Chemical modification of surfactant protein A alters high affinity binding to rat alveolar type II cells and regulation of phospholipid secretion

Alveolar type II cells express a high affinity receptor for pulmonary surfactant protein A (SP-A), and the interaction of SP-A with these cells leads to inhibition of surfactant lipid secretion. We have investigated the binding of native and modified forms of SP-A to isolated rat alveolar type II cells. Native and deglycosylated forms of SP-A readily competed with 125I-SP-A for cell surface binding. Alkylation of SP-A with excess iodoacetamide yielded forms of SP-A that did not inhibit surfactant lipid secretion and did not compete with 125I-SP-A for cell surface binding. Reductive methylation of SP-A with H2CO and NaCNBH3 yielded forms of SP-A with markedly reduced receptor binding activity that also exhibited significantly reduced capacity to inhibit lipid secretion. Modification of SP-A with cyclohexanedione reversibly altered cell surface binding and the activity of SP-A as an inhibitor of lipid secretion. Two monoclonal antibodies that block the function of SP-A as an inhibitor of lipid secretion completely prevented the high affinity binding of SP-A to type II cells. A monoclonal antibody that recognizes epitopes on SP-A but failed to block the inhibition of secretion also failed to completely attenuate high affinity binding to the receptor. Concanavalin A inhibits phospholipid secretion of type II cells by a mechanism that is reversed in the presence of excess alpha-methylmannoside. Concanavalin A did not block the high affinity binding of 125I-SP-A to the receptor. Neither the high affinity binding nor the inhibitor activity of SP-A was prevented by the presence of mannose or alpha-methylmannoside. The SP-A derived from humans with alveolar proteinosis is a potent inhibitor of surfactant lipid secretion but failed to completely displace 125I-SP-A binding from type II cells. From these data we conclude that: 1) cell surface binding activity of rat SP-A is directly related to its capacity to inhibit surfactant lipid secretion; 2) monoclonal antibodies directed against SP-A can be used to map binding domains for the receptor; 3) the lectin activity of SP-A against mannose ligands does not appear to be essential for cell surface binding; 4) concanavalin A does not compete with SP-A for receptor binding; and 5) the human SP-A derived from individuals with alveolar proteinosis exhibits different binding characteristics from rat SP-A.     

The major lung surfactant protein, SP 28-36, is a calcium-dependent, carbohydrate-binding protein

SP 28-36, a major protein of pulmonary surfactant, has striking amino acid sequence homology with soluble mannose-binding proteins isolated from rat liver and contains residues common to the carbohydrate-binding domains of other mammalian lectins. We have used carbohydrate-affinity chromatography to investigate carbohydrate-binding properties of SP 28-36 isolated from canine and human (alveolar proteinosis patients) lung lavage. SP 28-36 binds to immobilized D-mannose, L-fucose, D-galactose, and D-glucose. The protein binds only weakly to N-acetyl-D-galactosamine and N acetyl-D-glucosamine. Binding is Ca2+-dependent. The threshold Ca2+ concentration is 0.6 mM and maximal binding occurs with 1 mM Ca2+. Bound protein is quantitatively recovered by elution with 2 mM EDTA. Ba2+, Sr2+, and Mn2+, but not Mg2+, can substitute for Ca2+. Unlike some other mammalian lectins, SP 28-36 binds to carbohydrate at pH 5.0. Recombinant human SP 28-36 isolated from the media of Chinese hamster ovary cells, transfected with a DNA construct encoding SP 28-36, has similar carbohydrate-binding activity to the native proteins. Mannose affinity chromatography of the culture medium of Chinese hamster ovary cells results in an efficient purification of the secreted recombinant human SP 28-36.     

