Pathway to lamellar bodies for surfactant protein A.

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.

Adsorption behavior of a surfactant and a monoclonal antibody to sterilizing-grade filters.

Formulations of therapeutic proteins usually contain a surfactant such as polysorbate 80 to protect them against interfacial stresses. Since surfactants may interact with surfaces, the aim of the present work was to study the adsorption behavior of low concentrations of polysorbate 80 and of a monoclonal antibody during sterile filtration. Lab-scale tests were performed to study the adsorption behavior of a monoclonal antibody to different filter materials (PVDF, PES, CA, and Nylon) from different suppliers. Subsequently, protein and polysorbate 80 adsorption were tested in manufacturing scale experiments. It was found that the extent of protein adsorption differed with filter materials, but also with different suppliers. Prominently, Nylon filters showed the highest degree of protein adsorption. In manufacturing-scale filtration experiments, significant adsorption of polysorbate 80 to sterilizing-grade filters was found. Thus, the adsorption of both protein and polysorbate to filters should be taken into consideration in the formulation and manufacturing process and assessed on a case-by-case basis depending on the manufacturing process set-up.

Role of clathrin- and actin-dependent endocytotic pathways in lung phospholipid uptake.

We evaluated the contribution of endocytotic pathways to pulmonary uptake of surfactant lipids from the alveolar space. Resting and stimulated 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) uptake of unilamellar liposomes labeled with either [(3)H]dipalmitoylphosphatidylcholine ([(3)H]DPPC) or 1-palmitoyl-2-[12-(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] dodecanoyl-phosphatidylcholine (NBD-PC) was studied in isolated perfused rat lungs and isolated type II cells. Amantadine and phenylarsine oxide, inhibitors of clathrin-mediated endocytosis, each decreased [(3)H]DPPC uptake under resting conditions by approximately 40%; their combination had no additional effect. Cytochalasin D, an inhibitor of actin-dependent processes, reduced liposome uptake by 55% and potentiated the effect of either clathrin inhibitor alone. Relative inhibition for all agents was higher in the presence of 8-Br-cAMP. The effect of inhibitors was similar for liposomes labeled with [(3)H]DPPC or NBD-PC. By fluorescence microscopy, NBD-PC taken up by lungs was localized primarily to alveolar type II cells and was localized to lamellar bodies in both lungs and isolated cells. These studies indicate that both clathrin-mediated and actin-mediated pathways are responsible for endocytosis of DPPC-labeled liposomes by alveolar type II cells in the intact lung.