Regulation of SP-B and SP-C secretion in rat type II cells in primary culture

Laurice I. Gobran and Seamus A. Rooney

Division of Perinatal Medicine, Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06510


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Secretion of lung surfactant phospholipids is a highly regulated process. A variety of physiological and pharmacological agents stimulate surfactant phospholipid secretion in isolated type II cells. Although the lipid and hydrophobic protein components of surfactant are believed to be secreted together by exocytosis of lamellar body contents, regulation of surfactant protein (SP) B and SP-C secretion has not previously been examined. To address the question of whether secretion of SP-B and SP-C is stimulated by the same agonists that stimulate phospholipid secretion, we measured secretion of all four SPs under the same conditions used to measure phosphatidylcholine secretion. Freshly isolated rat type II cells were cultured overnight and exposed to known surfactant phospholipid secretagogues for 2.5 h, after which the amounts of SP-A, SP-B, SP-C, and SP-D in the medium were measured with immunoblotting. Secretion of SP-B and SP-C was stimulated three- to fivefold by terbutaline, 5'-(N-ethylcarboxyamido)adenosine, ATP, 12-O-tetradecanoylphorbol 13-acetate, and ionomycin. Similar to their effects on phospholipid secretion, the stimulatory effects of the agonists were abolished by Ro 31-8220. Secretion of SP-A and SP-D was not stimulated by the secretagogues tested. We conclude that secretion of the phospholipid and hydrophobic protein components of surfactant is similarly regulated, whereas secretion of the hydrophilic proteins is regulated differently.

surfactant protein; lung surfactant; regulated exocytosis; surfactant secretagogues; P2Y2 agonists; 12-O-tetradecanoylphorbol 13-acetate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LUNG SURFACTANT IS A COMPLEX of lipids, largely phospholipids, and proteins that is synthesized in the type II pneumocyte (36). Surfactant phospholipids are synthesized in the endoplasmic reticulum and stored in lamellar inclusion bodies, the secretory organelle characteristic of the type II cell, and finally secreted into the alveolar lumen by the process of regulated exocytosis (27, 31, 36). The phospholipid composition of isolated lamellar bodies is virtually identical to that of surfactant obtained by lung lavage (30), and there is abundant evidence that surfactant phospholipids are secreted together with lamellar body contents (3, 27, 31, 44).

Secretion of phosphatidylcholine, the principal lipid component of surfactant (30), has been extensively investigated in isolated type II cells (31, 32). There is considerable evidence that it is a regulated process; at least three distinct signaling mechanisms are known to regulate surfactant phospholipid secretion (32). Surfactant secretion in isolated type II cells can be stimulated and inhibited by a number of physiological and pharmacological agents (31, 32). Secretagogues include terbutaline, 5'-(N-ethylcarboxyamido)adenosine (NECA), and ATP, agents that activate beta -adrenergic, adenosine A2B, and purinergic P2Y2 receptors, respectively. Other surfactant secretagogues include 12-O-tetradecanoylphorbol 13-acetate (TPA), an agonist that directly activates protein kinase C, and ionomycin, an ionophore that promotes Ca2+ uptake.

In addition to the lipids that are its major component, surfactant contains four unique proteins: surfactant protein (SP) A, SP-B, SP-C, and SP-D (42). SP-A and SP-D are hydrophilic glycoproteins that have a role in innate lung immunity and host defense (6), whereas SP-B and SP-C are small hydrophobic proteins that are involved in the biophysical functions of surfactant (24). Secretion of SPs has been much less extensively investigated than that of the lipids. However, there is evidence that secretion of SP-A and SP-D occurs independently of lamellar bodies and is not regulated by the mechanisms that regulate surfactant phospholipid secretion (32). Lamellar bodies are enriched in SP-B and SP-C (28), and these organelles have a central role in the overall processing of both proteins (41). In addition, there is evidence that the in vivo secretion of SP-B and phosphatidylcholine has similar kinetics (20). Taken together, these data suggest that surfactant lipids and hydrophobic proteins are secreted together. Therefore, it is anticipated that secretion of phosphatidylcholine, SP-B, and SP-C is regulated by the same mechanisms.

