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The effect of isoprenaline on the ciliary activity of an in vitro preparation of rat trachea
European Journal of Pharmacology, 112 (1985) 429-432
429
Elsevier
Short communication T H E E F F E C T OF I S O P R E N A L I N E O N T H E CILIARY ACTIVITY O F AN IN V I T R O P R E P A R A T I O N O F RAT T R A C H E A M.T. LOPEZ-VIDRIERO 1, M. JACOBS 2,. and S.W. CLARKE 1
i Department of Thoracic Medicine, and 2 Academic Department of Pharmacology, Royal Free Hospital School of Medicine, London, NW3 2PF, England Received 15 April 1985, accepted 1 May 1985
M.T. LOPEZ-VIDRIERO, M. JACOBS and S.W. CLARKE, The effect of isoprenafine on the ciliary activity of an in vitro preparation of rat trachea, European J. Pharmacol. 112 (1985) 429-432. A dose dependent increase in the ciliary beat frequency of rat tracheal explants was found over a 10-7-10 -3 M concentration range of (+)-isoprenaline and was abolished by 10 -7 M propranolol. Furthermore (+)-isoprenaline, a drug which is much less potent in its action on fl-adrenoceptors than its ( - )-isomer, failed to cause a stimulation. We conclude that the acceleration in the intrinsic ciliary beat frequency by (-)-isoprenaline, although occurring at a relatively high concentration compared with the action of the drug on other tissues, is mediated by fl-adrenoceptors. Rat trachea
Isoprenaline
Ciliary activity
1. Introduction fl-Adrenoceptor agonists are known to increase mucociliary wave frequency both in vivo and in vitro in a variety of animal species (Melville et al., 1976; Van As and Parzyck, 1981; Hybbinette and Mercke, 1982). Mucociliary wave frequency, is, however, dependent on the mucus and ciliary components and their interactions. It is, therefore, not possible to distinguish the relative contributions of the two components in in vivo or in vitro experiments where mucus is present. The direct effect of drugs on the intrinsic ciliary activity can only be evaluated in in vitro tissue preparations where mucus is absent or very dilute. fl-Adrenoceptor agonist stimulation of ciliary beat frequency (CBF) was observed in such preparations of bronchial (Clarke and Lopez-Vidriero, 1983) and nasal mucosa (Konietzko and Kasparek, 1983). Although two agonists, terbutaline and isoprenaline, were used in these experiments, agonist * To whom all correspondence should be addressed: Academic Department of Pharmacology, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, U.K. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
activity alone is not sufficient evidence to prove an action at a specific receptor. In the only study so far done on animal cells using cultured rabbit ciliated epithelium (Verdugo et al., 1980), isoprenaline was found to mediate an increase in CBF which was antagonised by propranolol. This latter observation therefore confirms that isoprenaline was acting on/3-adrenoceptors. Unfortunately, these cultured cells are not well characterised and may not be at the same stage of development or have the same receptors as ciliated cells in intact rabbit trachea. The purpose of this current study was, therefore, to investigate the stimulation of CBF by isoprenaline in an in vitro preparation of trachea from a different species. Furthermore, previous studies have found a stimulation of CBF at relatively high isoprenaline concentrations (10-7-10 -4 M) compared with those required to elicit a response in the heart and tracheal smooth muscle (Buckner and Patil, 1971). We have therefore used additional pharmacological criteria to establish unequivocally that the stimulation of ciliary activity by isoprenaline is mediated by the activation of /3-adrenoceptors.
430
2. Materials and methods
2.1. General ( + )- and ( + )-isoprenaline and Hank's medium were purchased from Sigma (UK) and modified medium 199 with Earle's salts from Flow Laboratories (UK). (+)-Propranolol was obtained from Imperial Chemical Industries (UK). All drugs were dissolved in culture medium and the pH adjusted to 7.0; fresh solutions were prepared for each experiment.
2.2, Preparation of tissue Sprague-Dawley rats weighing 250 g were killed by a flow to the head. The trachea was immediately removed and washed several times in Hank's medium. The laryngeal segment, which contains almost all the submucosal glands and the upper third of the trachea, was discarded. The rest of the trachea was divided into small explants (5 mm × 5 mm) and washed several times with culture medium 199 at room temperature. Each explant was then placed in a Prior chamber and equilibrated in culture medium 199 at 20°C for 30 min.
