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Effect of artificial surfactant on ciliary beat frequency in guinea pig trachea
Respiration Physiology, 83 (! 991) 313-322 Elsevier
313
RESP 01747
Effect of artificial surfactant on ciliary beat frequency in guinea pig trachea Yasunori Kakuta, Hidetada Sasaki and Tamotsu Takishima The First Department of Internal Medicine, Tohoku University School of Medicine, Seiryo-machi, Aoba-ku, Sendal. Japan (Accepted 26 September 1990) Abstract. This study defines the effect of artificial surfactant on ciliary beat frequency. We employed guinea pig tracheal rings and a photomultiplier which allows in vitro measurement of ciliary beat frequency. The beat frequency without surfactant decreased continuously while surfactant caused a relative increase in beat frequency. The difference between the relative beat frequency in the presence and absence ofsurfactant was significant. The effect of surfactant was close dependent. Fifteen minute treatments with 2 mM hydrogen peroxide reduced the beat frequency. The reduction partially recovered in the absence of surfactant. In contrast, surfactant markedly accelerated the recovery of the beat frequency. Surfactant did not influence the effect of terbutaline (a/~,-adrenergic agonist) on beat frequency. These results indicate that artificial surfactant can, in some circumstances, promote ciliary beat frequency. This concept is important to the clearance mechanism of the airways, and its application to some pathophysiological states such as chronic bronchitis and bronchial asthma should be considered.
Airway clearance; Animal, guinea pig; Ciliary beat frequency, and artificial surfactant; Surfactant, artificial
Pulmonary alveoli are covered by a liquid film that reduces the surface tension. This material, called pulmonary surfactant, is essential for normal lung function (Tierney, 1989). Extensive studies on the secretion and role of surfactant in the alveoli have been performed, but only recently has the importance of surfactant on the airways become apparent (Widdicombe, 1985). Several studies have indicated the presence of surfactant on the airways (Gil and Weibel, 1970; Yoneda, 1976; Morgenroth and Bolz, 1985). In addition, surfactant has been reported to increase the rate of mucociliary transport in frog palate (Allegra et aL, 1985). This increase in the rate of mucociliary transport has been considered as a result of the reduction in the adhesive power of mucus to ciliated epithelium, but this increase may be, in part, caused by some direct action of surfactant on ciliary beat frequency. However, to the best of our knowledge, there has been no report of the influence of surfactant on ciliary beat frequency. The purpose of this study Correspondence to: T. Takishima, The First Department of Internal Medicine, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendal 980, Japan 0034-5687/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
314
Y. KAKUTA et al.
is to examine the effect of surfactant on ciliary beat frequency. For this purpose we applied artificial surfactant to guinea pig tracheal rings and measured the change ofbeat frequency. We sought to determine whether (1) artificial surfactant causes an increase in ciliary beat frequency, (2) artificial surfactant hastens the recovery of beat frequency from injury caused by hydrogen peroxide, and (3)there is an interaction between the effect of surfactant and that of/~e-adrenergic agonists on ciliary beat frequency.
Methods
Animals. Male guinea pigs weighing 200-250 g were anesthetized with 45 mg/kg sodium pentobarbital. After exsanguination a thoracotomy was performed. The trachea was excised and placed in standard externalsolution (SES) containing (mM): NaCI, 150; KCI, 4; calcium methane sulfonate, 2; N-2-hydroxyethyl-piperazine-N'-2-ethane sulfonic acid (Hepes), 20; glucose 5.6. SES was brought to pH 7.4 with tris (hydroxymethyl)-aminomethane (Tris) at 37 °C. The trachea was then put into a dissecting chamber. Small tracheal rings about 1-2 mm in width were dissected using a binocular stereomicroscope and knives made of pieces of razor blades. Three to four rings were obtained from each guinea pig. The internal surface of the rings was washed with SES, using a syringe to remove some tissue debris. By this procedure we could clean up the internal surface of the tracheal rings, which were sticked with some tissue debris. The procedure allowed us to perform stable measurement of ciliary beat frequency. Two opposite parts of each of these specimens were tied with silk threads (10 #m in diameter). The ring was then transfered to an experimental trough (1 ml capacity), through which solutions could be perfused rapidly. Most part of the trough was covered with thin glass plate. The segment was under slight tension and two silk threads were fixed with Scotch tape (3M) so that one of the cut ends of the ring could be surfaced to the bottom of the trough. Experiments were performed at 37 °C by using a heating plate (Microwarm plate 10, Kitazato). Measurement of the ciliary beat frequency. The frequency of ciliary beat was measured as previously reported (Kakuta et al., 1985) with several modifications. An inverted microscope (Diaphot, Nippon Kogaku) was employed. The image was magnified by a factor of 200. A photomultiplier (P 1, Nippon Kogaku) with a pin hole slit (0.2 mm in diameter) was attached to the microscope. The displacement of a specimen caused by exchanging a solution was less than 5 #m and we were able to observe the same portion of the segment during the course of a measurement. Fluctuations in light intensity corresponding to ciliary beat were conditioned by a iowpass filter (cut-off frequency --- 36 Hz) and 50 and 100 Hz notch filters. Since the change of light intensity which corresponded to ciliary movement was larger than the effect of light scattering caused by surfactant, we could measure the ciliary beat frequency in the presence of surfactant at concentrations examined in the present study. The output was displayed on a storage oscilloscope (5103N, Sony Tektronix) which was
SURFACTANT AND CILIARY BEAT FREQUENCY
315
used as a continuous monitor during the experiments. The signal was also recorded on a FM tape recorder (NFR 3000, Sony). The beat frequency was calculated from the signal which was displayed on the storage oscilloscope for 1 sec. An average of three displays was regarded as the frequency. All experiments except those of dose response experiments of surfactant were performed as follows. A specimen was first incubated in SES for 15 min and baseline frequency was measured. After the baseline measurement 10 ml of a test solution was applied in 20 sec. The flow was around 0.5 ml/sec. The exchange of the solution did not appreciably increase beat frequency. There was no continuous flow through the trough in between changes of bathing solution. Beat frequency was measured 15 rain after exchanging the solution. This procedure was repeated every 15 min. Data were expressed as percent change from initial baseline beat frequency. Total duration of these experiments were 60-75 min. Final beat frequencies of the experiments correspond to last plots of figures. In dose-response experiments of surfactant a specimen was first incubated in SES for 15 min and control beat frequency was measured. After that solutions containing various concentrations of surfactant were applied in random order of concentrations and beat frequency was measured 15 rain after exchanging the solution. After the measurement of beat frequencies at one or two different concentrations of surfactant, the solution was exchanged with 10 ml of S ES and control beat frequency was measured at 15 min after exchanging the solution. The application of 10 ml of a solution allowed us to decrease 500 ~g/ml Evans blue to 0.9/~g/ml, that was 1/500 of initial concentration. So, we presumed that the concentration of surfactant decreased to 1/500 by washout of surfactant with 10 ml of SES. Data were expressed as the percent change of beat frequency from the average of beat frequencies in S ES before addition and after washout of surfactant. If a remaining trace amount of surfactant has some effect on ciliary beat frequency, control beat frequency in SES after washout of surfactant would be affected by surfactant. So, this procedure may underestimate the effect of surfactant. We repeated these procedures two or three times in one experiment and total duration of the experiment was 105-120 min. The final control beat frequency in SES of these experiments was decreased by 3.5 + 1.9~o (mean + SEM, n = 9) as compared with that of initial control beat frequency.
