European Journal of Pharmacology, 212 (1992) 101-103
101
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 21008
Short communication
Both ¢1l- and ~2-adrenoceptors are involved in mediating phosphatidyicholine secretion in rat type II pneumocyte cultures Hirofumi Kai, Yoichiro Isohama, Kazunori Takaki, Yoshiaki Oda, Koichiro Murahara, Kazuo T a k a h a m a and Takeshi Miyata Department of Pharmacological Sciences. Faculty of Pharmaceutical Sciences, Kumamoto Unicersity, 5-I Oe-honmachi, Kumamoto 862, Japan Received 10 December 1991, accepted 7 January 1992
A primary culture of rat type II pneumocytes was used for the pharmacological and functional characterization of /3-adrenoceptor subtypes. The /3-adrenoceptor agonists, isoprenaline, dobutamine and procaterol concentration dependently increased the secretion of phosphatidylcholine. These effects were attenuated by propranolol. The effect of dobutamine was attenuated by atenolol, and that of procaterol by ICI 118,551. Isoprenaline-induced secretion was attenuated by the combination of the two blockers but not by each one alone. In conclusion, both /31- and /32-adrenoceptor subtypes mediate phosphatidylcholine secretion in rat type II pneumocytes. /3-Adrenoceptors; Pulmonary surfactant; Type II pneumocyte
1. Introduction
Pulmonary surfactant, which is composed of phospholipids and apoproteins and is produced in type II pneumocytes, lowers the surface tension at the airliquid interface in the lung and provides for alveolar stability. Insufficient surfactant at birth can lead to the respiratory distress syndrome, a leading cause of morbidity among premature infants. Maternal pretreatment with/3-adrenoceptor agonists has been shown to reduce respiratory distress in both prematurely delivered infants and rabbit fetuses (see Oyarzun and Clements, 1978). Our previous finding also indicated that mabuterol, a /32-adrenoceptor agonist, increased the amounts of pulmonary surfactant in rabbit respiratory tract fluid (Miyata et al., 1987). These effects of /3-adrenoceptor agonists are presumably mediated by their action on specific /3-adrenoceptors in type II pneumocytes. However, nothing is known about the functional contribution of the subtype of/3-adrenoceptors to pulmonary surfactant secretion, although several receptor binding studies have shown the presence of/3~- and /32-adrenoceptors (Sommers Smith and Giannopoulos, 1983; Smith and Sidhu, 1984; Das et al., 1987; Jones et al., 1987) in type II pneumocytes. There-
Correspondence to: H. Kai, Department of Pharmacological Sciences, Faculty of Pharmaceutical Sciences, Kumamoto University, 5-10e-honmachi, Kumamoto 862, Japan. Tel. 81.96.344 2111.
fore in the present study we examined the effects of /3I- and /32-adrenoceptor agonists and the antagonists on the secretion of phosphatidylcholine in rat type II pneumocytes.
