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1Institute for Environmental Medicine and 2Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
Submitted 21 August 2007 ; accepted in final form 5 December 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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type II pneumocytes; secretion; uptake; endocytosis; clathrin; actin
Clathrin-dependent pathways often involve specific receptor-protein interactions. SP-A plays an important role in surfactant lipid turnover in isolated type II pneumocytes, as this protein affects cellular secretion, uptake, and recycling of phospholipid (1, 15, 18, 20, 36). Secretagogues that stimulate phospholipid secretion also were found to enhance SP-A-mediated uptake of phospholipids (1, 19, 29). Clearance of SP-A from the lungs occurs via a classical receptor-mediated clathrin-coated pit pathway as uptake of SP-A by the lung was sensitive to clathrin inhibitors and gold-labeled SP-A was shown to bind to type II cells in coated plasma membrane invaginations (30, 43). Several candidate SP-A receptors on the surface of type II cells have been described, including the transmembrane protein P63 (13, 24, 33, 46, 47). Since secretagogue treatment enhanced calcium-dependent SP-A binding and recruitment of SP-A receptors to the type II cell membrane (11), it has been proposed that SP-A enhancement of phospholipid uptake occurs through this clathrin-dependent mechanism. Due to the high affinity of SP-A for surfactant DPPC, an SP-A/DPPC lipoprotein-type complex would interact with an SP-A receptor and be incorporated into type II cells via clathrin-coated pits (34).
The role of SP-A in phospholipid turnover was brought into question in view of the fact surfactant phospholipid metabolism in the SP-A –/– mouse did not differ appreciably from wild-type SP-A +/+ mice (27, 28, 32). Lung function, surfactant lipid pool sizes, and clearance of DPPC were similar (27, 31). However, with physiological challenges that normally enhanced surfactant uptake, such as hyperventilation or secretagogue treatment, the gene-targeted rodent lungs were unable to respond (29). In view of the importance of the regulation of surfactant pool size, compensation in the clearance pathway by the SP-A gene-targeted mouse lung due to the absence of SP-A was a possibility. We hypothesized that SP-A-independent liposome uptake by the lungs of SP-A –/– mice may occur via an actin-dependent cell membrane retrieval pathway (29). Thus, in the present study, we examined the pathways of surfactant lipid removal from the alveolar space of mouse lungs. We utilized inhibitors of clathrin-mediated and actin-mediated endocytosis to follow the uptake of intratracheally instilled labeled DPPC liposomes by the isolated, perfused intact lungs of wild-type and SP-A –/– mice.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), cytochalasin D, amantadine HCl, and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO); fatty acid free BSA was from Roche Diagnostics (Indianapolis, IN); [3H]methyl-choline chloride (specific activity = 90 mCi/mmol) and [choline-methyl-3H]DPPC were from Amersham (Arlington Heights, IL); and 125I was from Amersham Biosciences (Piscataway, NJ). Authentic lipids were obtained from Avanti (Birmingham, AL). All plastic tissue culture dishes (35 mm) were from Costar (Cambridge, MA). MEM and phosphate-buffered saline (PBS) were purchased from CellGro (Herndon, VA).
Mice
SP-A gene-targeted mice were initially obtained from the University of California, San Francisco (26). The SP-A –/– mice used in this study were the progeny of a breeding colony set up at the University of Pennsylvania (29). Analysis of SP-A –/– mice confirmed the absence of the normal SP-A (26, 29). Wild-type C57Bl6 mice were obtained from Jackson Laboratories (Bar Harbor, Maine). The mice were between 2–3 mo of age (body wt = 18–25 g). All procedures utilizing mice were approved by the Institutional Animal Care and Use Committees.