Reagents. Artificial surfactant, which is composed ofdipaimitoyl phosphatidylcholine, unsaturated phosphatidyl-glycerol and tripalmitin (65 : 25 : 10, w :w : w) was a kind gift from Teijin Institute for Biomedical Research. The surfactant was obtained as lyophilized powder. It was suspended with SES. The solutions were macroscopically homogeneous up to concentrations examined in the present experiments. Dipalmitoyl phosphatidylcholine and phosphatidyl-glycerol dipalmitoyl (unsaturated phosphatidylglycerol) was purchased from Funakoshi Pharmaceutical Co. Ltd. Tripalmitin was obtained from Sigma Chemical Co. Ltd.. Dipalmitoyl phosphatidylcholine and phosphatidyl-glycerol dipalmitoyl, 50 #g/ml, which was maximum concentration to be suspended, were sonicated and suspended in SES. These suspensions were macroscopically homogeneous, although each miccll was slightly larger than that of artificial
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surfactant. Tripalmitin was, however, not homogeneously suspended in S ES. So, we did not examine the effect of tripalmitin on ciliary beat frequency. Terbutaline (a fl2-adrenergic agonist) was a kind gift from Fujisawa Pharmaceutical Co. Ltd. Statistical methods. Each experimental value is expressed as the mean + SEM. Friedmann's test, Wilcoxon signed-rank test and Mann-Whitney's U-test were used for statistical analyses. P < 0.05 was considered to be significant.
Results
The average beat frequency from 68 specimens was 16.4 + 0.4 Hz (mean + SEM). Beat frequency was not appreciably changed in S ES with I mM magnesium methane sulfonate (16.1 + 1.1 Hz, n = 5). Total replacement of sodium with potassium in SES slightly increased beat frequency (17.6 + 1.5 Hz, n = 5), but it was not significant. In fig. 1 basal beat frequencies for surfactant and control groups in SES were 15.9 + 1.1 Hz and 15.8 + 1.3 Hz, respectively, which were not significantly different. Artificial surfactant, 100 #g/ml, caused a relative increase in beat frequency by 6.4 + 1.7Fo, 9.0 + 3.5~o, 7.8 + 4.3% and 5.2 + 2.3% at 15, 30, 45 and 60 min, respectively, after the application of surfactant (n = 7), while the beat frequency was slightly decreased in SES by 2.6 + 1.6Y/o,5.3 + 1.7~o, 5.1 + 1.5~ and 4.3 + 1.2Y/o,respectively (n = 5, fig. 1). Therefore, the difference between the mean beat frequency in the presence and absence of 100 #g/ml surfactant was significant (P < 0.05), as can be seen in fig. 1. In addition increase in beat frequency caused by 100 #g/ml surfactant was significant (P < 0.05) as compared with the initial beat frequency in SES. in fig. 2 dose-response effect of surfactant was examined. Initial beat frequency was
+10
ACiliary beat frequency
+5
(%) -5
-10
~
I
I
I
I
0
15
30
45
60
Time (min) Fig. I. Effect of 100 #g/ml surfactant on the ciliary beat frequency. After the baseline measurement solutions in the presence or absence ofsurfactant were exchanged every 15 min. Beat frequency was measured 15 min after exchanging a solution. The percent change of beat frequency from baseline was calculated and was plotted, n = 7 (with surfactant), ta = 5 (control). Values are mean + SEM. * P < 0.05 as compared with the frequency in the absence of surfactant.
317
SURFACTANT AND CILIARY BEAT FREQUENCY +10
z~Ciliary
i**
[,
+5
beat
frequency
T
o
(%)
* p
-5
-lO
i 1
I
I
I
10
100
1000
Surfactant (pg/ml) Fig. 2. Effect ofsurfactant on ciliary beat frequency. After the measurement ofcontrol, solutions containing various concentrations of artificial surfactant were applied in random order of concentrations and beat frequency was measured at 15 min after exchanging a solution. After the measurement of beat frequencies at one or two different concentrations of surfactant, the solution was exchanged to SES and control frequency was measured. The percent change of beat frequency from the average of control before addition and after washout ofsurfactant was calculated and was plotted, n = 9o Values are mean + SEM. * P < 0.05; "* P < 0.01 as compared with control.
