Synexin and GTP increase surfactant secretion in permeabilized alveolar type II cells

Avinash Chander, Namita Sen, and Alan R. Spitzer

Division of Neonatology, Department of Pediatrics, Thomas Jefferson University, Philadelphia, Pennsylvania 19107


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have previously suggested that synexin (annexin VII), a Ca2+-dependent phospholipid binding protein, may have a role in surfactant secretion, since it promotes membrane fusion between isolated lamellar bodies (the surfactant-containing organelles) and plasma membranes. In this study, we investigated whether exogenous synexin can augment surfactant phosphatidylcholine (PC) secretion in synexin-deficient lung epithelial type II cells. Isolated rat type II cells were cultured for 20-22 h with [3H]choline to label cellular PC. The cells were then treated with beta -escin, which forms pores in the cell membrane and releases cytoplasmic proteins including synexin. These cells, however, retained lamellar bodies. The permeabilized type II cells were evaluated for PC secretion during a 30-min incubation. Compared with PC secretion under basal conditions, the presence of Ca2+ (up to 10 µM) did not increase PC secretion. In the presence of 1 µM Ca2+, synexin increased PC secretion in a concentration-dependent manner, which reached a maximum at ~5 µg/ml synexin. The secretagogue effect of synexin was abolished when synexin was inactivated by heat treatment (30 min at 65°C) or by treatment with synexin antibodies. GTP or its nonhydrolyzable analog beta :gamma -imidoguanosine-5'-triphosphate also increased PC secretion in permeabilized type II cells. The PC secretion was further increased in an additive manner when a maximally effective concentration of synexin was added in the presence of 1 mM GTP, suggesting that GTP acts by a synexin-independent mechanism to increase membrane fusion. Thus our results support a direct role for synexin in surfactant secretion. Our study also suggests that membrane fusion during surfactant secretion may be mediated by two independent mechanisms.

membrane fusion; exocytosis; annexins; G proteins


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE LUNG SURFACTANT is a phospholipid-rich lipoprotein-like substance that is essential for lowering surface tension during end expiration to prevent alveolar collapse (reviewed in Ref. 2). The major component of lung surfactant is phosphatidylcholine (PC). The lung epithelial type II cells synthesize and store PC and other surfactant components in lamellar bodies, which are membrane-bound distinct surfactant storage organelles that are unique to type II cells. The secretion of lung surfactant occurs after membrane fusion between lamellar bodies and plasma membrane and release of lamellar body contents. Several agents modulate lung surfactant secretion by generating specific intracellular messengers (reviewed in Refs. 11 and 32). Although significant advances have been made in understanding the signal transduction processes leading to generation of second messengers, the mechanism(s) of membrane fusion and factors that regulate such membrane fusion during exocytosis of lamellar body contents have remained poorly investigated.

We have previously proposed that synexin (annexin VII), a member of the annexin family of proteins, is involved in membrane fusion between lamellar bodies and plasma membrane (14). Annexins are Ca2+-dependent phospholipid-binding proteins that share significant sequence homology in the carboxy-terminal domains but contain a unique amino-terminal domain with variable chain length (15, 19). These proteins have been invoked in Ca2+ homeostasis and in membrane fusion during exocytosis, endocytosis, and other intracellular membrane fusion events that may occur during vesicular trafficking. Our previous studies indicated that synexin can facilitate in vitro fusion of isolated lamellar bodies with plasma membrane (14). Our observations that certain stilbene disulfonic acids demonstrate similar inhibition potency and inhibition constants toward membrane fusion (23) and toward basal or stimulated PC secretion in type II cells (12) suggest that stilbene compounds may inhibit synexin-dependent membrane fusion during surfactant secretion. Our recent studies have shown that synexin can bind to specific proteins in the plasma membrane and the lamellar body fractions of lung (37). Together, these studies suggest a role for synexin in membrane fusion during surfactant secretion.