Surfactant peptides stimulate uptake of phosphatidylcholine by isolated cells

To determine whether small hydrophobic surfactant peptides (SP-B and SP-C) participate in recycling of pulmonary surfactant phospholipid, we determined the effect of these peptides on transfer of 3H- or 14C-labelled phosphatidylcholine from liposomes to isolated rat alveolar Type II cells and Chinese hamster lung fibroblasts. Both natural and synthetic SP-B and SP-C markedly stimulated phosphatidylcholine transfer to alveolar Type II cells and Chinese hamster lung fibroblasts in a dose- and time-dependent fashion. Effects of the peptides on phospholipid uptake were dose-dependent, but not saturable and occurred at both 4 and 37 degrees C. Uptake of labelled phospholipid into a lamellar body fraction prepared from Type II cells was augmented in the presence of SP-B. Neither SP-B nor SP-C augmented exchange of labelled plasma membrane phosphatidylcholine from isolated Type II cells or enhanced the release of surfactant phospholipid when compared to liposomes without SP-B or SP-C. Addition of native bovine SP-B and SP-C to the phospholipid vesicles perturbed the size and structure of the vesicles as determined by electron microscopy. To determine the structural elements responsible for the effect of the peptides on phospholipid uptake, fragments of SP-B were synthesized by solid-phase protein synthesis and their effects on phospholipid uptake assessed in Type II epithelial cells. SP-B (1-60) stimulated phospholipid uptake 7-fold. A smaller fragment of SP-B (15-60) was less active and the SP-B peptide (40-60) failed to augment phospholipid uptake significantly. Like SP-B and SP-C, surfactant-associated protein (SP-A) enhanced phospholipid uptake by Type II cells. However, SP-A failed to significantly stimulate phosphatidylcholine uptake by Chinese hamster lung fibroblasts. These studies demonstrate the independent activity of surfactant proteins SP-B and SP-C on the uptake of phospholipid by Type II epithelial cells and Chinese hamster lung fibroblasts in vitro.     

Purification and characterization of recombinant human testis angiotensin-converting enzyme expressed in Chinese hamster ovary cells.

Enzymatically active human testis angiotensin-converting enzyme (ACE) was expressed in Chinese hamster ovary (CHO) cells stably transfected with each of three vectors: p omega-ACE contains a full-length testis ACE cDNA under the control of a retroviral promoter; and pLEN-ACEVII and pLEN-ACE6/5, in which full-length and membrane anchor-minus testis ACE cDNAs, respectively, are under the control of the human metallothionein IIA promoter and SV40 enhancer. In every case, active recombinant human testis ACE (hTACE) was secreted in a soluble form into the culture media, up to 2.4 mg/liter in the media of metal-induced, high-producing clones transfected with one of the pLEN vectors. In addition, membrane-bound recombinant enzyme was recovered from detergent extracts of cell pellets of CHO cells transfected with either p omega-ACE or pLEN-ACE-VII. Recombinant converting enzyme was purified to homogeneity by single-step affinity chromatography of conditioned media and detergent-extracted cell pellets in 85 and 70% overall yield, respectively. Purified hTACE from all sources comigrated with the native testis isozyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r) approximately 100 kDa. The native and recombinant proteins cross-reacted equally with anti-human kidney ACE antiserum on Western blotting. The catalytic activity of recombinant angiotensin-converting enzyme, in terms of angiotensin I and 2-furanacryloyl-Phe-Gly-Gly hydrolysis, chloride activation, and lisinopril inhibition, was essentially identical to that of the native enzyme. The facile recovery in high yield of fully active hTACE from the media of stably transfected CHO cells provides a suitable system for investigating structure-function relationships in this enzyme.     

High-level expression of a codon optimized recombinant dust mite allergen, Blo t 5, in Chinese hamster ovary cells

Blo t 5 is a major allergen from house dust mite Blomia tropicalis. Purification of native Blo t 5 (nBlo t 5) from whole dust mite extract is tedious and gave low yield. In this study, we demonstrated that codon optimization facilitated high-level expression of Blo t 5 in Chinese hamster ovary (CHO)-K1 cells and thus allows production of sufficient recombinant cBlo t 5 for specific immunotherapy. A codon optimized Blo t 5 gene was synthesized by PCR and the codon optimized or wild-type Blo t 5 gene in pcDNA3.0 was transfected into CHO-K1 cells and stably selected with Geneticin (G418). Western-immunoblot analysis of spent culture media detected a positive band at 14kDa for the codon optimized but not wild-type gene transfectants. In addition, a stable CHO-K1 clone produced up to 13 mg/L of the cBlo t 5 protein having a high correlation of human IgE reactivities and allergenicity to the native Blo t 5, thus indicating proper conformation of this recombinant form.     

