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Interaction between β-adrenergic signaling and protein kinase C increases cytoplasmic Ca2+ in alveolar type II cells
Life Sciences 68 (2001) 2361–2371
Interaction between b-adrenergic signaling and protein kinase C increases cytoplasmic Ca21 in alveolar type II cells Yoichiro Isohama*, Miyuki Kanemaru, Hirofumi Kai, Kazuo Takahama, Takeshi Miyata Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe hon-machi, Kumamoto 862-0973, Japan Received 27 July 2000; accepted 27 September 2000
Abstract The interaction between b-adrenergic signaling and the activation of protein kinase C in alveolar type II cell plays an important role in the regulation of surfactant secretion because the combined application of b-adrenergic agonist with protein kinase C activator to the cells stimulates the secretion synergistically. However, the mechanisms underlying the interaction are not clear. In the present study, we examined the combined effect of terbutaline with phorbol 12-myristate 13-acetate (PMA) on cytoplasmic free Ca21 concentration ([Ca21]i) in rat alveolar type II cells. The combined application of terbutaline with PMA to the cells rapidly increased [Ca21]i, although neither of them affected it by itself. Similar increases of [Ca21]i were observed in other combinations, such as terbutaline with 1-oleoyl2-acetyl-sn-glycerol, and forskolin with PMA. Either the removal of extracellular Ca21 or the addition of Co21 remarkably suppressed the increase of [Ca21]i induced by the combination of terbutaline with PMA. In addition, Co21 inhibited the phosphatidylcholine secretion induced by the combination of terbutaline and PMA. These results suggested that the [Ca21]i increased as a result of the interaction between formation of cyclic AMP and activation of protein kinase C in alveolar type II cells, and that the increase in [Ca21]i was mediated by the Ca21 influx through the plasma membrane. This mechanism to modulate [Ca21]i may play a role in the regulation of surfactant secretion by alveolar type II cells. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Alveolar type II cell; Cytoplasmic Ca21; Pulmonary surfactant secretion; Cyclic AMP; Protein kinase C
Introduction The alveolar type II cell synthesizes and secretes pulmonary surfactant which lowers the surface tension at the air-liquid interface in the lung and provides for alveolar stability [see for review 1]. The secretion of pulmonary surfactant from alveolar type II cells is regulated * Corresponding author. Department of Pharmacological Sciences, Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe Hon-machi, Kumamoto 862-0973, Japan. Tel.: 81-96-371-4185; fax: 81-96-362-7795. E-mail address: [email protected] (Y. Isohama) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 0 2 8 -1
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by at least three different signal transduction pathways. These pathways involve different second messengers and protein kinases, i.e. the adenylate cyclase system with activation of cyclic AMP-dependent protein kinase (PKA) [2–5], the phosphoinositide-specific phospholipase C system with activation of protein kinase C (PKC) [6–9], and the Ca21/calmodulindependent system [10–12]. Several studies indicated that the interactions among these different signal transduction pathways played important roles in the overall regulation of surfactant secretion [10, 11, 14–16]. For instance, the stimulatory effect of the combination of an A2 adenosine agonist with phorbol 12-myristate 13-acetate (PMA) was significantly greater than the sum of the effects of individual secretagogues [15]. Similarly, the combination of terbutaline, a b-adrenergic agonist, with PMA or with 1-oleoyl-2-acetyl-sn-glycerol (OAG) stimulated the secretion synergistically [9, 16]. The synergism of the adenosine agonist and PMA is considered to be mediated by the increase of cyclic AMP formation [15]. However, the synergism of terbutaline and PKC activators seems to be mediated by other mechanisms, because cyclic AMP formation induced by terbutaline was not enhanced by the combination with PKC activators [16]. Because Ca21 is generally considered as an important second messenger for the surfactant secretion, we examined the combined effect of terbutaline with PMA on cytoplasmic free Ca21 concentration ([Ca21]i) in the present study. Interestingly, we found that the combination of terbutaline with PMA rapidly increased [Ca21]i in alveolar type II cells. Methods Cell culture Alveolar type II cells were isolated from the lungs of adult pathogen-free male Wistar rats (180–200 g), as described previously [5, 17, 18]. Briefly, trypsin was used to dissociate the cells from the lung tissues. The resultant cell suspension was incubated on rat IgGcoated plastic petri dishes for 30 min to remove the non-type II cells. The isolated alveolar type II cells were suspended at 1 3 106 cells/ml in Dulbecco’s modified Eagle’s medium (DMEM) containing 2 mM L-glutamate, 100 units/ml penicillin, 100 mg/ml streptomycin and 10% fetal bovine serum. This procedure routinely yielded about 1.0 3 107 cells/rat. The cells were dispensed onto glass bottom culture dishes (MatTek, Ashland MA, USA) at a density of 2 3 105 cells/cm2 and cultured in 5% CO2/95% air at 378C. In all experiments, nonadherent cells were removed from the dishes by washing with DMEM after 20–22 h cultivation. The purity of the overnight cultured type II cells was 95 6 3% (mean 6 SEM, n58), and the viability of the cells was 98 6 1% (mean 6 SEM, n58) as judged by the trypan blue exclusion test. Measurement of cytoplasmic Ca21 Cultured cells were washed and incubated in HEPES-buffer (140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 24 mM glucose and 10 mM HEPES, pH 7.4) containing 10 mM fura-2/AM and 2% BSA for 60 min at 378C. After fura-2 loading, the cells were washed twice with the HEPES-buffer and stored under light-free conditions until use. The [Ca21]i of a single cell was measured at 100 ms intervals with a digital imaging microscopic system