The regulation of secretion of SP-B and SP-C has not previously been investigated. The goal of the present study was to investigate if secretion of the hydrophobic components of surfactant is stimulated by agonists that are known to stimulate surfactant phospholipid secretion. We therefore examined the effects of TPA, ATP, NECA, terbutaline, and ionomycin on secretion of SP-B and SP-C in isolated type II cells. We also measured secretion of SP-A and SP-D for comparison.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals and reagents. Rabbit polyclonal antibodies against SP-A, SP-B, and SP-D (4, 43) were obtained from Dr. Samuel Hawgood (University of California, San Francisco, CA), and a similar antibody against mature SP-C (37) was obtained from Byk Gulden (Konstanz, Germany). Porcine pancreatic elastase was obtained from Elastin Products (Owensville, MO), rat IgG from Accurate (Westbury, NY), DMEM from GIBCO BRL (Rockville, MD), FCS from HyClone (Logan, UT), horseradish peroxidase-conjugated goat anti-rabbit IgG from Sigma (St. Louis, MO), and Western blot chemiluminescence reagent from PerkinElmer Life Sciences (Boston, MA). TPA, ATP (sodium salt), and NECA were obtained from Sigma, terbutaline sulfate (Brethine) from Geigy Pharmaceuticals (West Caldwell, NJ), ionomycin from Calbiochem (San Diego, CA), and Ro 31-8220 (bisindolylmaleimide IX) from Alexis Biochemicals (San Diego, CA).

Type II cell isolation and culture. Type II cells were isolated from adult male Sprague-Dawley rats as described previously (15). The isolation method involved digestion of the blood-free lungs with elastase and the separation of type II cells from contaminating cells by panning on bacteriological dishes coated with IgG (7). The freshly isolated cells were plated at a density of 4-5 × 106 cells/dish on 35-mm-diameter plastic tissue culture dishes (Falcon; Becton Dickinson, Lincoln Park, NJ) in 1.5 ml of DMEM containing 10% FCS, streptomycin (100 µg/ml), penicillin (100 U/ml), gentamicin (10 µg/ml), and amphotericin B (2.5 µg/ml) and cultured for 18-20 h in a humidified atmosphere of 90% air-10% CO2. We (15) previously reported that >95% of the attached cells were identifiable as type II pneumocytes at that stage.

Treatment with surfactant secretagogues. After overnight culture, the medium was removed, and the type II cell monolayers were washed with fresh DMEM without FCS or antibiotics. Fresh DMEM was then added, and the cells were returned to the incubator. After 30 min of preincubation in the fresh medium, agonists or solvent vehicle were added, and the incubation was continued for an additional 150 min (total 180 min) except in time-course experiments in which the total period of incubation varied from 30 to 240 min. The medium was then removed and centrifuged at 200 g for 10 min to remove any floating cells. The agonist concentrations used were those previously shown to optimally stimulate phosphatidylcholine secretion and to be nontoxic in type II cells (13-16).

Immunoblotting. Because an ELISA assay is currently not available for SP-C, we measured secretion of all four SPs by Western blotting. For analysis of SP-B and SP-C, the 200-g supernatant was centrifuged at 20,000 g for 1 h. The pellet was suspended in Laemmli sample buffer (25), heated at 37°C for 15 min, and subjected to electrophoresis under reducing conditions on Novex 16% Tricine gels (Invitrogen, Carlsbad, CA) for SP-B analysis or on NuPAGE 12% Bis-Tris gels in NuPAGE MES SDS buffer (Invitrogen) for SP-C analysis. For analysis of SP-A and SP-D, the 200-g supernatant was treated with cold 5% TCA. The precipitated proteins were then suspended in Laemmli sample buffer, boiled for 5 min, and subjected to electrophoresis under reducing conditions (25) on Novex 12% Tris-glycine gels with a 4% stacking gel overlay. The resolved proteins were electrotransferred to either nitrocellulose (SP-A, SP-B, and SP-D) or polyvinylidene difluoride (SP-C) membranes in 25 mM Tris containing 192 mM glycine and 20% methanol or NuPAGE transfer buffer (Invitrogen), respectively. The membranes were then sequentially incubated in Tris-buffered saline containing Tween 20 and nonfat dry milk for 2 h (46), a 1:2,000 (SP-A and SP-D) or 1:1,000 (SP-B and SP-C) dilution of primary antibody for 1 h, a 1:20,000 dilution of peroxidase-conjugated goat anti-rabbit IgG for 1 h, and Western blot chemiluminescence reagent (PerkinElmer Life Sciences) for 1 min. The blots were exposed to X-ray film for 15-30 s, and the autoradiographs were quantified by scanning densitometry as previously described (46).

