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α-adrenoceptors in human peripheral lung
83
European Journal of Pharmacology, 72 (1981) 83--86 Elsevier/North-Holland Biomedical Press
Short communication a-ADRENOCEFrORS IN HUMAN PERIPHERAL LUNG JUDITH BLACK, ALISON TURNER and JOHN SHAW
Department of Pharmacology, University of Sydney, Sydney, N.S. IV. 2006, Australia Received 10 February 1981, accepted 6 April 1981
J. BLACK, A. T U R N E R and J. SHAW, Cz-Adrenoceptors in human peripheral lung, European J. Pharmacol. 72 (1981) 83--86. In this study, strips of human peripheral lung tissue were used to investigate the presence of a population of ~-adrenoceptors. Lung strips contracted in response to stimulation with noradrenaline, adrenaline, methoxamine and phenylephrine. In the presence o f propranolol, responses to noradrenaline were antagonized by phentolamine and a PA2 of 7.29 was obtained. It is concluded that human peripheral lung tissue contains a pharmacologically distinct population of ~-adrenergic receptors.
c~-Adrenoceptors
H u m a n peripherallung
Asthma
1. Introduction
2. Materials and methods
The presence of a-adrenoceptors in human airways is disputed, as is their role in respiratory disease. Math~ et al. (1971) reported a sparse population of a-adrenoceptors in human bronchus in vitro, whilst in the in vivo study of Stone et al. (1973) there was evidence for their presence. There is evidence from receptor binding studies in guinea-pigs that a-adrenoceptor numbers may increase and ~-adrenoceptors decrease in an animal model of chronic asthma (Barnes et al., 1980). Henderson et al. (1979) suggested that patients with asthma exhibit enhanced a-adrenergic responses compared to normal subjects. Previous in vitro studies of human airways have concentrated on large, central airways. The major site of action of various agonists, however, lies in the peripheral airways (Colebatch et al., 1966). The aim of this study was to determine by specific functional quantitative pharmacological techniques whether there exists a population of a-adrenoceptors in human peripheral lung.
Specimens of human lung resected at thoracotomy, were cut into 4 strips (30 × 4 × 4 mm) and suspended under 1 g tension in a double-jacketed 20 ml organ bath containing Krebs-Henseleit solution maintained at 37°C and bubbled with carbogen (95% 02, 5% CO2). Changes in tension resulting from the addition of agonists to the bath fluid were measured isometrically using Grass FT03 transducers, and recorded on a Grass polygraph. After a period of equilibration (1-4 h) during which baseline tension stabilized, bolus doses of agonists were tested or concentration-response curves were obtained by addition of agonists in a cumulative fashion. In experiments in which blockade of responses to noradrenaline (NA) with the a-adrenoceptor antagonist phentolamine was assessed, all concentration-response curves to NA were obtained in the presence of propranolol (2.5 × 10 -6 tool. 1-1) to block agonist activity at ~-adrenoceptors. One or two strips in each experiment were exposed to NA alone,
0 014-2999/81/0000--0000/$02.50 © Elsevier/North-Holland Biomedical Press
84
J. BLACK ET AL.
and the remaining strips to different concentrations of phentolamine which was added 30 min prior to the c o m m e n c e m e n t of the NA curve. Responses to NA were expressed as a percentage of maximal responses and plotted against the log of its molar concentration. Dose ratios were determined at 50% of the maximal response (EDs0). All the dose ratios from all experiments were examined according to the method of Arunlakshana and Schild (1959). Least squares regression analysis was used to determine the relationship between log (dose ratio --1) and log of the antagonist concentration. The drugs used were: (--)-noradrenaline hydrogen tartrate, mol. wt. 319.3 (Sigma); histamine dihydrogen phosphate, mol. wt. 317.8 (Ciba); (--)phenylephrine hydrochloride, tool. wt. 203.67 (Sigma); (--)-adrenaline hydrogen tartrate, mol. wt. 333.3 (Sigma); methoxamine hydrochloride, mol. wt. 247.7 (Burroughs Wellcome); carbachol chloride, mol. wt. 182.6 (Sigma); propranolol hydrochloride, mol. wt. 295.8 (Sigma). Stock solutions were diluted with Krebs-Henseleit solution. Ascorbic acid 0 . 0 2 m g . m 1 - 1 was included in the Krebs-Henseleit solution whenever NA was used, and NA solutions were stored in ice throughout the experiments.