Several investigators have used permeabilized cells to evaluate the effects of membrane-impermeable agents on intracellular processes like protein trafficking and secretion (1, 3, 21, 24, 27, 29, 31). The permeabilized cells appear to support these physiological functions under specific conditions. Several agents (ATP4-, bacterial toxins, and detergents) can be used to form pores in the cell membrane. The choice of agent depends on the specific pore size required for passage of substances across the cell membrane. Previous studies have used ATP4- to form membrane pores for passage of low-molecular-mass substances (~1 kDa) into mast cells (3). However, this agent could not be used for our studies because type II cells could not be permeabilized with ATP4- (up to 5 µM ATP in divalent-free medium) and because we wished to introduce large-molecular-mass substances (synexin and its antibodies) into type II cells. In different types of cells, a bacterial toxin (streptolysin O) and a saponin (beta -escin) have been used to evaluate the role of Ca2+ (21, 31), GTP (21, 31), or annexin II (24) in exocytic secretion. Both streptolysin O and saponin can bind and remove cholesterol in the cell membrane and render the cells permeable to large molecules (5, 10a). Saponin treatment has been shown to form small or large pores in the cell membrane depending on its concentration (21). Liu and associates (24) have previously used beta -escin to introduce large molecules like annexin II into type II cells. In this study, we used beta -escin-permeabilized type II cells to demonstrate that purified bovine synexin or GTP can independently increase surfactant PC secretion. Thus our study shows a direct role of synexin in lung surfactant secretion. Our results also show the presence of two separate mechanisms for membrane fusion during surfactant secretion in type II cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GTP, beta :gamma -imidoguanosine-5'-triphosphate (NHpGpp), beta -escin, fatty acid-poor bovine serum albumin, antibiotics, and other standard chemicals were from Sigma Chemical (St. Louis, MO). SuperSignal reagent for chemiluminescence analysis of Western blots was purchased from Pierce (Rockford, IL). All plasticwares were obtained from Fisher Scientific (Pittsburgh, PA). All cell culture supplies were from GIBCO BRL (Gaithersburg, MD). [methyl-3H]choline and [14C]dipalmitoyl PC ([14C]DPPC) were obtained from Amersham (Arlington Heights, IL). The antibody to recombinant synexin peptide was a kind gift from Dr. A. Noegel (36).

Isolation of type II cells. Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 180-200 g were used for isolation of alveolar type II cells after elastase digestion of lungs (17) as described previously (12). The cells were plated in tissue culture plastic dishes for 20-22 h in minimum essential medium (MEM) containing 10% fetal calf serum at a cell density of 3 × 106 cells · 2 ml-1 · 35 mm-2. At the end of this culture period, the adherent cells were >95% type II cells as determined by phosphine fluorescence, and >97% of these cells excluded the vital dye erythrosin B.

Permeabilization of type II cells. The adherent type II cells were washed with buffer A (Krebs-Ringer bicarbonate buffer containing 10 mM glucose, 30 mM HEPES, pH 7.4, and 1 mM EGTA). Buffer A was equilibrated for >30 min with 5% CO2 in air before use. The cells were incubated for 10 min at 37°C in 1 ml of permeabilization buffer (buffer A containing indicated concentrations of beta -escin). At the end of this incubation, the permeabilization buffer was removed, and the cells were washed (3 times) with buffer A. In some experiments, the attached cells were scraped into buffer A, and the scraped cells and the permeabilization buffer were stored at -80°C until further analysis. The cells on the dishes were also examined for uptake of trypan blue (0.1%). At least 100 cells in four random fields were counted, and the percentage of trypan blue-stained (nonviable) cells was calculated.

Studies on PC secretion. In experiments designed to measure PC secretion, the cells were incubated for 20-22 h in MEM containing 10% fetal bovine serum and [3H-methyl]- choline (0.3 µCi/ml MEM). The cells were then permeabilized as described above. After the last wash, the cells were incubated for 5 min in 1 ml buffer A. In some experiments, EGTA in buffer A was replaced by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, with essentially similar results. In experiments evaluating the effect of Ca2+, appropriate amounts of CaCl2 were added to the medium to provide 0.5-10 µM Ca2+ buffered with 1 mM EGTA and accounted for the presence of other ions including Mg2+ (4). In each set of experiments, the medium and cells were collected from one plate after the 5-min incubation. This comprised the "zero- time" sample. For other plates, indicated agents or vehicle (<1% by vol) were added at the end of 5 min, and the incubations were continued for the next 30 min. Thereafter, the media were removed to terminate incubations. The medium samples were centrifuged to remove free cells, if any, which were pooled with the cells on plates. The media and the cells on plates were extracted for lipids (6) after addition of egg PC (0.4 mg each) and [14C]DPPC as described previously (12). Individual lipid samples were analyzed for the 3H radioactivity, which was corrected for the recovery of [14C]DPPC. The PC secretion was calculated from the corrected 3H radioactivity in the medium and cell samples [%PC secretion = (100 × 3H in the medium)/3H in the medium plus cells]. The changes in PC secretion are also expressed relative to the control secretion, which was expressed as 100%.

Purification of bovine synexin. Synexin was isolated from the soluble fraction (100,000 g for 60 min supernatant) of bovine lung homogenates as described (37). The purified protein migrated as a single band of ~47 kDa molecular mass on 10% SDS-PAGE. The purified protein was lyophilized and stored at -80°C. It was reconstituted in water at 0.1-1 mg/ml.