Data analysis and statistics. In each experiment, type II cells were isolated from the pooled lungs of eight rats. The cells were distributed among the various treatment groups, usually 12 dishes/group. Data are presented as means ± SE from the number of experiments indicated. The data were analyzed with ANOVA followed by the Student-Newman-Keuls test or Student's two-tailed t-test for paired samples. Instat 3.0 (GraphPad Software, San Diego, CA) was used for statistical analysis.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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The effects of known surfactant phospholipid secretagogues on secretion of SP-B and SP-C in isolated type II cells are shown in Fig 1. Secretion of both proteins was clearly increased by TPA, ATP, NECA, terbutaline, and ionomycin. SP-B secretion was increased three- to fivefold by the agonists, whereas that of SP-C was increased three- to fourfold. When analyzed by one-way ANOVA, there were no significant differences between the stimulatory effects of the various agonists. Only the mature forms of SP-B and SP-C were detected in the medium; there was no evidence that precursor forms were secreted. In contrast to that of SP-B and SP-C, secretion of SP-A and SP-D under the same experimental conditions was not increased by a combination of all of the agonists tested (Fig. 2).


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Fig. 1.   Effects of agonists on surfactant protein (SP) B (A) and SP-C (B) secretion in isolated type II cells. Cells were isolated from adult rats and cultured for 18-20 h in serum-containing medium. Medium was then removed and replaced with fresh serum-free medium. After 30 min of preincubation in fresh medium, 12-O-tetradecanoylphorbol 13-acetate (TPA; 10-7 M), ATP (10-3 M), 5'-(N-ethylcarboxyamido)adenosine (NECA; 10-5 M), terbutaline (3.5 × 10-5 M), ionomycin (5 × 10-8 M), or no agonist (controls) were added. Incubation was then continued for an additional 150 min, after which SPs in the medium were analyzed. Protein from equal aliquots of medium was applied to all lanes on each gel. Immunoblots were quantitated by densitometry. Values are means ± SE expressed as arbitrary densitometric units from 4-6 SP-B and 6-9 SP-C experiments. Data were analyzed statistically with Student's 2-tailed t-test for paired samples. Effects of all agonists were significant, P < 0.05.



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Fig. 2.   Secretion of SP-A and SP-D in response to secretagogues. Cells were treated with either no agonist (control) or with a combination of TPA (10-7 M), ATP (10-3 M), terbutaline (3.5 × 10-5 M), NECA (10-5 M), and ionomycin (5 × 10-8 M). Other experimental details are described in Fig. 1. These data are from typical experiments. Each SP-A lane is from a different pool of cells. An aliquot of rat lung lavage fluid protein (LL) was applied to each gel.

Secretion of SP-B and SP-C in response to TPA increased linearly with time for ~3 h, after which it plateaued (Fig. 3). Basal secretion had similar time courses (data not shown). Secretion in response to ATP and terbutaline exhibited a similar time course to that of TPA (Fig. 4).


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Fig. 3.   Time course of the effect of TPA on SP-B (A) and SP-C (B) secretion. Cells were cultured for 18-20 h in serum-containing medium as described in Fig. 1. They were then incubated in serum-free medium for times indicated. TPA (10-7 M) was added at 30 min. SP-B and SP-C in the medium were measured as in Fig. 1. The 30-min samples were taken before addition of TPA. Data are means ± SE from 5-6 SP-B and 2-3 SP-C experiments.



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Fig. 4.   Time course of the effects of TPA, ATP, and terbutaline on SP-B and SP-C secretion. Cells were cultured, and SP-B and SP-C were measured in the medium as described in Fig. 1. TPA (10-7 M), ATP (10-3 M), or terbutaline (3.5 × 10-5 M) was added at 30 min. Control (30 min) samples were taken before addition of agonists. Equal aliquots of medium were applied to each gel. Aliquots of rat lung lavage protein were also applied.