Liposome Preparation
Liposomes were prepared from L-
-DPPC, egg PC, egg phosphatidylglycerol, and cholesterol in molar ratio 10:5:2:3 with or without trace amounts of [3H]DPPC. The lipids were evaporated to dryness under nitrogen. The dried lipids were resuspended in PBS without calcium/magnesium. The mixture was frozen and thawed three times by alternating liquid nitrogen with a 50°C water bath and passed eight times at 50°C through 100 nm pore sized filters using an "Extruder" (Lipex Biomembranes Vancouver, BC). Liposomes were stored at 4°C and used within 24 h. This method resulted in unilamellar liposomes of 103 ± 8 nm (mean ± SE; n = 4) as assessed by light scattering using a 90Plus particle size analyzer (Brookhaven Instruments).
Isolated Lung Perfusion
Mice were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg body wt). The mice then were continuously ventilated, the thorax was incised, and the lungs were cleared of blood by perfusion through the pulmonary artery with buffer [Krebs' Ringer bicarbonate pH 7.4, with 3% BSA (KRB)]. The total isolation procedure required 
5 min. Isolated lungs were perfused with KRB containing 10 mM glucose in a closed circuit recirculating perfusion apparatus similar to that described previously for rat lungs (22). Lungs were perfused for 2 h with buffer alone (control) or with buffer containing 8-Br-cAMP (0.1 mM, stimulated), 10 mM amantadine, or 10 µM cytochalasin D. Perfusate was gassed constantly with 5% CO2 in air. Lungs were ventilated at 60 cycles/min, 0.3 ml tidal volume, and 2 cm H2O end-expiratory pressure. There was no significant change in the ventilation and perfusion pressures or overt evidence of lung edema during the experiments.
DPPC Uptake: Isolated Perfused Lung
To measure DPPC uptake, the trachea of anesthetized mice was cannulated. [3H]DPPC-labeled liposomes (10 nmol DPPC in 20 µl saline) were instilled into the lungs with a Hamilton syringe inserted into the trachea through the cannula to the level of carina. Then the lungs were perfused as described above. At the end of the 5-min period required for lung isolation (baseline) or the 2-h experimental perfusion, lungs were lavaged five times with 1-ml aliquots of ice-cold saline. Lung tissue was then homogenized in saline on ice by using a Polytron homogenizer followed by a motorized mortar and pestle. Lipid fractions from the lung homogenate were extracted using the Bligh and Dyer procedure (6). Lung homogenate and perfusate aliquots were counted for disintegrations per minute (dpm) to calculate lung uptake of [3H]DPPC as percentage of instilled [(dpm in the lung plus perfusate/total dpm instilled) x 100] (19). Uptake of liposomes over the initial 5 min was subtracted from the 2-h experimental perfusions. This baseline incorporation of [3H]DPPC liposomes did not differ between wild-type and SP-A gene-targeted mice and was 4.17 ± 0.04 and 4.25 ± 0.08% of instilled liposomes for wild-type and SP-A –/– mice, respectively (n = 6).
Distribution of the instilled liposomes in the mouse lung using the described protocol was determined. Twenty microliters of [3H]DPPC labeled liposomes (380,900 dpm) were instilled into each of two mouse lungs as described, and the lung perfusions were terminated after 15 min. The right and left lung lobes from each mouse were cut into 10 small segments per lobe (40 segments total), extracted, and analyzed for radioactivity and protein (8). For the two mice, 92 and 94% of the instilled [3H]DPPC was recovered from the lungs. The trachea and extra-pulmonary bronchi contained <2% of the total radioactivity in both cases. The radioactivity was equally divided between the right and left lungs with each containing 45.7 ± 0.5% (mean ± SE; n = 4) of the liposomes instilled. The 40 segments each contained 17,597 ± 385 dpm/mg protein or 4.6 ± 0.1% of instilled liposomes (mean ± SE- n = 40) with a range of 11,134 to 22,164 dpm/mg protein or 2.9 to 5.6% of instilled liposomes. The results indicate that the liposomes were distributed relatively evenly throughout the mouse pulmonary tissue.