16.5 + 1.1 Hz. As can be seen in fig. 2, the effect of artificial surfactant on ciliary beat frequency was dose dependent (P < 0.01, Friedman's test and Wilcoxon signed-rank test). In fig. 3 basal beat frequencies for surfactant and control groups were 16.9 _+ 1.3 Hz H202 I
0
+surfactant I
oe
I
.
+surfactant
~Ciliary beat -
t
frequency .so
(%)
*p
-100
I 0
, 15
, 30
J 45
Time (min) Fig. 3. Effect of surfactant on the recovery of beat frequency from injury caused by 2 mM hydrogen peroxide. After the measurement of control, ciliated epithelium was treated with 2 mM hydrogen peroxide for 15 rain. Then beat frequency was measured and expressed as percent change from control. After that the solution was exchanged to that either in the presence or absence of 100 #g/ml surfactant. Solutions were exchanged every 15 min and beat frequency was measured 15 min after exchanging the solution, n = 9 (with surfactant), n = 10 (control). Values are mean + SEM. * P < 0.05 as compared with the frequency in the absence of surfactant.
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Y. K A K U T A et al.
+50
Ciliary
T
-surfactant T
beat
frequency +2s
(%)
o[,
O.Ol
o.~
1.o
lO.O
Terbutaline (pM) Fig. 4. Effect of surfactant on response of beat frequency to terbutaline. After the measurement of control solutions in the presence or absence of 100 pg/ml surfactants were applied successively in the order of increasing terbutaline. Beat frequency was measured 15 rain after exchanging a solution, t~, = 7 (with surfactant), n = 8 (control). Values are mean _ SEM. No appreciable difference was observed between the two groups.
and 16.8 __ 1.3 Hz, respectively, which were not significantly different. A 15 min treatment with 2 mM hydrogen peroxide caused a decrease in beat frequency to 36.8 _ 6.8 of control (fig. 3). The beat frequency in SES partially recovered at 15 min and 30 rain after the treatment with hydrogen peroxide to 68.9 _+ 7.0 and 70.9 _+ 5.9~o of control, respectively (fig. 3). By contrast, the recovery of the beat frequency was markedly accelerated (P < 0.05) in the presence of 100 #g/ml surfactant and reached 93.7 _+ 5.4 and 90.0 +_.4.43/o of control, respectively (fig. 3). The recovery of beat frequency was also remarkably accelerated 15 rain after the treatment with 100 #g/ml of dipalmitoyl phosphatidylcholine (88.3 _ 3.3%, n = 7). The effect was, however, not significant. Phosphatidyl-glycerol dipalmitoyl, 50 #g/ml, did not appreciably affect the recovery of beat frequency (69.7 + 4.8%, n = 6). In fig. 4 basal beat frequencies for surfactant and control groups were 15,3 + 0.8 Hz and 17.4 + 0.7 Hz which were not significantly different. Terbutaline (a fl2-adrenergic agonist) caused an increase in beat frequency by 37.5 q_-4.8% at the concentration of 10 #M (fig. 4). This increase of beat frequency was not appreciably influenced by the presence of 100 #g/ml surfactant (fig. 4). Discussion There is growing evidence which indicates the presence of surfactant in the bronchioles and bronchi (Gil and Weibel, 1970; Yoneda, 1976; Morgenroth and Bolz, 1985). Gil and Weibei (1970) have observed surfactant in the bronchiolcs as well as the aiveoli in rats. Yoneda (1976) has observed osmiophilic lamellae (surfactant) in the lower zone (sol layer) of the mucus blanket in the tracheobronchial tree of rats. In addition, Morgenroth and Bolz (1985) have observed surfactant in the sol phase over the bronchial epithelium on human biopsy material.
SURFACTANT AND CILIARY BEAT FREQUENCY
319
Some physiological experiments have shown the usefulness of surfactant in the airways. Macklem et al. (1970) have reported that surfactant caused an increase in the stability of peripheral airways. Rensch et al. (1983)have speculated using an experimental model, that surfactant likely permits the clearance ofparticles from the peripheral airways. Moreover, Allega et al. (1985) have shown that surfactant caused an increase in mucociliary transport on frog palate preparations. So, surfactant may be useful in mucociliary transport in the airways. There has been, however, no report which examined the direct influence of surfactant on ciliary beat frequency. In this study we examined the effect of artificial surfactant on ciliary beat frequency. We found that (1) artificial surfactant increased ciliary beat frequency, (2)the effect of surfactant was dose dependent, (3)artificial surfactant accelerated the recovery ofbeat frequency from injury caused by hydrogen peroxide, and (4) the increase in beat frequency caused by p2-adrenergic agonist was not influenced with artificial surfactant. These results indicate that artificial surfactant can, in some circumstances, promote tracheal ciliary beat frequency. The average of initial beat frequency of our specimens was 16 Hz. The frequency was comparable with the beat frequency reported in other species (human, 10-14 Hz, Gail and Lenfant, 1983; cat and rat, 12 Hz and 22 Hz, respectively, Dalhamn and Rylander, 1962). It indicates that the initial beat frequency we observed was 'normal'. Application of surfactant caused significant increase in beat frequency. So, surfaetant has an effect to increase ciliary beat frequency. The beat frequency incubated in SES up to 60 min was slightly decreased. It might be due to evaporative loss of water from the trough. The possibility may be, however, very little since the solution was exchanged every 15 min and most part of the trough was covered with thin glass plate. Another explanation of the decline in beat frequency is that exchanging a solution causes loss of factors which prevent the decrease in ciliary beat frequency. This possibility is more likely since ciliary beat frequency was not decreased in the presence of surfactant. In addition prominent action of surfactant on beat frequency was observed in the recovery from injury caused by hydrogen peroxide. So, surfactant likely prevents loss of factors and protects to decrease ciliary beat frequency. We have not determined the exact mechanism of the promoting effect of surfactant on ciliary beat frequency. We can, however, propose several possibilities. First, a more likely explanation of this is that surfactant is changing the theological properties of periciliary fluid since the size of chaoges with surfactant were very small. Second, surfactant may be reversing epithelial damage since ciliary beat frequency was slightly decreased with several exchanges of solutions without surfactant while beat frequency was not decreased in solutions with surfactant. In addition the recovery of ciliary beat frequency from injury caused by hydrogen peroxide was accelerated in the presence of surfactant. Moreover, it should be noticed that dipalmitoyl phosphatidylcholine, which is a major component of the artificial surfactant, is one of important components of cell membrane. Third, the augmentation of ion transport by artificial surfactant is observed in canine tracheal epithelium (lkeda et al., 199,J). It suggests that surfactant acts on
