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Influence of substance P on ciliary beat frequency in airway isolated preparations
European Journal of Pharmacology, 130 (1986) 91-96
91
Elsevier EJP 00537
Influence of substance P on ciliary beat frequency in airway isolated preparations A. R a u f K h a n 1, Bengt Bengtsson 2 a n d Sven L i n d b e r g
3,.
I Clinical Pharmacological Laboratory, A B Draco, Lund, 2 Ferring Pharmaceuticals, Malmi~, and 3 Department of Oto-Rhino-Laryngology, University Hospital of Lund, Sweden Received 1 May 1986, revised MS received 3 July 1986, accepted 22 July 1986
The neuropeptide substance P (SP), which is released from sensory C-fibres after stimulation by chemical irritants, accelerates mucociliary activity in rabbit maxillary sinus in vivo. In order to investigate the mechanism behind this finding, we recorded ciliary beat frequency (CBF) in vitro from various respiratory explants by a photoelectric technique. It was noticed that ciliated cells from the maxillary sinus displayed a higher beating frequency than cells from the trachea and the bronchus, in that order. SP 10-1°-10-4 M did not influence the CBF in explants from rabbit maxillary sinus, rabbit trachea and main bronchi, guinea-pig trachea and human adenoids. Neurokinin A (NKA) 10-6-10 -5 M, which is also thought to be released from sensory C-fibres, did not influence the CBF in guinea-pig trachea. Neither did the C-fibre stimulant capsaicin 10-8-10 -4 M increase CBF in explants from human adenoids. The present findings indicate that the mucociliary effect of SP in vivo is not mediated by an effect on the cilia themselves. Cilia; Airway explants; Substance P; Neurokinin A; Capsaicin; Terbutaline; C-fibres 1. Introduction
Mucociliary (m.c.) activity is an important defence mechanism in the airways, cleaning the airway epithelium from contaminating particles, bacteria and cellular debris. An increased m.c. activity during inflammatory conditions seems desirable and several inflammatory mediators, e.g. prostaglandins, leukotriene C4 and histamine accelerate ciliary beat frequency in vitro (Wanner et al., 1983). Inflammatory reactions involve a neurogenic components (Jancs6 et al., 1980), probably mediated by substance P (SP) and related tachykinins, which are released from the peripheral end of sensory nerves (Lembeck and Holzer,
* To whom all correspondence should be addressed: Department of Oto-Rhino-Laryngology, University Hospital, S22185 Lund, Sweden. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
1979). The m.c. activity in the rabbit maxillary sinus is accelerated by i.a. injection of SP (Lindberg et al., 1986), an effect which is resistant to atropine pretreatment. Moreover, both pharmacological (bradykinin and capsaicin) and electrical stimulation of sensory C-fibres, supposed to release SP from the peripheral endings of these neurons, mimic the effect of exogenous SP on the m.c. activity and the triggered m.c. responses are inhibited by an SP antagonist (Lindberg and Mercke, 1986a,b,c). The mechanism of the in vivo effect of SP on the m.c. activity in the rabbit maxillary sinus could be elucidated by studying the effect of SP on the ciliary beating frequency (CBF) in airway explants. The purpose of the present investigation was therefore to study CBF in explants from the rabbit maxillary sinus, the rabbit trachea and main bronchi, the guinea-pig trachea and human epipharyngeal adenoids after challenges with SP, the
92 related tachykinin neurokinin A (NKA) or the C-fibre stimulant capsaicin.