Binding and uptake of pulmonary surfactant protein (SP-A) by pulmonary type II epithelial cells

A glycoprotein of Mr 26-36,000 (SP-A) is an abundant phospholipid-associated protein in pulmonary surfactant. SP-A enhances phospholipid reuptake and inhibits secretion by Type II epithelial cells in vitro. We have used two electron microscopic cytochemical methods to demonstrate selective binding and uptake of SP-A by rat pulmonary Type II epithelial cells. Using an immunogold bridging technique, we showed that SP-A binding was selective for Type II cell surfaces. Binding was dose dependent and saturable, reaching maximal binding at approximately 10 ng/ml. On warming to 23 degrees C, SP-A binding sites were clustered in coated pits on the cell surface. To characterize the internalization and intracellular routing of SP-A, we used the biotinyl ligand-avidin-gold technique. Biotinyl SP-A was bound by rat Type II epithelial cells as described above. On warming, biotinyl SP-A was seen in association with coated vesicles and was subsequently located in endosomes and multivesicular bodies. Biotinyl SP-A-gold complexes were seen in close approximation to lamellar bodies 10-60 min after warming. Binding of biotinyl SP-A was inhibited by competition with unlabeled SP-A. These results support the concept that Type II epithelial cells bind and internalize SP-A by receptor-mediated endocytosis. This newly described uptake system may play a role in the recycling of surfactant components or mediate the actions of SP-A on surfactant phospholipid secretion.     

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

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

Identification and characterization of p63 (CKAP4/ERGIC-63/CLIMP-63), a surfactant protein A binding protein, on type II pneumocytes

Surfactant protein A (SP-A) binds to alveolar type II cells through a specific high-affinity cell membrane receptor, although the molecular nature of this receptor is unclear. In the present study, we have identified and characterized an SP-A cell surface binding protein by utilizing two chemical cross-linkers: profound sulfo-SBED protein-protein interaction reagent and dithiobis(succinimidylpropionate) (DSP). Sulfo-SBED-biotinylated SP-A was cross-linked to the plasma membranes isolated from rat type II cells, and the biotin label was transferred from SP-A to its receptor by reduction. The biotinylated SP-A-binding protein was identified on blots by using streptavidin-labeled horseradish peroxidase. By using DSP, we cross-linked SP-A to intact mouse type II cells and immunoprecipitated the SP-A-receptor complex using anti-SP-A antibody. Both of the cross-linking approaches showed a major band of 63 kDa under reduced conditions that was identified as the rat homolog of the human type II transmembrane protein p63 (CKAP4/ERGIC-63/CLIMP-63) by matrix-assisted laser desorption ionization and nanoelectrospray tandem mass spectrometry of tryptic fragments. Thereafter, we confirmed the presence of p63 protein in the cross-linked SP-A-receptor complex by immunoprobing with p63 antibody. Coimmunoprecipitation experiments and functional assays confirmed specific interaction between SP-A and p63. Antibody to p63 could block SP-A-mediated inhibition of ATP-stimulated phospholipid secretion. Both intracellular and membrane localized pools of p63 were detected on type II cells by immunofluorescence and immunobloting. p63 colocalized with SP-A in early endosomes. Thus p63 closely interacts with SP-A and may play a role in the trafficking or the biological function of the surfactant protein.     

TGF-beta1 in SP-A preparations influence immune suppressive properties of SP-A on human CD4+ T lymphocytes