320
Y. KAKUTA et ai.
tracheal epithelium and allows increase in beat frequency. These possibilities likely work on ciliated epithelium and cause promotion of ciliary beat frequency. We studied which component of the artificial surfactant was effective on ciliary beat frequency. Tripalmitin is classified as 'triglyceride', which by itself did not fundamentally make micell in S ES. Phosphatidyl-glycerol dipalmitoyl did not appreciably affect the recovery of beat frequency from injury caused by hydrogen peroxide. Dipalmitoyl phosphatidylcholii:e remarkably accelerated the recovery of beat frequency, although the effect was not significant. So, we presume that dipalmitoyl phosphatidylcholine primarily acts on ciliary beat frequency. There is, however, considerable difference between the effect of artificial surfactant and dipalmitoyl phosphatidylcholine alone. Although we have not determined the exact mechanism of this difference, we can propose several possibilities. First, the micell of dipalmitoyl phosphatidylcholine was macroscopicaUy larger than that of artificial surfaetant. It may reduce the efficacy of the agent. Second, the three components of artificial surfactant make stable miceil and act cooperatively on ciliary beat frequency. We presume that second possibility is more plausible than the first one. The concentration of airway surfactant has not been reported to our knowledge. Slomiany et al. (1982) have reported that the proportion of phospholipid in aspirated tracheobronchial secretion is 2.8% of dry weight of the sample. Assuming 95 to 99% of airway mucus is water, the concentration of phospholipid in the airways is calculated to be 0.028% (280 #g/ml) to 0.14% (1400 #g/ml). The concentration of surfaetant applied for the present study was 500/~g/mi or less. It suggests that the effect of surfaetant observed in the present study is physiologically relevant. The effect of surfactant on the restoration of beat frequency from injury is important for the pathophysiology of airways, since ciliated epithelium is impaired by various toxic agents such as oxidant gases and smokings (Gail and Lenfant, 1983). In addition, it is likely that oxidants from neutrophils and eosinophils cause injury of ciliated epithelium. Therefore, restoration of beat frequency by surfactant is particulary important in the treatment of various kinds of airway disorders. Therefore, the concept that surfaetant has stabilizing effects on ciliary beat frequency is important to the clearance mechanism of the airways, and its application to some pathophysiological states such as chronic bronchitis and bronchial asthma has to be considered. Acknowledgements.We would like to thank Miss R. Haryu for typing the manuscript and Ms P. Tilla and Ms R. Snelling for the critical reading of the manuscript.
References Allegra, L., R. Bossi and P. Braga (1985). Influence of surfactant on mucociliary transport. Eur. J. Respir. Dis. Suppl. 142: 71-76. Dalhamn, T. and Rylander (1962). Frequency of ciliary beat measured with a photo-sensitive cell. Nature (London) 196: 592-593.
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Gail, D.B. and C lJ[ M. Lenfant (1983). Cells of the Lung: biology and clinical implications. Am. Rev. Respir. D/s. 127: 366-387. Gil, J. and E.R. Weibel (1970). Extracellular lining of bronchioles after perfusion-fixation of rat lungs for electron microscopy. Anat. Rec. 169: 185-200. Ikeda, K., T. Sasaki, S. Shimura, M. Sato, H. Ishihara, H. Sasaki, T. Takishima, Y. Saitoh and A. Nishiyama (1990). Effect of surfactant on bioelectric properties of canine tracheal epithelium. Respir. Physiol. 81: 41-49. Kakuta, Y., T. Kanno, H. Sasaki and T. Takishima (1985). Effect of Ca 2 + on the ciliary beat frequency of skinned dog tracheal epithelium. Respir. Physiol. 60: 9-19. Macklem, P.T., D.F. Proctor and J.C. Hogg (1970). The stability of peripheral airways. Respir. Physiol. 8: 191-203. Morgenroth, K. anJ J. Bolz (1985). Morphological features of the interaction between mucus and surfactant on the bronchial mucosa. Respiration 47: 225-231. Rensch, H., H. yon Seefeld, K. F. Gebhardt, D. Renzow and P.-J. Sell (1983). Stop and go particle transport in the peripheral airways'?. A model study. Respiration 44: 346-350. Slomiany, A., V. L. N. Murty, M. Aono, C. E. Snyder, A. Herp and B. L. Slomiany (1982). Lipid composition of tracheobronchial secretions from normal individuals and patients with cystic fibrosis. Bioc,~m. Biophys. Acta 710:106-111. Tierney, D.F. (1989). Lung surfactant: some historical perspectives leading to its cellular and molecular biology. Lung Cell. Mol. Physiol. 1: LI-LI2. Widdicombe, J.G. (1985). Introduction in endobronchial surface active phospholipids. Fur. J. Respir. Dis. Suppl. 142: 1-5. Yoneda, K. (1976). Mucus blanket of rat bronchus. An uitrastructural study. Am. Rev. Respir. D/s. 114: 837-842.