3. Results
A total of 200 strips from 40 patients (33 of w h o m had pulmonary carcinoma) were studied. More than 90% of the strips reacted with contraction to an a-adrenergic agonist (phenylephrine, methoxamine, adrenaline or noradrenaline) in the absence or presence of propranolol (2.5 X 10 -6 mol • 1-1). After initial testing with bolus doses of all agonists, NA was found to be the most potent and was used as the agonist in all subsequent experiments. Responses to NA were highly reproducible (mean EDs0, 3.5 × 10 -6 mol • 1-1; 95% confidence limits 2.8 X 10 -6 to 4.5 × 10-6; n = 92). What little variability there was apparent, existed between rather than within experiments, as the EDs0s of each of the 4 strips taken from the one patient were almost identical. However, when a second curve to NA was obtained, there was a loss of sensitivity which varied greatly between strips b o t h between and within experiments. Thus only single concentration-response curves in the presence or absence of antagonist were obtained in each lung strip. Maximum tension generated b y NA (145 ± 15 mg; mean ± S.E.M.) was found to be of the same order of magnitude as that obtained
96 E msx
phentolamine
0
10" 6
5 x 10-6
tool / I
100
50
0 k
10
I
-7
10
|
-6
10
-5
10
-4
Fig. 1. Graphs of concentration-response relationships for noradrenaline in human lung strips from a typical experiment using phentolamine at the indicated concentrations.
a-ADRENOCEPTORS
IN H U M A N
PERIPHERAL
LUNG
from supramaximal doses (1 X 10 -4 mol • 1-1) of histamine or carbachol. These were administered at the end of the NA concentration response curve when after repeated washing of the tissue, tension returned to baseline levels. Phentolamine, 5 × 10-7-5 × 10 -6 mol • 1-1 shifted concentration-response curves to NA to the right in a parallel fashion (fig. 1). The correlation coefficient of the regression line obtained from all the log (dose r a t i o - - 1 ) values plotted against log phentolamine concentration was 0.65 and was significant (n = 13, P < 0.05). The slope of the regression line was not significantly different from 1 (P < 0.05, Student's t-test for correlated data) indicating competitive antagonism. A pA2 of 7.29 -+ 0.53 (S.E.) was obtained.
4. Discussion
It is now recognized that the pharmacolo~cal responses of central large airways differ from those of the distal airways (Drazen and Schneider, 1978). The present study represents the first attempt to carry out a quantitative investigation of receptor populations in h u m a n peripheral lung. The fact that sever~ a-adrenergic agonists contracted the parenchymal strips, and that responses to NA were antagonized in a competitive manner by phentolamine with a pA2 similar to that found a t sites recognised as a-receptors, gives reasonable grounds for assumption that there is a population of a-adrenoceptors in this tissue. Moreover, stimulation of these receptors results in a substantial contraction of the lung strips, comparable to that produced by other agonists such as histamine and carbachol. The exact location of the a-receptors is not known. Drazen and Schneider (1978) demonstrated in guinea-pig lung strips that it is unlikely that cond_ucting airways and blood vessels are responsible for the contractions, and Colebatch et al. (1966) has observed that the time-course of action of
85
inhaled agonists is such as to suggest a direct action on respiratory rather than vascular smooth muscle. It is possible that alveolar d u c t smooth muscle or interstitial elements (Kapanci et al., 1974; Adler et al., 1980) are responsible for the c o n t r a c t i o n of the lung strips. Stone et al. (1973) failed to demonstrate any effect on airway conductance in nineteen subjects (15 healthy, 4 with chronic bronchitis) following parenteral administration of either noradrenaline or phentolamine. These authors considered that their negative results m a y have been a consequence of variation in drug effects due to route of administration, or to preferential uptake of adrenergic drug by cardiovascular receptor sites. Math~ et al. (1971) found only a sparse population of a-adrenoceptors and stimulation of these resulted in contraction only in the presence of propranolol. In the present study however, contraction was elicitedby several a-adrenergic agonists, whether or not propranolol was present. This m a y highlight the difference in receptor populations in different parts of the respiratory tree, i.e. peripheral as opposed to central. In a preliminary trialof the a-adrenoceptor antagonist indoramin in asthmatic patients (Black et al., 1978) beneficial therapeutic effects were obtained in some patients. The results of the present study raise the possibility that the therapeutic use of a-adrenoceptor antagonists in asthma and other pulmonary disease states m a y warrant further investigation.