Surfactant protein A (SP-A) and transforming growth factor-beta1 (TGF-beta1) have been shown to modulate the functions of different immune cells and specifically to inhibit T lymphocyte proliferation. The aim of the present study was to elucidate whether the Smad signaling pathway, which is activated by TGF-beta1, also plays a role in SP-A-mediated inhibition of CD4+ T lymphocyte activation. Recombinant human SP-A1 expressed in Chinese hamster ovary cells [rSP-A1m (mammalian)], but not recombinant Baculovirus-derived rSP-A1hyp (hydroxyproline-deficient), suppressed T lymphocyte proliferation and IL-2 mRNA expression. To test whether SP-A induced Smad signaling, a Smad3/4-specific reporter gene was transfected in primary human CD4+ T lymphocytes. Only rSP-A1m, but not rSP-A1hyp, induced Smad-specific reporter genes, Smad2 phosphorylation, and Smad7 mRNA expression. The effect of rSP-A1m was mediated through the TGF-betaRII and could be antagonized by anti-TGF-beta1 neutralizing antibodies and sTGF-betaRII. Western blot and ELISA analysis revealed that rSP-A1m, but not rSP-A1hyp, contained TGF-beta1. TGF-beta1 was responsible for the differences in inhibition of CD4+ T lymphocyte proliferation and activation of the Smad signaling pathway between rSP-A1m and rSP-A1hyp. After acidification, native SP-A, obtained from patients with alveolar proteinosis, also induced Smad signaling in human CD4+ T lymphocytes leading to an increased inhibition of T lymphocyte proliferation, thus indicating the presence of inactive, latent TGF-beta1 in native SP-A samples. Association between SP-A and latent TGF-beta1 provides a possible novel mechanism to regulate TGF-beta1-mediated inflammation and fibrosis reactions in the lung but also leads to possible misinterpretation of immune-modulator functions of SP-A. Monitoring of SP-A preparations for possible TGF-beta1 is essential.     

Role of P63 (CKAP4) in binding of surfactant protein-A to type II pneumocytes

We have recently described a putative receptor for lung surfactant protein-A (SP-A) on rat type II pneumocytes. The receptor, P63, is a 63-kDa type II transmembrane protein. Coincubation of type II cells with P63 antibody (Ab) reversed the inhibitory effect of SP-A on secretagogue-stimulated surfactant secretion from type II cells. To further characterize SP-A interactions with P63, we expressed recombinant P63 protein in Escherichia coli and generated antibodies to P63. Immunogold electron microscopy confirmed endoplasmic reticulum and plasma membrane localization of P63 in type II cells with prominent labeling of microvilli. Binding characteristics of iodinated SP-A to type II cells in the presence of P63 Ab were determined. Binding (4 degrees C, 1 h) of (125)I-SP-A to type II cells demonstrated both specific (calcium-dependent) and nonspecific (calcium-independent) components. Ab to P63 protein blocked the specific binding of (125)I-SP-A to type II cells and did not change the nonspecific SP-A association. A549 cells, a pneumocyte model cell line, expressed substantial levels of P63 and demonstrated specific binding of (125)I-SP-A that was inhibited by the P63 Ab. The secretagogue (cAMP)-stimulated increase in calcium-dependent binding of SP-A to type II cells was blocked by the presence of P63 Ab. Transfection of type II cells with small interfering RNA to P63 reduced P63 protein expression, attenuated P63-specific SP-A binding, and reversed the ability of SP-A to prevent surfactant secretion from the cells. Our results further substantiate the role of P63 as an SP-A receptor protein localized on the surface of lung type II cells.     

Expression and characterization of human recombinant and alpha-N-acetylglucosaminidase

Mucopolysaccharidosis type IIIB (MPS-IIIB, Sanfilippo type B Syndrome) is a heterosomal, recessive lysosomal storage disorder resulting from a deficiency of [alpha]-N-acetylglucosaminidase (NAGLU). To characterize this enzyme further and evaluate its potential for enzyme replacement studies we expressed the NAGLU-encoding cDNA in Chinese hamster ovary cells (CHO-K1 cells) and purified the recombinant enzyme from the medium of stably transfected cells by a two-step affinity chromatography. Two isoforms of recombinant NAGLU with apparent molecular weights of 89 and 79 kDa were purified and shown to differ in their glycosylation pattern. The catalytic parameters of both forms of the recombinant enzyme were indistinguishable from each other and similar to those of NAGLU purified from various tissues. However, compared to other recombinant lysosomal enzymes expressed from CHO-K1 cells, the mannose-6-phosphate receptor mediated uptake of the secreted form of recombinant NAGLU into cultured skin fibroblasts was considerably reduced. A small amount of phosphorylated NAGLU present in purified enzyme preparations was shown to be endocytosed by MPS-IIIB fibroblasts via the mannose-6-phosphate receptor-mediated pathway and transported to the lysosomes, where they corrected the storage phenotype. Direct metabolic labeling experiments with Na(2) (32)PO(4) confirmed that the specific phosphorylation of recombinant NAGLU secreted from transfected CHO cells is significantly lower when compared with a control lysosomal enzyme. These results suggest that the use of secreted NAGLU in future enzyme and gene replacement therapy protocols will be severely limited due to its small degree of mannose-6-phosphorylation.     