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Effect of artificial surfactant on ciliary beat frequency in guinea pig trachea Yasunori Kakuta, Hidetada Sasaki and Tamotsu Takishima The First Department of Internal Medicine, Tohoku University School of Medicine, Seiryo-machi, Aoba-ku, Sendal. Japan (Accepted 26 September 1990) Abstract. This study defines the effect of artificial surfactant on ciliary beat frequency. We employed guinea pig tracheal rings and a photomultiplier which allows in vitro measurement of ciliary beat frequency. The beat frequency without surfactant decreased continuously while surfactant caused a relative increase in beat frequency. The difference between the relative beat frequency in the presence and absence ofsurfactant was significant. The effect of surfactant was close dependent. Fifteen minute treatments with 2 mM hydrogen peroxide reduced the beat frequency. The reduction partially recovered in the absence of surfactant. In contrast, surfactant markedly accelerated the recovery of the beat frequency. Surfactant did not influence the effect of terbutaline (a/~,-adrenergic agonist) on beat frequency. These results indicate that artificial surfactant can, in some circumstances, promote ciliary beat frequency. This concept is important to the clearance mechanism of the airways, and its application to some pathophysiological states such as chronic bronchitis and bronchial asthma should be considered.
Airway clearance; Animal, guinea pig; Ciliary beat frequency, and artificial surfactant; Surfactant, artificial
Pulmonary alveoli are covered by a liquid film that reduces the surface tension. This material, called pulmonary surfactant, is essential for normal lung function (Tierney, 1989). Extensive studies on the secretion and role of surfactant in the alveoli have been performed, but only recently has the importance of surfactant on the airways become apparent (Widdicombe, 1985). Several studies have indicated the presence of surfactant on the airways (Gil and Weibel, 1970; Yoneda, 1976; Morgenroth and Bolz, 1985). In addition, surfactant has been reported to increase the rate of mucociliary transport in frog palate (Allegra et aL, 1985). This increase in the rate of mucociliary transport has been considered as a result of the reduction in the adhesive power of mucus to ciliated epithelium, but this increase may be, in part, caused by some direct action of surfactant on ciliary beat frequency. However, to the best of our knowledge, there has been no report of the influence of surfactant on ciliary beat frequency. The purpose of this study Correspondence to: T. Takishima, The First Department of Internal Medicine, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendal 980, Japan 0034-5687/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
314
Y. KAKUTA et al.
is to examine the effect of surfactant on ciliary beat frequency. For this purpose we applied artificial surfactant to guinea pig tracheal rings and measured the change ofbeat frequency. We sought to determine whether (1) artificial surfactant causes an increase in ciliary beat frequency, (2) artificial surfactant hastens the recovery of beat frequency from injury caused by hydrogen peroxide, and (3)there is an interaction between the effect of surfactant and that of/~e-adrenergic agonists on ciliary beat frequency.
Methods
Animals. Male guinea pigs weighing 200-250 g were anesthetized with 45 mg/kg sodium pentobarbital. After exsanguination a thoracotomy was performed. The trachea was excised and placed in standard externalsolution (SES) containing (mM): NaCI, 150; KCI, 4; calcium methane sulfonate, 2; N-2-hydroxyethyl-piperazine-N'-2-ethane sulfonic acid (Hepes), 20; glucose 5.6. SES was brought to pH 7.4 with tris (hydroxymethyl)-aminomethane (Tris) at 37 °C. The trachea was then put into a dissecting chamber. Small tracheal rings about 1-2 mm in width were dissected using a binocular stereomicroscope and knives made of pieces of razor blades. Three to four rings were obtained from each guinea pig. The internal surface of the rings was washed with SES, using a syringe to remove some tissue debris. By this procedure we could clean up the internal surface of the tracheal rings, which were sticked with some tissue debris. The procedure allowed us to perform stable measurement of ciliary beat frequency. Two opposite parts of each of these specimens were tied with silk threads (10 #m in diameter). The ring was then transfered to an experimental trough (1 ml capacity), through which solutions could be perfused rapidly. Most part of the trough was covered with thin glass plate. The segment was under slight tension and two silk threads were fixed with Scotch tape (3M) so that one of the cut ends of the ring could be surfaced to the bottom of the trough. Experiments were performed at 37 °C by using a heating plate (Microwarm plate 10, Kitazato). Measurement of the ciliary beat frequency. The frequency of ciliary beat was measured as previously reported (Kakuta et al., 1985) with several modifications. An inverted microscope (Diaphot, Nippon Kogaku) was employed. The image was magnified by a factor of 200. A photomultiplier (P 1, Nippon Kogaku) with a pin hole slit (0.2 mm in diameter) was attached to the microscope. The displacement of a specimen caused by exchanging a solution was less than 5 #m and we were able to observe the same portion of the segment during the course of a measurement. Fluctuations in light intensity corresponding to ciliary beat were conditioned by a iowpass filter (cut-off frequency --- 36 Hz) and 50 and 100 Hz notch filters. Since the change of light intensity which corresponded to ciliary movement was larger than the effect of light scattering caused by surfactant, we could measure the ciliary beat frequency in the presence of surfactant at concentrations examined in the present study. The output was displayed on a storage oscilloscope (5103N, Sony Tektronix) which was
SURFACTANT AND CILIARY BEAT FREQUENCY
315
used as a continuous monitor during the experiments. The signal was also recorded on a FM tape recorder (NFR 3000, Sony). The beat frequency was calculated from the signal which was displayed on the storage oscilloscope for 1 sec. An average of three displays was regarded as the frequency. All experiments except those of dose response experiments of surfactant were performed as follows. A specimen was first incubated in SES for 15 min and baseline frequency was measured. After the baseline measurement 10 ml of a test solution was applied in 20 sec. The flow was around 0.5 ml/sec. The exchange of the solution did not appreciably increase beat frequency. There was no continuous flow through the trough in between changes of bathing solution. Beat frequency was measured 15 rain after exchanging the solution. This procedure was repeated every 15 min. Data were expressed as percent change from initial baseline beat frequency. Total duration of these experiments were 60-75 min. Final beat frequencies of the experiments correspond to last plots of figures. In dose-response experiments of surfactant a specimen was first incubated in SES for 15 min and control beat frequency was measured. After that solutions containing various concentrations of surfactant were applied in random order of concentrations and beat frequency was measured 15 rain after exchanging the solution. After the measurement of beat frequencies at one or two different concentrations of surfactant, the solution was exchanged with 10 ml of S ES and control beat frequency was measured at 15 min after exchanging the solution. The application of 10 ml of a solution allowed us to decrease 500 ~g/ml Evans blue to 0.9/~g/ml, that was 1/500 of initial concentration. So, we presumed that the concentration of surfactant decreased to 1/500 by washout of surfactant with 10 ml of SES. Data were expressed as the percent change of beat frequency from the average of beat frequencies in S ES before addition and after washout of surfactant. If a remaining trace amount of surfactant has some effect on ciliary beat frequency, control beat frequency in SES after washout of surfactant would be affected by surfactant. So, this procedure may underestimate the effect of surfactant. We repeated these procedures two or three times in one experiment and total duration of the experiment was 105-120 min. The final control beat frequency in SES of these experiments was decreased by 3.5 + 1.9~o (mean + SEM, n = 9) as compared with that of initial control beat frequency.