Acknowledgement Judith Black is supported by the National Health and Medical Research Council of Australia.
References Adler, K.B., J. Kelley and J.N. Evans, 1980, Morphological and pharmac0mechanical evidence of increased contractility in human and rat fibrotic lungs, Am. Resp. Dis. 121,309.
86
Arunlakshana, O. and H.O. Schild, 1959, Some quantitative uses of drug antagonists, Br. J. Pharmacol. 14, 18. Barnes, P.J., C.T. Dollery and J. MacDermot, 1980, Increased pulmonary a-adrenergic and reduced ~-adrenergic receptors in experimental asthma, Nature 285, 569. Black, J.L., D.M. Temple and S.D. Anderson, 1978, Long term trial of an alpha adrenoceptor blocking drug (indoramin) in asthma, A preliminary report, Scand. J. Resp. Dis. 59, 307. Colebatch, H.J.H., C.R. Olsen and J.A. Nadel, 1966, Effect of histamine serotonin and acetylcholine on the peripheral airways, J. Appl. Physiol. 21, 217. Drazen, J.M. and M.W. Schneider, 1978, Comparative responses of tracheal spirals and parenchymal strips to histamine and carbachol in vitro, J. Clin. Invest. 61, 1441.
J. BLACK ET AL.
Henderson, W.R., J.H. Shelhamer, D.B. Reingold, L.J. Smith, R. Evans and M. Kaliner, 1979, Alpha adrenergic hyper-responsiveness in asthma, N e w Eng. J. Med. 300,642. Kapanci, Y., A. Assimacopoulos, C. Irle, A. Zwahlen and C. Gabbiani, 1974, 'Contractile interstitial cells'in pulmonary alveolar septa: a possible regulation of ventilation/perfusion ratios?, J. Cell Biol. 60,375. Math~, A.A., A. i~str~im and N.-A. Persson, 1971, Some bronchoconstricting and bronchodilating responses of human isolated bronchi: evidence for the existence of 0~-adrenoceptors, J. Pharm. Pharmacol. 23,905. Stone, D.J., T.K. Sarkar and H. Keltz, 1973, Effect of adrenergic stimulation and inhibition on human airways, J. Appl. Physiol. 34, 624.
European Journal of Pharmacology, 72 (1981) 83--86 Elsevier/North-Holland Biomedical Press
Short communication a-ADRENOCEFrORS IN HUMAN PERIPHERAL LUNG JUDITH BLACK, ALISON TURNER and JOHN SHAW
Department of Pharmacology, University of Sydney, Sydney, N.S. IV. 2006, Australia Received 10 February 1981, accepted 6 April 1981
J. BLACK, A. T U R N E R and J. SHAW, Cz-Adrenoceptors in human peripheral lung, European J. Pharmacol. 72 (1981) 83--86. In this study, strips of human peripheral lung tissue were used to investigate the presence of a population of ~-adrenoceptors. Lung strips contracted in response to stimulation with noradrenaline, adrenaline, methoxamine and phenylephrine. In the presence o f propranolol, responses to noradrenaline were antagonized by phentolamine and a PA2 of 7.29 was obtained. It is concluded that human peripheral lung tissue contains a pharmacologically distinct population of ~-adrenergic receptors.
c~-Adrenoceptors
H u m a n peripherallung
Asthma
1. Introduction
2. Materials and methods
The presence of a-adrenoceptors in human airways is disputed, as is their role in respiratory disease. Math~ et al. (1971) reported a sparse population of a-adrenoceptors in human bronchus in vitro, whilst in the in vivo study of Stone et al. (1973) there was evidence for their presence. There is evidence from receptor binding studies in guinea-pigs that a-adrenoceptor numbers may increase and ~-adrenoceptors decrease in an animal model of chronic asthma (Barnes et al., 1980). Henderson et al. (1979) suggested that patients with asthma exhibit enhanced a-adrenergic responses compared to normal subjects. Previous in vitro studies of human airways have concentrated on large, central airways. The major site of action of various agonists, however, lies in the peripheral airways (Colebatch et al., 1966). The aim of this study was to determine by specific functional quantitative pharmacological techniques whether there exists a population of a-adrenoceptors in human peripheral lung.