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Mu-1000 (Inter deck. Inc. Osaka, Japan). The ratio of the intensities of fluorescent emission at 510 nm with excitation at 340 nm and 380 nm was measured. The fluorescent excitation beam was led to drop on the cells, and images were recorded with videofilm and analyzed with a computer. Utilizing this protocol, cells were uniformly labeled when observed by fluorescence microscopy without evidence of the organellar sequestration which has been reported with longer incubation time [19]. [Ca21]i was calculated according to the method of Grynkiewicz et al. [20]. Phosphatidylcholine secretion The cells (5 3 105 cells/cm2) were cultured for 20 h with 74 kBq/mL [methyl-3H]choline to label the phosphatidylcholine pool, after which the medium was removed and the cells were washed with fresh DMEM. Fresh DMEM was then added, and the cells were returned to the incubator. After a 30-min preincubation in the fresh medium, agonists or solvent vehicle were added, and the incubation was continued for 90 min. The medium was then aspirated, and the cells were lysed with ice-cold 0.05% Triton X-100. Lipids were extracted from both cells and medium using the method of Folch et al. [21], and the amount of radioactivity was measured with a liquid scintillation counter. The secretion rate was expressed as the amount of [3H]phosphatidylcholine in the medium after 90-min incubation as a percentage of the total amount in cells plus medium. To assess cellular integrity, the activity of lactate dehydrogenase (LDH) in the cells and medium was measured with an LDH assay kit (Nippon Shoji Co., Ltd., Osaka, Japan). The amount of LDH released into the medium did not exceed 1% of the total cell content in any experiment. Materials The rats were purchased from Kyudo Farm (Fukuoka, Japan). Terbutaline sulfate, phorbol 12-myristate 13-acetate, forskolin, 1-oleoyl-2-acetyl-sn-glycerol and 4a-phorbol 12, 13didecanoate were from Sigma (St Louis, MO, USA). Fura-2/AM was from Dojin Chem. (Kumamoto. Japan). Fetal bovine serum was from JHR Bioscience (Lenexa, KS, USA). All drugs were applied to the incubation buffer. To examine the combined effect of different agonists, the first agonist was added 1 min prior to the addition of the second agonist. Terbutaline sulfate was dissolved in HEPES-buffer and other agents in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide in the buffer was less than 1%. Analyses of results Data are reported as the mean 6 SEM. The data were analyzed by Student’s t-test or analysis of variance (ANOVA) followed by Newman-Keuls test as shown in the results. Differences between means were considered significant at p value of ,0.05. Results ATP is the most effective stimulant to increase [Ca21]i in type II cells [12, 13], therefore, we determined first the effect of ATP on [Ca21]i using fura-2 technique. ATP rapidly in-
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Table 1 Effects of terbutaline, PMA and their combination on [Ca21]i in single rat alveolar type II cells
1st agonist
2nd agonist
ATP (0.1 mM) ATP (1 mM) Terbutaline (10 mM) PMA (10 nM) Terbutaline PMA
— — — — PMA Terbutaline
No. of cells examined (No. of experiments) 15 (4) 26 (7) 13 (4) 14 (4) 58 (10) 45 (8)
Basal [Ca21]i (nM)
Mean peak [Ca21]i (nM)
73 6 8 72 6 7 77 6 5 76 6 6 73 6 2 74 6 8
201 6 28a 471 6 22a 78 6 7 82 6 9 382 6 29a 178 6 23b
Values are mean 6 SEM for the number of cells indicated in 4–10 experiments (indicated in parentheses). In each experiment, alveolar type II cells were isolated from 2 rats and cultured overnight as shown in material and methods. The first agonist was added 1 min before the second. Significance was determined by paired Student’s t-test: a p,0.01, b p,0.05 for difference from the basal [Ca21]i.
creased [Ca21]i in a concentration-dependent manner (Table 1, Fig. 1). In 7 different experiments with a total of 26 cells, 1 mM of ATP increased [Ca21]i in 24 cells (92%). This high percentage of cells responding to 1 mM of ATP is in agreement with the estimated proportion of type II cells (<95%). Then, we examined the effects of terbutaline, PMA, and their combination on [Ca21]i (Table 1). Terbutaline (10 mM) or PMA (10 nM) alone had no significant
Fig. 1. Representative trace of the cytoplasmic free Ca21 concentration ([Ca21]i) in alveolar type II cells in response to terbutaline, PMA and their combination. Cultured alveolar type II cells were loaded with 10 mM fura2/AM for 60 min. One mM ATP (A), 10 mM terbutaline (B), 10 nM PMA (C) or combination of terbutaline and PMA (D) were applied to the cells, as indicated by the above tracings.
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Fig. 2. Combined effect of terbutaline and PMA on [Ca21]i in alveolar type II cells at various concentrations. Fura-2 loaded cells were incubated, as indicated above tracings. A: Cells were washed to remove the agonists, and second or third combinations were applied to the bath. The traces were from the same cell during the same experiment. B: Different concentrations of PMA were added to the bath in the presence of 10 mM of terbutaline.
effect on [Ca21]i. However, the addition of PMA 1 min after the application of terbutaline increased [Ca21]i which peaked at 382 6 29 nM. An example is shown in Fig. 1. This increase in [Ca21]i was dependent on the concentration of terbutaline (0.1–10 mM) and of PMA (0.1– 10 nM) (Fig. 2). Terbutaline following PMA also increased [Ca21]i, but the effect was weaker than that of PMA following terbutaline (Table 1). We next examined the effects of other combinations (Fig. 3). OAG (100 mM), another type of PKC activator, combined with terbutaline significantly increased [Ca21]i. Combination of forskolin (1 mM), an activator of adenylate cyclase, with PMA also increased [Ca21]i. In contrast, 4a-PDD (10 nM), an inactive phorbol ester, combined with terbutaline failed to increase [Ca21]i.
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Fig. 3. Combined effects of terbutaline with OAG, terbutaline with 4a-PDD and forskolin with PMA on [Ca21]i in alveolar type II cells. Fura-2-loaded cells were incubated with the combination of forskolin (1 mM) and PMA (10 nM) (A), terbutaline (10 mM) and OAG (100 mM) (B), or terbutaline (100 mM) and 4a-PDD (10 nM) (C), as indicated by the above tracings. D: Mean peak [Ca21]i 6 SEM elicited with indicated agonists. Each bar corresponds to 12 cells (3 different experiments). Significance was determined by ANOVA followed by NewmanKeuls test: * p,0.05 for difference from the basal [Ca21] i.