Surfactant Secretion: Isolated Perfused Lungs
Measurement of surfactant secretion from isolated mouse lungs was performed essentially as described for isolated rat lungs (9). Mouse lungs were perfused for 30 min with KRB buffer (pH 7.4) containing 10 µCi [3H]choline to label the lung PC. At the end of labeling period, 50-fold of excess unlabeled choline was added to the perfusion medium. Some perfusions were terminated at this stage to get zero time secretion data. Other lungs were perfused for 2 h with or without 1 mM ATP. Lungs were lavaged with 5 x 1 ml of cold saline, and the cell free lavaged fluid and postlavaged lungs were analyzed for radioactivity (6).
Isolation of Type II Cells
Type II cells were isolated from adult C57/BL6 wild-type or SP-A knockout (KO) mouse lungs using the previously described procedure of Bortnick et al. (7) that was modified from Warshamana et al. (49). After digestion of the lungs with dispase and treatment with DNase I, the cells from five sets of lungs were pooled, filtered through nylon mesh, and panned on mouse IgG-coated Petri dishes to remove macrophages. The nonadherent cells were seeded on 100-mm cell culture dishes at 37°C for 1 h in MEM with 10% FBS to remove fibroblasts. The final cell isolates were placed on type I collagen-coated 35-mm dishes in Ham's F-12 culture medium supplemented with 15 mM HEPES, 0.8 mM CaCl2, 0.25% BSA, 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodium selinite, and 2% mouse serum. After 24 h, the medium was changed to serum-free Ham's F-12 with the same additions as above. The isolated type II cells were >95% pure after 24 h in culture (7) as judged by cytokeratin staining and the presence of Nile red positive vacuoles.
Isolation and Iodination of SP-A
SP-A was purified from bronchoalveolar lavage fluid of alveolar proteinosis patients after therapeutic lavage at the Hospital of the University of Pennsylvania as described previously (1) using the method of Hawgood et al. (25). Endotoxin levels in SP-A preparations were 0.5 pg endotoxin/µg SP-A protein as measured by the Limulus Amebocyte Lysate Test performed by Lonza (Waldersville, MD). Iodination of SP-A was performed using Iodogen (Pierce, Rockford, IL) using the manufacturer's directions. One-hundred micrograms of SP-A were iodinated with 100 µCi of Na125I. Iodinated protein was dialyzed against 5 mM Tris buffer for 24 h with frequent buffer changes to remove free 125I. Specific activity for all preparations was 50–70 cpm/ng protein. The TCA precipitability ranged from 90 to 97%. The iodinated proteins were stored at 4°C and were used within 2–3 wk.
SP-A Binding by Type II Cells
After overnight culture, cells were placed on ice and washed two times with ice-cold MEM and once with MEM containing 0.1% fatty acid free BSA. Cells then were incubated at 4°C for 1 h with various concentrations of 125I-SP-A. To terminate the experiment, the media were removed and the cells were washed once with MEM + 0.3% fatty acid free BSA, twice with MEM + 0.1% fatty acid free BSA and twice with PBS. Cells were dissolved in 0.2 N NaOH, and aliquots taken for protein determination (39) and radioactive counts (Beckman).
PC Secretion
After isolation, type II cells were incubated overnight with 0.5 µCi/dish of [methyl-3H]choline in MEM containing 10% FCS to label cellular phospholipids. Cells were washed extensively and incubated for 30 min in MEM. One set of cells was harvested and served as control for phospholipid secretion associated with the medium change, as described previously (2). The remaining cells were preincubated with or without SP-A (1.0 µg/ml, 15 min), followed by addition of adenosine triphosphate (1 mM ATP, Sigma, St. Louis, MO) for 2 h. The media were removed and centrifuged to remove detached cells. Methanol was added to the cell monolayer, and the cells were scraped from the dish. The cells and the media were extracted using the Bligh and Dyer method (6). The amount of phospholipid secretion was calculated as the percentage of lipid counts per minute in the medium, relative to the total counts per minute lipid present in the cells plus the medium. All experiments were performed in duplicate or triplicate, and the values were averaged.
