INTRODUCTION

Pulmonary surfactant is a complex mixture of phospholipids and proteins secreted by the type II alveolar epithelial cells. Surfactant functions as the surface tension-reducing material at the air-liquid interface of the alveoli. In order to maintain an appropriate amount of functional surfactant in the alveolar space, surfactant turnover should be tightly regulated. The rate of surfactant lipid uptake by intact lungs and isolated type II cells has been shown to be stimulated by agents that promote surfactant secretion. Secretagogues have augmented the clearance of lipid from rabbit lungs (Pettenazzo et al., 1989) and the uptake of surfactant protein and dipalmitoylphosphatidylcholine into both isolated, perfused rat lungs (Fisher et al., 1985, 1989, 1991) and type II cells cultured on microporous membranes (Chinoy et al., 1993). Endocytosis of phospholipids is enhanced also by surfactant protein A (SP-A),¹ a major protein constituent of lung surfactant. SP-A (26-38 kDa, reduced) is a glycoprotein that shows calcium-dependent binding to phospholipid vesicles through an internal hydrophobic domain (Ross et al., 1986). Several studies have shown that SP-A stimulates the incorporation of phospholipid by primary cultures of type II cells (Wright et al., 1987; Tsuzuki et al., 1993; Bates et al., 1994). SP-A also functions to inhibit surfactant phospholipid secretion by type II cells (Dobbs et al., 1987; Kuroki et al., 1988). Thus, SP-A may play a pivotal role in the regulation of surfactant turnover. Type II cells express a high affinity receptor for SP-A (Kuroki et al., 1988, Wright et al., 1989) that recognizes the carboxyl-terminal portion of the SP-A molecule (Wright et al., 1989; Murata et al., 1993). Using an anti-idiotypic antibody approach, two groups have identified type II cell membrane proteins specific for SP-A with molecular masses of either 30-kDa (Strayer et al., 1993) or 55 kDa (Stevens et al., 1995). The cDNA for the 30-kDa receptor protein has been cloned and sequenced and the structure of the encoded protein deduced. Strayer et al. (1996) have shown that antibodies to the 30-kDa protein inhibit the binding of SP-A and mimic the functional ability of SP-A to down-regulate phospholipid secretion stimulated by secretagogues in isolated type II cells. Evidence has been presented that the receptors function physiologically since binding to type II cells isolated from rats after silica treatment showed a 2-fold increase in saturable SP-A binding over controls (Suwabe et al.,, 1991). In addition, SP-A metabolism was cell substrate-dependent. Type II cells plated on microporous membranes maintained their cuboidal shape and other differentiated characteristics and also demonstrated elevated binding and incorporation of SP-A as compared with cells plated on plastic tissue culture dishes (Bates et al., 1994; Chinoy et al., 1993). Since secretagogues stimulated the uptake of biosynthesized SP-A and phosphatidylcholine (Chinoy et al., 1993), in this report we have examined the possibility that secretagogues induce SP-A receptor activity and have found that secretagogues enhance SP-A binding to type II cells.

MATERIALS AND METHODS

Type II cells were isolated from anesthetized pathogen-free male Sprague-Dawley rats, weighing 200-250 g by the method of Dobbs et al. (1986), as described previously in detail (Chinoy et al., 1993). Briefly, after perfusion via the pulmonary artery and lavage through the tracheal cannula, lungs were digested with elastase and minced in the presence of deoxyribonuclease (DNase, Sigma) and fetal bovine serum (fetal calf serum, ICN/Flow Laboratories, ICN Biochemicals, Costa Mesa, CA). Cells were separated by filtration and enriched by plating on rat immunoglobulin G (IgG, Sigma)-coated Petri dishes. The purity of the freshly isolated type II cell preparation was routinely >80% by modified Papanicolaou stain, and the viability was >98% by vital dye exclusion.

Cells were plated at 5 × 10⁶ or 1.5 × 10⁶ type II cells in 24- or 12-mm inserts of Transwell microporous membranes (Costar, Cambridge, MA, 3-µm pore size), respectively, or 35-mm plastic tissue culture dishes (Costar) at 3 × 10⁶ cells/dish. Cells were cultured in 10% fetal calf serum in Eagle's minimal essential medium (MEM) at 37 °C in a humidified incubator with 5% CO₂ in air. After overnight culture and removal of nonadhered cells, the purity of the type II cells was >90%.