Further, we examined the influence of extracellular Ca21 on the increase in [Ca21]i induced by combination of terbutaline with PMA. The increase in [Ca21]i induced by the combination was dependent on the extracellular Ca21 concentration. (Fig. 4A, B and C). Several bivalent cations, such as Co21, Ni21, Mn21 and Cd21, bind too strongly with a group located inside the Ca21 channel, become competitive channel blockers [22]. Therefore, we examined the effect of Co21 on the increase in [Ca21]i. Co21 abolished the increase in [Ca21]i induced by the combination of terbutaline with PMA (Fig. 4D). Finally, we examined the effect of Co21 on [3H]phosphatidylcholine secretion induced by the combination of terbutaline and PMA. Pretreatment with Co21 did not affect the secretion induced by terbutaline or PMA alone. However, Co21 significantly decreased the secretion increased by the combination of terbutaline and PMA (Fig. 5).
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Fig. 4. Influence of extracellular Ca21 and Co21 on the increase of [Ca21]i induced by the combination of terbutaline with PMA in alveolar type II cells. Fura-2-loaded cells were incubated in control buffer containing 1 mM of Ca21 (A), in buffer containing 0.01 mM Ca21 (B), in Ca21-free buffer containing 1 mM EGTA (C), or in the buffer containing 1 mM Ca21 and 1 mM Co21 (D). Ten mM terbutaline and 10 nM PMA were applied to the cells, as indicated by the above tracings. E: Mean peak [Ca21]i 6 SEM elicited with indicated agonists. Each bar corresponds to 12 cells (3 different experiments). Significance was determined by ANOVA followed by NewmanKeuls test: * p,0.05 for difference from the basal [Ca21]I; † p,0.05 for difference from 1 mM Ca21 without Co21.
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Fig. 5. Effect of Co21 on phosphatidylcholine secretion induced by terbutaline, PMA and their combination. [3H]choline labeled cells were washed, and incubated with indicated agonists for 90 min. After incubation, lipids were extracted from medium and cells as described in materials and methods. Vehicle or 1 mM Co21 was pretreated 5 min prior to the addition of agonists. Secretion is expressed as amount of [3H]lipid in medium after 90min incubation as percentage of total in cell and medium. The data are mean 6 SEM from 4 different experiments. Significance was determined by paired Student’s t-test: * p,0.05 for difference.
Discussion Pulmonary surfactant secretion has been studied extensively in primary cultures of type II cells and shown to be stimulated by several physiological and pharmacological agents. Among these agents, terbutaline, a b-adrenergic agonist, acts on the membrane receptor coupled to activation of adenylate cyclase with generation of cyclic AMP and cyclic AMPdependent protein kinase. On the other hand, the effect of PMA on surfactant secretion is largely mediated by the activation of PKC. The current data show that the combined application of terbutaline and PMA increases [Ca21]i in alveolar type II cells. The result of the combination of forskolin with PMA suggested that the increase of [Ca21]i is dependent on cyclic AMP formation. Considering the finding that PMA was interchangeable with OAG, we assumed that the increase in [Ca21]i induced by the combination of terbutaline with PMA occurred as a result of the combination of cyclic AMP formation and activation of PKC. To our knowledge, similar regulation of [Ca21]i in any other cell system has not been reported. We therefore speculate that the increase in [Ca21]i by the combination is probably mediated by the mechanisms specified in type II cells. The increase in [Ca21]i induced by PMA following terbutaline was more marked than that by terbutaline following PMA (Table 1). This may occur through the PKC-mediated phosphorylation of b2-adrenergic receptor followed by desensitization of the receptor [23, 24]. This is supported by the finding that
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terbutaline-induced cyclic AMP formation was decreased by the pre-treatment of PMA in type II cells [16]. The increase in [Ca21]i induced by the combination of terbutaline and PMA seems to be mediated, at least in part, by the influx of extracellular Ca21 through Ca21 channels. This idea is supported by the following findings; 1) the increase of [Ca21]i induced by the combination was markedly suppressed by the removal of extracellular Ca21, 2) Co21 inhibited the increase of [Ca21]i, and 3) the combination of terbutaline with PMA did not affect inositol phosphates production in the cells (data not shown). There is little information about Ca21 channels in alveolar type II cells: L-type voltage-dependent channels are present in a pulmonary epithelial cell line (L2 cell) and indirect evidence suggests their presence in type II cells [25, 26], but their physiological significance remains to be established. However, Ca21 is considered as an important second messenger for the secretion of pulmonary surfactant. This is suggested by reports that several stimuli for surfactant secretion are accompanied by a Ca21 increase [27– 30], and strongly suggested by the use of Ca21 ionophores [10–12]. Therefore, we assumed that the increase in [Ca21]i by the combination of terbutaline and PMA is accompanied with synergistic secretion of phosphatidylcholine by these drugs. Supporting this hypothesis, Co21 significantly inhibited the phosphatidylcholine secretion induced by the combination of terbutaline and PMA, without affecting the effect of each drug alone. In the current study, neither terbutaline nor PMA changed the [Ca21]i by itself. These results are consistent with those of Rice et al [29]. These also consist with previous studies demonstrating terbutaline-induced activation of cyclic AMP-dependent kinase and PMAinduced