There are additive and synergistic interactions between agonists in the stimulation of phosphatidylcholine secretion in isolated type II cells. For instance, the effect of the combination of TPA, ATP, and NECA is significantly greater than that of any of these agonists alone (18). We therefore examined the effect of the same combination of agonists on secretion of SP-B and SP-C. As shown in Fig. 5, the combination of TPA, ATP, and NECA stimulated secretion of both proteins to a greater extent than did TPA alone. The combination stimulated secretion of both proteins to a significantly greater extent than did TPA alone (Fig. 6).


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Fig. 5.   Secretion of SP-B and SP-C in response to TPA alone and to the combination of TPA, ATP, and NECA. Cells were cultured for 18-20 h in serum-containing medium and then preincubated for 30 min in serum-free medium as in Fig. 1. TPA (10-7 M); a combination of TPA, ATP (10-3 M), and NECA (10-5 M); or no agonist (control) was then added, and the incubation was continued for 150 min. SP-B and SP-C in the medium were then measured. Equal aliquots of medium were applied to each gel. Aliquots of rat lung lavage protein were also applied.



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Fig. 6.   Quantitation of SP-B (A) and SP-C (B) secretion in response to TPA alone and to the combination of TPA, ATP, and NECA. Cells were cultured and treated with secretagogues as in Fig. 5. Data are means ± SE from 5 SP-B and 8 SP-C experiments and were analyzed statistically by ANOVA for repeated measures and the Student-Newman-Keuls test. Significantly different from control: *P < 0.05; **P < 0.01; ***P <0.001. Significantly different from TPA alone: **P < 0.05; ***P < 0.05.

Ro 31-8220 is a widely used inhibitor of protein kinase C (40), although it also inhibits other protein kinases (1, 2, 21) and has a number of other effects (2, 19, 26). We (34) previously reported that Ro 31-8220 virtually abolishes the stimulatory effects of TPA, ATP, NECA, terbutaline, and ionomycin on phosphatidylcholine secretion in type II cells. As shown in Fig. 7, it had a similar effect on SP-B and SP-C secretion. Ro 31-8220 completely abolished the stimulatory effects of the combination of TPA, ATP, and NECA. We (33) previously reported that 10-5 M Ro 31-8220, as determined by lactate dehydrogenase release, is not toxic to type II cells.


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Fig. 7.   Effect of Ro 31-8220 on agonist-stimulated SP-B and SP-C secretion. Cells were incubated with no agonist (control) or a combination of TPA, ATP, and NECA as in Figs. 5 and 6. Ro 31-8220 (10-5 M) was added 30 min before the agonists. +, Presence; -, absence. Data are means ± SE from 3 SP-B and 4 SP-C experiments and were analyzed statistically by ANOVA for repeated measures and the Student-Newman-Keuls test. Significantly different from control: *P < 0.001; **P < 0.05. Significantly different from agonists + Ro 31-8220 group: *P < 0.001; **P < 0.01. Results from control, Ro 31-8220 alone, and Ro 31-8220 + agonists groups were not significantly different from each other.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Most studies on the regulation of secretion of the phospholipid components of surfactant have been done on isolated rat type II cells in primary culture (31). We therefore investigated secretion of SPs in the same system. A problem with isolated type II cells, however, is that they rapidly differentiate in culture. Expression of SP-A, SP-B, SP-C, and SP-D declines with time in culture (10, 45) as does the response to surfactant phospholipid secretagogues (38). Nevertheless, secretion of phosphatidylcholine can be readily measured in freshly isolated type II cells cultured on plastic for 24 h, the system in which the effects of secretagogues have also been established. Secretion of SP-A has also been measured in type II cells cultured for 24 h (9, 35). Therefore, we postulated that secretion of SP-B and SP-C could be measured under the same conditions that are used to study secretion of the phospholipids if the hydrophobic SPs are secreted together with the lipids. We found that secretion of all four SPs could be measured in type II cells cultured for ~24 h.