Alveolar lavage fluid was obtained from the lungs of bovine (slaughterhouse) or alveolar proteinosis patients. The surfactant was purified using density gradient centrifugation followed by dialysis and lyophilization as described previously (Fisher et al., 1991). SP-A was isolated from surfactant by using 1-butanol and -D-glucopyranoside extraction, dialysis, and microconcentration according to the method of Hawgood et al. (1985). SP-A was iodinated using IODO-GEN (Pierce) according to the directions provided by the manufacturer, and the iodinated protein was dialyzed extensively against Tris buffer. An aliquot of ¹²⁵I-SP-A was analyzed for protein concentration (Bradford, 1976) and precipitability in 10% trichloroacetic acid. The specific activity was 1000 ± 420 cpm/ng protein, and 96.1 ± 1.4% (mean ± S.D., n = 19) of the radioactive protein was precipitable with trichloroacetic acid. The iodinated SP-A was used within 2 weeks.

The purity of the SP-A preparation was monitored by SDS-polyacrylamide gel electrophoresis run under reducing conditions and stained with Coomassie Blue (Laemmli, 1970). The radiolabeled SP-A was transferred to nitrocellulose paper and exposed to Kodak x-ray diagnostic film for 30 min at 80° C. As shown previously (Bates et al., 1996), there was a single predominant band at 32 kDa and a minor band at 64 kDa for human SP-A and a predominant band at 32 kDa for bovine SP-A. The radiolabeled SP-A showed the same profile as the unlabeled SP-A.

Type II cells were cultured for 18 h after isolation. To begin an experiment, the culture media were removed, and the cells were washed two times with MEM to remove the nonadhered cells. To study the effect of secretagogues on SP-A binding, type II cells were preincubated with various secretagogues for the indicated time at 37 °C, and the binding assay was performed at 4 °C in the presence of secretagogues. In order to evaluate the involvement of protein kinase C (PKC) in the process of secretagogue-induced SP-A binding, a model for desensitization of PKC was used (Dubrowin and Brown, 1992). Briefly, type II cells were preincubated with phorbol 12-myristate 13-acetate (PMA, 10 nM) at 37 °C for 2 h. PMA (40 µM) in dimethyl sulfoxide and 8-bromoadenosine 3,5-cyclic monophosphate (cAMP, 10 mM) in MEM were prepared as stock solutions and diluted with MEM. At the end of incubation, cells were washed and subsequently challenged with either cAMP (0.1 mM) or PMA (10 nM) at 37 °C for an additional 20 min before the SP-A binding assay was performed. To determine whether protein synthesis was involved in the secretagogue-induced elevation of SP-A binding, type II cells were preincubated with 50 µM cycloheximide at 37 °C for 10 min. The media were removed, and the cells were incubated with secretagogues in the presence of cycloheximide for an additional 20 min. To evaluate the effect of trypsin on the removal of putative receptor proteins from the cell surface, the adherent cells were pretreated with various concentrations of trypsin at room temperature for 20 min. In order to overcome the problem of detachment of cells from the dishes and minimize the damage of the cells by trypsin, 1% chicken serum was included in the trypsin solution because chicken serum does not contain antitrypsin activity. At the end of the treatment period, the medium with trypsin was removed, and 20% fetal calf serum in MEM was added to inhibit trypsinization.

For the SP-A binding assay, control and treated cells were transferred to 4 °C and washed with cold MEM. Next, the cells were incubated with ¹²⁵I-SP-A at 0.5 µg/ml and bovine serum albumin (0.1%) for 1 h at 4 °C since initial experiments had demonstrated that the binding of SP-A to type II cells at 4 °C reached maximal levels in that time. Bovine serum albumin was included in the binding medium to minimize SP-A binding to the plastic dishes. Incubations were terminated by removing the medium containing ¹²⁵I-SP-A. Cells then were washed three times with MEM and two times with phosphate-buffered saline. The cells were harvested by dissolution in 0.2 N NaOH, and the amount of ¹²⁵I-SP-A bound to the cells was determined with a gamma counter (WALLAC Wizard 1470-001, Turku, Finland). An aliquot of the samples was analyzed for protein concentration by the Lowry method (Lowry et al., 1951). Dishes or wells without cells were carried through the experiments concurrently, and the amount of background radioactivity was subtracted from the samples with cells. This correction varied between experiments and ranged from 10 to 30% of total counts. Each experiment was carried out with duplicate or triplicate samples.