Reagents. Artificial surfactant, which is composed ofdipaimitoyl phosphatidylcholine, unsaturated phosphatidyl-glycerol and tripalmitin (65 : 25 : 10, w :w : w) was a kind gift from Teijin Institute for Biomedical Research. The surfactant was obtained as lyophilized powder. It was suspended with SES. The solutions were macroscopically homogeneous up to concentrations examined in the present experiments. Dipalmitoyl phosphatidylcholine and phosphatidyl-glycerol dipalmitoyl (unsaturated phosphatidylglycerol) was purchased from Funakoshi Pharmaceutical Co. Ltd. Tripalmitin was obtained from Sigma Chemical Co. Ltd.. Dipalmitoyl phosphatidylcholine and phosphatidyl-glycerol dipalmitoyl, 50 #g/ml, which was maximum concentration to be suspended, were sonicated and suspended in SES. These suspensions were macroscopically homogeneous, although each miccll was slightly larger than that of artificial
316
Y. KAKUTA etal.
surfactant. Tripalmitin was, however, not homogeneously suspended in S ES. So, we did not examine the effect of tripalmitin on ciliary beat frequency. Terbutaline (a fl2-adrenergic agonist) was a kind gift from Fujisawa Pharmaceutical Co. Ltd. Statistical methods. Each experimental value is expressed as the mean + SEM. Friedmann's test, Wilcoxon signed-rank test and Mann-Whitney's U-test were used for statistical analyses. P < 0.05 was considered to be significant.
Results
The average beat frequency from 68 specimens was 16.4 + 0.4 Hz (mean + SEM). Beat frequency was not appreciably changed in S ES with I mM magnesium methane sulfonate (16.1 + 1.1 Hz, n = 5). Total replacement of sodium with potassium in SES slightly increased beat frequency (17.6 + 1.5 Hz, n = 5), but it was not significant. In fig. 1 basal beat frequencies for surfactant and control groups in SES were 15.9 + 1.1 Hz and 15.8 + 1.3 Hz, respectively, which were not significantly different. Artificial surfactant, 100 #g/ml, caused a relative increase in beat frequency by 6.4 + 1.7Fo, 9.0 + 3.5~o, 7.8 + 4.3% and 5.2 + 2.3% at 15, 30, 45 and 60 min, respectively, after the application of surfactant (n = 7), while the beat frequency was slightly decreased in SES by 2.6 + 1.6Y/o,5.3 + 1.7~o, 5.1 + 1.5~ and 4.3 + 1.2Y/o,respectively (n = 5, fig. 1). Therefore, the difference between the mean beat frequency in the presence and absence of 100 #g/ml surfactant was significant (P < 0.05), as can be seen in fig. 1. In addition increase in beat frequency caused by 100 #g/ml surfactant was significant (P < 0.05) as compared with the initial beat frequency in SES. in fig. 2 dose-response effect of surfactant was examined. Initial beat frequency was
+10
ACiliary beat frequency
+5
(%) -5
-10
~
I
I
I
I
0
15
30
45
60
Time (min) Fig. I. Effect of 100 #g/ml surfactant on the ciliary beat frequency. After the baseline measurement solutions in the presence or absence ofsurfactant were exchanged every 15 min. Beat frequency was measured 15 min after exchanging a solution. The percent change of beat frequency from baseline was calculated and was plotted, n = 7 (with surfactant), ta = 5 (control). Values are mean + SEM. * P < 0.05 as compared with the frequency in the absence of surfactant.
317
SURFACTANT AND CILIARY BEAT FREQUENCY +10
z~Ciliary
i**
[,
+5
beat
frequency
T
o
(%)
* p
-5
-lO
i 1
I
I
I
10
100
1000
Surfactant (pg/ml) Fig. 2. Effect ofsurfactant on ciliary beat frequency. After the measurement ofcontrol, solutions containing various concentrations of artificial surfactant were applied in random order of concentrations and beat frequency was measured at 15 min after exchanging a solution. After the measurement of beat frequencies at one or two different concentrations of surfactant, the solution was exchanged to SES and control frequency was measured. The percent change of beat frequency from the average of control before addition and after washout ofsurfactant was calculated and was plotted, n = 9o Values are mean + SEM. * P < 0.05; "* P < 0.01 as compared with control.
