Br. J. Pharmacol. (1 992), 106, 373 - 379
(D Macmillan Press Ltd,
1992
Cibacron blue stimulation of surfactant secretion in rat type II pneumocytes 'Matthias Griese, Laurice
I.
Gobran & 2Seamus A. Rooney
Division of Perinatal Medicine, Department of Pediatrics, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510, U.S.A. 1 The effect of cibacron blue, a selective P2y-purinoceptor antagonist in some systems, on the stimulatory effect of adenosine 5'-triphosphate (ATP) on [3H]-phosphatidylchloine secretion was examined in primary cultures of rat type II pneumocytes prelabelled by overnight culture with [3H]-choline. 2 Cibacron blue alone stimulated phosphatidylcholine secretion in a concentration-dependent manner in the range 10--10-3 M. At a concentration of 10- or lower, cibacron blue had no effect on ATP-induced phosphatidylcholine secretion but at 10-4-10-3 M it increased the effect of ATP. Enhancement of the ATP effect was apparent whether cibacron blue was added before or together with ATP. Cibacron blue also increased ATP-induced secretion in the presence of the PI-purinoceptor antagonist, xanthine amine congener (10-5 M). 3 The stimulatory effect of cibacron blue on phosphatidylcholine secretion was additive to those of 5' (N-ethylcarboxyamido) adenosine (NECA) and terbutaline but less than additive to that of ATP. 4 Cibacron blue alone had no effect on formation of cyclic AMP or inositol phosphate and when added stimultaneously with ATP it did not affect the ATP-induced increase in these second messengers. Preincubation of the cells with cibacron blue before addition of ATP, however, resulted in antagonism of the ATP-induced increase in cyclic AMP and inositol phosphates. Preincubation with ATP had the same effect. The stimulatory effects of NECA and terbutaline on cyclic AMP formation were enhanced by preincubation with cibacron blue. 5 Thus, ATP-induced phosphatidylcholine secretion in type II cells is not diminished by the P2yantagonist, cibacron blue. On the contrary, cibacron blue stimulates phosphatidylcholine secretion. Cibacron blue may act as a P2-agonist in type II pneumocytes. Keywords: Pulmonary surfactant; lung; phosphatidylcholine secretion; P2-purinoceptor; ATP; inositol trisphosphate; cyclic AMP; suramin
Introduction Lung surfactant, phospholipid-rich material lining the alveolar surface, is synthesized and stored in the type II pneumocyte and secreted by exocytosis (Chander & Fisher, 1990; Haagsman & van Golde, 1991; Wright & Dobbs, 1991). Secretion of surfactant phospholipids has been shown to be influenced by a variety of physiological and pharmacological agents (Chander & Fisher, 1990; Wright & Dobbs, 1991). A role for purinoceptor agonists in the regulation of surfactant secretion is suggested by the findings that secretion of phosphatidylcholine, its major lipid component (Haagsman & van Golde, 1991), was stimulated by adenosine, adenosine analogues and adenine nucleotides in primary cultures of type II cells (Gilfillan & Rooney, 1987a,b; 1988; Gobran & Rooney, 1990; Griese et al., 1991a,c; Rice & Singleton, 1986; 1987; Warburton et al., 1989) while surfactant secretion in vivo was stimulated by adenosine (Ekelund et al., 1985) and inhibited by an adenosine receptor antagonist (Rooney & Gobran, 1988). There is functional evidence for both P1- (adenosine) and P2- (adenosine 5'-triphosphate, ATP) purinoceptors on the type II cell. The stimulatory effects of adenosine and its analogues on phosphatidylcholine secretion were shown to be mediated by the A2 subtype of the PI-receptor (Gilfillan & Rooney, 1987b; Griese et al., 1991a) and there is also evidence of an inhibitory Al-receptor on the type II cell (Gobran & Rooney, 1990). We recently found that a large part of the action of ATP on the type II cell was mediated by the P1 receptor since as much as 75% of its effect on phosphatidyl-
' Present address: Ludwig-Maximilians-Universitat, Kinderpoliklinik, PettenkoferstraBe 8a, 8000 Munchen 2, Germany. Author for correspondence.