2. Materials and methods
2.1. Preparations Human adenoids were obtained from children (age between 2.5 and 8 years) undergoing adenoidectomy at the E N T department of the University Hospital in Lund. After removal, the tissues were placed in Krebs solution and transported to the laboratory where they were thoroughly washed with aerated (95% 02 + 5% CO2) Krebs solution. Small areas (about 0.5 mm square) of ciliated epithelium were dissected under the microscope and transferred to a microscopic coverslip in 30 /~1 Krebs solution. The coverslip was then placed upside down on a glass slide, 2 mm thick with a 1.2 mm deep cavity in the centre, giving a preparation hanging freely in a drop of Krebs solution. To obtain guinea-pig and rabbit preparations the animals were killed by a blow on the head. The trachea were removed immediately, along with the main bronchi, and placed in aerated Krebs solution. Connective tissues were carefully removed and the trachea or bronchus was cut into small rings of about 0.5-0.8 mm width. Adhering mucus was washed off with Krebs solution. The tracheal preparation in a drop of Krebs solution was transferred to a coverslip as described above. Rabbit maxillary epithelium was obtained by cutting through the bone and prepared for recording of CBF as described for human adenoids. The hanging drop preparation was placed on a microscope stage maintained at room temperature (22-23°C). The beating cilia were viewed at 400 × magnification in a Nikon-Optiphoto CF (chromatic aberration-free) microscope. The cilia were oriented to interrupt the passage of light through a slit in a diaphragm (0.2 mm) into the photometer (Nikon photometer P1), which transduced the light energy into an electrical signal. The electrical signal generated was converted into a reading of ciliary beat frequency displayed on the screen of a Nicolet 3091 oscilloscope. The signals displayed
on the oscilloscope were also recorded on paper by means of an X-Y BD 90 recorder. CBF in each preparation was recorded from 6 different sites and a mean frequency was calculated. The results were expressed as means +__S.E.M. Statistical evaluations were performed by means of Student's t-test for paired observations. 2.2. Solutions Krebs solution had the following composition: (mM) NaC1 120, KC1 4.0, N a H C O 3 20, N a H 2 P O 4 1.5, MgSO 4 1.5, Na acetate 20, CaC12 1.5, glucose 10, pH 7.4. The following drugs were used: SP (Beckman Bioproducts, Switzerland), N K A (Peninsula, USA), capsaicin (Sigma, USA) and terbutaline (Draco, Sweden). The drugs were diluted in Krebs solution and the effect on CBF was compared with controls containing Krebs solution only. The CBF was measured 2 min after the administration of a drug. The experiments with terbutaline were included for control purposes only in order to check the responsiveness of the preparations. As the 10 - 4 M dilutions of substance P were made in Krebs solution containing 0.5 m g / m l of albumin, the control CBF in these experiments were recorded in Krebs with albumin. The preparation was kept under the microscope for at least 15 min before the start of the experiment.
3. Results
Figure 1 shows the concentration-effect relationship for terbutaline on CBF in preparations from human adenoids. Terbutaline (5 × 10-5 M) produced a 30% increase in CBF, proving that the preparations were able to respond to agents which increase CBF. Figure 2 illustrates the CBF in preparations from different levels in the rabbit airways. The ciliated cells from the maxillary sinus had a considerably higher CBF than those from the trachea, which in turn showed a higher frequency than the bronchial cilia. After exposure to substance P there was a very slight reduction of the CBF which was not statistically significant.
93
15 30
u. m 0
-
10 20 0
10
0
I
I
5 ~ 1 0 -9
I
t
I
5 , 1 0 -T
5 , 1 0 -e
5 x 1 0 -s
Terbutaline concentration
(M)
Fig. 1. The effect of various doses of terbutaline on CBF in explants from human adenoids. The results are expressed as means and S.E.M. n = 5-7 for the various concentrations.
Maxillary sinus
Tracheal ring
Bronchial ring
Fig. 2. The effect of 10 -4 M SP on the CBF in three different airway preparations from 6 rabbits. [] initial CBF, [] CBF after 2-10 min exposure to SP 10 -4 M. The results are expressed as means and S.E.M. Note the high CBF in explants from the maxillary sinus compared with the tracheo-bronchial specimens.
TABLE 1 Effect of substance P on ciliary beat frequency (CBF) in vitro. Values are means + S.E.M.; n is the number of individuals examined at each concentration. Statistical evaluations by Student's t-test for paired observations. Substance P concentration
Initial CBF Hz + S.E.M.
CBF after 2-10 min exposure H z + S.E.M.
Effect in % of initial CBF
P
11.7+0.3 11.5 +0.4 11.0+0.6 10.3+0.9 11.7
11.6-t-0.4 11.5 -t-0.4 11.0+0.6 11.1+0.8 11.8
-0.9 -I-00.0 +00.0 +7.8 +0.9
NS NS NS NS NS
11.5+0.6 10.8+0.2 11.2+0.8
10.8+0.4 11.7+0.2 10.9+0.6
-6.1 +8.5 -2.7
Human adenoids 6 7 7 4 2
10 -1° M 10 - s M 10 -6 M 5 x 1 0 -5 M 10 -4 M
Guinea-pig trachea 5 5 6
10 -1° M 10 -6 M 10 -4 M
NS < 0.05 NS
TABLE 2 Effect of neurokinin A on ciliary beat frequency (CBF) in vitro, guinea-pig trachea. Values are means+S.E.M.; n is the number of individuals examined at each concentration. Statistical evaluation by Student's t-test for paired observations. n
Neurokinin A concentration
Initial CBF Hz + S.E.M.
CBF after 2-10 min exposure Hz + S.E.M.
Effect in % of initial CBF
P
6 6
5 × 10-6 M 10 -5 M
9.3 + 0.9 10.8+0.5
9.0 + 0.7 10.3+0.5
- 3.2 -4.6
NS NS
94 TABLE 3 Effect of capsaicin on ciliary beat frequency (CBF) in vitro, human adenoids. Values are means-+S.E.M.; n is the number of individuals examined at each concentration. Statistical evaluation by Student's t-test for paired observations. n
Capsaicin concentration
Initial CBF Hz + S.E.M.
CBF after 2-10 min exposure H z _ S.E.M.