Statistical Analysis
Results are reported as means ± SE unless otherwise stated. Results were analyzed statistically by t-test or paired t-test using SigmaStat for Windows (Jandel, San Rafael, CA) where statistical significance is taken as P < 0.05. Multiple group comparisons were done by one-way ANOVA with Bonferroni's correction. When SE bars are not visible in the figures, they are contained within the symbols.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Using the SP-A gene-targeted mouse, we have shown that basal uptake of liposomes into the lung was similar between wild-type and SP-A gene-targeted mice, while the presence of SP-A was necessary for the increased clearance of alveolar DPPC liposomes after secretagogue treatment (29). To characterize the endocytic pathways involved, we adapted procedures previously utilized in isolated, perfused rat lungs to this mouse system (42). Amantadine is a cationic amphiphilic agent that prevents budding of clathrin-coated vesicles and is used to block clathrin-mediated processes. Cytochalasin D is an agent that destabilizes the actin cytoskeleton and inhibits actin-mediated endocytosis. In previous studies using amantadine and cytochalasin D, we demonstrated that both clathrin-mediated and actin-mediated pathways were utilized for endocytosis of DPPC-labeled liposomes by granular pneumocytes in the intact rat lung, while SP-A was cleared from the rat lung by a clathrin-mediated pathway that required the polymerization of actin (30, 42).
Inhibition of the uptake of intratracheally instilled labeled liposomes by amantadine and cytochalasin D in wild-type (SP-A +/+) and SP-A gene-targeted (SP-A –/–) mouse lungs was examined under unstimulated (basal) or cAMP-stimulated conditions. The recovery of [3H]DPPC-labeled liposomes from the lung at the end of the 2-h experiment is shown in Figure 1. Table 1 documents the amount of liposome clearance inhibited by the drug treatment and was determined by subtraction of the residual [3H]DPPC liposome uptake that occurred in the presence of inhibitors from that which occurred without inhibition (control). As reported previously, under unstimulated basal conditions, uptake of [3H]DPPC-labeled liposomes over the 2-h perfusion period was similar in the wild-type and SP-A –/– mice (5.7 ± 0.1 vs. 5.8 ± 0.2% of instilled, respectively) in the absence of inhibitors (control). The addition of amantadine plus cytochalasin D to the perfusate reduced liposome uptake by 74% in both mouse groups (Fig. 1A; Table 1). However, blocking just the clathrin-mediated pathway using amantadine alone in wild-type mice reduced liposome uptake 70%, while having only a minimal effect (19%) on liposome clearance in the SP-A –/– mice.
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Uptake of liposomes into the lungs of SP-A –/– mice is unresponsive to secretagogue treatment unlike the stimulatory effect on liposome clearance seen in SP-A +/+ mice (29). Those observations were reconfirmed by the experimental data in Fig. 1B and Table 1 where the total uptake of liposomes with cAMP increased by 1.9-fold over basal conditions in the wild-type mice with no effect in the SP-A –/– mice. Both the amantadine (clathrin) and cytochalasin D (actin)-sensitive pathways in the wild-type mice were stimulated due to secretagogue treatment with the former making a larger contribution. Inclusion of both amantadine and cytochalasin D in the perfusate reduced liposomal PC incorporation into cAMP-stimulated SP-A +/+ or SP-A –/– lung tissue from 10.5% or 5.6% of instilled, respectively, to 2% of instilled in both groups of mice (Fig. 1B), blocking 82 or 63% of instilled liposomes in wild-type or SP-A –/– mice, respectively (Table 1). In the SP-A –/– mice there were no significant changes in either the clathrin- or actin-sensitive pathways with cAMP present (Fig. 1; Table 1). Thus, amantadine-sensitive liposome uptake increases 1.7-fold in the wild-type mice from 4.0% to 7.0% of instilled liposomes and remained unchanged in the SP-A –/– mice (Table 1).