Other methods. Protein samples were separated by 10% SDS-PAGE under reducing conditions and stained with Coomassie blue to visualize proteins. For Western analysis, the protein samples were separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes. These membranes were probed with monoclonal antibody to recombinant synexin peptide (36) and visualized by chemiluminescence using SuperSignal reagent. For pretreatment of synexin with the antibody, aliquots containing indicated amounts of synexin were incubated for 20 h at 4°C with the synexin antibody (10 µl). An equal volume of the antibody without synexin or synexin without the antibody was incubated in parallel. These were then evaluated for effects on surfactant PC secretion in permeabilized cells. For heat inactivation of synexin, appropriate amounts of synexin were incubated for 30 min at 65°C, cooled to room temperature, and evaluated for secretagogue effects in permeabilized type II cells. Lactate dehydrogenase (LDH) activity was assayed by monitoring the reduction of NAD at 340 nm (22). Proteins were measured by reaction with the protein-binding dye reagent (Bio-Rad Laboratories, Richmond, CA) using bovine gamma -globulin as a standard (7).

Statistical analysis. Results were analyzed for statistical significance by Student's t-test for experiments paired with respect to cell preparations. For comparison of results from multiple groups, the results were analyzed by ANOVA followed by Newman-Keuls post hoc analysis. All differences were considered significant at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell permeabilization. The permeabilization of type II cells was evaluated by measuring cell uptake of the vital dye trypan blue and by following the release of LDH, cellular proteins, and synexin from type II cells. After a 10-min incubation with 10 µg/ml beta -escin, a significantly higher proportion of cells was stained with trypan blue, while there was only a slight increase in the LDH release when compared with the controls (Fig. 1). These findings suggested that low concentrations of beta -escin formed small pores in type II cell membranes as previously shown for mast cells (20). When incubated with increasingly higher concentrations of beta -escin, the LDH release and trypan blue uptake increased in a concentration-dependent manner. Cells treated with 40 µg/ml beta -escin released ~70% of cellular LDH, and ~90% of these cells could be stained with trypan blue (Fig. 1). In parallel with the release of LDH, other cellular proteins (Fig. 2A) and synexin were also released (Fig. 2B). The release of these cell constituents also increased with beta -escin concentration. Type II cells treated with 40 µg/ml beta -escin, however, retained lamellar bodies as shown by phosphine staining of lamellar bodies (Fig. 3). Because phosphine binds to lipids in the lamellar bodies, these findings suggest that the lamellar bodies in these cells were intact. In light of previous reports that digitonin-permeabilized type II cells do not show regulated secretion (24, 27), we elected to use only beta -escin for cell permeabilization.


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Fig. 1.   Release of lactate dehydrogenase (LDH) and uptake of trypan blue in type II cells treated with beta -escin. Isolated type II cells were cultured for 20-22 h, and the adherent cells were treated for 10 min with indicated amounts of beta -escin. At the end of this period, the media and the cells were collected and analyzed for LDH activity. The LDH release () is expressed as percent of total (cell plus medium) LDH activity. Trypan blue-stained cells () are expressed as percent of the total cells on the plate. The LDH release and the trypan blue uptake were dependent on beta -escin concentration. Results are means ± SE of analysis in 3 separate cell preparations. Some error bars are masked by the symbols.



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Fig. 2.   The release of cellular proteins and synexin during cell permeabilization with beta -escin. Isolated adherent type II cells were incubated for 10 min with indicated amounts of beta -escin. The media and the cells were collected, and the release of cellular proteins and synexin were evaluated by SDS-PAGE and by Western blot analysis. A: medium samples (equal volumes) were lyophilized and analyzed for protein content before separation of proteins by SDS-PAGE. The proteins on the gel were stained with Coomassie blue. Lane 1, prestained standards with molecular mass in kDa indicated on the left; lanes 2-5, medium samples from cells treated with 0, 10, 20, and 40 µg/ml beta -escin, respectively. Note that the protein content increased with the beta -escin concentration. B: Western blot of synexin in cells and medium samples of cells treated with indicated concentrations of beta -escin.



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Fig. 3.   Permeabilized type II cells retain lamellar bodies. Isolated and adherent type II cells were permeabilized for 10 min with 40 µg/ml beta -escin, washed with MEM, treated with 0.01% phosphine, and viewed with a fluorescence microscope. Fluorescence micrographs of the control (A) and beta -escin-treated (B) cells show bright fluorescence in vesicles (lamellar bodies). Magnification, ×600.