6 5 6
10 8 M 10 6 M 10 -4 M
11.9_+0.6 11.8+0.8 11.0_+0.5
12.1 _+0.5 12.1_+1.5 9.6 _+0.5
The CBF from human adenoids and guinea-pig trachea were very similar (about 11 Hz). The differences in CBF seen after exposure to substance P were small and not consistent (table 1). Neither did N K A influence the CBF in preparations from guinea-pig trachea (table 2). Capsaicin at concentrations of 10 -8 and 10 -6 M did not affect the CBF whereas the highest concentration (10 -4 M) used reduced the CBF (table 3).
4. Discussion
Mucociliary (m.c.) transport is dependent on both the ciliary beats and the properties of the periciliary fluid. Although the frequency of the ciliary beats is often taken as a measure of the mechanical activity of the cilia, it should be emphasized that the viscosity of the periciliary fluid also has an influence on the CBF. However, measuring under both in vitro and in vivo conditions provides a possibility of distinguishing between effects on CBF which are exerted directly on the ciliated cells and effects induced through changes of the fluid surrounding the cilia. The present finding that SP did not influence CBF in in vitro preparations from several species contrasts with the effects of the peptide in the rabbit maxillary sinus in vivo, where i.a. injections produced a significant m.c. response (Lindberg et al., 1986). The related tachykinin N K A also did not influence CBF in airway isolated preparation, although N K A stimulates m.c. activity in vivo (Lindberg et al., unpublished observations). In line with these results, the SP releasing drug
Effect in % of initial CBF +1.9+2.2 +2.9-+1.9 13.1 -+ 1.6
P
NS NS < 0.001
capsaicin, which has been shown to have a marked effect in vivo (Lindberg and Mercke, 1986b) did not increase CBF in vitro. The decrease of CBF after the administration of capsaicin 10 - 4 M is probably explained by a toxic effect of the drug. Irritants are known to cause ciliostasis on intense or prolonged exposure (Dalhamn, 1956). Even if the present method does not allow recordings to be made directly after administration of a drug and transient changes in the CBF immediately after administration could thus have been overlooked, our results indicate that there is no sustained in vitro effect of SP on the cilia themselves. Thus it is unlikely that the effect of SP on m.c. activity in vivo is due to a direct action on the cilia. The cilia beat in the periciliary fluid, and changes in the composition of this fluid are critical for m.c. function. The secretions forming the periciliary layer probably originate from the ciliated cells themselves (Reid et al., 1982) and both ciliated and non-ciliated cells in the airways are provided with microvilli sharing characteristics with other secretory cells elsewhere in the body (Rhodin, 1966; Petruson et al., 1984). The periciliary fluid is probably the result of active ion transport by epothelial cells (Olver et al., 1975). In this context, some previous reports have shown in vitro effects of SP on respiratory secretions which are relevant to the control of m.c. function. SP stimulates mucin production in canine tracheal explants (Baker et al., 1977). It also elicits a rapid rise of chloride luminal flux (Al-Bazzaz et al., 1985), creating an osmotic gradient which increases the water content of the respiratory secretions, and in turn affects m.c. function (Nadel and
95
Davis, 1978; Widdicombe and Welsh, 1980). Taking into account the results of the present investigation, it is likely that the rapid increase in m.c. activity in vivo seen after exogenously applied or endogenously released SP reflects a rapid change in the periciliary fluid. Thus the increased water content produced by SP might induce a more serous periciliary fluid, allowing an increased frequency for the m.c. wave movements. The increased ciliary beat frequency in the trachea compared with the main bronchi in the rabbit agrees with previous results obtained in the rat (Iravani and Van As, 1972; Iravani and Melville, 1976) and in man (Morrow et al., 1967). It is generally accepted that the different frequencies and transport velocities in the lower respiratory tract reflect a corresponding heavier load on the m.c. system in the trachea than in the more peripheral airways. The frequency in the rabbit maxillary sinus was even higher than in the rabbit trachea, and the basal ciliary beat frequency of 13.7 _+ 1.2 Hz in the sinus explants at room temperature could be compared with the basal m.c. wave frequency of about 20 waves/s recorded in vivo in most rabbits (Hybbinette and Mercke, 1982). The ostium between the nasal cavity and the maxillary sinus is small in rabbits, as in humans, with an average size of about 0.5 × 4 mm cross-section and 2.6 mm length (Kumlien and Schiratzki, 1985). It is therefore unlikely that the sinus mucosa would be contaminated with inhaled particles and irritants under normal conditions. One may speculate that the high capacity of the m.c. system in the sinus, including a prompt reflex acceleration of the m.c. activity after exposure to cigarette smoke, a common irritant (Hybbinette, 1982; Lindberg, 1986), is to provide a continuous supply of a fresh mucus blanket in the nasal cavity as suggested by Drettner (1982). The need of a well-functioning m.c. system in the nose seems particularly important, since mammals in general are nose-breathers, and thus the nasal mucosa constitutes the first line of airway defence. References Al-Bazzaz, F.J., J.G. Kelsey and W.D. Kaage, 1985, Substance P stimulation of chloride secretion by canine tracheal mucosa, Am. Rev. Respir. Dis. 131, 86.