2
choline secretion was antagonized by 8-[4-(2-aminoethylaminocarbonylmethyloxy)phenyl]-1,3-dipropylxanthine (xanthine amine congener, XAC) and 8-phenyltheophylline (Griese et al., 1991c). However, there is evidence that ATP and ATP analogues also promote phosphatidylcholine secretion by action at a P2-receptor (Gilfillan & Rooney, 1988; Greise et al., 1991c; Rice & Singleton, 1987) that is coupled to activation of phosphoinositide-specific phospholipase C (Warburton et al., 1989; Rice et al., 1990; Griese et al., 199 1b). P2-receptors have been subdivided into P2,- and P2y-types (Burnstock & Kennedy, 1985) although at least three other subtypes have also been proposed (Gordon, 1986; Wiklund & Gustafsson, 1988; Stone, 1991). However, the receptor mediating the effect of ATP and its analogues in the type II cell does not fit the potency orders described for any of those (Rooney, 1990). Rice & Singleton (1987) proposed a classical P2y-subtype for the type II cell P2-receptor. This, however, is not supported by the potency order since 2-methylthio ATP is reported to be the most potent agonist at the P2y-receptor (Burnstock & Kennedy, 1985) and it was considerably less potent than ATP in the type II cell (Gilfillan & Rooney, 1988; Griese et al., 1991c). Cibacron blue, previously termed reactive blue 2 (Sigma Chemical Co., St. Louis, MO, U.S.A.), was reported to be a specific antagonist of the P2y-receptor in some systems (Burnstock & Warland, 1987; Hopwood & Burnstock, 1987). Rice & Singleton (1989) found that cibacron blue antagonized the effect of ATP in the type II cell but in a preliminary study we were unable to confirm that finding (Rooney, 1990; Rooney et al., 1988; 1990). We have now examined the effect of cibacron blue in more detail and show that it has unexpected actions in combination with other agents on both phosphatidylcholine secretion and second messenger formation.
374
M. GRIESE et al.
Methods
Isolation and culture to type II cells Type II cells were isolated from the lungs of adult rats by the method of Dobbs et al. (1986) as described previously (Gobran & Rooney, 1990). This method involves digestion of the blood-free lungs with elastase and separation of type II pneumocytes from contaminating cells by panning on bacteriological dishes coated with IgG. The freshly isolated cells were plated at a density of 2-5 x 106 cells per dish on 35 mm i.d. plastic dishes and cultured in 1.5 ml Dulbecco's Modified Eagle's Medium (DMEM) containing 10% foetal bovine serum, streptomycin (10 tg ml-'), penicillin (100 u ml-') and, when indicated, radiochemicals for 18-20 h at 370C in a humidified atmosphere of 90% air: 10% CO2. At least 95% of the cells attached to the dishes after this period of culture were identifiable as type II cells (Gobran & Rooney, 1990).
Adenosine 3':5'-cyclic monophosphate (cyclic AMP) assay The medium was rapidly aspirated and 1 ml of ice-cold 0.1 M HC1 was added to the dish which was placed on an ice-cooled surface. The HCl extract was evaporated to dryness at 40'C under a stream of air and the sample was stored at - 70C. After reconstitution and acetylation the cyclic AMP content was determined following the procedure described in the RIA kit.
Lactate dehydrogenase assay The rate of lactate dehydrogenase release into the medium was determined to assess cellular integrity. The cells were cultured as in the secretion experiments but in the absence of radioactivity after which lactate dehydrogenase activity in the cells and medium was assayed by measuring disappearance of NADH at 340nm (Wroblewski & LaDue, 1955).
Drug treatment
Materials
After culture of the cells for 18-20 h the medium was removed and the cells were rinsed with fresh DMEM without serum, antibiotics or radiochemicals. Fresh DMEM was then added and the dishes were returned to the incubator for a 30 min preincubation period. Secretagogues were then added and the incubation continued for various time periods. Antagonists were generally added at the beginning of the preincubation period. Where indicated, adenosine deaminase (3 u ml-') was also added at this stage. XAC was dissolved in dimethylsulphoxide (1% final concentration) and this solvent was added to the corresponding control dishes. The other drugs were dissolved in DMEM.
Adult male Sprague-Dawley rats (200-300 g) were purchased from Charles River Breeding Laboratories (Kingston, NY, U.S.A.), rat IgG from Calbiochem (San Diego, CA, U.S.A.), porcine pancreatic elastase from Elastin Products (Owensville, MO, U.S.A.), foetal bovine serum from HyClone Laboratories (Logan, UT, U.S.A.), Dulbecco's modified Eagle's medium (DMEM) and myo-inositol-free DMEM from GIBCO, Grand Island, NY, U.S.A.; [methyl-3H]-choline chloride from NEN-du Pont (Wilmington, DE, U.S.A.), myo-[2-3H]-inositol from Amersham (Arlington Heights, IL, U.S.A.), cyclic AMP RIA kits from Biomedical Technologies (Cambridge, MA, U.S.A), XAC from Research Biochemicals (Natick, MA, U.S.A.), terbutaline sulphate (Brethine) from Geigy Pharmaceuticals (West Caldwell, NJ, U.S.A.), 5' (Nethylcarboxyamido)adenosine (NECA), disodium ATP, a, Pmethylene ATP, cibacron blue 3GA (Lot 50F-03341), calf intestinal mucosal adenosine deaminase (type VIII) and other biochemicals from Sigma (St. Louis, MO, U.S.A.). Suramin was a gift from Dr John Becher, Centers for Disease Control (Atlanta, GA, U.S.A.).