2. Materials and methods
Type II pneumocytes were isolated from the lungs of adult specific-pathogen-free male Wistar rats (180200 g) according to the method of Dobbs et al. (1986). This method routinely yielded 107 cells per rat. The cells were suspended at 106 cells/ml in Dulbecco's modified Eagle's medium supplemented with 10% featal bovine serum, 2 # C i / m l [methyl-3H]choline (specific activity, 80.0 Ci/mmol), 100 units/ml penicillin and 100 /~g/ml streptomycin, and plated on 24-well tissue culture plate (Falcon 3047) then cultured at 37°C in a 5%CO2-air for 18 h. Non-adherent cells were removed from the wells by washing before the assay. The purity of the type II pneumocytes monolayer was 95 ± 3%. For cellular identification, the sample was stained with a tannic acid and polychrome stain and alkaline phosphatase stain. The viability of type II pneumocytes was 98 ± 2% as judged by the trypan blue exclusion test. Secretion of phosphatidylcholine by cultured type II pneumocytes was determined as follows. The cells were rinsed with fresh serum- and antibiotic-free medium to remove [3H]choline and unattached cells; the test
102
agcnts were addcd aftcr 30 min and the incubation was continued for 90 min. Antagonists wcre added 5 min before the addition of agonists. At the end of the incubation period, the medium was aspirated off, thc cells were lysed with 2 ml ice-cold 0.05% triton X-100 solution and lipids were extracted from both ceils and medium with chloroform and methanol (2: 1, v/v). Phosphatidylcholine was separated from the othcr phospholipids by thin-layer chromatography (Miyata et al., 1987), and its radioactivity was measured with a liquid scintillation counter after the addition of 5 ml Aquasol I1 to each sample. Secretion was expressed as the amount of [3H]phosphatidylcholinc in thc medium after the 90-min incubation, as a percentage of that in cells plus medium. To assess cellular integrity, the activity of lactate dehydrogenase (LDH) in the cells and medium was measured with an LDH kit-s (Nippon Shoji Co., Ltd.). The LDH activity released into the medium did not exceed 1% of the total cell content in all experiments. Statistical analysis was performed by means of Duncan's multiple-range test. The rats werc purchased from Kyudo Farm (Fukuoka, Japan), tissue culture medium from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan) and fetal bovine serum from Sera-Lab Ltd. (Sussex, England). [Methyl-3H]choline, Aquasol II w a s . o b t a i n e d from NEN Research Products (Boston, MA). Procaterol hydrochloridc was from Otuka Pharmaceuticals (Tokushima, Japan), ICI 118, 551 was a kind gift from Dr. Misu at Yokohama City University and other reagents and biochemicals were from Sigma Chemical Co. (St. Louis, MO).
3. Results
Figure 1 shows the effect of increasing concentrations of three/3-adrenoceptor agonists on the secretion of phosphatidyicholine. In these experiments, the mean rate of basal secretion was 0.69 + 0.09% of total cellular phospholipids per 90 min. There were no significant differences in maximum responsiveness to isoprenaline, dobutamine or procaterol. The ECs0 value of isoprenaline was 0.01 /.tM and that of dobutamine and procaterol was 0.15 and 0.10 /zM, respectively. The decrease in stimulation at the highest concentrations (termed 'autoinhibition') of the agonists was observed, as indicated by Dobbs and Mason (1979), but we do not know the reason in this case. Table 1 shows the effects of/31- or fl2-adrenoceptor agonists and each antagonist on phosphatidyicholine secretion in rat type II pneumocytes. At concentration used the antagonists had no effect on the basal secretion of phosphatidylcholine (data not shown). The concentrations of the antagonists were based on PA2 vai-
~
120
o
loo 80
~;' // ~ 40' ~ 20 m
0
9
8 7 g -toO~0[agonistl (M)
g
Fig. 1. Concentration-response curves for the effect of isoprenaline (o), dobutamine ( a ) and procaterol (11) on phosphatidylcholine secretion in type II pneumocytes. The release of [3H]phosphatidylcholine induced by 0.1 /xM isoprenaline minus the basal secretion at the end of a 90-min incubation was normalized to 100% and is indicated on the ordinate. The concentration of isoprenaline, dobufamine and procaterol in samples of cells is expressed on the abscissa. The data are means _+S.E. (bar) from more than six experiments.