Baker, A.P., L.M. Hillegass, D.A. Holden and W.J. Smith, 1977, Effect of kallidin, substance P, and other basic polypeptides on the production of respiratory macromolecules, Am. Rev. Respir. Dis. 115, 811. Dalhamn, T., 1956, Mucous flow and ciliary activity in the trachea of healthy rats and rats exposed to respiratory irritant gases (SO2, H3N, HCHO). A functional and morphologic (light microscopic and electron microscopic) study, with special reference to technique, Acta Pysiol. Scand. 36 (Suppl.), 123. Drettner, B., 1982, The paranasal sinuses, in: The Nose Upper Airway Physiology and the Atmospheric Environment, eds. D.F. Proctor and I. Andersen (Elsevier Biomedical Press, Amsterdam) p. 145. Hybbinette, J.-C., 1982, A pharmacological evaluation of the short-term effect of cigarette smoke on mucociliary activity, Acta Otolaryngol. (Stockholm) 94, 351. Hybbinette, J.-C. and U. Mercke, 1982, A method for evaluating the effects of pharmacological substances on mucociliary activity in vivo, Acta Otolaryngol. (Stockholm) 93, 151. Iravani, J. and G.N. Melville, 1976, Mucociliary function in the respiratory tract influenced by physicochemical factors, Pharmacol. Ther. 2, 471. Iravani, J. and A. Van As, 1972, Mucus transport in the tracheobronchial tree of normal and bronchitic rats, J. Pathol. 106, 81. Jancz6, G., E. Kirhly and A. Jancsr-Ghbor, 1980, Chemosensitive pain fibres and inflammation, Int. J. Tissue React. 2, 57. Kumlien, J. and H. Schiratzki, 1985, The vascular arrangement of the sinus mucosa, Acta Otolaryngol. (Stockholm) 99, 122. Lembeck, F. and P. Holzer, 1979, Substance P as neurogenic mediator of antidromic vasodilation and neurogenic plasma extravasation, Naunyn-Schmiedeb. Arch. Pharmacol. 310, 175. Lindberg, S., 1986, Reflex-induced acceleration of mucociliary activity in rabbit after short-term exposure to cigarette smoke, Clin. Respir. Physiol. 22, 273. Lindberg, S. and U. Mercke, 1986a, Bradykinin accelerates mucociliary activity in rabbit maxillary sinus, Acta Otolaryngol (Stockholm) 101,114. Lindberg, S. and U. Mercke, 1986b, Capsaicin stimulates mucociliary activity by releasing substance P and acetylcholine, European J. Respir. Dis. 68, 96. Lindberg, S. and U. Mercke, 1986c, Antidromic nerve stimulation accelerates mucociliary activity in rabbit maxillary sinus, Acta Otolaryngol. (Stockholm) 101,484. Lindberg, S., J.-C. Hybbinette and U. Mercke, 1986, Effects of neuropeptides on mucociliary activity, Ann. Otol. Rhinol. Laryngol. 95, 94. Morrow, P.E., F.R. Gibb and K.M. Gazioglu, 1967, A study of particulate clearance from the human lungs, Am. Rev. Respir. Dis. 96, 1209. Nadel, J.A. and B. Davis, 1978, Regulation of Na + and CItransport and mucous gland secretion in airway epithelium, in: Respiratory Tract Mucus, Ciba Foundation Symposium 54 (new series) (Elsevier, Excerpta Medica, North-Holland, Amsterdam) p. 133.
96 Olver, R.E,, B. Davis, M.G. Marin and J.A. Nadek 1975, Active transport of Na + and C1 across the canine tracheal epithelium in vitro, Am. Rev. Respir. dis. 112, 811. Petruson, B., H.-A. Hansson and G. Karlsson, 1984, Structural and functional aspects of cells in the nasal mucociliary system, Arch. Otolaryngol. 110, 576. Reid, L., K. Bhaskar and S. Coles, 1982, Clinical aspects of respiratory mucus, in: Mucus in Health and Disease II, Advances in Experimental Medicine and Biology, Vol. 144,
eds. E.N. Chantler, J.B. Elder and M. Elstein (Plenum Press, New York and London) p. 369. Rhodin, J.A.G., 1966, Ultrastructure and function of the hum a n tracheal mucosa, Am. Rev. Respir. Dis. 93, 1. Wanner, A., D. Maurer, W.M. Abraham, Z. Szepfalusi and M. Sielczak, 1983, Effects of chemical mediators of anaphylaxis on ciliary function, J. Allergy Clin. Immunol. 72, 663. Widdicombe, J.H. and M.J. Welsh, 1980, Ion transport by dog tracheal epithelium, Fed. Proc. 39, 3062.