Phosphatidylcholine secretion [3H]-choline (2 1Ci ml-) was included in the medium during the overnight culture of the cells (2 x 106 per dish). With the exception of the time course experiments, secretion was measured 90 min after addition of the secretagogues. The medium was aspirated and centrifuged at 200g for 10min while the attached cells were lysed by addition of ice-cold water. Lipids were extracted from both cells and medium with a mixture of chloroform and methanol by the method of Bligh & Dyer (1959) and total lipid radioactivity was quantified by liquid scintillation counting (Gobran & Rooney, 1990). The total amount of lipid radioactivity ranged from 150,000 to 700,000 c.p.m. per dish. We previously reported that under these conditions phosphatidylcholine accounts for more than 95% of the lipid radioactivity in both cells and medium (Gobran & Rooney, 1990) while sphingomyelin and lysophosphatidylcholine account for the remainder. Phosphatidylcholine secretion is therefore expressed as the amount of lipid radioactivity in the medium after the 2 h incubation as a percentage of the total in cells and medium combined.
Measurement of [3HJ-inositol phosphates The overnight culture of the cells (3-5 x 106 per dish) was carried out in inositol-free DMEM to which [3H]-inositol (20 pCi ml', 80-120 Ci mmol-') was added (Griese et al., 1991b). Formation of inositol phosphates was measured in the presence of 20 mM LiCl as described previously in detail (Griese et al., 1991b). Briefly, the reaction was terminated at various times after addition of the secretagogues by rapid aspiration of the medium and addition of ice-cold perchloric acid. [3H]-inositol phosphates were then extracted, fractionated into individual components on ion exchange columns by a modification of the method of Berridge (1986) and quantified by liquid scintillation counting.
Statistics In the secretion experiments type II cells were isolated from the pooled lungs of three rats and distributed among the various treatment groups, three dishes per group. The dishes were processed separately and the mean values calculated to yield a single data point per group per experiment. In the second messenger experiments the cells were isolated from the pooled lungs of four rats and one dish was used at each time point per experiment. Cyclic AMP extracts were analyzed in duplicate and the mean values calculated to yield a single data point per time point per experiment. All data are mean ± s.e.mean from the number of individual experiments indicated. As the cyclic AMP values were log normally distributed, they were normalized by a log transfusion prior to statistical analysis. The data were analyzed statistically by analysis of variance (ANOVA) followed by Fisher's least significance difference test or by Student's two tailed t test for paired samples with correction for multiple comparisons when necessary (Rohlf & Sokal, 1981).
Results
Phosphatidylcholine secretion Cibacron blue alone at low concentrations had no effect on phosphatidylcholine secretion in type II cells but stimulated in a concentration-dependent manner at higher concentrations (Figure 1). As shown previously (Rooney et al., 1988;
CIBACRON BLUE AND SURFACTANT SECRETION 600
I
500 c
o 400 =
0
300)I
i,
co
'--
200)I 100
0 6
7
5
4
3
Cibacron blue (-log M) Figure 1 Phosphatidylcholine secretion in type II cells in response to various concentrations of cibacron blue alone and in combination with ATP and ATP+ xanthine amine congener (XAC). The cells were cultured for 18-20h in the presence of [3H]-choline, washed with fresh medium and preincubated with the indicated concentrations of cibacron blue with and without XAC (10-5 M) for 30 min. ATP (10-I M) was then added and the incubation continued for 90 min after which phosphatidylcholine secretion was measured. When the effect of cibacron blue alone was to be examined it was added at the end of the preincubation period: (0) cibacron blue alone; (0) cibacron blue + ATP; (A) cibacron blue + ATP + XAC. The data are expressed as % stimulation over the basal rate of secretion in control cells incubated with vehicle only and are means ± s.e.mean (bar) from 3 experiments except for 10- M and 5 x 10- M cibacron blue + ATP which are from I and 9 experiments, respectively, and 0-7I M to 5 x 0-5I M cibacron blue alone, 6 x 0-5I M cibacron blue + ATP and 5 x 10-5 M cibacron blue + ATP + XAC which are means ± range from 2 experiments. The basal rate of secrtion was 1.13 ± 0.07% of cellular [3H]-phosphatidylcholine secreted in 2 h (n = 14). The rate of secretion in cells treated with 10-4 M, 5 X 10-5 M and 10-3 M cibacron blue was significantly different from that in the controls (P ,-methylene ATP in the type II cells (Gilfillan & Rooney, 1988). Such differences suggest that the P2-receptors in these two different respiratory tissue cells are not identical. Our data are different from those of Rice & Singleton (1989) who found that cibacron blue inhibited ATP-stimulated phosphatidylcholine secretion in type II cells. The inhibition was reported to be dependent on concentration with an IC50 of approx. 