mobilization of PKC directly in association with surfactant secretion [9, 31]. However, Sano et al. have reported a small increase in the [Ca21]i induced by terbutaline or forskolin with quin2-loaded type II cells [11]. They described that terbutaline-induced increase in [Ca21]i occurred in the absence of extracellular Ca21 and that PMA inhibited this increase [11]. The discrepancy between the two results might be due to some methodological difference, e.g., fluorescent indicator or in the procedure for cell culture. On the other hand, ATP effectively increased [Ca21]i in type II cells, in current and previous studies [12, 13]. ATPinduced increase in [Ca21]i is believed to be mediated by IP3 formation and Ca21 release from intracellular store site. However, Dorn et al. showed that ATP-induced increase in [Ca21]i was composed of two phases, transient and prolonged plateau phases, and that the plateau phase was abolished by chelation of extracellular Ca21 [13]. We are interested in their findings, because ATP, by itself stimulates the formation of both cyclic AMP and diacylglycerol in type II cells [7, 14]. In addition, in our preliminary study, the increase in [Ca21]i by combined application of three agents, terbutaline, PMA and ATP, was considerably less than sum of the effect of ATP alone and combination of terbutaline with PMA (data not shown). Therefore, it might be possible that ATP increases [Ca21]i not only through IP3dependent Ca21 release but also through Ca21 influx mediated by the interaction between cyclic AMP- and PKC-dependent pathway. However, further studies are needed to establish this idea. In conclusion, our findings provide the first evidence that the [Ca21]i is increased by the combined application of terbutaline and PMA, and that the activation of both cyclic AMPand PKC-dependent signaling pathway are necessary for this stimulation. The present and previous studies indicate that there are several interactions between different signal-transduction
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mechanisms for surfactant secretion, including mobilization of [Ca21]i and enhancement of cyclic AMP formation. Considering that alveolar type II cells in vivo are exposed to various mediators simultaneously, the secretion of surfactant might be influenced by these interactions. Acknowledgments We thank W. Hirakawa and K. Kurita for their technical assistance. We also thank Dr. SA. Rooney in Yale University for his critical review of this manuscript. This study was supported by Grants-in-Aid 05857258 and 07772175 (to Y. Isohama) from the Ministry of Education, Sciences, Sports and Culture of Japan. References 1. Rooney SA. Regulation of surfactant secretion. In: Rooney SA, editor. Lung surfactant: Cellular and molecular processing. Austin. TX: Landes, 1998, pp.139–163. 2. Brown LAS, Longmore WJ. Adrenergic and cholinergic regulation of lung surfactant secretion in the isolated perfused rat lung and in the alveolar type II cell in culture. J. Biol. Chem. 1981; 256: 66–72. 3. Dobbs LG, Mason RJ. Pulmonary alveolar type II cells isolated from rats. Release of phosphatidylcholine in response to beta-adrenergic stimulation. J. Clin. Invest. 1979; 63: 378–87. 4. Gilfillan AM, Rooney SA. Functional evidence for adenosine A2 receptor regulation of phosphatidylcholine secretion in cultured type II pneumocytes. J. Pharmacol. Exp. Ther. 1988; 241: 907–14. 5. Kai H, Isohama Y, Takaki K, Oda Y, Murahara K, Takahama K, Miyata T. Both b1- and b 2-adrenoceptors are involved in mediating phosphatidylcholine secretion in rat type II pneumocyte cultures. Eur. J. Pharmacol. 1992; 212: 101–3. 6. Chander A, Sen N, Wu AM, Spitzer AR. Protein kinase C in ATP regulation of lung surfactant secretion in type II cells. Am. J. Physiol. 1995; 268: L108–16. 7. Griese M, Gobran LI, Rooney SA. ATP-stimulated inositol phospholipid metabolism and surfactant secretion in rat type II pneumocytes. Am. J. Physiol. 1991; 260: L586–93. 8. Rice WR, Dorn CC, Singleton FM. P2-purinoceptor regulation of surfactant phosphatidylcholine secretion. Relative roles of calcium and protein kinase C. Biochem. J. 1990; 266: 407–13. 9. Sano K, Voelker DR, Mason RJ. Involvement of protein kinase C in pulmonary surfactant secretion from alveolar type II cells. J. Biol. Chem. 1985; 260: 12725–29. 10. Dobbs LG, Gonzalez RF, Marinari LA, Mescher EJ, Hawgood S. The role of calcium in the secretion of surfactant by rat alveolar type II cells. Biochim. Biophys. Acta. 1986; 877: 305–313. 11. Sano K, Voelker DR, Mason RJ. Effect of secretagogues on cytoplasmic free calcium in alveolar type II epithelial cells. Am. J. Physiol. 1987; 253: C679–86. 12. Voyno-Yasenetskaya TA, Dobbs LG, Williams MC. Regulation of ATP-dependent surfactant secretion and activation of second-messenger systems in alveolar type II cells. Am. J. Physiol. 1991; 261:105–9. 13. Dorn CC, Rice WR, Singleton FM. Calcium mobilization and response recovery following P2-purinergic stimulation of rat isolated alveolar type II cells. Br. J. Pharmacol. 1989; 97: 163–70. 14. Griese M, Gobran LI, Rooney SA. A2 and P2 purine receptor interactions and surfactant secretion in primary cultures of type II cells. Am. J. Physiol. 1991; 261: L140–7. 15. Griese M, Gobran LI, Rooney SA. Potentiation of A2 purinoceptor-stimulated surfactant phospholipid secretion in primary cultures of rat type II pneumocytes. Lung 1993; 171: 75–86. 16. Isohama Y, Takahama K, Kai H, Miyata T. Cross-talk between b-adrenergic and other signal transduction pathways in pulmonary surfactant secretion from cultured alveolar type II cells. Jpn. J. Pharmacol. 1993; 61 (suppl.): 82p. 17. Isohama Y, Matsuo T, Kai H, Takahama K, Miyata T. Changes in b1- and b2-adrenoceptor mRNA levels in alveolar type II cells during cultivation. Biochem. Mol. Biol. Int. 1995; 36: 561–8.