Results are expressed as mean ± S.E. for the number of experiments indicated (n). Statistical analysis was performed using one-way analysis of variance followed by Dunnett's test and Student's t test. Significance was set at p < 0.05.

RESULTS

Our previous reports had shown an increase in the binding of SP-A and in the uptake of surfactant phospholipid, liposomal phospholipid, and bovine SP-A by type II cells on Transwell membrane as compared with cells on plastic dishes (Bates et al., 1994; Chinoy et al., 1993). In the present study, the binding at 4 °C of human ¹²⁵I-SP-A to type II cells was examined. Type II cells plated on plastic dishes bound 5.7 ± 0.7 ng of SP-A/mg of cell protein (n = 8), whereas the binding of SP-A to cells plated on Transwell membranes was 27.7 ± 2.7 ng of SP-A/mg of protein (n = 13), approximately five times higher than that for cells on plastic (p < 0.01). Upon exposure of cells on Transwell membranes to 8-bromo-cAMP (0.1 mM), terbutaline (0.1 mM), or ATP (1 mM) for 2 h, the binding of SP-A increased 1.4-2-fold, over controls, as shown in Fig. 1A. There were no significant changes in SP-A binding in the presence of PMA (10 nM). In contrast, type II cells plated on plastic did not show significant changes in SP-A binding upon exposure to any of the secretagogues (Fig. 1B).

To determine the time of incubation necessary for the augmentation of SP-A binding, a time course of the effect of cAMP and PMA on the stimulation of SP-A binding was performed. Cells cultured on Transwell membranes were incubated with cAMP or PMA for the indicated time at 37 °C and washed, and the binding assay was performed at 4 °C. After 20 min, cAMP exposure increased SP-A binding by 2-fold over control values, and SP-A binding remained elevated at 2 h (Fig. 2A). Type II cells incubated with PMA also showed a 2-fold elevation in SP-A binding at 20 min (p < 0.01). The increased level with PMA was not maintained, and after 60 min of exposure to PMA, SP-A binding had returned essentially to control level (125.7 ± 4.2% of control, n = 3). As shown in Fig. 2B, type II cells treated with PMA for 2 h, washed, and reincubated with PMA did not show increased SP-A binding in response to the second PMA treatment. However, cAMP was able to induce a 2-fold increase in binding of SP-A in the PMA-pretreated type II cells.

The increase in SP-A binding to type II cells on Transwell membranes was dependent on the concentration of cAMP or PMA, as shown in Fig. 3. Type II cells were exposed to 0.1-150 µM cAMP or 0.1-50 nM PMA for 20 min, cooled to 4 °C, and the SP-A binding assay performed. The secretagogue concentrations that resulted in maximal binding of SP-A to type II cells were 100 µM cAMP or 10 nM PMA.

Short term inhibition of protein synthesis with cycloheximide did not affect the cellular binding of SP-A. Type II cells were incubated without or with 50 µM cycloheximide for 30 min at 37 °C. The cells were cooled and the SP-A binding assay was performed. Cycloheximide treatment did not significantly alter the binding of SP-A (96.8 ± 1.4%, n = 3 of control values). In the presence of cycloheximide, the cells responded to a 20-min exposure to cAMP (193.7 ± 8.1%, n = 3) or PMA (199.5 ± 4.4%, n = 3) in a similar manner as type II cells incubated with secretagogues when protein synthesis was not inhibited (Table I, Expt. 1).