Specimens of human lung resected at thoracotomy, were cut into 4 strips (30 × 4 × 4 mm) and suspended under 1 g tension in a double-jacketed 20 ml organ bath containing Krebs-Henseleit solution maintained at 37°C and bubbled with carbogen (95% 02, 5% CO2). Changes in tension resulting from the addition of agonists to the bath fluid were measured isometrically using Grass FT03 transducers, and recorded on a Grass polygraph. After a period of equilibration (1-4 h) during which baseline tension stabilized, bolus doses of agonists were tested or concentration-response curves were obtained by addition of agonists in a cumulative fashion. In experiments in which blockade of responses to noradrenaline (NA) with the a-adrenoceptor antagonist phentolamine was assessed, all concentration-response curves to NA were obtained in the presence of propranolol (2.5 × 10 -6 tool. 1-1) to block agonist activity at ~-adrenoceptors. One or two strips in each experiment were exposed to NA alone,
0 014-2999/81/0000--0000/$02.50 © Elsevier/North-Holland Biomedical Press
84
J. BLACK ET AL.
and the remaining strips to different concentrations of phentolamine which was added 30 min prior to the c o m m e n c e m e n t of the NA curve. Responses to NA were expressed as a percentage of maximal responses and plotted against the log of its molar concentration. Dose ratios were determined at 50% of the maximal response (EDs0). All the dose ratios from all experiments were examined according to the method of Arunlakshana and Schild (1959). Least squares regression analysis was used to determine the relationship between log (dose ratio --1) and log of the antagonist concentration. The drugs used were: (--)-noradrenaline hydrogen tartrate, mol. wt. 319.3 (Sigma); histamine dihydrogen phosphate, mol. wt. 317.8 (Ciba); (--)phenylephrine hydrochloride, tool. wt. 203.67 (Sigma); (--)-adrenaline hydrogen tartrate, mol. wt. 333.3 (Sigma); methoxamine hydrochloride, mol. wt. 247.7 (Burroughs Wellcome); carbachol chloride, mol. wt. 182.6 (Sigma); propranolol hydrochloride, mol. wt. 295.8 (Sigma). Stock solutions were diluted with Krebs-Henseleit solution. Ascorbic acid 0 . 0 2 m g . m 1 - 1 was included in the Krebs-Henseleit solution whenever NA was used, and NA solutions were stored in ice throughout the experiments.
3. Results
A total of 200 strips from 40 patients (33 of w h o m had pulmonary carcinoma) were studied. More than 90% of the strips reacted with contraction to an a-adrenergic agonist (phenylephrine, methoxamine, adrenaline or noradrenaline) in the absence or presence of propranolol (2.5 X 10 -6 mol • 1-1). After initial testing with bolus doses of all agonists, NA was found to be the most potent and was used as the agonist in all subsequent experiments. Responses to NA were highly reproducible (mean EDs0, 3.5 × 10 -6 mol • 1-1; 95% confidence limits 2.8 X 10 -6 to 4.5 × 10-6; n = 92). What little variability there was apparent, existed between rather than within experiments, as the EDs0s of each of the 4 strips taken from the one patient were almost identical. However, when a second curve to NA was obtained, there was a loss of sensitivity which varied greatly between strips b o t h between and within experiments. Thus only single concentration-response curves in the presence or absence of antagonist were obtained in each lung strip. Maximum tension generated b y NA (145 ± 15 mg; mean ± S.E.M.) was found to be of the same order of magnitude as that obtained
96 E msx
phentolamine
0
10" 6
5 x 10-6
tool / I
100
50
0 k
10
I
-7
10
|
-6
10
-5
10
-4
Fig. 1. Graphs of concentration-response relationships for noradrenaline in human lung strips from a typical experiment using phentolamine at the indicated concentrations.
a-ADRENOCEPTORS
IN H U M A N
PERIPHERAL
LUNG
from supramaximal doses (1 X 10 -4 mol • 1-1) of histamine or carbachol. These were administered at the end of the NA concentration response curve when after repeated washing of the tissue, tension returned to baseline levels. Phentolamine, 5 × 10-7-5 × 10 -6 mol • 1-1 shifted concentration-response curves to NA to the right in a parallel fashion (fig. 1). The correlation coefficient of the regression line obtained from all the log (dose r a t i o - - 1 ) values plotted against log phentolamine concentration was 0.65 and was significant (n = 13, P < 0.05). The slope of the regression line was not significantly different from 1 (P < 0.05, Student's t-test for correlated data) indicating competitive antagonism. A pA2 of 7.29 -+ 0.53 (S.E.) was obtained.
