perfused airways (34). They report that smaller degrees of smooth muscle contraction are required to produce large changes in resistance than are conventionally used in airway strips. Could it be that the in vitro preparations that are used to investigate hyperresponsiveness. are inappropriate? Dr. Black: This is certainly possible. These experiments are very interesting, and I think we will obtain most valuable information from these investigations. Perhaps in future the use of this type of preparation will allow
PHARMACOLOGY OF AIRWAY SMOOTH MUSCLE IN COPO AND ASTHMA
the demonstration of in vitro differences in pharmacologic responsiveness of muscle from different patients. Dr. O'Byrne: Did PGE 2 have any postsynaptic effects on human airway smooth muscle in vitro? Dr. Black: Yes, PGE 2 has a biphasic action on human airway smooth muscle, relaxation at low, contraction at high concentrations. Dr. Pare: (1) Why did you in your initial statements rule out the study of maximal tension generated by human smooth muscle preparations? (2) In vitro studies of asthmatic airway smooth muscle by de Jongste and Schellenberg showed an increased tension generation. Dr. Black: I did not rule out the importance of maximal tension. I said it was extremely difficult to normalize maximal tension values between different patient groups. It is a question of how you express the data. Several
groups have tried to normalize for wet weight, dry weight, mg of muscle or mg of protein, largely without success. There is little relationship between amount of smooth muscle and maximal responses. (2) These studies are on small numbers of subjects. de Jongste studied only one asthmatic subject and Schellenberg two. Their results may not necessarily be confirmed in larger numbers of patients. Dr. Corrigan: How far is the capacity of airway smooth muscle to cause bronchoconstriction affected by the elastic recoil pressure of the tissue in which the muscle is situated? Dr. Black: Certainly this is not affected to a great extent when we study airways in vitro. In vivo it is probably important. Dr. Bleecker: Most comparisons of in vivo hyperresponsiveness with in vitro smooth muscle function have been performed in specimens obtained from long-standing cigarette smokers who are having a thoracotomy for
1181
lung cancer. Are there still additional studies that should be performed or have you put this topic to rest? Specifically, should smooth muscle from normal lungs and from asthmatics be studied? Dr. Black: I believe we need more studies on well-characterized patient groups, especially asthmatics. Perhaps a good method for comparing maximal responses in tissues from different patients could be established, and this would be very valuable. Dr. Skoogh: How do we know that not all airway smooth muscle tissue is hyperresponsive in vitro? Dr. Black: Wedo not know whether all tissue is hyperresponsive. There is some evidence that transplanted lungs are more responsive in vivo, which would indicate that a centrally emanating inhibitory pathway has been severed or lost.