91
Elsevier EJP 00537
Influence of substance P on ciliary beat frequency in airway isolated preparations A. R a u f K h a n 1, Bengt Bengtsson 2 a n d Sven L i n d b e r g
3,.
I Clinical Pharmacological Laboratory, A B Draco, Lund, 2 Ferring Pharmaceuticals, Malmi~, and 3 Department of Oto-Rhino-Laryngology, University Hospital of Lund, Sweden Received 1 May 1986, revised MS received 3 July 1986, accepted 22 July 1986
The neuropeptide substance P (SP), which is released from sensory C-fibres after stimulation by chemical irritants, accelerates mucociliary activity in rabbit maxillary sinus in vivo. In order to investigate the mechanism behind this finding, we recorded ciliary beat frequency (CBF) in vitro from various respiratory explants by a photoelectric technique. It was noticed that ciliated cells from the maxillary sinus displayed a higher beating frequency than cells from the trachea and the bronchus, in that order. SP 10-1°-10-4 M did not influence the CBF in explants from rabbit maxillary sinus, rabbit trachea and main bronchi, guinea-pig trachea and human adenoids. Neurokinin A (NKA) 10-6-10 -5 M, which is also thought to be released from sensory C-fibres, did not influence the CBF in guinea-pig trachea. Neither did the C-fibre stimulant capsaicin 10-8-10 -4 M increase CBF in explants from human adenoids. The present findings indicate that the mucociliary effect of SP in vivo is not mediated by an effect on the cilia themselves. Cilia; Airway explants; Substance P; Neurokinin A; Capsaicin; Terbutaline; C-fibres 1. Introduction
Mucociliary (m.c.) activity is an important defence mechanism in the airways, cleaning the airway epithelium from contaminating particles, bacteria and cellular debris. An increased m.c. activity during inflammatory conditions seems desirable and several inflammatory mediators, e.g. prostaglandins, leukotriene C4 and histamine accelerate ciliary beat frequency in vitro (Wanner et al., 1983). Inflammatory reactions involve a neurogenic components (Jancs6 et al., 1980), probably mediated by substance P (SP) and related tachykinins, which are released from the peripheral end of sensory nerves (Lembeck and Holzer,
* To whom all correspondence should be addressed: Department of Oto-Rhino-Laryngology, University Hospital, S22185 Lund, Sweden. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
1979). The m.c. activity in the rabbit maxillary sinus is accelerated by i.a. injection of SP (Lindberg et al., 1986), an effect which is resistant to atropine pretreatment. Moreover, both pharmacological (bradykinin and capsaicin) and electrical stimulation of sensory C-fibres, supposed to release SP from the peripheral endings of these neurons, mimic the effect of exogenous SP on the m.c. activity and the triggered m.c. responses are inhibited by an SP antagonist (Lindberg and Mercke, 1986a,b,c). The mechanism of the in vivo effect of SP on the m.c. activity in the rabbit maxillary sinus could be elucidated by studying the effect of SP on the ciliary beating frequency (CBF) in airway explants. The purpose of the present investigation was therefore to study CBF in explants from the rabbit maxillary sinus, the rabbit trachea and main bronchi, the guinea-pig trachea and human epipharyngeal adenoids after challenges with SP, the
92 related tachykinin neurokinin A (NKA) or the C-fibre stimulant capsaicin.
2. Materials and methods
2.1. Preparations Human adenoids were obtained from children (age between 2.5 and 8 years) undergoing adenoidectomy at the E N T department of the University Hospital in Lund. After removal, the tissues were placed in Krebs solution and transported to the laboratory where they were thoroughly washed with aerated (95% 02 + 5% CO2) Krebs solution. Small areas (about 0.5 mm square) of ciliated epithelium were dissected under the microscope and transferred to a microscopic coverslip in 30 /~1 Krebs solution. The coverslip was then placed upside down on a glass slide, 2 mm thick with a 1.2 mm deep cavity in the centre, giving a preparation hanging freely in a drop of Krebs solution. To obtain guinea-pig and rabbit preparations the animals were killed by a blow on the head. The trachea were removed immediately, along with the main bronchi, and placed in aerated Krebs solution. Connective tissues were carefully removed and the trachea or bronchus was cut into small rings of about 0.5-0.8 mm width. Adhering mucus was washed off with Krebs solution. The tracheal preparation in a drop of Krebs solution was transferred to a coverslip as described above. Rabbit maxillary epithelium was obtained by cutting through the bone and prepared for recording of CBF as described for human adenoids. The hanging drop preparation was placed on a microscope stage maintained at room temperature (22-23°C). The beating cilia were viewed at 400 × magnification in a Nikon-Optiphoto CF (chromatic aberration-free) microscope. The cilia were oriented to interrupt the passage of light through a slit in a diaphragm (0.2 mm) into the photometer (Nikon photometer P1), which transduced the light energy into an electrical signal. The electrical signal generated was converted into a reading of ciliary beat frequency displayed on the screen of a Nicolet 3091 oscilloscope. The signals displayed
on the oscilloscope were also recorded on paper by means of an X-Y BD 90 recorder. CBF in each preparation was recorded from 6 different sites and a mean frequency was calculated. The results were expressed as means +__S.E.M. Statistical evaluations were performed by means of Student's t-test for paired observations. 2.2. Solutions Krebs solution had the following composition: (mM) NaC1 120, KC1 4.0, N a H C O 3 20, N a H 2 P O 4 1.5, MgSO 4 1.5, Na acetate 20, CaC12 1.5, glucose 10, pH 7.4. The following drugs were used: SP (Beckman Bioproducts, Switzerland), N K A (Peninsula, USA), capsaicin (Sigma, USA) and terbutaline (Draco, Sweden). The drugs were diluted in Krebs solution and the effect on CBF was compared with controls containing Krebs solution only. The CBF was measured 2 min after the administration of a drug. The experiments with terbutaline were included for control purposes only in order to check the responsiveness of the preparations. As the 10 - 4 M dilutions of substance P were made in Krebs solution containing 0.5 m g / m l of albumin, the control CBF in these experiments were recorded in Krebs with albumin. The preparation was kept under the microscope for at least 15 min before the start of the experiment.