2.3. Measurement of ciliary beat frequency Ciliary beat frequency (CBF) was measured by the photometric technique (Dalham and Rylander, 1962; Lopez-Vidriero and Clarke, 1982) at 20°C. Baseline CBF measurements were taken from 4-6 different sites on each explant and the same sites were measured during drug treatment. Experiments to determine dose response curves for (_+)isoprenaline or (+)-isoprenaline were performed by perfusing the chamber with increasing concentrations of drug. The explant was incubated for 15 min at each concentration before CBF measurements were taken. In between concentrations, the chamber was perfused for 30 rain with fresh culture medium, at a flow rate of 0.15 ml/min. In experiments with (+)-propranol, fresh explants were pre-incubated with drug for 30 min and then perfused with the medium containing the initial concentrations of antagonist and the appropriate
concentrations of isoprenaline. Propranolol was also present in the washing solution. In all experiments, untreated control explants showed no change in basal CBF or sensitivity to (+)-isoprenaline.
2.4. Viscosity measurements A separate experiment was performed to estimate the apparent viscosity changes expected during the treatment of the tracheal explants with isoprenaline. Four explants were used to maximise the viscosity changes. These explants were suspended in culture medium and then challenged with 10 - 4 M isoprenaline, the highest concentration of the drug used for the dose response curve. Measurements were performed on ten aliquots of culture medium before and after treatment. The apparent viscosities were 0.872 + 0.005 mPas. s -1 and 0.877 + 0.015 mPas. s- 1 respectively.
2.5. Statistics The results were expressed as mean + S.E. All straight lines were drawn by linear regression using the Minitab statistical programme and tested for deviation from linearity by analysis of variance by regression. Statistical difference between two means were determined by Student's t-test by testing for overlap of 95% confidence limits.
3. Results
3.1. Effect of isomers of isoprenaline on CBF The effect of (+)-isoprenaline was studied in 5 tracheal explants and that of ( + )-isoprenaline in 4 explants, all from different animals. A dose dependent increase in CBF compared with baseline values (7.4+ 0.3 s -1) was observed o v e r 1 0 - 7 - 1 0 - 3 M range of (+)-isoprenaline concentrations (fig. 1). The increase in CBF was constant over 45 min, suggesting that tachyphylaxis was not a problem. A maximal stimulation of 50% over the control was found at 10 - 4 M , this CBF corresponds to 10.8 + 0.8 s -1. A half maximal stimulation was achieved at 7.46 x 10 -7 M concentration. In con-
431 CBF %CONTROL 160.
3.2. The effect of ( +- )-propranolol on ( +_ )isoprenaline stimulation of CBF
[/
140
I/
120
]b) 100
~Z......._ ~ CONTROL
CBF %CONTROL
"~
-LOG ISOPRENALINE
4. Discussion (c)
11° t,00~--';--;~;~-L-.._..~ CONTROL
The effect of ( + ) - p r o p r a n o l o l at 10 7 M concentration was investigated in 4 tracheal explants from different tracheas. The effect of 10 - 9 M concentrations was studied only in 2 explants and one C B F measurement was taken during baseline and isoprenaline stimulation. ( + ) - P r o p r a n o l o l at 10 -7 M concentration had no effect on the baseline CBE but completely inhibited the stimulation observed with (+)-isoprenaline (fig. 1). In contrast, 10 - 9 M concentration did not block the stimulatory effect of ( _+)-isoprenaline (not shown).
-LOG ISOPRENALINE
Fig. 1. (a) The effect of (+)-isoprenaline on ciliary beat frequency. Rat tracheal explant was perfused with increasing concentrations of (+)-isoprenaline as shown. Plot is the mean + S.E. of 5 explants from different animals measured at 5 sites per explant. (+)-Isoprenaline • i. (b) The explant was preincubated with 10 -7 M propranolol for 30 min and then perfused with increasing concentrations of (_+)-isoprenaline plus 10 -v M propranolol. Plot is the mean + S.E. of 4 explants from different animals, with measurements taken at 6 sites per explant. (+)-lsoprenaline + 10-7 M propranolol • •. (c) The effect of (+)-isoprenaline on ciliary beat frequency. Experiment performed as in (a). Plot is the mean_+S.E. of 4 explants from different animals, with measurements from 5 sites per explant. • •. All measurements were performed at 20° and commenced 15 •in after perfusion with drugs.
