Pathway to lamellar bodies for surfactant protein A

Abstract

Alveolar surfactant protein A (SP-A) is endocytosed by type II epithelial cells through clathrin-dependent uptake and targeted to lamellar bodies for resecretion. However, the mechanism for secretion of newly synthesized SP-A, whether regulated exocytosis of lamellar bodies or constitutive secretion, is unresolved. If it is the latter, lamellar body SP-A would represent endocytosed protein. Amantadine, an inhibitor of clathrin-coated vesicle budding, was used to evaluate the role of endocytosis in accumulation of SP-A in lamellar bodies. In isolated rat lungs, amantadine (10 mM) inhibited uptake of endotracheally instilled (35)S-labeled biosynthesized surfactant proteins by >80%. To study trafficking of newly synthesized SP-A, lungs were perfused for up to 6 h with [(35)S]methionine, and surfactant was isolated from lung lavage fluid and lamellar bodies were isolated from lung homogenate. With control lungs, the mean specific activity of [(35)S]SP-A (disintegrations per minute per microgram of SP-A) increased linearly with time of perfusion: it was significantly higher in isolated lamellar bodies than in surfactant and was increased in both compartments by 50-60% in the presence of 0.1 mM 8-bromo-cAMP. These results suggest a precursor-product relationship between lamellar body and extracellular [(35)S]SP-A. Specific activities in both compartments were unaffected by addition of amantadine (10 mM) to the lung perfusate, indicating that uptake from the alveolar space was not responsible for the increase in lamellar body [(35)S]SP-A. Thus the pathway for secretion of newly synthesized SP-A is by transfer from the site of synthesis to the storage/secretory organelle prior to lamellar body exocytosis.

Fig. 1.

Representative SDS-polyacrylamide gel with Coomassie blue staining and Western blot analysis using anti-surfactant protein A (SP-A) antibody for lamellar bodies and lung surfactant. Samples were isolated following a 6-h perfusion period under basal conditions without (lane 1) or with (lane 2) addition of 10 mM amantadine to the perfusate. Images were scanned using the Odyssey system, and results are shown as “Quantitation.” MW lane shows molecular mass standards, with size shown in kDa.

Fig. 2.

Immunofluorescent localization of SP-A in lamellar bodies of isolated type II cells. Type II cells were fixed and permeabilized using methods 1, 2, and 3 (see Table 1). Cells were labeled with the primary antibodies to ABCA3 [monoclonal antibody 3C9 (A, E, and I)] or to SP-A [rabbit polyclonal anti-rat SP-A (C, G, and K)] and secondary antibodies FITC-goat anti-mouse antibody (green) or Texas red-goat anti-rabbit antibody (red). Insets (B, F, and J): phase images of matching cell samples. ABCA3/SP-A, merged images of ABCA3 and SP-A (D, H, and L). A–D correspond to method 1, E–H to method 2, and I–L to method 3. Scale bar, 10 μm.

Fig. 3.

SP-A protein in rat isolated type II (TII) cells and lung lamellar bodies (LBs). A: freshly isolated type II cells and lamellar bodies isolated from whole rat lung were run on 10% gels using SDS-PAGE techniques under reducing conditions and transferred to nitrocellulose membranes. Amount of protein loaded per lane is indicated. Membranes were probed with an antibody against SP-A using Western blot procedures. B: lamellar bodies were lysed in hypertonic sucrose by freeze-thawing and subfractionated by centrifugation over a sucrose cushion. Soluble fraction (sol), lipid band, and pellet were run on an SDS-polyacrylamide gel and probed with an antibody against SP-A. Type II whole cell lysate was used as a standard (TII std).

Fig. 4.

Effect of varying concentrations of amantadine in lung perfusate on uptake of ³⁵S-labeled surfactant during 2 h of isolated rat lung perfusion. Uptake was calculated from disintegrations per minute (dpm) present in lung tissue following lavage as a percentage of initial dpm instilled into the lungs. Each point represents a separate isolated perfused lung preparation.

Fig. 5.

Time course for the effect of amantadine on incorporation of [³⁵S]methionine into the SP-A fraction of lamellar bodies (A) and surfactant (B). Lungs were perfused for 1–6 h following the addition of ³⁵S-translabel to the lung perfusate in the absence (control) or presence (+Amantadine) of 10 mM amantadine. Lung perfusate contained 100 μM 8-bromo-cAMP. At the end of perfusion, lung surfactant was isolated from lung lavage fluid and lamellar bodies from postlavage lung homogenate. Fractions were analyzed for SP-A content and dpm for calculation of specific activity. Each point is a separate lung perfusion experiment. Separate regression lines for values obtained in the absence (solid line) and presence of amantadine (dashed line) are drawn by the least-mean-squares method.

Fig. 6.

Effect of amantadine (10 mM) on incorporation of [³⁵S]SP-A into lamellar bodies or surfactant. Lungs were perfused for 6 h in the absence (A) or presence (B) of a secretagogue, 8-bromo-AMP (100 μM), and in the absence (control) or presence of amantadine (10 mM). SP-A specific activity was calculated as described in Fig. 5 legend. Values are means ± SE (n = 6 for basal and n = 3 for cAMP). For cAMP values, results include 6-h perfusion experiments shown in Fig. 5. *P < 0.05 vs. corresponding value for surfactant.