1.5 x 10-4 M. However, some inhibition was observed even at the lowest concentration examined (Rice & Singleton, 1989). Whereas we found that the stimulatory effects of cibacron blue and terbutaline were additive, Rice & Singleton (1989) reported that cibacron blue did not enhance the effect of terbutaline on phosphatidylcholine secretion. The reason for the different findings are not clear. They are not due to differences in the purity of cibacron blue since its source and lot number were the same in both studies (Rice, W.R., personal communication). Rice & Singleton (1989) did not examine the effect of cibacron blue alone or the time
course of its action. Our data show that the onset of the effect of cibacron blue in combination with ATP was very rapid and that the effects of ATP and cibacron blue were not additive in contrast to those of terbutaline and cibacron blue which were. As Rice & Singleton (1989) subtracted the amount of [3H]-phosphatidylcholine that was released during the 30 min preincubation from the total released at the completion of the incubation, an apparent inhibition of the effect of ATP and no enhancement of the effect of terbutaline might have been noted. Other possible differences include use of collagen-coated tissue culture dishes in the study of Rice & Singleton (1989) and our inclusion of adenosine deaminase in the terbutaline experiments. The mechanism by which cibacron blue stimulates phosphatidylcholine secretion remains to be elucidated. That its effect on secretion was additive to those of NECA and terbutaline suggests that its action does not involve activation of adenylate cyclase. This conclusion is supported by the finding that cibacron blue did not increase cellular cyclic AMP levels. That its effect on ATP-stimulated secretion was apparent in the presence of XAC further shows that it does not act via a PI-receptor. That the stimulatory effects of cibacron blue and ATP were not completely additive suggests that the mechanism by which these two agents stimulate phosphatidylcholine secretion are not independent of each other. ATP has been found to increase formation of cyclic AMP (Gilfillan & Rooney, 1987b; Warburton et al., 1989; Griese et al., 1991c), IP3 (Warburton et al., 1989; Rice et al., 1990; Griese et al., 1991b) and diacylglycerols (Rice et al., 1990; Griese et al., 1991b) as well as promote mobilization of intracellular Ca2" (Rice & Singleton, 1987) in type II cells. In addition to the lack of an increase in cyclic AMP, cibacron blue also did not increase the formation of IP2 or IP3 but it had a small stimulatory effect on IPI formation. It is possible, therefore, that cibacron blue causes a small and probably delayed activation of phosphoinositide-specific phospholipase C. Cibacron blue activation of other phospholipases potentially involved in mediating the effect of ATP on phosphatidylcholine secretion (Griese et al., 1991b) or action via additional mechanisms are also possible. Although cibacron blue did not diminish the stimulatory effects of ATP on cyclic AMP and inositol phosphate formation when the two agents were added simultaneously, preincubation with cibacron blue before addition of ATP led to inhibition of the ATP effects on formation of these second messengers. Such interactions could account for the finding that the effects of ATP and cibacron blue on phosphatidylcholine secretion were less than additive. It is noteworthy Table 6 Effect of pretreatment with cibacron blue and P2-agonists on ATP-induced formation of inositol phosphates in type II cells Pretreatment
IP,
IP2
IP3
% of control
Vehicle Cibacron blue (10-4 M) ATP (5x 10-4M) a,-methylene ATP (5 x 10-4 M)
232 ± 16 477 ± 73 359 ± 30 200 ± 21 276 ± 33 276 ± 22*
257± 111 274±93*249± 75 319±44 457± 69 442±81
The cells were cultured for 18 h with [3H]-inositol, washed and preincubated for 30 min in fresh medium containing 20 mM LiCl. ATP (10-I M) was then added and the incubation continued for 2 min after which inositol phosphates were measured. The data are means ± s.e.mean from 4 experiments except for the 1P1 data from the cells pretreated with ATP which are from 3 experiments. [3H]-inositol phosphate levels in control cells were as indicated in Figure 4. *Significantly different from the value in the cells pretreated with vehicle (P
(D Macmillan Press Ltd,
1992
Cibacron blue stimulation of surfactant secretion in rat type II pneumocytes 'Matthias Griese, Laurice
I.