3. Results
Figure 1 shows the concentration-effect relationship for terbutaline on CBF in preparations from human adenoids. Terbutaline (5 × 10-5 M) produced a 30% increase in CBF, proving that the preparations were able to respond to agents which increase CBF. Figure 2 illustrates the CBF in preparations from different levels in the rabbit airways. The ciliated cells from the maxillary sinus had a considerably higher CBF than those from the trachea, which in turn showed a higher frequency than the bronchial cilia. After exposure to substance P there was a very slight reduction of the CBF which was not statistically significant.
93
15 30
u. m 0
-
10 20 0
10
0
I
I
5 ~ 1 0 -9
I
t
I
5 , 1 0 -T
5 , 1 0 -e
5 x 1 0 -s
Terbutaline concentration
(M)
Fig. 1. The effect of various doses of terbutaline on CBF in explants from human adenoids. The results are expressed as means and S.E.M. n = 5-7 for the various concentrations.
Maxillary sinus
Tracheal ring
Bronchial ring
Fig. 2. The effect of 10 -4 M SP on the CBF in three different airway preparations from 6 rabbits. [] initial CBF, [] CBF after 2-10 min exposure to SP 10 -4 M. The results are expressed as means and S.E.M. Note the high CBF in explants from the maxillary sinus compared with the tracheo-bronchial specimens.
TABLE 1 Effect of substance P on ciliary beat frequency (CBF) in vitro. Values are means + S.E.M.; n is the number of individuals examined at each concentration. Statistical evaluations by Student's t-test for paired observations. Substance P concentration
Initial CBF Hz + S.E.M.
CBF after 2-10 min exposure H z + S.E.M.
Effect in % of initial CBF
P
11.7+0.3 11.5 +0.4 11.0+0.6 10.3+0.9 11.7
11.6-t-0.4 11.5 -t-0.4 11.0+0.6 11.1+0.8 11.8
-0.9 -I-00.0 +00.0 +7.8 +0.9
NS NS NS NS NS
11.5+0.6 10.8+0.2 11.2+0.8
10.8+0.4 11.7+0.2 10.9+0.6
-6.1 +8.5 -2.7
Human adenoids 6 7 7 4 2
10 -1° M 10 - s M 10 -6 M 5 x 1 0 -5 M 10 -4 M
Guinea-pig trachea 5 5 6
10 -1° M 10 -6 M 10 -4 M
NS < 0.05 NS
TABLE 2 Effect of neurokinin A on ciliary beat frequency (CBF) in vitro, guinea-pig trachea. Values are means+S.E.M.; n is the number of individuals examined at each concentration. Statistical evaluation by Student's t-test for paired observations. n
Neurokinin A concentration
Initial CBF Hz + S.E.M.
CBF after 2-10 min exposure Hz + S.E.M.
Effect in % of initial CBF
P
6 6
5 × 10-6 M 10 -5 M
9.3 + 0.9 10.8+0.5
9.0 + 0.7 10.3+0.5
- 3.2 -4.6
NS NS
94 TABLE 3 Effect of capsaicin on ciliary beat frequency (CBF) in vitro, human adenoids. Values are means-+S.E.M.; n is the number of individuals examined at each concentration. Statistical evaluation by Student's t-test for paired observations. n
Capsaicin concentration
Initial CBF Hz + S.E.M.
CBF after 2-10 min exposure H z _ S.E.M.