ues showing the selectivity against each receptor subtype according to several reports (O'Donnell and Wanstall, 1980; Mahe et al., 1991). The stimulation of phosphatidylcholine secretion induced by 0.1 IzM isoprenaline was significantly attenuated by propranolol at 0.1 IzM, but was not blocked by either 1.0 /.tM atenolol, a selective flt-adrenoceptor antagonist, or 0.1 /xM ICI 118,551, a selective fl2-adrenoceptor antagonist. However, the combination of atenolol and ICI 118,551 significantly attenuated the isoprenaline-induced secretion as potently as did propranoloi. The
TABLE 1 Effect of/3-adrenoceptor antagonists on /3-adrenoceptor agonist-induced secretion of [3H]phosphatidylcholine in type II pneumocytes. Secretion is expressed as the amount of [3Hlphosphatidylcholine in the medium as a percentage of that in cells plus medium at the end of the incubation period. The % change in the secretion attenuated by antagonists was obtained from the comparison with the effect of agonists alone as shown in parentheses. Antagonists were added 5 min before the addition of agonists. The basal secretion represents the 90-min secretion without agonists and antagonists: isoprenalinc, 0.45_+ 0.05; dobutamine, 0.39_+ 0.04; procaterol, 0.505- 0.07%. The data are shown as means _+S.E. from four to six experiments and were analyzed statistically with Duncan's multiple-range test. a p < 0.05 as compared to control group values. N.D.: not done. Antagonists No antagonist
lsoprenaline Dobutamine Procaterol (0.1 /zM) (1.0/~M) (1.0 p.M)
1.22 + 0.08 0.96 5- 0.09 1.24 5- 0.11 (100) (100) (100) Propranolol (0.1 ~M) 0.78+0.08 a 0.54_+0.03 " 0.65_+0.08 a (42.9) (26.3) (20.3) Atenolol (1.0 ~ M ) 1.00+0.09 0.56_+0.05 " 1.32-+0.06 (71.4) (29.8) (114) ICI 118,551 (0.1 IzM) 0.94_+0.07 0.86-+0.04 0.61 -+0.16 ~ (63.6) (82.5) (14.9) Atenolol (1.0 p.M) 0.68+_0.18 " N.D. N.D. +ICI 118,551 (0.1 /zM) (29.9)
103
stimulation by dobutamine 1.0 /xM was attenuated by atenolol but not by ICI 118,551. On the other hand, the procaterol-induced secretion was attenuated by ICI 118,551 but not by atenolol.
4. Discussion
Regarding the stimulatory effect of/3-adrenoceptor agonists, Dobbs and Mason (1979) previously reported that the secretion of phosphatidylcholine was the result of /32- rather than /31-adrenoceptor stimulation based on the effect of a selective /32-adrenoceptor agonist, terbutaline, and suggested that this issue would be best decided by the results of experiments with selective/31and /32-adrenoceptor antagonists. Therefore, to explore the functional roles of/3-adrenoceptor subtypes in phosphatidylcholine secretion in type II pneumocytes, we used both atenolol, a selective/31-adrenoceptor antagonist, and ICI 118,551, a selective/32-adrenoceptor antagonist, as well as several selective/3-adrenoceptor agonists. The present study indicated that both /3-adrenoceptors contribute almost equally to phosphatidylcholine secretion. Isoprenaline, which generally interacts with both /3-adrenoceptors, stimulated the release of phosphatidylcholine. The stimulatory effect was blocked by the combination of atenolol and ICI 118,551, as well as by propranolol, a non-selective adrenoceptor antagonist. The effect of either atenolol or ICI 118,551 alone was not sufficient to block the stimulation. This suggests that each receptor has a compensatory function. However, the mechanism still remains unclear. It is likely that isoprenaline stimulates phosphatidylcholine secretion via both /3-adrenoceptors. This possibility is supported by the present findings that the effect of dobutamine was attenuated by atenolol but not by ICI 118,551 and in contrast, that the effect of procaterol, a more selective /32-adrenoceptor agonist than terbutaline, was attenuated reversibly. Additionally, the selective actions of the antagonists suggest that in the present study the concentration of drugs used