Gobran & 2Seamus A. Rooney
Division of Perinatal Medicine, Department of Pediatrics, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510, U.S.A. 1 The effect of cibacron blue, a selective P2y-purinoceptor antagonist in some systems, on the stimulatory effect of adenosine 5'-triphosphate (ATP) on [3H]-phosphatidylchloine secretion was examined in primary cultures of rat type II pneumocytes prelabelled by overnight culture with [3H]-choline. 2 Cibacron blue alone stimulated phosphatidylcholine secretion in a concentration-dependent manner in the range 10--10-3 M. At a concentration of 10- or lower, cibacron blue had no effect on ATP-induced phosphatidylcholine secretion but at 10-4-10-3 M it increased the effect of ATP. Enhancement of the ATP effect was apparent whether cibacron blue was added before or together with ATP. Cibacron blue also increased ATP-induced secretion in the presence of the PI-purinoceptor antagonist, xanthine amine congener (10-5 M). 3 The stimulatory effect of cibacron blue on phosphatidylcholine secretion was additive to those of 5' (N-ethylcarboxyamido) adenosine (NECA) and terbutaline but less than additive to that of ATP. 4 Cibacron blue alone had no effect on formation of cyclic AMP or inositol phosphate and when added stimultaneously with ATP it did not affect the ATP-induced increase in these second messengers. Preincubation of the cells with cibacron blue before addition of ATP, however, resulted in antagonism of the ATP-induced increase in cyclic AMP and inositol phosphates. Preincubation with ATP had the same effect. The stimulatory effects of NECA and terbutaline on cyclic AMP formation were enhanced by preincubation with cibacron blue. 5 Thus, ATP-induced phosphatidylcholine secretion in type II cells is not diminished by the P2yantagonist, cibacron blue. On the contrary, cibacron blue stimulates phosphatidylcholine secretion. Cibacron blue may act as a P2-agonist in type II pneumocytes. Keywords: Pulmonary surfactant; lung; phosphatidylcholine secretion; P2-purinoceptor; ATP; inositol trisphosphate; cyclic AMP; suramin
Introduction Lung surfactant, phospholipid-rich material lining the alveolar surface, is synthesized and stored in the type II pneumocyte and secreted by exocytosis (Chander & Fisher, 1990; Haagsman & van Golde, 1991; Wright & Dobbs, 1991). Secretion of surfactant phospholipids has been shown to be influenced by a variety of physiological and pharmacological agents (Chander & Fisher, 1990; Wright & Dobbs, 1991). A role for purinoceptor agonists in the regulation of surfactant secretion is suggested by the findings that secretion of phosphatidylcholine, its major lipid component (Haagsman & van Golde, 1991), was stimulated by adenosine, adenosine analogues and adenine nucleotides in primary cultures of type II cells (Gilfillan & Rooney, 1987a,b; 1988; Gobran & Rooney, 1990; Griese et al., 1991a,c; Rice & Singleton, 1986; 1987; Warburton et al., 1989) while surfactant secretion in vivo was stimulated by adenosine (Ekelund et al., 1985) and inhibited by an adenosine receptor antagonist (Rooney & Gobran, 1988). There is functional evidence for both P1- (adenosine) and P2- (adenosine 5'-triphosphate, ATP) purinoceptors on the type II cell. The stimulatory effects of adenosine and its analogues on phosphatidylcholine secretion were shown to be mediated by the A2 subtype of the PI-receptor (Gilfillan & Rooney, 1987b; Griese et al., 1991a) and there is also evidence of an inhibitory Al-receptor on the type II cell (Gobran & Rooney, 1990). We recently found that a large part of the action of ATP on the type II cell was mediated by the P1 receptor since as much as 75% of its effect on phosphatidyl-
' Present address: Ludwig-Maximilians-Universitat, Kinderpoliklinik, PettenkoferstraBe 8a, 8000 Munchen 2, Germany. Author for correspondence.