PC secretion. Because permeabilized type II cells retained lamellar bodies, we evaluated them for PC secretion. During the 30-min incubation in beta -escin-free medium, these cells released ~1% of the cellular PC into the medium (Table 1). The control secretion, however, showed variation between different cell preparations. Some of this variation is inherent to the cell preparation. In our previous studies with nonpermeabilized cells, we have observed that the control secretion can vary by ~100% (13). The permeabilization of type II cells with a detergent (beta -escin) and the variation in the number of cells rendered permeable to large molecules, as indicated by the LDH release (Fig. 1), also likely contribute to the variations in control secretion and in synexin-dependent secretion (see below). Nevertheless, the PC secretion under the indicated experimental conditions showed a consistent pattern of changes in different cell preparations. To minimize the effect of such variation, the PC secretion under all experimental conditions of a group was measured in the same cell preparations whenever possible. In our initial studies, we evaluated the effects of varying free Ca2+ concentrations on PC secretion. The PC secretion was unchanged when low concentrations of Ca2+ (0.5-10 µM) buffered with EGTA (4) were present in the incubation buffer (Fig. 4). These studies suggested that some of the factors, possibly proteins, had leaked out of the cells and, therefore, Ca2+ was unable to increase PC secretion in these cells. Next, we evaluated the effects of purified synexin on PC secretion in the absence or presence of 1 µM Ca2+. The addition of synexin increased the secretion of PC in the presence but not in the absence of 1 µM Ca2+ (Table 1). The Ca2+ requirement for synexin effect on PC secretion is consistent with the previously described Ca2+ requirement for synexin-dependent membrane aggregation and fusion activity (37). This increase in PC secretion was dependent on synexin concentration (Fig. 5). The maximal increase in secretion was observed at ~5 µg/ml synexin.

                              
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Table 1.   Effect of Ca2+ and synexin in permeabilized type II cells



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Fig. 4.   Effect of Ca2+ on phosphatidylcholine (PC) secretion in permeabilized type II cells. Isolated type II cells were incubated for 20-22 h with [3H]choline, and the adherent cells were permeabilized for 10 min with 40 µg/ml beta -escin and evaluated for PC secretion during a 30-min incubation in the absence and presence of indicated concentrations of Ca2+ buffered with 1 mM EGTA. Results are expressed as percentage of secretion in the absence of Ca2+ and are means ± SE of experiments in 3-5 cell preparations. Ca2+ did not increase PC secretion at any of the indicated concentrations.



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Fig. 5.   Synexin increases PC secretion in a concentration-dependent manner. Permeabilized type II cells were evaluated for PC secretion in the absence and presence of 1 µM Ca2+ and in the absence or presence of indicated amounts of purified bovine synexin. The addition of synexin increased PC secretion in a concentration-dependent manner. Results are means ± SE of experiments in 5-9 separate cell preparations. Statistical significance was evaluated by ANOVA followed by Newman-Keuls post hoc test. *P < 0.05 when compared with secretion in the absence of synexin or 1 µM Ca2+. dagger P < 0.05 when compared with secretion in the absence or presence of 1 µM Ca2+.

Specificity of synexin effect on PC secretion. Next, we investigated the specificity of synexin effects on surfactant secretion. Addition of bovine serum albumin at concentrations comparable with those of synexin did not increase PC secretion (Table 2). The effect of synexin could be abolished with heat inactivation of synexin (30 min at 65°C). These results suggest that the synexin effect on PC secretion is not nonspecific. In another set of experiments, we evaluated whether antibody treatment of synexin would abolish the synexin effect on PC secretion. For these studies, indicated amounts of synexin were incubated for 18-20 h at 4°C with the synexin antibody. The antibody-synexin mixture was then used to evaluate its effect on PC secretion. An equivalent amount of synexin antibody was similarly treated in parallel but without synexin and evaluated for its effect on PC secretion. The antibody or the antibody-synexin mixture did not increase PC secretion (Table 3). These observations suggest that the effect of synexin on PC secretion in type II cells was specific. Furthermore, because synexin antibody alone did not decrease PC secretion when compared with control secretion, we suggest that the residual (possibly cell membrane-associated) synexin does not mediate the control (basal) secretion (Fig. 2B). However, we cannot exclude the possibility that synexin antibody may not be able to react with membrane-associated synexin and, therefore, is unable to inhibit its activity.