Expression of biologically active human follitropin in Chinese hamster ovary cells

To study the structure-function relationships of follitropin (FSH), we expressed the hormone in a heterologous cell system. A genomic clone bearing a 3.7-kilobase FSH beta insert containing the entire coding sequence was transfected alone or together with the alpha subunit gene into Chinese hamster ovary cells and stable lines expressing either FSH beta or FSH dimer were selected. Pulse-chase experiments revealed that, when transfected alone FSH beta was very slowly secreted similar to lutropin beta and thyrotropin beta but unlike choriogonadotropin beta which is efficiently secreted. However, cotransfection of the FSH beta and alpha subunit genes resulted in "rescue" of the beta subunit and rapid secretion of dimer. These data support the hypothesis that the glycoprotein hormones of pituitary origin have determinants for secretion that differ from those on the placental hormone, choriogonadotropin. Recombinant FSH stimulated steroidogenesis comparable to purified human FSH isolated from pituitaries in an in vitro rat granulosa cell assay and appears more homogeneous by chromatofocusing. Human FSH produced by this cell line provides a source of bioactive FSH for experimental and clinical use.     

Proteomic profiling of secreted proteins from CHO cells using Surface-Enhanced Laser desorption ionization time-of-flight mass spectrometry

Chinese hamster ovary (CHO) cells are the most commonly used host cell line for the production of recombinant biopharmaceuticals. These biopharmaceuticals are typically secreted from CHO cells and purified from harvested cell culture media. The purpose of this study was to investigate changes in the secreted proteome of CHO cells over the various stages of the growth cycle using Surface Enhanced Laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS). Conditioned media samples were collected each day over a 6 day growth period from CHO-K1 cells grown in low serum (0.5% FBS) conditions in monolayer culture. Samples were profiled on a number of ProteinChip arrays with different chromatographic surfaces. From this study, 24 proteins were found to be differentially regulated at different phases of the growth cycle in CHO-K1 cells, when profiled on two chromatographic surfaces, Q10 (anionic) and IMAC30 (metal affinity) ProteinChip arrays.     

Purification of recombinant activin A using the second follistatin domain of follistatin-related gene (FLRG)

Activins are multifunctional growth factors belonging to the transforming growth factor-beta superfamily. Isolation of activins from natural sources requires many steps and only produces limited quantities. Even though recombinant preparations have been used in recent studies, purification of recombinant activins still requires multiple steps. To purify recombinant activin A, we have developed a simple method using the second follistatin domain of an activin-binding protein follistatin-related gene (FLRG). An affinity column was prepared with a partial FLRG fusion protein. The partial FLRG protein contained the second follistatin domain and the C-terminus acidic domain, and was tagged with six histidine residues at its N-terminus. The fusion protein was expressed in Escherichia coli and purified with nickel affinity column. Thereafter, the purified fusion protein was coupled to NHS-activated column. Recombinant activin A was produced in Chinese hamster ovary (CHO) cells, which were stably transfected with rat inhibin/activin betaA-subunit cDNA. After 48-h suspension culture of the cells in a serum free medium, the culture media was recovered and passed through the FLRG-coupled column. After washing with phosphate-buffered saline, bound protein was eluted out with an acidic buffer. Any significant contaminations were not detected when the purified protein was analyzed by SDS-PAGE. Apparent sizes of the protein were 14 and 28 kDa under the reduced and non-reduced conditions, respectively. Western blot analysis confirmed that the purified protein was activin A. The purified recombinant activin stimulated p3TP-lux reporter activity in CHO cells and follicle-stimulating hormone secretion from rat pituitary cells.     