2
choline secretion was antagonized by 8-[4-(2-aminoethylaminocarbonylmethyloxy)phenyl]-1,3-dipropylxanthine (xanthine amine congener, XAC) and 8-phenyltheophylline (Griese et al., 1991c). However, there is evidence that ATP and ATP analogues also promote phosphatidylcholine secretion by action at a P2-receptor (Gilfillan & Rooney, 1988; Greise et al., 1991c; Rice & Singleton, 1987) that is coupled to activation of phosphoinositide-specific phospholipase C (Warburton et al., 1989; Rice et al., 1990; Griese et al., 199 1b). P2-receptors have been subdivided into P2,- and P2y-types (Burnstock & Kennedy, 1985) although at least three other subtypes have also been proposed (Gordon, 1986; Wiklund & Gustafsson, 1988; Stone, 1991). However, the receptor mediating the effect of ATP and its analogues in the type II cell does not fit the potency orders described for any of those (Rooney, 1990). Rice & Singleton (1987) proposed a classical P2y-subtype for the type II cell P2-receptor. This, however, is not supported by the potency order since 2-methylthio ATP is reported to be the most potent agonist at the P2y-receptor (Burnstock & Kennedy, 1985) and it was considerably less potent than ATP in the type II cell (Gilfillan & Rooney, 1988; Griese et al., 1991c). Cibacron blue, previously termed reactive blue 2 (Sigma Chemical Co., St. Louis, MO, U.S.A.), was reported to be a specific antagonist of the P2y-receptor in some systems (Burnstock & Warland, 1987; Hopwood & Burnstock, 1987). Rice & Singleton (1989) found that cibacron blue antagonized the effect of ATP in the type II cell but in a preliminary study we were unable to confirm that finding (Rooney, 1990; Rooney et al., 1988; 1990). We have now examined the effect of cibacron blue in more detail and show that it has unexpected actions in combination with other agents on both phosphatidylcholine secretion and second messenger formation.
374
M. GRIESE et al.
Methods
Isolation and culture to type II cells Type II cells were isolated from the lungs of adult rats by the method of Dobbs et al. (1986) as described previously (Gobran & Rooney, 1990). This method involves digestion of the blood-free lungs with elastase and separation of type II pneumocytes from contaminating cells by panning on bacteriological dishes coated with IgG. The freshly isolated cells were plated at a density of 2-5 x 106 cells per dish on 35 mm i.d. plastic dishes and cultured in 1.5 ml Dulbecco's Modified Eagle's Medium (DMEM) containing 10% foetal bovine serum, streptomycin (10 tg ml-'), penicillin (100 u ml-') and, when indicated, radiochemicals for 18-20 h at 370C in a humidified atmosphere of 90% air: 10% CO2. At least 95% of the cells attached to the dishes after this period of culture were identifiable as type II cells (Gobran & Rooney, 1990).
Adenosine 3':5'-cyclic monophosphate (cyclic AMP) assay The medium was rapidly aspirated and 1 ml of ice-cold 0.1 M HC1 was added to the dish which was placed on an ice-cooled surface. The HCl extract was evaporated to dryness at 40'C under a stream of air and the sample was stored at - 70C. After reconstitution and acetylation the cyclic AMP content was determined following the procedure described in the RIA kit.
Lactate dehydrogenase assay The rate of lactate dehydrogenase release into the medium was determined to assess cellular integrity. The cells were cultured as in the secretion experiments but in the absence of radioactivity after which lactate dehydrogenase activity in the cells and medium was assayed by measuring disappearance of NADH at 340nm (Wroblewski & LaDue, 1955).
Drug treatment
Materials
After culture of the cells for 18-20 h the medium was removed and the cells were rinsed with fresh DMEM without serum, antibiotics or radiochemicals. Fresh DMEM was then added and the dishes were returned to the incubator for a 30 min preincubation period. Secretagogues were then added and the incubation continued for various time periods. Antagonists were generally added at the beginning of the preincubation period. Where indicated, adenosine deaminase (3 u ml-') was also added at this stage. XAC was dissolved in dimethylsulphoxide (1% final concentration) and this solvent was added to the corresponding control dishes. The other drugs were dissolved in DMEM.
Adult male Sprague-Dawley rats (200-300 g) were purchased from Charles River Breeding Laboratories (Kingston, NY, U.S.A.), rat IgG from Calbiochem (San Diego, CA, U.S.A.), porcine pancreatic elastase from Elastin Products (Owensville, MO, U.S.A.), foetal bovine serum from HyClone Laboratories (Logan, UT, U.S.A.), Dulbecco's modified Eagle's medium (DMEM) and myo-inositol-free DMEM from GIBCO, Grand Island, NY, U.S.A.; [methyl-3H]-choline chloride from NEN-du Pont (Wilmington, DE, U.S.A.), myo-[2-3H]-inositol from Amersham (Arlington Heights, IL, U.S.A.), cyclic AMP RIA kits from Biomedical Technologies (Cambridge, MA, U.S.A), XAC from Research Biochemicals (Natick, MA, U.S.A.), terbutaline sulphate (Brethine) from Geigy Pharmaceuticals (West Caldwell, NJ, U.S.A.), 5' (Nethylcarboxyamido)adenosine (NECA), disodium ATP, a, Pmethylene ATP, cibacron blue 3GA (Lot 50F-03341), calf intestinal mucosal adenosine deaminase (type VIII) and other biochemicals from Sigma (St. Louis, MO, U.S.A.). Suramin was a gift from Dr John Becher, Centers for Disease Control (Atlanta, GA, U.S.A.).