                              
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Table 2.   Specificity of synexin effect on PC secretion in permeabilized type II cells


                              
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Table 3.   Effect of synexin antibody on synexin-stimulated PC secretion

Synexin and GTP. In several cell types, GTP is known to promote exocytic secretion by mechanisms involving members of heterotrimeric or low-molecular-weight G proteins. Other investigators have postulated a role for G proteins in membrane fusion during exocytosis of lung surfactant. Previous studies on GTP binding to lamellar bodies revealed the presence of several low-molecular-mass G proteins, some of which could be related to the Ras family of proteins (34). By Western blot analysis, these authors reported the presence of Ras p21 and Rho proteins in the lamellar bodies. It is likely that some of these low-molecular-mass G proteins regulate lung surfactant secretion. Synexin has also been postulated to act as a Ca2+ sensor in the presence of GTP, since it could bind and hydrolyze GTP (10). Therefore, we investigated the effects of GTP and its nonhydrolyzable analog NHpGpp on PC secretion in permeabilized type II cells. The effects of GTP were investigated in the presence of 1 µM Ca2+. The PC secretion in the presence of 1 mM GTP was elevated in the presence of Ca2+ when compared with controls (Table 4). The hydrolysis of GTP was apparently not required because NHpGpp also increased PC secretion in permeabilized type II cells. Compared with PC secretion in control cells, the secretion was increased by 152 ± 42% in the presence of 0.1 mM NHpGpp (control, 0.91 ± 0.19; NHpGpp, 2.13 ± 0.26%, n = 3, P < 0.05). The presence of GTP and synexin further increased PC secretion when compared with GTP or synexin alone (Table 4). Thus synexin and GTP appear to increase PC secretion in an additive manner, suggesting that these two agents act through different mechanisms to increase membrane fusion for PC secretion.

                              
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Table 4.   Synexin effects on GTP-stimulated secretion in permeabilized type II cells


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The fusion of lamellar bodies with the plasma membrane is an obligatory step for the exocytic release of lung surfactant from lung epithelial type II cells. In our previous studies, we had postulated that synexin may play an important role in such membrane fusion during surfactant secretion because of the following observations: 1) in vitro studies showed that synexin can augment fusion of lamellar bodies with plasma membrane (13), 2) synexin binds to specific proteins in the plasma membrane and lamellar body fractions of lung (37), and 3) inhibitors of synexin activity can inhibit surfactant secretion in isolated type II cells (12, 23). The present study provides direct evidence for a role of synexin in surfactant secretion, since exogenous synexin could increase PC secretion in type II cells that were made synexin deficient by cell permeabilization. Although the secretion response to synexin varied between cell preparations, probably because of variation in the number of cells rendered permeable to large molecules (Fig. 1), the effect of synexin was consistent in all cell preparations. This effect of synexin was specific because it could be abolished after inactivation of synexin by two independent protocols (Tables 2 and 3). However, our studies suggest that alternate synexin-independent mechanisms also mediate membrane fusion for surfactant secretion. One of these is GTP dependent and possibly involves G proteins. The effects of synexin and GTP appear additive, suggesting that these two apparently function independently of each other to mediate membrane fusion for surfactant secretion.

Previous studies with streptolysin O-permeabilized type II cells showed that GTP can both stimulate and inhibit surfactant secretion and that the GTP effect was dependent on Ca2+ concentration in the assay medium (27). Later studies showed that activation of heterotrimeric G proteins may stimulate adenyl cyclase (28), which increases the cAMP levels and is associated with the increased secretion of lung surfactant (8, 26). More recently, exocytosis in type II cells was studied by measuring membrane capacitance and by following changes in the fluorescence of a dye, FM1-43, on binding of lipids in the lamellar body (25). These studies showed that guanosine 5'-O-(3-thiotriphosphate), a nonhydrolyzable analog of GTP, increases the formation of exocytic fusion pores in type II cells, suggesting increased secretion of lung surfactant. Our results of increased secretion with GTP and NHpGpp also suggest the involvement of G proteins in lamellar body exocytosis. The GTP-dependent mechanisms do not seem to require synexin for augmentation of PC secretion, since the effects of GTP were additive with those of synexin and the GTP modulation of PC secretion was observed in synexin-deficient type II cells (Fig. 2B and Table 4).

Several members of the low-molecular-mass G proteins, Ras, Rho, ADP-ribosylation factor (ARF), Rab, and Ran, which are active in GTP-bound form, are invoked to play a major role in various cellular processes like growth, morphogenesis, vesicle trafficking, and exocytosis (35). The soluble N-ethylmaleimide-sensitive factor attachment protein recepter (SNARE) complex is postulated to form the basic machinery for vesicle docking and membrane fusion during vesicle trafficking and exocytosis (33, 38). GTP is suggested to promote exocytic secretion in several cell types by acting on the low-molecular-mass G proteins such as Rab and ARF. It is suggested that the GTP-bound form of Rab recognizes the target SNAREs and facilitates their pairing with vesicle SNAREs to promote vesicle docking and membrane fusion (35). The low-molecular-mass G proteins are also present in type II cells and at least two of these, Ras p21 and Rho, and the substrates for ARF are present in lamellar bodies (34). If the GTP effect on PC secretion in type II cells is mediated by facilitating the SNARE pathway, then our observation of apparently additive effects of synexin and GTP would suggest that synexin acts independently of the SNARE pathway.