The contribution of actin polymerization to the liposome uptake process in these experiments was determined by subtraction (Table 1). To confirm the role of actin in a clathrin-independent pathway as well as confirm an actin contribution to the coated pit system, cytochalasin D was added to the perfusate of the mouse lungs under all experimental conditions. The data in Fig. 1 and Table 2 indicate that the actin-mediated pathway functioned independently from both the presence of SP-A and the stimulatory effect of cAMP as the addition of cytochalasin D alone blocked 60–70% of liposome clearance under all experimental conditions.
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Isolated perfused lung. Since surfactant clearance from the SP-A –/– mice lungs was unresponsive to secretagogue treatment, we determined whether secretion of surfactant was similarly affected. Surfactant PC secretion from the lungs of SP-A +/+ and -/- mice after perfusion with [3H]choline for 2 h was examined. The amount of PC synthesis ([3H]choline incorporation into PC) that occurred over this time period did not differ with genotype or secretagogue treatment (Table 3). Secretion is expressed as the percentage of the total [3H]PC (lung tissue plus lavage) that is found in the lavage itself (10, 21) after a subsequent 1-h period. As shown in Fig. 2A and Table 4, the wild-type mouse lung responded to secretagogue treatment (1 mM ATP) with an approximately twofold increase in release of PC. While the SP-A KO mice responded to ATP treatment in a similar fashion as the wild-type mice, surfactant secretion was significantly higher than that seen with wild-type mice lungs under both basal and stimulated conditions (Fig. 2A; Table 4).
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Binding of 125I-SP-A to Mouse Type II Cells
To determine the status of SP-A binding proteins on the type II cells from SP-A –/– mice, binding and uptake studies were performed using 125I-SP-A. Type II cells isolated from the lungs of SP-A +/+ or SP-A –/– mice were incubated at 4°C for 1 h with increasing concentrations of iodinated SP-A. Figure 3 demonstrates that the trypsin-releasable binding of 125I-SP-A to type II cells showed saturation kinetics and the extent of binding did not differ between the alveolar cells from different genotypes. Examination of the total cell association (binding plus uptake) of iodinated SP-A in the specific portion of the SP-A binding curve (at 0.5 and 1 µg SP-A/ml) showed comparable cell association of SP-A to the mouse pneumocytes regardless of genotype (Table 5).
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Since SP-A binding to the type II cells from SP-A –/– mice appeared to be normal, "rescue" experiments were performed to determine whether the clearance of liposomes from the alveolar space of SP-A negative mice could be enhanced by the addition of SP-A to the instilled liposomes. [3H]DPPC liposomes without or with SP-A (10 µg/lung) were instilled into the lungs of SP-A –/– mice and uptake of liposomes into the lungs was measured after 2 h of perfusion. The addition of SP-A to liposomes enhanced liposome uptake by the SP-A –/– lungs 1.7-fold under basal conditions (control, Fig. 4A). Further, the clearance of the SP-A/liposome complex by the SP-A –/– lungs became sensitive to secretagogue treatment. As shown in Fig. 4A, addition of cAMP or TPA to the perfusate resulted in a threefold higher lipid uptake with SP-A-enriched liposomes compared with liposomes alone. As seen previously in the SP-A –/– mice, liposome uptake was unaffected by secretagogue treatment. With SP-A-enriched liposomes, cAMP treatment enhanced [3H]DPPC uptake by SP-A –/– mice lungs 1–9-fold over controls (Fig. 4A), values comparable to the effect of cAMP on liposome uptake in SP-A +/+ wild-type mice (1.8-fold; Table 1; Fig. 4B). In the presence of normal levels of surfactant SP-A, as in the wild-type mouse, the addition of SP-A to the instilled liposomes had no further effect on liposome uptake under either unstimulated or stimulated (cAMP) conditions (Fig. 4B).