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18. Isohama Y, Kumanda Y, Tanaka K, Kai H, Takahama K, Miyata T. Dexamethasone increases b2-adrenoceptorregulated phosphatidylcholine secretion in rat alveolar type II cells. Jpn. J. Pharmacol. 1997; 73: 163–9. 19. Pian MS, Dobbs LG, Düzgünes L. Positive correlation between cytosolic free calcium and surfactant secretion in culrured rat alveolar type II cells. Bichem. Biophys. Acta. 1988; 960: 43–53. 20. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca21 indicators with greatly improved fluorescence properties. J. Biol. Chem. 1985; 260: 3440–50. 21. Folch J, Less M, Stanley GHS. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 1957; 226: 497–509. 22. Kostyuk PG, Calcium channels in the cell membrane. Neurosci. Behav. Physiol. 1986; 16: 401–10. 23. Yoshimasa T, Sibley DR, Bouvier M, Lefkowitz RJ, Caron MG. Cross-talk between cellular signaling pathways suggested by phorbol-ester-induced adenylate cyclase phosphorylation. Nature 1987; 327: 67–70. 24. Yuan N, Friedman J, Whaley BS, Clark RB. cAMP-dependent protein kinase and protein kinase C consensus site mutations of the beta-adrenergic receptor. Effect on desensitization and stimulation of adenylylcyclase. J. Biol. Chem. 1994; 269: 23032–8. 25. Dietl P, Haller T, Wirleitner B, Volkl H, Friedrich F, Striessnig J. Activation of L-type Ca21 channels after purinoceptor stimulation by ATP in an alveolar epithelial cell (L2). Am. J. Physiol. 1995; 269: L873–83. 26. Sen N, Grubstein MM, Chander A. Stimulation of lung surfactant secretion bt endothelin-1 from rat alveolar type II cells. Am. J. Physiol. 1994; 266: L255–62. 27. Gerboth GD, Effros RM, Roma RJ, Jacobs ER. pH-induced calcium transients in type II alveolar epithelial cells. Am. J. Physiol. 1993; 264: L448–57. 28. Hinman LE, Beilman GE, Groehler KE, Sammak PJ. Wound-induced calcium waves in alveolar type II cells. Am. J. Physiol. 1997, 273: L1242–8. 29. Rice WR, Singleton FM. P2Y-purinoceptor regulation of surfactant secretion from rat isolated alveolar type II cells is associated with mobilization of intracellular calcium. Br. J. Pharmacol. 1987, 91: 833–8. 30. Wirtz HR, Dobbs LG. Calcium mobilization and exocytosis after one mechanical stretch of lung epithelial cells. Science 1990; 250: 1266–9. 31. Rice WR, Hull WM, Dion CA, Hollinger BA, Whitsett JA. Activation of cAMP dependent protein kinase during surfactant release from type II pneumocytes. Exp. Lung Res. 1985; 9: 135–49.
Interaction between b-adrenergic signaling and protein kinase C increases cytoplasmic Ca21 in alveolar type II cells Yoichiro Isohama*, Miyuki Kanemaru, Hirofumi Kai, Kazuo Takahama, Takeshi Miyata Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe hon-machi, Kumamoto 862-0973, Japan Received 27 July 2000; accepted 27 September 2000
Abstract The interaction between b-adrenergic signaling and the activation of protein kinase C in alveolar type II cell plays an important role in the regulation of surfactant secretion because the combined application of b-adrenergic agonist with protein kinase C activator to the cells stimulates the secretion synergistically. However, the mechanisms underlying the interaction are not clear. In the present study, we examined the combined effect of terbutaline with phorbol 12-myristate 13-acetate (PMA) on cytoplasmic free Ca21 concentration ([Ca21]i) in rat alveolar type II cells. The combined application of terbutaline with PMA to the cells rapidly increased [Ca21]i, although neither of them affected it by itself. Similar increases of [Ca21]i were observed in other combinations, such as terbutaline with 1-oleoyl2-acetyl-sn-glycerol, and forskolin with PMA. Either the removal of extracellular Ca21 or the addition of Co21 remarkably suppressed the increase of [Ca21]i induced by the combination of terbutaline with PMA. In addition, Co21 inhibited the phosphatidylcholine secretion induced by the combination of terbutaline and PMA. These results suggested that the [Ca21]i increased as a result of the interaction between formation of cyclic AMP and activation of protein kinase C in alveolar type II cells, and that the increase in [Ca21]i was mediated by the Ca21 influx through the plasma membrane. This mechanism to modulate [Ca21]i may play a role in the regulation of surfactant secretion by alveolar type II cells. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Alveolar type II cell; Cytoplasmic Ca21; Pulmonary surfactant secretion; Cyclic AMP; Protein kinase C
Introduction The alveolar type II cell synthesizes and secretes pulmonary surfactant which lowers the surface tension at the air-liquid interface in the lung and provides for alveolar stability [see for review 1]. The secretion of pulmonary surfactant from alveolar type II cells is regulated * Corresponding author. Department of Pharmacological Sciences, Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe Hon-machi, Kumamoto 862-0973, Japan. Tel.: 81-96-371-4185; fax: 81-96-362-7795. E-mail address: [email protected] (Y. Isohama) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 0 2 8 -1
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by at least three different signal transduction pathways. These pathways involve different second messengers and protein kinases, i.e. the adenylate cyclase system with activation of cyclic AMP-dependent protein kinase (PKA) [2–5], the phosphoinositide-specific phospholipase C system with activation of protein kinase C (PKC) [6–9], and the Ca21/calmodulindependent system [10–12]. Several studies indicated that the interactions among these different signal transduction pathways played important roles in the overall regulation of surfactant secretion [10, 11, 14–16]. For instance, the stimulatory effect of the combination of an A2 adenosine agonist with phorbol 12-myristate 13-acetate (PMA) was significantly greater than the sum of the effects of individual secretagogues [15]. Similarly, the combination of terbutaline, a b-adrenergic agonist, with PMA or with 1-oleoyl-2-acetyl-sn-glycerol (OAG) stimulated the secretion synergistically [9, 16]. The synergism of the adenosine agonist and PMA is considered to be mediated by the increase of cyclic AMP formation [15]. However, the synergism of terbutaline and PKC activators seems to be mediated by other mechanisms, because cyclic AMP formation induced by terbutaline was not enhanced by the combination with PKC activators [16]. Because Ca21 is generally considered as an important second messenger for the surfactant secretion, we examined the combined effect of terbutaline with PMA on cytoplasmic free Ca21 concentration ([Ca21]i) in the present study. Interestingly, we found that the combination of terbutaline with PMA rapidly increased [Ca21]i in alveolar type II cells. Methods Cell culture Alveolar type II cells were isolated from the lungs of adult pathogen-free male Wistar rats (180–200 g), as described previously [5, 17, 18]. Briefly, trypsin was used to dissociate the cells from the lung tissues. The resultant cell suspension was incubated on rat IgGcoated plastic petri dishes for 30 min to remove the non-type II