16.5 + 1.1 Hz. As can be seen in fig. 2, the effect of artificial surfactant on ciliary beat frequency was dose dependent (P < 0.01, Friedman's test and Wilcoxon signed-rank test). In fig. 3 basal beat frequencies for surfactant and control groups were 16.9 _+ 1.3 Hz H202 I
0
+surfactant I
oe
I
.
+surfactant
~Ciliary beat -
t
frequency .so
(%)
*p
-100
I 0
, 15
, 30
J 45
Time (min) Fig. 3. Effect of surfactant on the recovery of beat frequency from injury caused by 2 mM hydrogen peroxide. After the measurement of control, ciliated epithelium was treated with 2 mM hydrogen peroxide for 15 rain. Then beat frequency was measured and expressed as percent change from control. After that the solution was exchanged to that either in the presence or absence of 100 #g/ml surfactant. Solutions were exchanged every 15 min and beat frequency was measured 15 min after exchanging the solution, n = 9 (with surfactant), n = 10 (control). Values are mean + SEM. * P < 0.05 as compared with the frequency in the absence of surfactant.
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Y. K A K U T A et al.
+50
Ciliary
T
-surfactant T
beat
frequency +2s
(%)
o[,
O.Ol
o.~
1.o
lO.O
Terbutaline (pM) Fig. 4. Effect of surfactant on response of beat frequency to terbutaline. After the measurement of control solutions in the presence or absence of 100 pg/ml surfactants were applied successively in the order of increasing terbutaline. Beat frequency was measured 15 rain after exchanging a solution, t~, = 7 (with surfactant), n = 8 (control). Values are mean _ SEM. No appreciable difference was observed between the two groups.
and 16.8 __ 1.3 Hz, respectively, which were not significantly different. A 15 min treatment with 2 mM hydrogen peroxide caused a decrease in beat frequency to 36.8 _ 6.8 of control (fig. 3). The beat frequency in SES partially recovered at 15 min and 30 rain after the treatment with hydrogen peroxide to 68.9 _+ 7.0 and 70.9 _+ 5.9~o of control, respectively (fig. 3). By contrast, the recovery of the beat frequency was markedly accelerated (P < 0.05) in the presence of 100 #g/ml surfactant and reached 93.7 _+ 5.4 and 90.0 +_.4.43/o of control, respectively (fig. 3). The recovery of beat frequency was also remarkably accelerated 15 rain after the treatment with 100 #g/ml of dipalmitoyl phosphatidylcholine (88.3 _ 3.3%, n = 7). The effect was, however, not significant. Phosphatidyl-glycerol dipalmitoyl, 50 #g/ml, did not appreciably affect the recovery of beat frequency (69.7 + 4.8%, n = 6). In fig. 4 basal beat frequencies for surfactant and control groups were 15,3 + 0.8 Hz and 17.4 + 0.7 Hz which were not significantly different. Terbutaline (a fl2-adrenergic agonist) caused an increase in beat frequency by 37.5 q_-4.8% at the concentration of 10 #M (fig. 4). This increase of beat frequency was not appreciably influenced by the presence of 100 #g/ml surfactant (fig. 4). Discussion There is growing evidence which indicates the presence of surfactant in the bronchioles and bronchi (Gil and Weibel, 1970; Yoneda, 1976; Morgenroth and Bolz, 1985). Gil and Weibei (1970) have observed surfactant in the bronchiolcs as well as the aiveoli in rats. Yoneda (1976) has observed osmiophilic lamellae (surfactant) in the lower zone (sol layer) of the mucus blanket in the tracheobronchial tree of rats. In addition, Morgenroth and Bolz (1985) have observed surfactant in the sol phase over the bronchial epithelium on human biopsy material.
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Some physiological experiments have shown the usefulness of surfactant in the airways. Macklem et al. (1970) have reported that surfactant caused an increase in the stability of peripheral airways. Rensch et al. (1983)have speculated using an experimental model, that surfactant likely permits the clearance ofparticles from the peripheral airways. Moreover, Allega et al. (1985) have shown that surfactant caused an increase in mucociliary transport on frog palate preparations. So, surfactant may be useful in mucociliary transport in the airways. There has been, however, no report which examined the direct influence of surfactant on ciliary beat frequency. In this study we examined the effect of artificial surfactant on ciliary beat frequency. We found that (1) artificial surfactant increased ciliary beat frequency, (2)the effect of surfactant was dose dependent, (3)artificial surfactant accelerated the recovery ofbeat frequency from injury caused by hydrogen peroxide, and (4) the increase in beat frequency caused by p2-adrenergic agonist was not influenced with artificial surfactant. These results indicate that artificial surfactant can, in some circumstances, promote tracheal ciliary beat frequency. The average of initial beat frequency of our specimens was 16 Hz. The frequency was comparable with the beat frequency reported in other species (human, 10-14 Hz, Gail and Lenfant, 1983; cat and rat, 12 Hz and 22 Hz, respectively, Dalhamn and Rylander, 1962). It indicates that the initial beat frequency we observed was 'normal'. Application of surfactant caused significant increase in beat frequency. So, surfaetant has an effect to increase ciliary beat frequency. The beat frequency incubated in SES up to 60 min was slightly decreased. It might be due to evaporative loss of water from the trough. The possibility may be, however, very little since the solution was exchanged every 15 min and most part of the trough was covered with thin glass plate. Another explanation of the decline in beat frequency is that exchanging a solution causes loss of factors which prevent the decrease in ciliary beat frequency. This possibility is more likely since ciliary beat frequency was not decreased in the presence of surfactant. In addition prominent action of surfactant on beat frequency was observed in the recovery from injury caused by hydrogen peroxide. So, surfactant likely prevents loss of factors and protects to decrease