6 5 6
10 8 M 10 6 M 10 -4 M
11.9_+0.6 11.8+0.8 11.0_+0.5
12.1 _+0.5 12.1_+1.5 9.6 _+0.5
The CBF from human adenoids and guinea-pig trachea were very similar (about 11 Hz). The differences in CBF seen after exposure to substance P were small and not consistent (table 1). Neither did N K A influence the CBF in preparations from guinea-pig trachea (table 2). Capsaicin at concentrations of 10 -8 and 10 -6 M did not affect the CBF whereas the highest concentration (10 -4 M) used reduced the CBF (table 3).
4. Discussion
Mucociliary (m.c.) transport is dependent on both the ciliary beats and the properties of the periciliary fluid. Although the frequency of the ciliary beats is often taken as a measure of the mechanical activity of the cilia, it should be emphasized that the viscosity of the periciliary fluid also has an influence on the CBF. However, measuring under both in vitro and in vivo conditions provides a possibility of distinguishing between effects on CBF which are exerted directly on the ciliated cells and effects induced through changes of the fluid surrounding the cilia. The present finding that SP did not influence CBF in in vitro preparations from several species contrasts with the effects of the peptide in the rabbit maxillary sinus in vivo, where i.a. injections produced a significant m.c. response (Lindberg et al., 1986). The related tachykinin N K A also did not influence CBF in airway isolated preparation, although N K A stimulates m.c. activity in vivo (Lindberg et al., unpublished observations). In line with these results, the SP releasing drug
Effect in % of initial CBF +1.9+2.2 +2.9-+1.9 13.1 -+ 1.6
P
NS NS < 0.001
capsaicin, which has been shown to have a marked effect in vivo (Lindberg and Mercke, 1986b) did not increase CBF in vitro. The decrease of CBF after the administration of capsaicin 10 - 4 M is probably explained by a toxic effect of the drug. Irritants are known to cause ciliostasis on intense or prolonged exposure (Dalhamn, 1956). Even if the present method does not allow recordings to be made directly after administration of a drug and transient changes in the CBF immediately after administration could thus have been overlooked, our results indicate that there is no sustained in vitro effect of SP on the cilia themselves. Thus it is unlikely that the effect of SP on m.c. activity in vivo is due to a direct action on the cilia. The cilia beat in the periciliary fluid, and changes in the composition of this fluid are critical for m.c. function. The secretions forming the periciliary layer probably originate from the ciliated cells themselves (Reid et al., 1982) and both ciliated and non-ciliated cells in the airways are provided with microvilli sharing characteristics with other secretory cells elsewhere in the body (Rhodin, 1966; Petruson et al., 1984). The periciliary fluid is probably the result of active ion transport by epothelial cells (Olver et al., 1975). In this context, some previous reports have shown in vitro effects of SP on respiratory secretions which are relevant to the control of m.c. function. SP stimulates mucin production in canine tracheal explants (Baker et al., 1977). It also elicits a rapid rise of chloride luminal flux (Al-Bazzaz et al., 1985), creating an osmotic gradient which increases the water content of the respiratory secretions, and in turn affects m.c. function (Nadel and
95
Davis, 1978; Widdicombe and Welsh, 1980). Taking into account the results of the present investigation, it is likely that the rapid increase in m.c. activity in vivo seen after exogenously applied or endogenously released SP reflects a rapid change in the periciliary fluid. Thus the increased water content produced by SP might induce a more serous periciliary fluid, allowing an increased frequency for the m.c. wave movements. The increased ciliary beat frequency in the trachea compared with the main bronchi in the rabbit agrees with previous results obtained in the rat (Iravani and Van As, 1972; Iravani and Melville, 1976) and in man (Morrow et al., 1967). It is generally accepted that the different frequencies and transport velocities in the lower respiratory tract reflect a corresponding heavier load on the m.c. system in the trachea than in the more peripheral airways. The frequency in the rabbit maxillary sinus was even higher than in the rabbit trachea, and the basal ciliary beat frequency of 13.7 _+ 1.2 Hz in the sinus explants at room temperature could be compared with the basal m.c. wave frequency of about 20 waves/s recorded in vivo in most rabbits (Hybbinette and Mercke, 1982). The ostium between the nasal cavity and the maxillary sinus is small in rabbits, as in humans, with an average size of about 0.5 × 4 mm cross-section and 2.6 mm length (Kumlien and Schiratzki, 1985). It is therefore unlikely that the sinus mucosa would be contaminated with inhaled particles and irritants under normal conditions. One may speculate that the high capacity of the m.c. system in the sinus, including a prompt reflex acceleration of the m.c. activity after exposure to cigarette smoke, a common irritant (Hybbinette, 1982; Lindberg, 1986), is to provide a continuous supply of a fresh mucus blanket in the nasal cavity as suggested by Drettner (1982). The need of a well-functioning m.c. system in the nose seems particularly important, since mammals in general are nose-breathers, and thus the nasal mucosa constitutes the first line of airway defence. References Al-Bazzaz, F.J., J.G. Kelsey and W.D. Kaage, 1985, Substance P stimulation of chloride secretion by canine tracheal mucosa, Am. Rev. Respir. Dis. 131, 86.