was optimal for distinguishing the share of each /3-adrenoceptor in the effect. The coexistence of/31- and /3~-adrenoceptors in the same tissue is not exceptional, as demonstrated in the guinea-pig trachea and other tissues (see Mahe et al., 1991, for more examples). The presence of both /31and /32-adrenoceptors in type II pneumocytes was shown using a receptor binding assay. Sommers Smith and Giannopoulos (1983) showed the presence of specific, high-affinity, low-capacity /3-adrenoceptors in rabbit type II pneumocytes, and suggested that the receptors exhibited binding characteristics consistent with the /31-adrenoceptor subtype. Das et al. (1987) also reported that [3H]dihydroalprenolol binding to guinea-pig type II pneumocytes revealed the presence
of both high- and low-affinity fl-adrenoceptors, and that the low-affinity site (fit) had a higher binding capacity than the high-affinity site (/32). Furthermore, a 1:3 ratio of /3t- to /32-adrenoceptor was reported by Jones et al. (1987) for rat type II pneumocytes. The present results distinguished functional roles for both /3-adrenoceptors in the secretion of phosphatidylcholine. However, it remains possible that proteolytic enzymes used in cell isolation procedures may alter the cellular response a n d / o r the receptor structure and, in addition, that variation of the receptor population may occur during culture. However, Smith and Sidhu (1984) described the existence of specific/3-adrenoceptors on adult rat type II pneumocytes based on their experiments with in vivo autoradiographic demonstration of /3-adrenergic binding sites. Further, there are several reports that /3-adrenoceptor agonists stimulated pulmonary surfactant secretion in vivo (Oyarzun and Clements, 1978; Miyata et al., 1987). Therefore, we believe that the response to /3-adrenergic stimuli is unlikely to be merely an artifact due to the isolation and culture procedure. In conclusion, both /31- and /32-adrenoceptor subtypes are present in rat type II pneumocytes, each one mediating the secretion of phosphatidyicholine. References Das, S.K., M.O. Sikpi and P. Skolnick, 1987, Heterogeneity of fl-adrenoceptors in guinea pig alveolar type II cells, Biochem. Biophys. Res. Commun. 142, 898. Dobbs, L.G. and R.J. Mason, 1979, Pulmonary alveolar type II cells isolated from rats. Release of phosphatidylcholine in response to ~-adrenergic stimulation, J. Clin. Invest. 63, 378. Dobbs, L.G., R. Gonzalez and M.C. Williams, 1986, An improved method for isolating type II cells in high yield and purity, Am. Rev. Respir. Dis. 134, 141. Jones, L.M., M.E. Gray, A.J.J. Wood and V.S. LeQiure, 1987, Beta-adrenergic receptor properties of a pulmonary alveolar type II cell preparation from the adult rat, Lung 165, 201. Mahe, L., B. Chapelain, Y.-M. Gargouil and G. Neliat, 1991, Characterization of/3-adrenoceptor subtypes and indications for two cell populations in isolated bovine mesenteric lymphatic vessels, Eur. J. Pharmacol. 199, 19. Miyata, T., H. Kai, K. Furusawa, H. Nakamura, M. Saito, Y. Okano and K. Takahama, 1987, Secretomotor and mucolytic effects of mabuterol, a novel bronchodilator, Arch. Int. Pharmacodyn, Ther. 288, 147. O'Donnell, S.R. and J.C. Wanstall, 1980, Evidence that ICI 118,551 is a potent, highly, beta2-selective adrenoceptor antagonist and can be used to characterize beta-adrenoceptor populations in tissues, Life Sci. 27, 671. Oyarzun, M.J. and R.J. Clements, 1978, Control of lung surfactant by ventilation, adrenergic mediators, and prostaglandins in the rabbits, Am. Rev. Respir. Dis. 117, 879. Smith, D.M. and N.K. Sidhu, 1984, In vivo autoradiographic demonstration of/3-adrenergic binding sites in adult rat type II alveolar epithelial cells, Life Sci. 34, 519. Sommers Smith, S.K. and G. Giannopoulos, 1983, Identification of /3-adrenergic receptors in pulmonary alveolar type II cells, Life Sci. 33, 2071.