trast, (+)-isoprenaline caused no increase in C B F (fig. 1), in fact a small inhibition was observed at high concentrations. When the presence of the ( + ) - i s o m e r is taken into account, the calculated value of the - l o g EDs0 for the stimulation of the C B F by ( - ) - i s o p r e n a l i n e is 6.43 + 1.1 c o m p a r e d with 8.50 _+ 0.06 and 8.65 -t- 0.05 found for guinea pig tracheal smooth muscle and atria respectively (Buckner and Patti, 1971).
A dose dependent increase in ciliary activity was found when the rat trachea was treated with ( ___)-isoprenaline. It was unlikely that this increased response was due to mucus, since the upper one third of the trachea and the submucosal gland, which is the main source of mucus in the rat, was excluded from the preparation. Furthermore, a change of approximately 5 m P a s . s ] is required to produce a ten per cent reduction in ciliary activity (Luk and Dulfano, 1982). A change of less than 0.01 m P a s . s-1 was observed in the apparent viscosity of the culture medium when a large tracheal explant was challenged with - - 1 0 - 4 M isoprenaline. Although the release of mucus cannot be absolutely excluded, this insignificant change in viscosity indicates that mucus was not a factor during our experiments. EDs0 obtained for the stimulation of ciliary beat frequency by ( - ) - i s o p r e n a l i n e in this study, is comparable to the value estimated from the data of Verdugo et al. (1980) using cultured rabbit epithelium. This value is at least an order of magnitude higher than typical EDs0 values found for isoprenaline in tracheal smooth muscle and the heart (Buckner and Patil, 1971). In spite of this high value, our data proves conclusively that ( - ) isoprenaline was acting specifically on /~-adrenoceptors. Firstly, the increase in ciliary beat frequency was completely inhibited by propranolol, at a 10-7 M concentration which is consistent with the pA2
432
value of the antagonist and is in agreement with the findings of Verdugo et al. (1980). Secondly, the ( + )-isomer of isoprenaline which is known to have a very low potency at fl-adrenoceptors on the heart and trachea (Buckner and Patil, 1971), also failed to stimulate CBF. The reason for the high EDs0 value found for the stimulation of CBF is not known but may be due to the involvement of a fl-adrenoceptor subtype or less efficient coupling of the receptor to pathways within the ciliated cell.
References Buckner, C.K. and P.N. Patil, 1971, Steric aspects of adrenergic drugs, XVI, Beta adrenergic receptors of guinea pig atria and trachea, J. Pharmacol. Exp. Ther. 176, 634. Clarke, S.W. and M.T. Lopez-Vidriero, 1983, The effect of beta 2 agonists on the activity of human bronchial cilia in vitro, J. Physiol. 336, 40P. Dalham, T. and R. Rylander, 1962, Frequency of ciliary beat measured with a photo-sensitive cell, Nature 196, 592.
Hybbinette, J.C. and U. Mercke, 1982, Effects of sympathomimetic agonists and antagonists on mucociliary activity, Acta Otolaryngol. 94, 121. Konietzko, N. and R. Kasparek, 1983, Effect of adrenergic, cholinergic and histaminergic stimulation on ciliary beat frequency of human nasal mucosa in vitro, Am. Rev. Respir. Dis. 127, 294. Lopez-Vidriero, M.T. and S.W. Clarke, 1982, Objective criteria for measuring ciliary beat frequency in vitro, Am. Rev. Respir. Dis. 125, 244. Luk, C.K.A. and M.J. Dulfano, 1983, Effect of pH, viscosity and ionic-strength changes in ciliary beat frequency of human bronchial explants, Clin. Sci. 64, 449. Melville, G.N., G. Horstmann and J. Irvani, 1976, Adrenergic compounds and the respiratory tract, Respiration 33, 261. Van As, A. and T. Parzyck, 1981, The role of beta receptor function in the control of ciliary frequency, Am. Rev. Respir, Dis. 12, 215. Verdugo, P., N.T. Johnson and P.Y. Tam, 1980, Adrenergic stimulation of respiratory ciliary activity, J. Appl. Physiol.: Respirat. Environ. Exerc. Physiol. 48, 868.