A previous study reported that only annexins I, II, III, and VI were present in type II cells (24). Our study shows that synexin is also present in type II cells. Like our study showing augmentation of surfactant secretion by synexin, the study by Liu et al. (24) showed that annexin II tetramer also increased PC secretion in permeabilized type II cells. These authors suggested that annexin II was necessary for surfactant PC exocytosis. However, our studies show that synexin can also increase PC secretion in beta -escin-permeabilized type II cells that were shown to be deficient in annexin II (24). In PC12 cells also, the expression of structurally modified annexin II, which caused its aggregation and functional depletion, or overexpression of annexin II did not affect Ca2+-dependent dopamine secretion (20). Thus it is unlikely that annexin II is necessary for membrane fusion during exocytic secretion. Our studies also suggest that type II cells may contain annexin-dependent and annexin-independent (some of which may be GTP-dependent) mechanisms for secretion. Because of similar characteristics of synexin and annexin II (Ca2+-dependent phospholipid binding, augmentation of surfactant PC secretion), we suggest that synexin and annexin II can substitute for each other during annexin-dependent membrane fusion for PC secretion in type II cells.

The annexin-independent mechanism of membrane fusion may function during control (basal) secretion and during GTP-dependent secretion. Because of similar conditions for cell permeabilization (this study and Ref. 24), we assume that the beta -escin-permeabilized type II cells were deficient in both synexin and annexin II. Thus the control secretion in these cells was through the annexin-independent mechanism. Because synexin antibodies did not inhibit PC secretion under control conditions (Table 3), we suggest that residual synexin in permeabilized type II cells did not mediate the control PC secretion. However, we cannot rule out that synexin antibody may not be able to inactivate cell-associated residual synexin. The GTP stimulation of PC secretion in synexin-deficient cells (Table 4) would also support the presence of an annexin-independent mechanism. It is likely that the proteins of the SNARE complex that are associated with the lamellar bodies and the plasma membrane bring about the control secretion, which is further facilitated with the addition of GTP because of its postulated role in the vesicle docking and membrane fusion process (35).

The synexin-dependent mechanism of membrane fusion and PC secretion requires Ca2+ (Table 1), which is consistent with previously described properties of synexin and other annexins (15, 19). In the studies reported here, we employed low concentrations of Ca2+ to evaluate synexin effects on surfactant PC secretion because of the low Ca2+ threshold for synexin binding to biological membranes (37). Although our earlier studies on membrane fusion between lamellar bodies and plasma membranes were carried out with high (1 mM) Ca2+ (14), our later studies suggest that synexin binding to biological membranes, which can occur at low Ca2+, may be the rate-limiting step in synexin-dependent membrane fusion (37). Therefore, we used low Ca2+ concentrations to evaluate PC secretion in permeabilized type II cells. Because synexin increased PC secretion at low Ca2+, we suggest that the threshold Ca2+ concentrations for synexin binding are sufficient to facilitate membrane fusion during surfactant secretion. The underlying reasons for different Ca2+ requirements for the in vitro fusion (14) and for PC secretion in permeabilized type II cells (this study) are not clear but may be related to the presence of membrane proteins, which may be lost during isolation of subcellular fractions.

The mechanism of synexin action during surfactant secretion is not clear. In vitro studies have shown that synexin can undergo Ca2+-dependent self-association (16, 23). The self-associated form of synexin may bind to membranes and establish intermembrane contact, which is postulated as an essential step in the hydrophobic bridge hypothesis for membrane fusion (30). In analogy with the SNARE mechanism of membrane fusion, which involves several proteins, the synexin-dependent membrane fusion may also involve other proteins. It is noteworthy that some proteins can bind to synexin and thus may regulate its activity. We have previously shown that synexin binding to a specific protein (~76 kDa) in the lung lamellar body and plasma membrane increases in a Ca2+-dependent manner (37). Studies with recombinant synexin peptide showed that sorcin (soluble resistance-related Ca2+-binding protein) can bind to the amino domain of synexin in a Ca2+-dependent manner and inhibit synexin-mediated chromaffin granule aggregation (9). Thus the presence of these synexin-binding proteins suggests that synexin may not function alone in the membrane fusion process during surfactant secretion.