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Figure 5 summarizes the contribution of the various pathways to the clearance of liposomes from the lungs of the two mice genotypes. Uptake that was not inhibited by amantadine or cytochalasin D (neither, Fig. 5) was an unidentified pathway that remains fairly constant under all situations and may represent DPPC adsorbed to the surface of the lung past the initial 5-min period (42). Under basal conditions, it is apparent that the clathrin-mediated pathway, presumably via SP-A/DPPC receptor interactions, was responsible for the bulk of the lipid uptake in the SP-A +/+ mice. To determine the fractional contribution of SP-A to liposome clearance, we subtracted the amantadine-sensitive liposome uptake in the SP-A –/– mouse lung (clathrin-mediated lipid uptake that occurs in the absence of SP-A) from the amantadine-sensitive liposome uptake in the wild-type mouse. The results indicate that 75% of the clathrin-mediated uptake of liposomes was mediated by SP-A under basal situations and 81% under stimulated conditions in the wild-type mice. Both clathrin-mediated and nonclathrin, actin-mediated pathways were stimulated with secretagogue addition, but the majority of liposome clearance utilized the clathrin dependent receptor-mediated process. In the absence of SP-A (SP-A –/– mice) the nonclathrin, actin-mediated pathway predominated under basal and stimulated conditions. As demonstrated in rats (42), the present study in mice reconfirms the presence of both clathrin- and actin-dependent pathways that account for 65% or more of the lipid uptake.
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The polymerization of the actin network plays a role in several endocytic pathways including the clathrin-mediated endocytosis (5, 38). Our current and previous data on the mechanism for clearance of surfactant were consistent with actin contributing to clathrin-mediated lipid incorporation into the lung since the inhibitory effects of amantadine and cytochalasin D in combination (4.2% of instilled) were not the sum of each drug alone [amantadine (4.0% of instilled) plus cytochalasin D (3.8% of instilled) equals 7.8% of instilled]. Thus, the effects of the inhibitor cytochalasin D were not solely on actin-mediated pathways but also on clathrin-mediated pathways, complicating the interpretation of the cytochalasin D data. To determine the contribution of the actin-dependent but clathrin-independent pathway, we subtracted the amount of lipid uptake inhibited by amantadine and cytochalasin D instilled together from that by amantadine alone, termed the "nonclathrin, actin-dependent" pathway. To confirm an effect of actin-polymerization on liposome uptake, we used cytochalasin D alone. The extent of the inhibition of liposome uptake in the presence of cytochalasin D alone was the same or greater than that attributed to the nonclathrin, cytochalasin D-sensitive pathway. The results confirm the findings from our laboratory and others that actin filaments make an important contribution to clathrin-mediated endocytosis (5, 23, 42).
The precise nature of the clathrin-independent actin-sensitive pathway of liposome uptake into the lung was not examined in further detail. However, the polymerization of the actin network plays a role in several endocytic pathways including two that are sensitive to secretagogues, lipid rafts and lamellar body membrane retrieval. Caveolae are considered to be lipid rafts enriched in cholesterol, glycosphingolipids, sphingomyelin, glycosylphosphatidylinositol-linked proteins, and several other proteins including caveolin and flotillin. Although type II cells lack caveolin 1, flotillin-positive lipid rafts have been isolated from type II cells, were found to play a role in surfactant secretion, and were sensitive to secretagogues (12). With the observed coordination between secretion and reuptake (16), lipid rafts may function in surfactant uptake as well. We have previously examined the retrieval of plasma membrane patches that contain ABCA3, a 180-kDa protein that is associated with lamellar body membranes (52). ABCA3 is expressed on the cell surface of the rat type II cell after secretion in association with membrane patches that were subsequently endocytosed in small vesicles and fused with existing lamellar body organelles inside the cell (45). NBD-PC liposomes added to the type II cells in culture are reinserted in the same intracellular compartment as ABCA3, the lamellar body (44). Using monoclonal antibody 3C9 that recognizes ABCA3, we followed trafficking of lamellar bodies and determined that stimulation of secretion enhanced the appearance and retrieval of ABCA3 from the surface of the type II cell. This pathway required the active participation of actin since it was inhibited by cytochalasin D but was insensitive to inhibitors of clathrin-mediated endocytosis (3).