429
Elsevier
Short communication T H E E F F E C T OF I S O P R E N A L I N E O N T H E CILIARY ACTIVITY O F AN IN V I T R O P R E P A R A T I O N O F RAT T R A C H E A M.T. LOPEZ-VIDRIERO 1, M. JACOBS 2,. and S.W. CLARKE 1
i Department of Thoracic Medicine, and 2 Academic Department of Pharmacology, Royal Free Hospital School of Medicine, London, NW3 2PF, England Received 15 April 1985, accepted 1 May 1985
M.T. LOPEZ-VIDRIERO, M. JACOBS and S.W. CLARKE, The effect of isoprenafine on the ciliary activity of an in vitro preparation of rat trachea, European J. Pharmacol. 112 (1985) 429-432. A dose dependent increase in the ciliary beat frequency of rat tracheal explants was found over a 10-7-10 -3 M concentration range of (+)-isoprenaline and was abolished by 10 -7 M propranolol. Furthermore (+)-isoprenaline, a drug which is much less potent in its action on fl-adrenoceptors than its ( - )-isomer, failed to cause a stimulation. We conclude that the acceleration in the intrinsic ciliary beat frequency by (-)-isoprenaline, although occurring at a relatively high concentration compared with the action of the drug on other tissues, is mediated by fl-adrenoceptors. Rat trachea
Isoprenaline
Ciliary activity
1. Introduction fl-Adrenoceptor agonists are known to increase mucociliary wave frequency both in vivo and in vitro in a variety of animal species (Melville et al., 1976; Van As and Parzyck, 1981; Hybbinette and Mercke, 1982). Mucociliary wave frequency, is, however, dependent on the mucus and ciliary components and their interactions. It is, therefore, not possible to distinguish the relative contributions of the two components in in vivo or in vitro experiments where mucus is present. The direct effect of drugs on the intrinsic ciliary activity can only be evaluated in in vitro tissue preparations where mucus is absent or very dilute. fl-Adrenoceptor agonist stimulation of ciliary beat frequency (CBF) was observed in such preparations of bronchial (Clarke and Lopez-Vidriero, 1983) and nasal mucosa (Konietzko and Kasparek, 1983). Although two agonists, terbutaline and isoprenaline, were used in these experiments, agonist * To whom all correspondence should be addressed: Academic Department of Pharmacology, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, U.K. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
activity alone is not sufficient evidence to prove an action at a specific receptor. In the only study so far done on animal cells using cultured rabbit ciliated epithelium (Verdugo et al., 1980), isoprenaline was found to mediate an increase in CBF which was antagonised by propranolol. This latter observation therefore confirms that isoprenaline was acting on/3-adrenoceptors. Unfortunately, these cultured cells are not well characterised and may not be at the same stage of development or have the same receptors as ciliated cells in intact rabbit trachea. The purpose of this current study was, therefore, to investigate the stimulation of CBF by isoprenaline in an in vitro preparation of trachea from a different species. Furthermore, previous studies have found a stimulation of CBF at relatively high isoprenaline concentrations (10-7-10 -4 M) compared with those required to elicit a response in the heart and tracheal smooth muscle (Buckner and Patil, 1971). We have therefore used additional pharmacological criteria to establish unequivocally that the stimulation of ciliary activity by isoprenaline is mediated by the activation of /3-adrenoceptors.
430
2. Materials and methods
2.1. General ( + )- and ( + )-isoprenaline and Hank's medium were purchased from Sigma (UK) and modified medium 199 with Earle's salts from Flow Laboratories (UK). (+)-Propranolol was obtained from Imperial Chemical Industries (UK). All drugs were dissolved in culture medium and the pH adjusted to 7.0; fresh solutions were prepared for each experiment.
2.2, Preparation of tissue Sprague-Dawley rats weighing 250 g were killed by a flow to the head. The trachea was immediately removed and washed several times in Hank's medium. The laryngeal segment, which contains almost all the submucosal glands and the upper third of the trachea, was discarded. The rest of the trachea was divided into small explants (5 mm × 5 mm) and washed several times with culture medium 199 at room temperature. Each explant was then placed in a Prior chamber and equilibrated in culture medium 199 at 20°C for 30 min.