Table I. The additive effect of cAMP and PMA on the binding of SP-A to type II cells The data are the ng of 125I-SP-A/mg of cell protein bound expressed as the % of control value for the number of experiments (mean ± S.E.). Expt. 1, type II cells on Transwell membranes were incubated with cAMP, PMA, or cAMP plus PMA at the indicated concentrations at 37 °C for 20 min. The cells were cooled, and the SP-A binding assay was performed. Expt. 2, the same experiment as designed in Expt. 1 with different concentrations of additions. n refers to the number of experiments. Additions Concentrations Binding Control n ng 125I-SP-A/mg of cell protein % Expt. 1  Control 27.1  ± 1.6 100.0 3  cAMP 0.1 mM 54.3  ± 4.3a 200.2 3  PMA 10 nM 55.7  ± 1.3a 205.4 3  cAMP + PMA 0.1 mM + 10 nM 55.9  ± 3.9a 206.3 3 Expt. 2  Control 34.8  ± 3.3 100.0 8  cAMP 0.1 µM 50.5  ± 3.9a 145.2 8  PMA 1 nM 48.5  ± 4.3a 139.4 6  cAMP + PMA 0.1 µM + 1 nM 70.2  ± 7.6a,b 201.6 5 a  Significantly different from control values at p < 0.05. b  Significantly different from cAMP or PMA group at p < 0.05.

The data indicated that both cAMP and PMA increase SP-A binding but probably function via two different pathways. The effect of cAMP on surfactant secretion by type II cells has been shown to act via protein kinase A (PKA) while PMA activates PKC (Griese et al., 1993). Thus, the two secretagogues were tested for additive effects on SP-A binding. Fig. 3 indicates that a concentration of 0.1 mM cAMP and 10 nM PMA stimulated SP-A binding to the maximal extent. As shown in Table I (Expt. 1), the exposure of type II cells on Transwell membranes to a combination of 0.1 mM cAMP and 10 nM PMA did not enhance SP-A binding above the 2-fold stimulation produced by the secretagogues alone. Due to the possibility that the number of SP-A receptors was limited and that a 2-fold stimulation was maximal, experiments were performed using concentrations of secretagogues that produced a half-maximal response. Either 0.1 µM cAMP or 1 nM PMA stimulated SP-A binding by approximately 140% over controls, after 20 min of incubation (Table I, Expt. 2). Exposure of type II cells to both 0.1 µM cAMP and 1 nM PMA for 20 min increased SP-A binding to 202%, consistent with the possibility that the effects of the secretagogues were additive under these conditions.

To determine whether SP-A was binding to a trypsin-sensitive site on the cell membrane, type II cells plated on plastic dishes were incubated at room temperature for 20 min with increasing concentrations of trypsin from 12 to 250 µg/ml, washed with ice-cold media, and assayed for ¹²⁵I-SP-A binding at 4 °C (see ``Materials and Methods''). Trypsin at 12 µg/ml had no effect on SP-A binding. However, trypsin at 50 µg/ml reduced SP-A binding to 42 ± 5% (n = 6) of control values. Increasing the trypsin concentration up to 250 µg/ml did not reduce binding further (43 ± 5%, n = 2).

The reduction of type II cell surface SP-A binding sites by trypsin was reversible as shown in Fig. 4A. After trypsin treatment (50 µg/ml), the cells were incubated in trypsin-free media with or without secretagogues for an additional 20 min. Trypsin-treated type II cells (no additions) showed a return of SP-A binding to resting values (106.3 ± 7.7%, n = 3). Type II cells that were exposed to cAMP or PMA following trypsin treatment were able to enhance SP-A binding to levels that were significantly higher than cells with no additions (p < 0.005) and comparable with SP-A binding levels seen in nontrypsin-treated cells incubated with secretagogues. The SP-A binding sites stimulated by secretagogues were trypsin-sensitive, as shown in Fig. 4B. After exposure of type II cells to cAMP or PMA, treatment with trypsin reduced SP-A binding to levels below control indicating that both the secretagogue-stimulated sites and the constitutive SP-A binding sites on the cells had been removed.