Our data on the synthesis of PC using the incorporation of [3H]choline into the PC of the isolated perfused mouse lung after 2 h of perfusion agree with those of Ikegami et al. (27) who followed the incorporation of [3H]choline or [14C]palmitate into saturated PC in lung slices. Both groups showed no difference in lung PC synthesis between wild-type and SP-A –/– mice. We found significantly more (35–90% greater) secretion of PC from isolated, perfused lungs, and isolated type II cells from SP-A –/– mice than wild-type mice. Such data support a role for SP-A in the inhibition of surfactant secretion. In contrast to our results, the previous study found no significant difference between SP-A +/+ and SP-A –/– mice in DPPC secretion (27). This latter result may reflect the relative difficulty in the study of surfactant secretion in intact mice where compensatory mechanisms may overshadow the primary effects. In any case, it is clear from our data that PC secretion from type II pneumocytes isolated from either wild-type or SP-A –/– mice are sensitive to the presence of SP-A. Basal and stimulated PC secretion was inhibited by exposure to SP-A in pneumocytes from either source. Since it is well established that the ability of SP-A to regulate secretion requires SP-A-receptor interactions, the data imply that the level of SP-A receptors is normal in both cases and binding studies utilizing iodinated SP-A confirmed the equivalent status of the SP-A receptor.
The binding kinetics of SP-A to wild-type mouse type II cells were similar to those seen with rat type II cells (1, 35, 51). Further, binding of 125I-SP-A to the surface of type II cells isolated from the SP-A +/+ and SP-A –/– mice was comparable, indicating that the absence of SP-A did not affect the status of SP-A binding proteins although the cells have never interacted with SP-A. The presence of SP-A receptors that function via a clathrin-dependent pathway in the SP-A –/– was demonstrated in the "rescue" experiments using liposomes in conjunction with SP-A. In the SP-A –/– mice lungs, instillation of an SP-A/liposome complex resulted in enhanced lung internalization of surfactant liposomes and recovery of sensitivity to secretagogue challenge through increased lipid clearance, uptake that was similar to that previously seen in the SP-A +/+ mice. The data indicate an important role for the uptake of an SP-A/DPPC complex via clathrin- (and presumably SP-A receptor-) mediated endocytosis in wild-type mice. Further studies will determine whether the putative SP-A receptor we have recently described, P63, plays a role in mediating the uptake of SP-A/DPPC lipoprotein-like liposomes (24).
In the absence of SP-A, there remained a minor amantadine-sensitive liposome uptake pathway in SP-A –/– mice (19% of total uptake). This clathrin-mediated clearance of liposomes was unaffected by secretagogue exposure. There are several possible reasons for this finding including association of other surfactant proteins with the liposomes and utilization of different receptor-mediated, clathrin-sensitive pathways (31). SP-B and SP-C have been reported to stimulate liposome uptake by type II cells in culture although the uptake was not saturable, data that would not be compatible with receptor-mediated events (41). In addition, since both alveolar macrophages and type II cells could contribute to all of the observed findings, studies on the role of the macrophages in surfactant clearance are necessary to fully define possible mechanisms involved in this alternate pathway.
In summary, the SP-A-dependent clathrin-mediated pathway was of major importance for the uptake of phospholipid from the isolated perfused lung of wild-type mice. In the absence of this pathway due to deletion of SP-A, an actin-dependent pathway is utilized, which is insensitive to the physiologic challenge of secretagogue treatment. Thus, SP-A is necessary for the normal clearance of surfactant lipid from the alveolar space. Characterization of the interaction of SP-A with the SP-A receptor will be important to further the understanding of the regulation of surfactant turnover.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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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.
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