2.3. Measurement of ciliary beat frequency Ciliary beat frequency (CBF) was measured by the photometric technique (Dalham and Rylander, 1962; Lopez-Vidriero and Clarke, 1982) at 20°C. Baseline CBF measurements were taken from 4-6 different sites on each explant and the same sites were measured during drug treatment. Experiments to determine dose response curves for (_+)isoprenaline or (+)-isoprenaline were performed by perfusing the chamber with increasing concentrations of drug. The explant was incubated for 15 min at each concentration before CBF measurements were taken. In between concentrations, the chamber was perfused for 30 rain with fresh culture medium, at a flow rate of 0.15 ml/min. In experiments with (+)-propranol, fresh explants were pre-incubated with drug for 30 min and then perfused with the medium containing the initial concentrations of antagonist and the appropriate
concentrations of isoprenaline. Propranolol was also present in the washing solution. In all experiments, untreated control explants showed no change in basal CBF or sensitivity to (+)-isoprenaline.
2.4. Viscosity measurements A separate experiment was performed to estimate the apparent viscosity changes expected during the treatment of the tracheal explants with isoprenaline. Four explants were used to maximise the viscosity changes. These explants were suspended in culture medium and then challenged with 10 - 4 M isoprenaline, the highest concentration of the drug used for the dose response curve. Measurements were performed on ten aliquots of culture medium before and after treatment. The apparent viscosities were 0.872 + 0.005 mPas. s -1 and 0.877 + 0.015 mPas. s- 1 respectively.
2.5. Statistics The results were expressed as mean + S.E. All straight lines were drawn by linear regression using the Minitab statistical programme and tested for deviation from linearity by analysis of variance by regression. Statistical difference between two means were determined by Student's t-test by testing for overlap of 95% confidence limits.
3. Results
3.1. Effect of isomers of isoprenaline on CBF The effect of (+)-isoprenaline was studied in 5 tracheal explants and that of ( + )-isoprenaline in 4 explants, all from different animals. A dose dependent increase in CBF compared with baseline values (7.4+ 0.3 s -1) was observed o v e r 1 0 - 7 - 1 0 - 3 M range of (+)-isoprenaline concentrations (fig. 1). The increase in CBF was constant over 45 min, suggesting that tachyphylaxis was not a problem. A maximal stimulation of 50% over the control was found at 10 - 4 M , this CBF corresponds to 10.8 + 0.8 s -1. A half maximal stimulation was achieved at 7.46 x 10 -7 M concentration. In con-
431 CBF %CONTROL 160.
3.2. The effect of ( +- )-propranolol on ( +_ )isoprenaline stimulation of CBF
[/
140
I/
120
]b) 100
~Z......._ ~ CONTROL
CBF %CONTROL
"~
-LOG ISOPRENALINE
4. Discussion (c)
11° t,00~--';--;~;~-L-.._..~ CONTROL
The effect of ( + ) - p r o p r a n o l o l at 10 7 M concentration was investigated in 4 tracheal explants from different tracheas. The effect of 10 - 9 M concentrations was studied only in 2 explants and one C B F measurement was taken during baseline and isoprenaline stimulation. ( + ) - P r o p r a n o l o l at 10 -7 M concentration had no effect on the baseline CBE but completely inhibited the stimulation observed with (+)-isoprenaline (fig. 1). In contrast, 10 - 9 M concentration did not block the stimulatory effect of ( _+)-isoprenaline (not shown).
-LOG ISOPRENALINE
Fig. 1. (a) The effect of (+)-isoprenaline on ciliary beat frequency. Rat tracheal explant was perfused with increasing concentrations of (+)-isoprenaline as shown. Plot is the mean + S.E. of 5 explants from different animals measured at 5 sites per explant. (+)-Isoprenaline • i. (b) The explant was preincubated with 10 -7 M propranolol for 30 min and then perfused with increasing concentrations of (_+)-isoprenaline plus 10 -v M propranolol. Plot is the mean + S.E. of 4 explants from different animals, with measurements taken at 6 sites per explant. (+)-lsoprenaline + 10-7 M propranolol • •. (c) The effect of (+)-isoprenaline on ciliary beat frequency. Experiment performed as in (a). Plot is the mean_+S.E. of 4 explants from different animals, with measurements from 5 sites per explant. • •. All measurements were performed at 20° and commenced 15 •in after perfusion with drugs.
trast, (+)-isoprenaline caused no increase in C B F (fig. 1), in fact a small inhibition was observed at high concentrations. When the presence of the ( + ) - i s o m e r is taken into account, the calculated value of the - l o g EDs0 for the stimulation of the C B F by ( - ) - i s o p r e n a l i n e is 6.43 + 1.1 c o m p a r e d with 8.50 _+ 0.06 and 8.65 -t- 0.05 found for guinea pig tracheal smooth muscle and atria respectively (Buckner and Patti, 1971).