In summary, our studies show the presence of synexin in alveolar type II cells. Using permeabilized alveolar type II cells, we have provided direct evidence for involvement of synexin in surfactant secretion. Our studies also suggest that the membrane fusion for PC secretion in type II cells may be mediated by synexin (annexin)-dependent and -independent mechanisms. The presence of several independent mechanisms for membrane fusion during surfactant secretion possibly protects against defects in one pathway and underscores the importance of the secretion process. Although each mechanism can support secretion, the maximal secretion of surfactant may require that both mechanisms function properly.


    ACKNOWLEDGEMENTS

We acknowledge the expert technical assistance of Ai-Min Wu and Graham Vigliotta. We also acknowledge a generous gift of synexin antibodies from Dr. A. Noegel (Max-Planck Institute for Biochemistry, Martinstried, Germany).


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-49959.

Some of these studies were presented at the International Conference of The American Lung Association in San Francisco, CA, in May 1997.

Address for reprint requests and other correspondence: A. Chander, Dept. of Pediatrics, Division of Neonatology, SUNY at Stony Brook, School of Medicine, Health Sciences Center, Stony Brook, NY 11794 (E-mail: achander{at}mail.som.sunysb.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 September 2000; accepted in final form 30 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ahnert-Hilger, G, Mach W, Fohr KJ, and Gratzl M. Poration by alpha -toxin and streptolysin O: an approach to analyze intracellular processes. Methods Cell Biol 31: 63-90, 1989[ISI][Medline].

2.   Batenburg, JJ, and Haagsman HP. The lipids of pulmonary surfactant: dynamics and interactions with proteins. Prog Lipid Res 37: 235-276, 1998[ISI][Medline].

3.   Bennet, JP, Cockroft S, and Gomperts BD. Rat mast cells permeabilized with ATP secrete histamine in response to calcium ions buffered in the micromolar range. J Physiol (Lond) 317: 335-345, 1981[Abstract].

4.   Bers, DM. A simple method for accurate determination of free [Ca] in Ca-EGTA solutions. Am J Physiol Cell Physiol 242: C404-C408, 1982[Abstract].

5.   Bhakdi, S, Tranum-Jensen J, and Sziegoleit A. Mechanism of membrane damage by streptolysin O. Infect Immun 47: 52-60, 1985[ISI][Medline].

6.   Bligh, EG, and Dyer WJ. A rapid method for total lipid extraction and purification. Can J Biochem Physiol 37: 911-917, 1959[ISI].

7.   Bradford, MM. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976[ISI][Medline].

8.   Brown, LAS, and Longmore WJ. Adrenergic and cholinergic regulation of lung surfactant secretion in the isolated perfused lung and in the alveolar type II cells in culture. J Biol Chem 256: 66-72, 1981[Abstract/Free Full Text].

9.   Brownawell, AM, and Creutz CE. Calcium-dependent binding of sorcin to the N-terminal domain of synexin (annexin VII). J Biol Chem 272: 22182-22190, 1997[Abstract/Free Full Text].

10.   Cahouy, H, Srivastava M, and Pollard HB. Membrane fusion protein synexin (annexin VII) as a Ca2+/GTP sensor in exocytotic secretion. Proc Natl Acad Sci USA 93: 10797-10802, 1996[Abstract/Free Full Text].

11.   Chander, A, and Fisher AB. Regulation of lung surfactant secretion. Am J Physiol Lung Cell Mol Physiol 258: L241-L253, 1990[Abstract/Free Full Text].

12.   Chander, A, and Sen N. Inhibition of phosphatidylcholine secretion by stilbene disulfonates in alveolar type II cells. Biochem Pharmacol 45: 1905-1912, 1993[ISI][Medline].

13.   Chander, A, Sen N, Wu A-I, and Spitzer AR. Protein kinase C in ATP regulation of lung surfactant secretion in type II cells. Am J Physiol Lung Cell Mol Physiol 268: L108-L116, 1995[Abstract/Free Full Text].

14.   Chander, A, and Wu R-D. In vitro fusion of lung lamellar bodies and plasma membrane is augmented by lung synexin. Biochim Biophys Acta 1086: 157-166, 1991[ISI][Medline].

15.   Creutz, CE. The annexins and exocytosis. Science 258: 924-930, 1992[ISI][Medline].

16.   Creutz, CE, Pazoles CJ, and Pollard HB. Self-association of synexin in the presence of calcium: correlation with synexin-induced membrane fusion and examination of the structure of synexin aggregates. J Biol Chem 254: 553-558, 1979[Abstract].

17.   Dobbs, LG, Gonzalez R, and Williams MC. An improved method for isolation of type II pneumocytes in high yield and purity. Am Rev Respir Dis 134: 141-145, 1986[ISI][Medline].