cells. The isolated alveolar type II cells were suspended at 1 3 106 cells/ml in Dulbecco’s modified Eagle’s medium (DMEM) containing 2 mM L-glutamate, 100 units/ml penicillin, 100 mg/ml streptomycin and 10% fetal bovine serum. This procedure routinely yielded about 1.0 3 107 cells/rat. The cells were dispensed onto glass bottom culture dishes (MatTek, Ashland MA, USA) at a density of 2 3 105 cells/cm2 and cultured in 5% CO2/95% air at 378C. In all experiments, nonadherent cells were removed from the dishes by washing with DMEM after 20–22 h cultivation. The purity of the overnight cultured type II cells was 95 6 3% (mean 6 SEM, n58), and the viability of the cells was 98 6 1% (mean 6 SEM, n58) as judged by the trypan blue exclusion test. Measurement of cytoplasmic Ca21 Cultured cells were washed and incubated in HEPES-buffer (140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 24 mM glucose and 10 mM HEPES, pH 7.4) containing 10 mM fura-2/AM and 2% BSA for 60 min at 378C. After fura-2 loading, the cells were washed twice with the HEPES-buffer and stored under light-free conditions until use. The [Ca21]i of a single cell was measured at 100 ms intervals with a digital imaging microscopic system
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Mu-1000 (Inter deck. Inc. Osaka, Japan). The ratio of the intensities of fluorescent emission at 510 nm with excitation at 340 nm and 380 nm was measured. The fluorescent excitation beam was led to drop on the cells, and images were recorded with videofilm and analyzed with a computer. Utilizing this protocol, cells were uniformly labeled when observed by fluorescence microscopy without evidence of the organellar sequestration which has been reported with longer incubation time [19]. [Ca21]i was calculated according to the method of Grynkiewicz et al. [20]. Phosphatidylcholine secretion The cells (5 3 105 cells/cm2) were cultured for 20 h with 74 kBq/mL [methyl-3H]choline to label the phosphatidylcholine pool, after which the medium was removed and the cells were washed with fresh DMEM. Fresh DMEM was then added, and the cells were returned to the incubator. After a 30-min preincubation in the fresh medium, agonists or solvent vehicle were added, and the incubation was continued for 90 min. The medium was then aspirated, and the cells were lysed with ice-cold 0.05% Triton X-100. Lipids were extracted from both cells and medium using the method of Folch et al. [21], and the amount of radioactivity was measured with a liquid scintillation counter. The secretion rate was expressed as the amount of [3H]phosphatidylcholine in the medium after 90-min incubation as a percentage of the total amount in cells plus medium. To assess cellular integrity, the activity of lactate dehydrogenase (LDH) in the cells and medium was measured with an LDH assay kit (Nippon Shoji Co., Ltd., Osaka, Japan). The amount of LDH released into the medium did not exceed 1% of the total cell content in any experiment. Materials The rats were purchased from Kyudo Farm (Fukuoka, Japan). Terbutaline sulfate, phorbol 12-myristate 13-acetate, forskolin, 1-oleoyl-2-acetyl-sn-glycerol and 4a-phorbol 12, 13didecanoate were from Sigma (St Louis, MO, USA). Fura-2/AM was from Dojin Chem. (Kumamoto. Japan). Fetal bovine serum was from JHR Bioscience (Lenexa, KS, USA). All drugs were applied to the incubation buffer. To examine the combined effect of different agonists, the first agonist was added 1 min prior to the addition of the second agonist. Terbutaline sulfate was dissolved in HEPES-buffer and other agents in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide in the buffer was less than 1%. Analyses of results Data are reported as the mean 6 SEM. The data were analyzed by Student’s t-test or analysis of variance (ANOVA) followed by Newman-Keuls test as shown in the results. Differences between means were considered significant at p value of ,0.05. Results ATP is the most effective stimulant to increase [Ca21]i in type II cells [12, 13], therefore, we determined first the effect of ATP on [Ca21]i using fura-2 technique. ATP rapidly in-
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Table 1 Effects of terbutaline, PMA and their combination on [Ca21]i in single rat alveolar type II cells
1st agonist
2nd agonist
ATP (0.1 mM) ATP (1 mM) Terbutaline (10 mM) PMA (10 nM) Terbutaline PMA
— — — — PMA Terbutaline
No. of cells examined (No. of experiments) 15 (4) 26 (7) 13 (4) 14 (4) 58 (10) 45 (8)
Basal [Ca21]i (nM)
Mean peak [Ca21]i (nM)
73 6 8 72 6 7 77 6 5 76 6 6 73 6 2 74 6 8
201 6 28a 471 6 22a 78 6 7 82 6 9 382 6 29a 178 6 23b
Values are mean 6 SEM for the number of cells indicated in 4–10 experiments (indicated in parentheses). In each experiment, alveolar type II cells were isolated from 2 rats and cultured overnight as shown in material and methods. The first agonist was added 1 min before the second. Significance was determined by paired Student’s t-test: a p,0.01, b p,0.05 for difference from the basal [Ca21]i.
creased [Ca21]i in a concentration-dependent manner (Table 1, Fig. 1). In 7 different experiments with a total of 26 cells, 1 mM of ATP increased [Ca21]i in 24 cells (92%). This high percentage of cells responding to 1 mM of ATP is in agreement with the estimated proportion of type II cells (<95%). Then, we examined the effects of terbutaline, PMA, and their combination on [Ca21]i (Table 1). Terbutaline (10 mM) or PMA (10 nM) alone had no significant
Fig. 1. Representative trace of the cytoplasmic free Ca21 concentration ([Ca21]i) in alveolar type II cells in response to terbutaline, PMA and their combination. Cultured alveolar type II cells were loaded with 10 mM fura2/AM for 60 min. One mM ATP (A), 10 mM terbutaline (B), 10 nM PMA (C) or combination of terbutaline and PMA (D) were applied to the cells, as indicated by the above tracings.
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Fig. 2. Combined effect of terbutaline and PMA on [Ca21]i in alveolar type II cells at various concentrations. Fura-2 loaded cells were incubated, as indicated above tracings. A: Cells were washed to remove the agonists, and second or third combinations were applied to the bath. The traces were from the same cell during the same experiment. B: Different concentrations of PMA were added to the bath in the presence of 10 mM of terbutaline.
effect on [Ca21]i. However, the addition of PMA 1 min after the application of terbutaline increased [Ca21]i which peaked at 382 6 29 nM. An example is shown in Fig. 1. This increase in [Ca21]i was dependent on the concentration of terbutaline (0.1–10 mM) and of PMA (0.1– 10 nM) (Fig. 2). Terbutaline following PMA also increased [Ca21]i, but the effect was weaker than that of PMA following terbutaline (Table 1). We next examined the effects of other combinations (Fig. 3). OAG (100 mM), another type of PKC activator, combined with terbutaline significantly increased [Ca21]i. Combination of forskolin (1 mM), an activator of adenylate cyclase, with PMA also increased [Ca21]i. In contrast, 4a-PDD (10 nM), an inactive phorbol ester, combined with terbutaline failed to increase [Ca21]i.