101
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 21008
Short communication
Both ¢1l- and ~2-adrenoceptors are involved in mediating phosphatidyicholine secretion in rat type II pneumocyte cultures Hirofumi Kai, Yoichiro Isohama, Kazunori Takaki, Yoshiaki Oda, Koichiro Murahara, Kazuo T a k a h a m a and Takeshi Miyata Department of Pharmacological Sciences. Faculty of Pharmaceutical Sciences, Kumamoto Unicersity, 5-I Oe-honmachi, Kumamoto 862, Japan Received 10 December 1991, accepted 7 January 1992
A primary culture of rat type II pneumocytes was used for the pharmacological and functional characterization of /3-adrenoceptor subtypes. The /3-adrenoceptor agonists, isoprenaline, dobutamine and procaterol concentration dependently increased the secretion of phosphatidylcholine. These effects were attenuated by propranolol. The effect of dobutamine was attenuated by atenolol, and that of procaterol by ICI 118,551. Isoprenaline-induced secretion was attenuated by the combination of the two blockers but not by each one alone. In conclusion, both /31- and /32-adrenoceptor subtypes mediate phosphatidylcholine secretion in rat type II pneumocytes. /3-Adrenoceptors; Pulmonary surfactant; Type II pneumocyte
1. Introduction
Pulmonary surfactant, which is composed of phospholipids and apoproteins and is produced in type II pneumocytes, lowers the surface tension at the airliquid interface in the lung and provides for alveolar stability. Insufficient surfactant at birth can lead to the respiratory distress syndrome, a leading cause of morbidity among premature infants. Maternal pretreatment with/3-adrenoceptor agonists has been shown to reduce respiratory distress in both prematurely delivered infants and rabbit fetuses (see Oyarzun and Clements, 1978). Our previous finding also indicated that mabuterol, a /32-adrenoceptor agonist, increased the amounts of pulmonary surfactant in rabbit respiratory tract fluid (Miyata et al., 1987). These effects of /3-adrenoceptor agonists are presumably mediated by their action on specific /3-adrenoceptors in type II pneumocytes. However, nothing is known about the functional contribution of the subtype of/3-adrenoceptors to pulmonary surfactant secretion, although several receptor binding studies have shown the presence of/3~- and /32-adrenoceptors (Sommers Smith and Giannopoulos, 1983; Smith and Sidhu, 1984; Das et al., 1987; Jones et al., 1987) in type II pneumocytes. There-
Correspondence to: H. Kai, Department of Pharmacological Sciences, Faculty of Pharmaceutical Sciences, Kumamoto University, 5-10e-honmachi, Kumamoto 862, Japan. Tel. 81.96.344 2111.
fore in the present study we examined the effects of /3I- and /32-adrenoceptor agonists and the antagonists on the secretion of phosphatidylcholine in rat type II pneumocytes.