Phosphatidylcholine secretion [3H]-choline (2 1Ci ml-) was included in the medium during the overnight culture of the cells (2 x 106 per dish). With the exception of the time course experiments, secretion was measured 90 min after addition of the secretagogues. The medium was aspirated and centrifuged at 200g for 10min while the attached cells were lysed by addition of ice-cold water. Lipids were extracted from both cells and medium with a mixture of chloroform and methanol by the method of Bligh & Dyer (1959) and total lipid radioactivity was quantified by liquid scintillation counting (Gobran & Rooney, 1990). The total amount of lipid radioactivity ranged from 150,000 to 700,000 c.p.m. per dish. We previously reported that under these conditions phosphatidylcholine accounts for more than 95% of the lipid radioactivity in both cells and medium (Gobran & Rooney, 1990) while sphingomyelin and lysophosphatidylcholine account for the remainder. Phosphatidylcholine secretion is therefore expressed as the amount of lipid radioactivity in the medium after the 2 h incubation as a percentage of the total in cells and medium combined.
Measurement of [3HJ-inositol phosphates The overnight culture of the cells (3-5 x 106 per dish) was carried out in inositol-free DMEM to which [3H]-inositol (20 pCi ml', 80-120 Ci mmol-') was added (Griese et al., 1991b). Formation of inositol phosphates was measured in the presence of 20 mM LiCl as described previously in detail (Griese et al., 1991b). Briefly, the reaction was terminated at various times after addition of the secretagogues by rapid aspiration of the medium and addition of ice-cold perchloric acid. [3H]-inositol phosphates were then extracted, fractionated into individual components on ion exchange columns by a modification of the method of Berridge (1986) and quantified by liquid scintillation counting.
Statistics In the secretion experiments type II cells were isolated from the pooled lungs of three rats and distributed among the various treatment groups, three dishes per group. The dishes were processed separately and the mean values calculated to yield a single data point per group per experiment. In the second messenger experiments the cells were isolated from the pooled lungs of four rats and one dish was used at each time point per experiment. Cyclic AMP extracts were analyzed in duplicate and the mean values calculated to yield a single data point per time point per experiment. All data are mean ± s.e.mean from the number of individual experiments indicated. As the cyclic AMP values were log normally distributed, they were normalized by a log transfusion prior to statistical analysis. The data were analyzed statistically by analysis of variance (ANOVA) followed by Fisher's least significance difference test or by Student's two tailed t test for paired samples with correction for multiple comparisons when necessary (Rohlf & Sokal, 1981).
Results
Phosphatidylcholine secretion Cibacron blue alone at low concentrations had no effect on phosphatidylcholine secretion in type II cells but stimulated in a concentration-dependent manner at higher concentrations (Figure 1). As shown previously (Rooney et al., 1988;
CIBACRON BLUE AND SURFACTANT SECRETION 600
I
500 c
o 400 =
0
300)I
i,
co
'--
200)I 100
0 6
7
5
4
3
Cibacron blue (-log M) Figure 1 Phosphatidylcholine secretion in type II cells in response to various concentrations of cibacron blue alone and in combination with ATP and ATP+ xanthine amine congener (XAC). The cells were cultured for 18-20h in the presence of [3H]-choline, washed with fresh medium and preincubated with the indicated concentrations of cibacron blue with and without XAC (10-5 M) for 30 min. ATP (10-I M) was then added and the incubation continued for 90 min after which phosphatidylcholine secretion was measured. When the effect of cibacron blue alone was to be examined it was added at the end of the preincubation period: (0) cibacron blue alone; (0) cibacron blue + ATP; (A) cibacron blue + ATP + XAC. The data are expressed as % stimulation over the basal rate of secretion in control cells incubated with vehicle only and are means ± s.e.mean (bar) from 3 experiments except for 10- M and 5 x 10- M cibacron blue + ATP which are from I and 9 experiments, respectively, and 0-7I M to 5 x 0-5I M cibacron blue alone, 6 x 0-5I M cibacron blue + ATP and 5 x 10-5 M cibacron blue + ATP + XAC which are means ± range from 2 experiments. The basal rate of secrtion was 1.13 ± 0.07% of cellular [3H]-phosphatidylcholine secreted in 2 h (n = 14). The rate of secretion in cells treated with 10-4 M, 5 X 10-5 M and 10-3 M cibacron blue was significantly different from that in the controls (P ,-methylene ATP in the type II cells (Gilfillan & Rooney, 1988). Such differences suggest that the P2-receptors in these two different respiratory tissue cells are not identical. Our data are different from those of Rice & Singleton (1989) who found that cibacron blue inhibited ATP-stimulated phosphatidylcholine secretion in type II cells. The inhibition was reported to be dependent on concentration with an IC50 of approx. 