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Fig. 3. Combined effects of terbutaline with OAG, terbutaline with 4a-PDD and forskolin with PMA on [Ca21]i in alveolar type II cells. Fura-2-loaded cells were incubated with the combination of forskolin (1 mM) and PMA (10 nM) (A), terbutaline (10 mM) and OAG (100 mM) (B), or terbutaline (100 mM) and 4a-PDD (10 nM) (C), as indicated by the above tracings. D: Mean peak [Ca21]i 6 SEM elicited with indicated agonists. Each bar corresponds to 12 cells (3 different experiments). Significance was determined by ANOVA followed by NewmanKeuls test: * p,0.05 for difference from the basal [Ca21] i.
Further, we examined the influence of extracellular Ca21 on the increase in [Ca21]i induced by combination of terbutaline with PMA. The increase in [Ca21]i induced by the combination was dependent on the extracellular Ca21 concentration. (Fig. 4A, B and C). Several bivalent cations, such as Co21, Ni21, Mn21 and Cd21, bind too strongly with a group located inside the Ca21 channel, become competitive channel blockers [22]. Therefore, we examined the effect of Co21 on the increase in [Ca21]i. Co21 abolished the increase in [Ca21]i induced by the combination of terbutaline with PMA (Fig. 4D). Finally, we examined the effect of Co21 on [3H]phosphatidylcholine secretion induced by the combination of terbutaline and PMA. Pretreatment with Co21 did not affect the secretion induced by terbutaline or PMA alone. However, Co21 significantly decreased the secretion increased by the combination of terbutaline and PMA (Fig. 5).
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Fig. 4. Influence of extracellular Ca21 and Co21 on the increase of [Ca21]i induced by the combination of terbutaline with PMA in alveolar type II cells. Fura-2-loaded cells were incubated in control buffer containing 1 mM of Ca21 (A), in buffer containing 0.01 mM Ca21 (B), in Ca21-free buffer containing 1 mM EGTA (C), or in the buffer containing 1 mM Ca21 and 1 mM Co21 (D). Ten mM terbutaline and 10 nM PMA were applied to the cells, as indicated by the above tracings. E: Mean peak [Ca21]i 6 SEM elicited with indicated agonists. Each bar corresponds to 12 cells (3 different experiments). Significance was determined by ANOVA followed by NewmanKeuls test: * p,0.05 for difference from the basal [Ca21]I; † p,0.05 for difference from 1 mM Ca21 without Co21.
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Fig. 5. Effect of Co21 on phosphatidylcholine secretion induced by terbutaline, PMA and their combination. [3H]choline labeled cells were washed, and incubated with indicated agonists for 90 min. After incubation, lipids were extracted from medium and cells as described in materials and methods. Vehicle or 1 mM Co21 was pretreated 5 min prior to the addition of agonists. Secretion is expressed as amount of [3H]lipid in medium after 90min incubation as percentage of total in cell and medium. The data are mean 6 SEM from 4 different experiments. Significance was determined by paired Student’s t-test: * p,0.05 for difference.
Discussion Pulmonary surfactant secretion has been studied extensively in primary cultures of type II cells and shown to be stimulated by several physiological and pharmacological agents. Among these agents, terbutaline, a b-adrenergic agonist, acts on the membrane receptor coupled to activation of adenylate cyclase with generation of cyclic AMP and cyclic AMPdependent protein kinase. On the other hand, the effect of PMA on surfactant secretion is largely mediated by the activation of PKC. The current data show that the combined application of terbutaline and PMA increases [Ca21]i in alveolar type II cells. The result of the combination of forskolin with PMA suggested that the increase of [Ca21]i is dependent on cyclic AMP formation. Considering the finding that PMA was interchangeable with OAG, we assumed that the increase in [Ca21]i induced by the combination of terbutaline with PMA occurred as a result of the combination of cyclic AMP formation and activation of PKC. To our knowledge, similar regulation of [Ca21]i in any other cell system has not been reported. We therefore speculate that the increase in [Ca21]i by the combination is probably mediated by the mechanisms specified in type II cells. The increase in [Ca21]i induced by PMA following terbutaline was more marked than that by terbutaline following PMA (Table 1). This may occur through the PKC-mediated phosphorylation of b2-adrenergic receptor followed by desensitization of the receptor [23, 24]. This is supported by the finding that
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terbutaline-induced cyclic AMP formation was decreased by the pre-treatment of PMA in type II cells [16]. The increase in [Ca21]i induced by the combination of terbutaline and PMA seems to be mediated, at least in part, by the influx of extracellular Ca21 through Ca21 channels. This idea is supported by the following findings; 1) the increase of [Ca21]i induced by the combination was markedly suppressed by the removal of extracellular Ca21, 2) Co21 inhibited the increase of [Ca21]i, and 3) the combination of terbutaline with PMA did not affect inositol phosphates production in the cells (data not shown). There is little information about Ca21 channels in alveolar type II cells: L-type voltage-dependent channels are present in a pulmonary epithelial cell line (L2 cell) and indirect evidence suggests their presence in type II cells [25, 26], but their physiological significance remains to be established. However, Ca21 is considered as an important second messenger for the secretion of pulmonary surfactant. This is suggested by reports that several stimuli for surfactant secretion are accompanied by a Ca21 increase [27– 30], and strongly suggested by the use of Ca21 ionophores [10–12]. Therefore, we assumed that the increase in [Ca21]i by the combination of terbutaline and PMA is accompanied with synergistic secretion of phosphatidylcholine by these drugs. Supporting this hypothesis, Co21 significantly inhibited the phosphatidylcholine secretion induced by the combination of terbutaline and PMA, without affecting the effect