2. Materials and methods
Type II pneumocytes were isolated from the lungs of adult specific-pathogen-free male Wistar rats (180200 g) according to the method of Dobbs et al. (1986). This method routinely yielded 107 cells per rat. The cells were suspended at 106 cells/ml in Dulbecco's modified Eagle's medium supplemented with 10% featal bovine serum, 2 # C i / m l [methyl-3H]choline (specific activity, 80.0 Ci/mmol), 100 units/ml penicillin and 100 /~g/ml streptomycin, and plated on 24-well tissue culture plate (Falcon 3047) then cultured at 37°C in a 5%CO2-air for 18 h. Non-adherent cells were removed from the wells by washing before the assay. The purity of the type II pneumocytes monolayer was 95 ± 3%. For cellular identification, the sample was stained with a tannic acid and polychrome stain and alkaline phosphatase stain. The viability of type II pneumocytes was 98 ± 2% as judged by the trypan blue exclusion test. Secretion of phosphatidylcholine by cultured type II pneumocytes was determined as follows. The cells were rinsed with fresh serum- and antibiotic-free medium to remove [3H]choline and unattached cells; the test
102
agcnts were addcd aftcr 30 min and the incubation was continued for 90 min. Antagonists wcre added 5 min before the addition of agonists. At the end of the incubation period, the medium was aspirated off, thc cells were lysed with 2 ml ice-cold 0.05% triton X-100 solution and lipids were extracted from both ceils and medium with chloroform and methanol (2: 1, v/v). Phosphatidylcholine was separated from the othcr phospholipids by thin-layer chromatography (Miyata et al., 1987), and its radioactivity was measured with a liquid scintillation counter after the addition of 5 ml Aquasol I1 to each sample. Secretion was expressed as the amount of [3H]phosphatidylcholinc in thc medium after the 90-min incubation, as a percentage of that in cells plus medium. To assess cellular integrity, the activity of lactate dehydrogenase (LDH) in the cells and medium was measured with an LDH kit-s (Nippon Shoji Co., Ltd.). The LDH activity released into the medium did not exceed 1% of the total cell content in all experiments. Statistical analysis was performed by means of Duncan's multiple-range test. The rats werc purchased from Kyudo Farm (Fukuoka, Japan), tissue culture medium from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan) and fetal bovine serum from Sera-Lab Ltd. (Sussex, England). [Methyl-3H]choline, Aquasol II w a s . o b t a i n e d from NEN Research Products (Boston, MA). Procaterol hydrochloridc was from Otuka Pharmaceuticals (Tokushima, Japan), ICI 118, 551 was a kind gift from Dr. Misu at Yokohama City University and other reagents and biochemicals were from Sigma Chemical Co. (St. Louis, MO).
3. Results
Figure 1 shows the effect of increasing concentrations of three/3-adrenoceptor agonists on the secretion of phosphatidyicholine. In these experiments, the mean rate of basal secretion was 0.69 + 0.09% of total cellular phospholipids per 90 min. There were no significant differences in maximum responsiveness to isoprenaline, dobutamine or procaterol. The ECs0 value of isoprenaline was 0.01 /.tM and that of dobutamine and procaterol was 0.15 and 0.10 /zM, respectively. The decrease in stimulation at the highest concentrations (termed 'autoinhibition') of the agonists was observed, as indicated by Dobbs and Mason (1979), but we do not know the reason in this case. Table 1 shows the effects of/31- or fl2-adrenoceptor agonists and each antagonist on phosphatidyicholine secretion in rat type II pneumocytes. At concentration used the antagonists had no effect on the basal secretion of phosphatidylcholine (data not shown). The concentrations of the antagonists were based on PA2 vai-
~
120
o
loo 80
~;' // ~ 40' ~ 20 m
0
9
8 7 g -toO~0[agonistl (M)
g
Fig. 1. Concentration-response curves for the effect of isoprenaline (o), dobutamine ( a ) and procaterol (11) on phosphatidylcholine secretion in type II pneumocytes. The release of [3H]phosphatidylcholine induced by 0.1 /xM isoprenaline minus the basal secretion at the end of a 90-min incubation was normalized to 100% and is indicated on the ordinate. The concentration of isoprenaline, dobufamine and procaterol in samples of cells is expressed on the abscissa. The data are means _+S.E. (bar) from more than six experiments.