To determine whether the increase in SP-A binding to type II cells in the presence of secretagogues might be due to a change in receptor affinity or in nonspecific binding, the dose-response for SP-A binding to type II cells was characterized after secretagogue treatment. As shown in Fig. 5, the binding of SP-A varied with the exogenous SP-A levels and showed saturation at an SP-A concentration of 1 µg/ml. After exposure to either cAMP or PMA, the binding was significantly greater than untreated controls at each concentration of SP-A tested, but the concentration required for saturation was similar. The extent of SP-A binding was comparable for both secretagogues. To evaluate the amount of calcium-dependent specific binding, the binding assay was performed in the presence or absence of 10 mM EGTA. Specific binding was determined by subtracting the calcium-independent binding (with 10 mM EGTA) from the total binding value (no EGTA). Of the total binding of SP-A to type II cells, the calcium-dependent binding of SP-A was 68.2 ± 4.4% for control cells, 69.6 ± 4.2% for PMA, and 68.1 ± 1.5% for cAMP-treated cells (mean ± S.E., n = 3-4) as shown in Fig. 6. Both PMA and cAMP stimulated specific SP-A binding 2-fold while also increasing calcium-independent binding. With increasing levels of SP-A in the media (Fig. 7), both the calcium-dependent (Fig. 7A) and the calcium-independent (Fig. 7B) binding were saturable and showed a similar binding pattern as the total binding values presented in Fig. 5. Although the calcium-independent binding of SP-A was enhanced with secretagogue treatment, the bulk of the observed increases in total binding were due to a rise in the specific calcium-dependent binding.

DISCUSSION

As a major surfactant protein constituent, SP-A has been proposed to have several functions in the alveoli of the lung. The capacity of SP-A to function as the modulator of surfactant secretion and turnover appears to involve the binding of SP-A to type II cells, probably via specific cell surface receptors (Murata et al., 1993; Wright et al.,, 1989; Kuroki et al., 1988). Previously we have reported that secretagogues enhanced the uptake of lipid and protein components of lung surfactant by isolated perfused lungs (Fisher et al., 1991) and by type II cells (Chinoy et al., 1993). This effect also may be associated with an increase in binding sites for SP-A. Therefore, our present effort characterized the cell surface receptor for SP-A and evaluated the effect of secretagogues on the activity of the receptor.

Previously, we reported that the secretagogues, cAMP and terbutaline, significantly stimulated the uptake of surfactant ³⁵S-SP-A protein and [³H]phosphatidylcholine by type II cells, whereas PMA did not. This stimulation was cell substrate-dependent, as type II cells plated on plastic were not affected by any secretagogues. In the present study, we found that a 2-h exposure of type II cells on Transwell membranes to cAMP or terbutaline increased the binding of SP-A, whereas PMA did not show significant stimulation, which paralleled the results seen for surfactant uptake (Chinoy et al., 1993). Since the amount of surfactant uptake is likely to be proportional to its binding, the data support the hypothesis that changes in SP-A binding due to secretagogues account for the observed enhancement of surfactant incorporation.

The effect of secretagogues on SP-A binding showed that cAMP and PMA followed a different time course. 20 min of exposure to both secretagogues was sufficient to promote SP-A binding. However, the PMA-induced elevation was not maintained as SP-A binding returned to control levels within 2 h. This finding is consistent with a rapid desensitization of PKC by PMA as was reported in studies with a variety of cell types (Philips and Jaken, 1983; Solanki et al., 1981). In response to PMA treatment, type II cell PKC activity increased rapidly in the membranes and decreased in the cytosol fraction. The membrane-associated PKC activity reached a maximum by 30 min and declined to control levels after 2 h (Chander et al., 1995). Such results would suggest that the reason for our failure to detect stimulation of natural surfactant uptake by PMA after a 2-h exposure seen in our previous report (Chinoy et al., 1993) may have been that the SP-A binding sites were not elevated for a sufficient period. The cAMP-induced increase in SP-A binding to type II cells after 20 min exposure was sustained for up to 2 h and, thus, could have been present long enough to produce a detectable increase in surfactant uptake by cAMP.