of each drug alone. In the current study, neither terbutaline nor PMA changed the [Ca21]i by itself. These results are consistent with those of Rice et al [29]. These also consist with previous studies demonstrating terbutaline-induced activation of cyclic AMP-dependent kinase and PMAinduced mobilization of PKC directly in association with surfactant secretion [9, 31]. However, Sano et al. have reported a small increase in the [Ca21]i induced by terbutaline or forskolin with quin2-loaded type II cells [11]. They described that terbutaline-induced increase in [Ca21]i occurred in the absence of extracellular Ca21 and that PMA inhibited this increase [11]. The discrepancy between the two results might be due to some methodological difference, e.g., fluorescent indicator or in the procedure for cell culture. On the other hand, ATP effectively increased [Ca21]i in type II cells, in current and previous studies [12, 13]. ATPinduced increase in [Ca21]i is believed to be mediated by IP3 formation and Ca21 release from intracellular store site. However, Dorn et al. showed that ATP-induced increase in [Ca21]i was composed of two phases, transient and prolonged plateau phases, and that the plateau phase was abolished by chelation of extracellular Ca21 [13]. We are interested in their findings, because ATP, by itself stimulates the formation of both cyclic AMP and diacylglycerol in type II cells [7, 14]. In addition, in our preliminary study, the increase in [Ca21]i by combined application of three agents, terbutaline, PMA and ATP, was considerably less than sum of the effect of ATP alone and combination of terbutaline with PMA (data not shown). Therefore, it might be possible that ATP increases [Ca21]i not only through IP3dependent Ca21 release but also through Ca21 influx mediated by the interaction between cyclic AMP- and PKC-dependent pathway. However, further studies are needed to establish this idea. In conclusion, our findings provide the first evidence that the [Ca21]i is increased by the combined application of terbutaline and PMA, and that the activation of both cyclic AMPand PKC-dependent signaling pathway are necessary for this stimulation. The present and previous studies indicate that there are several interactions between different signal-transduction
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mechanisms for surfactant secretion, including mobilization of [Ca21]i and enhancement of cyclic AMP formation. Considering that alveolar type II cells in vivo are exposed to various mediators simultaneously, the secretion of surfactant might be influenced by these interactions. Acknowledgments We thank W. Hirakawa and K. Kurita for their technical assistance. We also thank Dr. SA. Rooney in Yale University for his critical review of this manuscript. This study was supported by Grants-in-Aid 05857258 and 07772175 (to Y. Isohama) from the Ministry of Education, Sciences, Sports and Culture of Japan. References 1. Rooney SA. Regulation of surfactant secretion. In: Rooney SA, editor. Lung surfactant: Cellular and molecular processing. Austin. TX: Landes, 1998, pp.139–163. 2. Brown LAS, Longmore WJ. Adrenergic and cholinergic regulation of lung surfactant secretion in the isolated perfused rat lung and in the alveolar type II cell in culture. J. Biol. Chem. 1981; 256: 66–72. 3. Dobbs LG, Mason RJ. Pulmonary alveolar type II cells isolated from rats. Release of phosphatidylcholine in response to beta-adrenergic stimulation. J. Clin. Invest. 1979; 63: 378–87. 4. Gilfillan AM, Rooney SA. Functional evidence for adenosine A2 receptor regulation of phosphatidylcholine secretion in cultured type II pneumocytes. J. Pharmacol. Exp. Ther. 1988; 241: 907–14. 5. Kai H, Isohama Y, Takaki K, Oda Y, Murahara K, Takahama K, Miyata T. Both b1- and b 2-adrenoceptors are involved in mediating phosphatidylcholine secretion in rat type II pneumocyte cultures. Eur. J. Pharmacol. 1992; 212: 101–3. 6. Chander A, Sen N, Wu AM, Spitzer AR. Protein kinase C in ATP regulation of lung surfactant secretion in type II cells. Am. J. Physiol. 1995; 268: L108–16. 7. Griese M, Gobran LI, Rooney SA. ATP-stimulated inositol phospholipid metabolism and surfactant secretion in rat type II pneumocytes. Am. J. Physiol. 1991; 260: L586–93. 8. Rice WR, Dorn CC, Singleton FM. P2-purinoceptor regulation of surfactant phosphatidylcholine secretion. Relative roles of calcium and protein kinase C. Biochem. J. 1990; 266: 407–13. 9. Sano K, Voelker DR, Mason RJ. Involvement of protein kinase C in pulmonary surfactant secretion from alveolar type II cells. J. Biol. Chem. 1985; 260: 12725–29. 10. Dobbs LG, Gonzalez RF, Marinari LA, Mescher EJ, Hawgood S. The role of calcium in the secretion of surfactant by rat alveolar type II cells. Biochim. Biophys. Acta. 1986; 877: 305–313. 11. Sano K, Voelker DR, Mason RJ. Effect of secretagogues on cytoplasmic free calcium in alveolar type II epithelial cells. Am. J. Physiol. 1987; 253: C679–86. 12. Voyno-Yasenetskaya TA, Dobbs LG, Williams MC. Regulation of ATP-dependent surfactant secretion and activation of second-messenger systems in alveolar type II cells. Am. J. Physiol. 1991; 261:105–9. 13. Dorn CC, Rice WR, Singleton FM. Calcium mobilization and response recovery following P2-purinergic stimulation of rat isolated alveolar type II cells. Br. J. Pharmacol. 1989; 97: 163–70. 14. Griese M, Gobran LI, Rooney SA. A2 and P2 purine receptor interactions and surfactant secretion in primary cultures of type II cells. Am. J. Physiol. 1991; 261: L140–7. 15. Griese M, Gobran LI, Rooney SA. Potentiation of A2 purinoceptor-stimulated surfactant phospholipid secretion in primary cultures of rat type II pneumocytes. Lung 1993; 171: 75–86. 16. Isohama Y, Takahama K, Kai H, Miyata T. Cross-talk between b-adrenergic and other signal transduction pathways in pulmonary surfactant secretion from cultured alveolar type II cells. Jpn. J. Pharmacol. 1993; 61 (suppl.): 82p. 17. Isohama Y, Matsuo T, Kai H, Takahama K, Miyata T. Changes in b1- and b2-adrenoceptor mRNA levels in alveolar type II cells during cultivation. Biochem. Mol. Biol. Int. 1995; 36: 561–8.
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