ues showing the selectivity against each receptor subtype according to several reports (O'Donnell and Wanstall, 1980; Mahe et al., 1991). The stimulation of phosphatidylcholine secretion induced by 0.1 IzM isoprenaline was significantly attenuated by propranolol at 0.1 IzM, but was not blocked by either 1.0 /.tM atenolol, a selective flt-adrenoceptor antagonist, or 0.1 /xM ICI 118,551, a selective fl2-adrenoceptor antagonist. However, the combination of atenolol and ICI 118,551 significantly attenuated the isoprenaline-induced secretion as potently as did propranoloi. The
TABLE 1 Effect of/3-adrenoceptor antagonists on /3-adrenoceptor agonist-induced secretion of [3H]phosphatidylcholine in type II pneumocytes. Secretion is expressed as the amount of [3Hlphosphatidylcholine in the medium as a percentage of that in cells plus medium at the end of the incubation period. The % change in the secretion attenuated by antagonists was obtained from the comparison with the effect of agonists alone as shown in parentheses. Antagonists were added 5 min before the addition of agonists. The basal secretion represents the 90-min secretion without agonists and antagonists: isoprenalinc, 0.45_+ 0.05; dobutamine, 0.39_+ 0.04; procaterol, 0.505- 0.07%. The data are shown as means _+S.E. from four to six experiments and were analyzed statistically with Duncan's multiple-range test. a p < 0.05 as compared to control group values. N.D.: not done. Antagonists No antagonist
lsoprenaline Dobutamine Procaterol (0.1 /zM) (1.0/~M) (1.0 p.M)
1.22 + 0.08 0.96 5- 0.09 1.24 5- 0.11 (100) (100) (100) Propranolol (0.1 ~M) 0.78+0.08 a 0.54_+0.03 " 0.65_+0.08 a (42.9) (26.3) (20.3) Atenolol (1.0 ~ M ) 1.00+0.09 0.56_+0.05 " 1.32-+0.06 (71.4) (29.8) (114) ICI 118,551 (0.1 IzM) 0.94_+0.07 0.86-+0.04 0.61 -+0.16 ~ (63.6) (82.5) (14.9) Atenolol (1.0 p.M) 0.68+_0.18 " N.D. N.D. +ICI 118,551 (0.1 /zM) (29.9)
103
stimulation by dobutamine 1.0 /xM was attenuated by atenolol but not by ICI 118,551. On the other hand, the procaterol-induced secretion was attenuated by ICI 118,551 but not by atenolol.
4. Discussion
Regarding the stimulatory effect of/3-adrenoceptor agonists, Dobbs and Mason (1979) previously reported that the secretion of phosphatidylcholine was the result of /32- rather than /31-adrenoceptor stimulation based on the effect of a selective /32-adrenoceptor agonist, terbutaline, and suggested that this issue would be best decided by the results of experiments with selective/31and /32-adrenoceptor antagonists. Therefore, to explore the functional roles of/3-adrenoceptor subtypes in phosphatidylcholine secretion in type II pneumocytes, we used both atenolol, a selective/31-adrenoceptor antagonist, and ICI 118,551, a selective/32-adrenoceptor antagonist, as well as several selective/3-adrenoceptor agonists. The present study indicated that both /3-adrenoceptors contribute almost equally to phosphatidylcholine secretion. Isoprenaline, which generally interacts with both /3-adrenoceptors, stimulated the release of phosphatidylcholine. The stimulatory effect was blocked by the combination of atenolol and ICI 118,551, as well as by propranolol, a non-selective adrenoceptor antagonist. The effect of either atenolol or ICI 118,551 alone was not sufficient to block the stimulation. This suggests that each receptor has a compensatory function. However, the mechanism still remains unclear. It is likely that isoprenaline stimulates phosphatidylcholine secretion via both /3-adrenoceptors. This possibility is supported by the present findings that the effect of dobutamine was attenuated by atenolol but not by ICI 118,551 and in contrast, that the effect of procaterol, a more selective /32-adrenoceptor agonist than terbutaline, was attenuated reversibly. Additionally, the selective actions of the antagonists suggest that in the present study the concentration of drugs used