ciliary beat frequency. We have not determined the exact mechanism of the promoting effect of surfactant on ciliary beat frequency. We can, however, propose several possibilities. First, a more likely explanation of this is that surfactant is changing the theological properties of periciliary fluid since the size of chaoges with surfactant were very small. Second, surfactant may be reversing epithelial damage since ciliary beat frequency was slightly decreased with several exchanges of solutions without surfactant while beat frequency was not decreased in solutions with surfactant. In addition the recovery of ciliary beat frequency from injury caused by hydrogen peroxide was accelerated in the presence of surfactant. Moreover, it should be noticed that dipalmitoyl phosphatidylcholine, which is a major component of the artificial surfactant, is one of important components of cell membrane. Third, the augmentation of ion transport by artificial surfactant is observed in canine tracheal epithelium (lkeda et al., 199,J). It suggests that surfactant acts on
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tracheal epithelium and allows increase in beat frequency. These possibilities likely work on ciliated epithelium and cause promotion of ciliary beat frequency. We studied which component of the artificial surfactant was effective on ciliary beat frequency. Tripalmitin is classified as 'triglyceride', which by itself did not fundamentally make micell in S ES. Phosphatidyl-glycerol dipalmitoyl did not appreciably affect the recovery of beat frequency from injury caused by hydrogen peroxide. Dipalmitoyl phosphatidylcholii:e remarkably accelerated the recovery of beat frequency, although the effect was not significant. So, we presume that dipalmitoyl phosphatidylcholine primarily acts on ciliary beat frequency. There is, however, considerable difference between the effect of artificial surfactant and dipalmitoyl phosphatidylcholine alone. Although we have not determined the exact mechanism of this difference, we can propose several possibilities. First, the micell of dipalmitoyl phosphatidylcholine was macroscopicaUy larger than that of artificial surfaetant. It may reduce the efficacy of the agent. Second, the three components of artificial surfactant make stable miceil and act cooperatively on ciliary beat frequency. We presume that second possibility is more plausible than the first one. The concentration of airway surfactant has not been reported to our knowledge. Slomiany et al. (1982) have reported that the proportion of phospholipid in aspirated tracheobronchial secretion is 2.8% of dry weight of the sample. Assuming 95 to 99% of airway mucus is water, the concentration of phospholipid in the airways is calculated to be 0.028% (280 #g/ml) to 0.14% (1400 #g/ml). The concentration of surfaetant applied for the present study was 500/~g/mi or less. It suggests that the effect of surfaetant observed in the present study is physiologically relevant. The effect of surfactant on the restoration of beat frequency from injury is important for the pathophysiology of airways, since ciliated epithelium is impaired by various toxic agents such as oxidant gases and smokings (Gail and Lenfant, 1983). In addition, it is likely that oxidants from neutrophils and eosinophils cause injury of ciliated epithelium. Therefore, restoration of beat frequency by surfactant is particulary important in the treatment of various kinds of airway disorders. Therefore, the concept that surfaetant has stabilizing effects on ciliary beat frequency is important to the clearance mechanism of the airways, and its application to some pathophysiological states such as chronic bronchitis and bronchial asthma has to be considered. Acknowledgements.We would like to thank Miss R. Haryu for typing the manuscript and Ms P. Tilla and Ms R. Snelling for the critical reading of the manuscript.
References Allegra, L., R. Bossi and P. Braga (1985). Influence of surfactant on mucociliary transport. Eur. J. Respir. Dis. Suppl. 142: 71-76. Dalhamn, T. and Rylander (1962). Frequency of ciliary beat measured with a photo-sensitive cell. Nature (London) 196: 592-593.
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Gail, D.B. and C lJ[ M. Lenfant (1983). Cells of the Lung: biology and clinical implications. Am. Rev. Respir. D/s. 127: 366-387. Gil, J. and E.R. Weibel (1970). Extracellular lining of bronchioles after perfusion-fixation of rat lungs for electron microscopy. Anat. Rec. 169: 185-200. Ikeda, K., T. Sasaki, S. Shimura, M. Sato, H. Ishihara, H. Sasaki, T. Takishima, Y. Saitoh and A. Nishiyama (1990). Effect of surfactant on bioelectric properties of canine tracheal epithelium. Respir. Physiol. 81: 41-49. Kakuta, Y., T. Kanno, H. Sasaki and T. Takishima (1985). Effect of Ca 2 + on the ciliary beat frequency of skinned dog tracheal epithelium. Respir. Physiol. 60: 9-19. Macklem, P.T., D.F. Proctor and J.C. Hogg (1970). The stability of peripheral airways. Respir. Physiol. 8: 191-203. Morgenroth, K. anJ J. Bolz (1985). Morphological features of the interaction between mucus and surfactant on the bronchial mucosa. Respiration 47: 225-231. Rensch, H., H. yon Seefeld, K. F. Gebhardt, D. Renzow and P.-J. Sell (1983). Stop and go particle transport in the peripheral airways'?. A model study. Respiration 44: 346-350. Slomiany, A., V. L. N. Murty, M. Aono, C. E. Snyder, A. Herp and B. L. Slomiany (1982). Lipid composition of tracheobronchial secretions from normal individuals and patients with cystic fibrosis. Bioc,~m. Biophys. Acta 710:106-111. Tierney, D.F. (1989). Lung surfactant: some historical perspectives leading to its cellular and molecular biology. Lung Cell. Mol. Physiol. 1: LI-LI2. Widdicombe, J.G. (1985). Introduction in endobronchial surface active phospholipids. Fur. J. Respir. Dis. Suppl. 142: 1-5. Yoneda, K. (1976). Mucus blanket of rat bronchus. An uitrastructural study. Am. Rev. Respir. D/s. 114: 837-842.