Phosphatidylcholine secretion by type II cells is usually expressed as the amount in the medium as a percentage of total phosphatidylcholine in the cells and medium combined (3, 27, 31, 44). However, in the current study, we did not express secretion of SP-B and SP-C in that form for a number of reasons. The cellular proteins consist of the precursor proteins as well as the mature peptides, whereas only the mature proteins are found in the medium. In addition, because there are large disparities between the amount of the proteins in the cells and in the medium, accurate quantitation of the amount in the medium as a percentage of the total is difficult given the methodology used.

The data show that secretion of SP-B and SP-C is regulated by the same mechanisms previously established for phosphatidylcholine. First, secretion of both proteins and lipids is stimulated by beta -adrenergic, adenosine A2B, and purinergic P2Y2 receptor agonists as well as by TPA and ionomycin. Second, a combination of TPA, ATP, and NECA stimulates secretion of both proteins and lipids to a greater extent than TPA alone. Finally, the stimulatory effects of agonists on secretion of both proteins and lipids are abolished by Ro 31-8220.

There are some differences, however, between secretion of surfactant lipids and hydrophobic proteins. One difference relates to the extent of stimulation in response to the different agonists. Whereas there was no significant difference between the different agonists in the extent to which they increased SP-B or SP-C secretion, they increased phosphatidylcholine secretion to different extents (30). In previous studies (15, 17, 18) in which the effects of all agonists were measured simultaneously, phosphatidylcholine secretion was doubled by terbutaline and NECA and increased two- to threefold by ionomycin, fourfold by ATP, and as much as fivefold by TPA. In contrast, all agonists stimulate SP-B and SP-C secretion to essentially the same extent. A second difference is in the time course of secretion. Secretion of phosphatidylcholine in response to a variety of agonists is linear for at least 4 h (8, 14), whereas that of SP-B and SP-C reached a plateau after 3 h. Currently, we have no explanation for these differences. They cannot be due to exhaustion of the SP-B and SP-C pools because secretion was measured while the rate of secretion was increasing linearly with time. In addition, further amounts of both SP-B and SP-C were secreted in response to a combination of agonists. They may be attributable to differences between the rates of reuptake of the lipids and proteins and the extent to which such rates are affected by secretagogues (11).

In contrast to SP-B and SP-C, secretion of SP-A and SP-D was not increased by the surfactant phospholipid secretagogues. This finding is consistent with previous reports that suggest that secretion of the hydrophilic SPs is regulated differently from that of the phospholipids. There is substantial evidence that SP-A is largely secreted independently of lamellar bodies and by a constitutive mechanism rather than by regulated exocytosis (27, 31, 32). In previous studies, SP-A secretion in isolated type II cells was not generally stimulated by agonists that stimulate phospholipid secretion (12, 35, 45), although it was stimulated by TPA in two studies (9, 45). Furthermore, both in vivo (22, 23) and tissue culture (29) experiments suggested that newly synthesized SP-A is secreted independently of lamellar bodies. Lamellar bodies do not contain SP-D (5, 39), which means that it must also be secreted independently of the lipids. Consistent with this, our data show that SP-D secretion is not stimulated by a combination of agonists. Similarly, secretion of SP-D was not stimulated by TPA or terbutaline in type II cells cultured under conditions that maintain their phenotype for several days (45).

In summary, we have shown that surfactant phospholipid secretagogues also stimulate SP-B and SP-C secretion in isolated type II cells. This is consistent with the notion that secretion of the phospholipid and hydrophobic protein components of surfactant are similarly regulated.


    ACKNOWLEDGEMENTS

We thank Dr. Samuel Hawgood (University of California, San Francisco, CA) and Dr. Wolfram Steinhilber (Byk Gulden, Konstanz, Germany) for generously providing the antibodies.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-31175 and HL-43320.

A preliminary report of these data was presented at the annual meeting of the American Thoracic Society in San Francisco, CA, in May 2001. An abstract has been published (Am J Respir Crit Care Med 163: A329, 2001).

Address for reprint requests and other correspondence: S. A. Rooney, Dept. of Pediatrics, Yale Univ. School of Medicine, PO Box 208064, New Haven, CT 06520-8064 (E-mail: seamus.rooney{at}yale.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 June 2001; accepted in final form 2 August 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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