The stimulation of SP-A binding to alveolar cells by cAMP and PMA probably occurred through two different pathways. First, the stimulation of SP-A binding by PMA was not prolonged while cAMP enhanced binding for up to 2 h. Second, PMA-desensitized type II cells no longer responded to a further exposure to PMA while continuing to respond to cAMP. Third, exposure of type II cells to a combination of cAMP and PMA showed an additive effect at half-maximal concentrations of drugs. The lack of a synergistic effect with maximal doses of cAMP or PMA may have been due to a limited number of SP-A receptors available for recruitment to the plasma membrane. It is well-known that cAMP and PMA act via the activation of two different kinases, PKA and PKC, respectively. It will be of interest to determine whether PKA and PKC phosphorylate the receptor at two different sites on the protein as has been the case with other proteins such as lipocortin I (Vaticovski et al., 1988). Dwyer-Nield et al. (1996) recently reported that PMA altered the architecture of epithelial cells cultured on plastic dishes. The enhancement of SP-A binding by PMA-treated type II cells is probably not due to changes in the architecture of these cells, because the cells also responded to cAMP, terbutaline, and ATP, secretagogues which do not alter cell shape. In addition, only the type II cells plated on Transwell membrane responded to PMA while the cells plated on plastic did not.

The regulation of cell surface receptors can be controlled through four general mechanisms, changes in receptor protein synthesis, affinity, internalization, and externalization. Synthesis of the receptor protein molecule seems unlikely to be involved in the regulation of SP-A binding to type II cell, since acceleration in SP-A binding occurred too rapidly to be regulated by new protein production and cycloheximide did not prevent the increase of SP-A binding by secretagogues. As the targeting material of two major signal transduction pathways, cAMP and PMA have been reported to regulate the cycling of a variety of cell surface receptors through serine/threonine phosphorylation mediated by an activation of PKA or PKC, respectively (Hu et al., 1990; Larose et al., 1992; Zacharias et al., 1995). The effects of phosphorylation have been shown to be either stimulatory or inhibitory. Insulin growth factor II/mannose 6-phosphate receptor in microvascular endothelial cells moved from an intracellular pool to the cell surface when treated with PMA, causing an elevation of ligand binding accompanied by a reduction of intracellular receptor stores (Hu et al., 1990). On the other hand, exposure to PMA led to an a absolute decrease of surface asialoglycoprotein receptor number in a hepatoma cell line (Hep G₂ cells) due to internalization of receptors. Finally, transferrin binding on these cells was down-regulated by a reduction in the binding affinity of the transferrin receptor for the ligand after incubation with PMA (Fallon and Schwartz, 1986). By measuring the characteristics of binding in the presence of secretagogues, we determined that the affinity of the receptors was not changed, whereas the amount of specific, calcium-dependent binding was significantly elevated. Thus, the most likely mechanism whereby cAMP and PMA enhanced the binding of SP-A to type II cells was that these two secretagogues phosphorylated the SP-A receptors, stimulating their movement from the cytosol to the plasma membrane, resulting in an increase in SP-A binding sites on the type II cell surface.

The SP-A binding site on the type II cells shows the characteristics of a protein in that it was sensitive to trypsin treatment. Previous studies on the effects of trypsin on the binding of SP-A to type II cells have been inconclusive, probably due to the various concentrations of trypsin used. The present study confirmed the observations of Kuroki et al. (1988) that treatment of type II cells with 12 µg/ml trypsin does not reduce the SP-A binding, and also confirmed the observations of Wright et al. (1989) that 250 µg/ml trypsin reduced binding by approximately 50%. Data presented here determined that 50 µg/ml trypsin was sufficient to produce the maximum removal of binding sites, since higher concentrations of trypsin did not reduce binding further. The data indicated that trypsin treatment did not damage cells because the cells were able to respond normally to subsequent treatment with secretagogues by increasing SP-A binding sites. Our results also suggest that approximately half of the SP-A binding sites were trypsin-sensitive, in that increasing trypsin to concentrations where cell detachment occurred did not reduce the binding further. In addition, all of the SP-A binding sites stimulated by cAMP or PMA were trypsin-sensitive providing evidence that these binding sites were proteins. Our results correspond with the observations of Kuroki et al. (1988) and Wright et al. (1989) that SP-A binding to type II cells was dependent on calcium and that calcium-independent binding accounted for approximately one-third of the total binding.

In summary, the present work suggests that the secretagogues increased the binding of SP-A to type II cells by increasing SP-A receptor activity on the plasma membrane of type II cells. The enhancement of surfactant uptake due to either secretagogues or to the use of microporous membranes as a cell substrate has been shown to occur in conjunction with an increase in the number of SP-A binding sites. Regulation of these binding sites may well be the mechanism whereby the turnover of SP-A, and perhaps surfactant, occurs in the intact lung.