1.5 x 10-4 M. However, some inhibition was observed even at the lowest concentration examined (Rice & Singleton, 1989). Whereas we found that the stimulatory effects of cibacron blue and terbutaline were additive, Rice & Singleton (1989) reported that cibacron blue did not enhance the effect of terbutaline on phosphatidylcholine secretion. The reason for the different findings are not clear. They are not due to differences in the purity of cibacron blue since its source and lot number were the same in both studies (Rice, W.R., personal communication). Rice & Singleton (1989) did not examine the effect of cibacron blue alone or the time
course of its action. Our data show that the onset of the effect of cibacron blue in combination with ATP was very rapid and that the effects of ATP and cibacron blue were not additive in contrast to those of terbutaline and cibacron blue which were. As Rice & Singleton (1989) subtracted the amount of [3H]-phosphatidylcholine that was released during the 30 min preincubation from the total released at the completion of the incubation, an apparent inhibition of the effect of ATP and no enhancement of the effect of terbutaline might have been noted. Other possible differences include use of collagen-coated tissue culture dishes in the study of Rice & Singleton (1989) and our inclusion of adenosine deaminase in the terbutaline experiments. The mechanism by which cibacron blue stimulates phosphatidylcholine secretion remains to be elucidated. That its effect on secretion was additive to those of NECA and terbutaline suggests that its action does not involve activation of adenylate cyclase. This conclusion is supported by the finding that cibacron blue did not increase cellular cyclic AMP levels. That its effect on ATP-stimulated secretion was apparent in the presence of XAC further shows that it does not act via a PI-receptor. That the stimulatory effects of cibacron blue and ATP were not completely additive suggests that the mechanism by which these two agents stimulate phosphatidylcholine secretion are not independent of each other. ATP has been found to increase formation of cyclic AMP (Gilfillan & Rooney, 1987b; Warburton et al., 1989; Griese et al., 1991c), IP3 (Warburton et al., 1989; Rice et al., 1990; Griese et al., 1991b) and diacylglycerols (Rice et al., 1990; Griese et al., 1991b) as well as promote mobilization of intracellular Ca2" (Rice & Singleton, 1987) in type II cells. In addition to the lack of an increase in cyclic AMP, cibacron blue also did not increase the formation of IP2 or IP3 but it had a small stimulatory effect on IPI formation. It is possible, therefore, that cibacron blue causes a small and probably delayed activation of phosphoinositide-specific phospholipase C. Cibacron blue activation of other phospholipases potentially involved in mediating the effect of ATP on phosphatidylcholine secretion (Griese et al., 1991b) or action via additional mechanisms are also possible. Although cibacron blue did not diminish the stimulatory effects of ATP on cyclic AMP and inositol phosphate formation when the two agents were added simultaneously, preincubation with cibacron blue before addition of ATP led to inhibition of the ATP effects on formation of these second messengers. Such interactions could account for the finding that the effects of ATP and cibacron blue on phosphatidylcholine secretion were less than additive. It is noteworthy Table 6 Effect of pretreatment with cibacron blue and P2-agonists on ATP-induced formation of inositol phosphates in type II cells Pretreatment
IP,
IP2
IP3
% of control
Vehicle Cibacron blue (10-4 M) ATP (5x 10-4M) a,-methylene ATP (5 x 10-4 M)
232 ± 16 477 ± 73 359 ± 30 200 ± 21 276 ± 33 276 ± 22*
257± 111 274±93*249± 75 319±44 457± 69 442±81
The cells were cultured for 18 h with [3H]-inositol, washed and preincubated for 30 min in fresh medium containing 20 mM LiCl. ATP (10-I M) was then added and the incubation continued for 2 min after which inositol phosphates were measured. The data are means ± s.e.mean from 4 experiments except for the 1P1 data from the cells pretreated with ATP which are from 3 experiments. [3H]-inositol phosphate levels in control cells were as indicated in Figure 4. *Significantly different from the value in the cells pretreated with vehicle (P