18.   Gennis, RB. Pores, channels, and transporters. In: Biomembranes. Molecular Structure and Function. New York: Springer-Verlag, 1988, chapt. 8, p. 270-322. (Springer Advanced Texts Chem Ser).

19.   Gerke, V, and Moss SE. Annexins and membrane dynamics. Biochim Biophys Acta 1357: 129-154, 1997[ISI][Medline].

20.   Graham, ME, Gerke V, and Burgoyne RD. Modification of annexin II expression in PC12 cell line does not affect Ca2+-dependent exocytosis. Mol Biol Cell 8: 431-442, 1997[Abstract].

21.   Izushi, K, and Tasaka K. Histamine release from beta -escin-permeabilized rat peritoneal mast cells and its inhibition by intracellular Ca2+ blockers, calmodulin inhibitors, and cAMP. J Immunopharmacol 18: 177-186, 1989.

22.   Lee, YP, and Lardy HA. Influence of thyroid hormones on L-alpha -glycerophosphate dehydrogenase in various tissues of rat. J Biol Chem 240: 427-436, 1965.

23.   Liu, L, and Chander A. Stilbene disulfonic acids inhibit synexin-mediated membrane aggregation and fusion. Biochim Biophys Acta 1254: 274-282, 1995[ISI][Medline].

24.   Liu, L, Wang M, Fisher AB, and Zimmerman UJ. Involvement of annexin II in exocytosis of lamellar bodies from alveolar type II cells. Am J Physiol Lung Cell Mol Physiol 270: L668-L676, 1996[Abstract/Free Full Text].

25.   Mair, N, Haller T, and Dietl P. Exocytosis in alveolar type II cells revealed by cell capacitance and fluorescence measurements. Am J Physiol Lung Cell Mol Physiol 276: L376-L382, 1999[Abstract/Free Full Text].

26.   Mescher, EJ, Dobbs LG, and Mason RJ. Cholera toxin stimulates secretion of saturated phosphatidylcholine and increases cellular cyclic AMP in isolated rat alveolar type II cells. Exp Lung Res 5: 173-182, 1983[ISI][Medline].

27.   Pian, MS, and Dobbs LG. Activation of G proteins may inhibit or stimulate surfactant secretion in alveolar type II cells. Am J Physiol Lung Cell Mol Physiol 266: L375-L381, 1994[Abstract/Free Full Text].

28.   Pian, MS, and Dobbs LG. Evidence for G beta gamma-mediated cross-talk in primary cultures of lung alveolar cells: pertussis toxin-sensitive production of cAMP. J Biol Chem 270: 7427-7430, 1995[Abstract/Free Full Text].

29.   Pimplikar, SW, Ikonen E, and Simons K. Basolateral protein transport in streptolysin O-permeabilized MDCK cells. J Cell Biol 125: 1025-1035, 1994[Abstract].

30.   Pollard, HB, Burns AL, and Rojas E. A molecular basis for synexin-driven, calcium dependent membrane fusion. J Exp Biol 139: 267-286, 1988[Abstract].

31.   Prentki, M, Wolheim SG, and Lew PD. Ca2+ homeostasis in permeabilized human neutrophils. J Biol Chem 259: 13777-13782, 1984[Abstract/Free Full Text].

32.   Rooney, SA, Young SL, and Mendelson CR. Molecular and cellular processing of lung surfactant. FASEB J 8: 957-967, 1993[Abstract/Free Full Text].

33.   Rothman, JE. Mechanisms of intracellular protein transport. Nature 372: 55-63, 1994[ISI][Medline].

34.   Rubins, JB, Panchenko M, Shannon TM, and Dickey BF. Identification of ras and ras-related low-molecular-mass GTP-binding proteins associated with rat lung lamellar bodies. Am J Respir Cell Mol Biol 6: 253-259, 1992[ISI][Medline].

35.   Schimmöller, F, Simon I, and Pfeffer SR. Rab GTPases, directors of vesicle docking. J Biol Chem 273: 22161-22164, 1999[Free Full Text].

36.   Selbert, S, Fischer P, Pongratz D, Stewart M, and Noegel A. Expression and localization of annexin VII (synexin) in muscle cells. J Cell Sci 108: 85-95, 1995[Abstract/Free Full Text].

37.   Sen, N, Spitzer AR, and Chander A. Calcium-dependence of synexin binding may determine aggregation and fusion of lamellar bodies. Biochem J 322: 103-109, 1997[ISI][Medline].

38.   Weber, T, Zemelman BV, McNew JA, Westermann B, Gmachl M, Parlati F, Sollner TH, and Rothman JE. SNAREpins: minimal machinery for membrane fusion. Cell 92: 759-772, 1998[ISI][Medline].


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