was optimal for distinguishing the share of each /3-adrenoceptor in the effect. The coexistence of/31- and /3~-adrenoceptors in the same tissue is not exceptional, as demonstrated in the guinea-pig trachea and other tissues (see Mahe et al., 1991, for more examples). The presence of both /31and /32-adrenoceptors in type II pneumocytes was shown using a receptor binding assay. Sommers Smith and Giannopoulos (1983) showed the presence of specific, high-affinity, low-capacity /3-adrenoceptors in rabbit type II pneumocytes, and suggested that the receptors exhibited binding characteristics consistent with the /31-adrenoceptor subtype. Das et al. (1987) also reported that [3H]dihydroalprenolol binding to guinea-pig type II pneumocytes revealed the presence
of both high- and low-affinity fl-adrenoceptors, and that the low-affinity site (fit) had a higher binding capacity than the high-affinity site (/32). Furthermore, a 1:3 ratio of /3t- to /32-adrenoceptor was reported by Jones et al. (1987) for rat type II pneumocytes. The present results distinguished functional roles for both /3-adrenoceptors in the secretion of phosphatidylcholine. However, it remains possible that proteolytic enzymes used in cell isolation procedures may alter the cellular response a n d / o r the receptor structure and, in addition, that variation of the receptor population may occur during culture. However, Smith and Sidhu (1984) described the existence of specific/3-adrenoceptors on adult rat type II pneumocytes based on their experiments with in vivo autoradiographic demonstration of /3-adrenergic binding sites. Further, there are several reports that /3-adrenoceptor agonists stimulated pulmonary surfactant secretion in vivo (Oyarzun and Clements, 1978; Miyata et al., 1987). Therefore, we believe that the response to /3-adrenergic stimuli is unlikely to be merely an artifact due to the isolation and culture procedure. In conclusion, both /31- and /32-adrenoceptor subtypes are present in rat type II pneumocytes, each one mediating the secretion of phosphatidyicholine. References Das, S.K., M.O. Sikpi and P. Skolnick, 1987, Heterogeneity of fl-adrenoceptors in guinea pig alveolar type II cells, Biochem. Biophys. Res. Commun. 142, 898. Dobbs, L.G. and R.J. Mason, 1979, Pulmonary alveolar type II cells isolated from rats. Release of phosphatidylcholine in response to ~-adrenergic stimulation, J. Clin. Invest. 63, 378. Dobbs, L.G., R. Gonzalez and M.C. Williams, 1986, An improved method for isolating type II cells in high yield and purity, Am. Rev. Respir. Dis. 134, 141. Jones, L.M., M.E. Gray, A.J.J. Wood and V.S. LeQiure, 1987, Beta-adrenergic receptor properties of a pulmonary alveolar type II cell preparation from the adult rat, Lung 165, 201. Mahe, L., B. Chapelain, Y.-M. Gargouil and G. Neliat, 1991, Characterization of/3-adrenoceptor subtypes and indications for two cell populations in isolated bovine mesenteric lymphatic vessels, Eur. J. Pharmacol. 199, 19. Miyata, T., H. Kai, K. Furusawa, H. Nakamura, M. Saito, Y. Okano and K. Takahama, 1987, Secretomotor and mucolytic effects of mabuterol, a novel bronchodilator, Arch. Int. Pharmacodyn, Ther. 288, 147. O'Donnell, S.R. and J.C. Wanstall, 1980, Evidence that ICI 118,551 is a potent, highly, beta2-selective adrenoceptor antagonist and can be used to characterize beta-adrenoceptor populations in tissues, Life Sci. 27, 671. Oyarzun, M.J. and R.J. Clements, 1978, Control of lung surfactant by ventilation, adrenergic mediators, and prostaglandins in the rabbits, Am. Rev. Respir. Dis. 117, 879. Smith, D.M. and N.K. Sidhu, 1984, In vivo autoradiographic demonstration of/3-adrenergic binding sites in adult rat type II alveolar epithelial cells, Life Sci. 34, 519. Sommers Smith, S.K. and G. Giannopoulos, 1983, Identification of /3-adrenergic receptors in pulmonary alveolar type II cells, Life Sci. 33, 2071.