Insulin action on activity and cell surface disposition of human HepG2 glucose transporters expressed in Chinese hamster ovary cells

Complementary DNA encoding a facilitative glucose transporter was isolated from a human hepatoma cell line (HepG2) cDNA library and subcloned into a metal-inducible mammalian expression vector, pLEN (California Biotechnology) containing human metallothionein gene II promoter sequences. Chinese hamster ovary (CHO) cells transfected with this transporter expression vector, pLENGT, exhibited a 2-17-fold increase in immunoreactive HepG2-type glucose transporter protein, as measured by protein immunoblotting with antipeptide antibodies directed against the HepG2-type glucose transporter C-terminal domain. Expression of the human glucose transporter was verified by protein immunoblotting with a mouse polyclonal antiserum that recognizes the human but not the rodent HepG2-type transporter. 2-Deoxy-D-glucose uptake was increased 2-7-fold in transfected cell lines. Polyclonal antisera directed against purified red blood cell glucose transporter were raised in several rabbits. Antiserum from one rabbit, delta, was found to bind to the surface of intact red cells but not to inside-out red cell ghosts. Using this delta-antiserum in intact cell-binding assays, 1.6-9-fold increases in cell surface expression of the human glucose transporter were measured in CHO-K1 cell lines transfected with the transporter expression vector. Measurements of total cellular glucose transporter immunoreactive protein using anti-HepG2 transporter C-terminal peptide serum, cell surface glucose transporter protein using delta-antiserum and 2-deoxyglucose uptake revealed proportional relationships among these parameters in transfected cell lines expressing different levels of transporter protein. Insulin increased 2-deoxyglucose uptake 40% in control CHO-K1 cells and in CHO-K1 cells expressing modest levels of the human glucose transporter protein. However, stimulation of sugar-uptake by insulin was only 10% in cells overexpressing human glucose transporter protein 9-fold, and no effect of insulin on sugar uptake was detected in several cell lines expressing very high levels (12-17-fold over controls) of human HepG2 glucose transporter protein. No insulin stimulation of anti-cell surface glucose transporter antibody binding was detected in any control or transfected CHO-K1 cell lines. These data indicate that a glucose transporter protein that is insensitive to insulin in HepG2 cells is regulated by insulin when expressed at low but not at high levels in insulin-response CHO-K1 cells. Additionally, the results suggest that insulin does not increase 2-deoxyglucose uptake by increasing the number of cell surface HepG2-type glucose transporters in CHO-K1 fibroblasts.     

Sequence and expression of rat ICAM-1.

We have isolated cDNA clones-coding for rat intercellular adhesion molecule-1 (RICAM-1) from a cDNA library constructed from rat Ax cells stimulated with IL-1 beta using the mouse ICAM-1 cDNA as a hybridization probe. The RICAM-1 sequence shows 79.1% homology with mouse ICAM-1 and 55.6% homology with human ICAM-1 at the nucleic acid level. In order to examine the expression of RICAM-1 on Chinese hamster ovary (CHO) cells, we constructed the vector, pSV-RICAM1-neo, containing the SV40 promoter. Flowcytometric analysis showed that CHO-K1 cells transfected with pSV-RICAM1-neo expressed high amounts of RICAM-1 on their surfaces.     

Effect of surfactant protein A on granular pneumocyte surfactant secretion in vitro

Surfactant secretion by lung type II cells occurs when lamellar bodies (LBs) fuse with the plasma membrane and surfactant is released into the alveolar lumen. Surfactant protein A (SP-A) blocks secretagogue-stimulated phospholipid (PL) release, even in the presence of surfactant-like lipid. The mechanism of action is not clear. We have shown previously that an antibody to LB membranes (MAb 3C9) can be used to measure LB membrane trafficking. Although the ATP-stimulated secretion of PL was blocked by SP-A, the cell association of iodinated MAb 3C9 was not altered, indicating no effect on LB movement. FM1-43 is a hydrophobic dye used to monitor the formation of fusion pores. After secretagogue exposure, the threefold enhancement of the number of FM1-43 fluorescent LBs (per 100 cells) was not altered by the presence of SP-A. Finally, there was no evidence of a large PL pool retained on the cell surface through interaction with SP-A. Thus SP-A exposure does not affect these stages in the surfactant secretory pathway of type II cells.