4. Discussion
It is now recognized that the pharmacolo~cal responses of central large airways differ from those of the distal airways (Drazen and Schneider, 1978). The present study represents the first attempt to carry out a quantitative investigation of receptor populations in h u m a n peripheral lung. The fact that sever~ a-adrenergic agonists contracted the parenchymal strips, and that responses to NA were antagonized in a competitive manner by phentolamine with a pA2 similar to that found a t sites recognised as a-receptors, gives reasonable grounds for assumption that there is a population of a-adrenoceptors in this tissue. Moreover, stimulation of these receptors results in a substantial contraction of the lung strips, comparable to that produced by other agonists such as histamine and carbachol. The exact location of the a-receptors is not known. Drazen and Schneider (1978) demonstrated in guinea-pig lung strips that it is unlikely that cond_ucting airways and blood vessels are responsible for the contractions, and Colebatch et al. (1966) has observed that the time-course of action of
85
inhaled agonists is such as to suggest a direct action on respiratory rather than vascular smooth muscle. It is possible that alveolar d u c t smooth muscle or interstitial elements (Kapanci et al., 1974; Adler et al., 1980) are responsible for the c o n t r a c t i o n of the lung strips. Stone et al. (1973) failed to demonstrate any effect on airway conductance in nineteen subjects (15 healthy, 4 with chronic bronchitis) following parenteral administration of either noradrenaline or phentolamine. These authors considered that their negative results m a y have been a consequence of variation in drug effects due to route of administration, or to preferential uptake of adrenergic drug by cardiovascular receptor sites. Math~ et al. (1971) found only a sparse population of a-adrenoceptors and stimulation of these resulted in contraction only in the presence of propranolol. In the present study however, contraction was elicitedby several a-adrenergic agonists, whether or not propranolol was present. This m a y highlight the difference in receptor populations in different parts of the respiratory tree, i.e. peripheral as opposed to central. In a preliminary trialof the a-adrenoceptor antagonist indoramin in asthmatic patients (Black et al., 1978) beneficial therapeutic effects were obtained in some patients. The results of the present study raise the possibility that the therapeutic use of a-adrenoceptor antagonists in asthma and other pulmonary disease states m a y warrant further investigation.
Acknowledgement Judith Black is supported by the National Health and Medical Research Council of Australia.
References Adler, K.B., J. Kelley and J.N. Evans, 1980, Morphological and pharmac0mechanical evidence of increased contractility in human and rat fibrotic lungs, Am. Resp. Dis. 121,309.
86
Arunlakshana, O. and H.O. Schild, 1959, Some quantitative uses of drug antagonists, Br. J. Pharmacol. 14, 18. Barnes, P.J., C.T. Dollery and J. MacDermot, 1980, Increased pulmonary a-adrenergic and reduced ~-adrenergic receptors in experimental asthma, Nature 285, 569. Black, J.L., D.M. Temple and S.D. Anderson, 1978, Long term trial of an alpha adrenoceptor blocking drug (indoramin) in asthma, A preliminary report, Scand. J. Resp. Dis. 59, 307. Colebatch, H.J.H., C.R. Olsen and J.A. Nadel, 1966, Effect of histamine serotonin and acetylcholine on the peripheral airways, J. Appl. Physiol. 21, 217. Drazen, J.M. and M.W. Schneider, 1978, Comparative responses of tracheal spirals and parenchymal strips to histamine and carbachol in vitro, J. Clin. Invest. 61, 1441.
J. BLACK ET AL.
Henderson, W.R., J.H. Shelhamer, D.B. Reingold, L.J. Smith, R. Evans and M. Kaliner, 1979, Alpha adrenergic hyper-responsiveness in asthma, N e w Eng. J. Med. 300,642. Kapanci, Y., A. Assimacopoulos, C. Irle, A. Zwahlen and C. Gabbiani, 1974, 'Contractile interstitial cells'in pulmonary alveolar septa: a possible regulation of ventilation/perfusion ratios?, J. Cell Biol. 60,375. Math~, A.A., A. i~str~im and N.-A. Persson, 1971, Some bronchoconstricting and bronchodilating responses of human isolated bronchi: evidence for the existence of 0~-adrenoceptors, J. Pharm. Pharmacol. 23,905. Stone, D.J., T.K. Sarkar and H. Keltz, 1973, Effect of adrenergic stimulation and inhibition on human airways, J. Appl. Physiol. 34, 624.