A dose dependent increase in ciliary activity was found when the rat trachea was treated with ( ___)-isoprenaline. It was unlikely that this increased response was due to mucus, since the upper one third of the trachea and the submucosal gland, which is the main source of mucus in the rat, was excluded from the preparation. Furthermore, a change of approximately 5 m P a s . s ] is required to produce a ten per cent reduction in ciliary activity (Luk and Dulfano, 1982). A change of less than 0.01 m P a s . s-1 was observed in the apparent viscosity of the culture medium when a large tracheal explant was challenged with - - 1 0 - 4 M isoprenaline. Although the release of mucus cannot be absolutely excluded, this insignificant change in viscosity indicates that mucus was not a factor during our experiments. EDs0 obtained for the stimulation of ciliary beat frequency by ( - ) - i s o p r e n a l i n e in this study, is comparable to the value estimated from the data of Verdugo et al. (1980) using cultured rabbit epithelium. This value is at least an order of magnitude higher than typical EDs0 values found for isoprenaline in tracheal smooth muscle and the heart (Buckner and Patil, 1971). In spite of this high value, our data proves conclusively that ( - ) isoprenaline was acting specifically on /~-adrenoceptors. Firstly, the increase in ciliary beat frequency was completely inhibited by propranolol, at a 10-7 M concentration which is consistent with the pA2
432
value of the antagonist and is in agreement with the findings of Verdugo et al. (1980). Secondly, the ( + )-isomer of isoprenaline which is known to have a very low potency at fl-adrenoceptors on the heart and trachea (Buckner and Patil, 1971), also failed to stimulate CBF. The reason for the high EDs0 value found for the stimulation of CBF is not known but may be due to the involvement of a fl-adrenoceptor subtype or less efficient coupling of the receptor to pathways within the ciliated cell.
References Buckner, C.K. and P.N. Patil, 1971, Steric aspects of adrenergic drugs, XVI, Beta adrenergic receptors of guinea pig atria and trachea, J. Pharmacol. Exp. Ther. 176, 634. Clarke, S.W. and M.T. Lopez-Vidriero, 1983, The effect of beta 2 agonists on the activity of human bronchial cilia in vitro, J. Physiol. 336, 40P. Dalham, T. and R. Rylander, 1962, Frequency of ciliary beat measured with a photo-sensitive cell, Nature 196, 592.
Hybbinette, J.C. and U. Mercke, 1982, Effects of sympathomimetic agonists and antagonists on mucociliary activity, Acta Otolaryngol. 94, 121. Konietzko, N. and R. Kasparek, 1983, Effect of adrenergic, cholinergic and histaminergic stimulation on ciliary beat frequency of human nasal mucosa in vitro, Am. Rev. Respir. Dis. 127, 294. Lopez-Vidriero, M.T. and S.W. Clarke, 1982, Objective criteria for measuring ciliary beat frequency in vitro, Am. Rev. Respir. Dis. 125, 244. Luk, C.K.A. and M.J. Dulfano, 1983, Effect of pH, viscosity and ionic-strength changes in ciliary beat frequency of human bronchial explants, Clin. Sci. 64, 449. Melville, G.N., G. Horstmann and J. Irvani, 1976, Adrenergic compounds and the respiratory tract, Respiration 33, 261. Van As, A. and T. Parzyck, 1981, The role of beta receptor function in the control of ciliary frequency, Am. Rev. Respir, Dis. 12, 215. Verdugo, P., N.T. Johnson and P.Y. Tam, 1980, Adrenergic stimulation of respiratory ciliary activity, J. Appl. Physiol.: Respirat. Environ. Exerc. Physiol. 48, 868.