Baker, A.P., L.M. Hillegass, D.A. Holden and W.J. Smith, 1977, Effect of kallidin, substance P, and other basic polypeptides on the production of respiratory macromolecules, Am. Rev. Respir. Dis. 115, 811. Dalhamn, T., 1956, Mucous flow and ciliary activity in the trachea of healthy rats and rats exposed to respiratory irritant gases (SO2, H3N, HCHO). A functional and morphologic (light microscopic and electron microscopic) study, with special reference to technique, Acta Pysiol. Scand. 36 (Suppl.), 123. Drettner, B., 1982, The paranasal sinuses, in: The Nose Upper Airway Physiology and the Atmospheric Environment, eds. D.F. Proctor and I. Andersen (Elsevier Biomedical Press, Amsterdam) p. 145. Hybbinette, J.-C., 1982, A pharmacological evaluation of the short-term effect of cigarette smoke on mucociliary activity, Acta Otolaryngol. (Stockholm) 94, 351. Hybbinette, J.-C. and U. Mercke, 1982, A method for evaluating the effects of pharmacological substances on mucociliary activity in vivo, Acta Otolaryngol. (Stockholm) 93, 151. Iravani, J. and G.N. Melville, 1976, Mucociliary function in the respiratory tract influenced by physicochemical factors, Pharmacol. Ther. 2, 471. Iravani, J. and A. Van As, 1972, Mucus transport in the tracheobronchial tree of normal and bronchitic rats, J. Pathol. 106, 81. Jancz6, G., E. Kirhly and A. Jancsr-Ghbor, 1980, Chemosensitive pain fibres and inflammation, Int. J. Tissue React. 2, 57. Kumlien, J. and H. Schiratzki, 1985, The vascular arrangement of the sinus mucosa, Acta Otolaryngol. (Stockholm) 99, 122. Lembeck, F. and P. Holzer, 1979, Substance P as neurogenic mediator of antidromic vasodilation and neurogenic plasma extravasation, Naunyn-Schmiedeb. Arch. Pharmacol. 310, 175. Lindberg, S., 1986, Reflex-induced acceleration of mucociliary activity in rabbit after short-term exposure to cigarette smoke, Clin. Respir. Physiol. 22, 273. Lindberg, S. and U. Mercke, 1986a, Bradykinin accelerates mucociliary activity in rabbit maxillary sinus, Acta Otolaryngol (Stockholm) 101,114. Lindberg, S. and U. Mercke, 1986b, Capsaicin stimulates mucociliary activity by releasing substance P and acetylcholine, European J. Respir. Dis. 68, 96. Lindberg, S. and U. Mercke, 1986c, Antidromic nerve stimulation accelerates mucociliary activity in rabbit maxillary sinus, Acta Otolaryngol. (Stockholm) 101,484. Lindberg, S., J.-C. Hybbinette and U. Mercke, 1986, Effects of neuropeptides on mucociliary activity, Ann. Otol. Rhinol. Laryngol. 95, 94. Morrow, P.E., F.R. Gibb and K.M. Gazioglu, 1967, A study of particulate clearance from the human lungs, Am. Rev. Respir. Dis. 96, 1209. Nadel, J.A. and B. Davis, 1978, Regulation of Na + and CItransport and mucous gland secretion in airway epithelium, in: Respiratory Tract Mucus, Ciba Foundation Symposium 54 (new series) (Elsevier, Excerpta Medica, North-Holland, Amsterdam) p. 133.
96 Olver, R.E,, B. Davis, M.G. Marin and J.A. Nadek 1975, Active transport of Na + and C1 across the canine tracheal epithelium in vitro, Am. Rev. Respir. dis. 112, 811. Petruson, B., H.-A. Hansson and G. Karlsson, 1984, Structural and functional aspects of cells in the nasal mucociliary system, Arch. Otolaryngol. 110, 576. Reid, L., K. Bhaskar and S. Coles, 1982, Clinical aspects of respiratory mucus, in: Mucus in Health and Disease II, Advances in Experimental Medicine and Biology, Vol. 144,
eds. E.N. Chantler, J.B. Elder and M. Elstein (Plenum Press, New York and London) p. 369. Rhodin, J.A.G., 1966, Ultrastructure and function of the hum a n tracheal mucosa, Am. Rev. Respir. Dis. 93, 1. Wanner, A., D. Maurer, W.M. Abraham, Z. Szepfalusi and M. Sielczak, 1983, Effects of chemical mediators of anaphylaxis on ciliary function, J. Allergy Clin. Immunol. 72, 663. Widdicombe, J.H. and M.J. Welsh, 1980, Ion transport by dog tracheal epithelium, Fed. Proc. 39, 3062.