Lamellar body membrane turnover is stimulated by secretagogues

Abstract

Lamellar bodies are specialized cellular organelles used for storage of surfactant by alveolar type II cells of the lung. We utilized monoclonal antibody (MAb) 3C9, which recognizes an integral lamellar body-limiting membrane protein of 180 kDa, to follow lamellar body trafficking. (125)I-labeled MAb 3C9 bound to the surface of type II cells and was internalized by the cells in a time- and concentration-dependent manner that was inhibitable by excess unlabeled antibody. The internalized antibody remained undegraded over a 4-h time period. The L2 rat lung cell line that does not have lamellar bodies did not bind iodinated 3C9. Exposure of type II cells to the secretagogues ATP, phorbol 12-myristate 13-acetate, and cAMP resulted in a 1.5- to 2-fold enhancement of binding and uptake of MAb 3C9. Calphostin C inhibited phorbol 12-myristate 13-acetate-stimulated phospholipid secretion and also reduced binding and uptake of MAb 3C9 by type II cells. Treatment of type II cells with phenylarsine oxide to obstruct clathrin-mediated endocytosis had no effect on the internalization of MAb 3C9 while markedly blocking the uptake of surfactant protein A and transferrin. An actin-mediated process was important for lamellar body membrane uptake because incubation with cytochalasin D partially inhibited MAb 3C9 incorporation by type II cells. These studies are compatible with enhanced lamellar body membrane turnover associated with surfactant secretion and indicate that this process can be monitored by the trafficking of the antigen reporter MAb 3C9.

Pathways for clearance of surfactant protein A from the lung

“…After 1 h of incubation with 125 I-SP-A, PAO decreased SP-A uptake in type II cells to ϳ50% of the control value (Fig. 2), confirming previous results (8). Amantadine and CytoD were as effective as PAO in inhibiting SP-A uptake by type II cells in culture (Fig.…”

“…SP-A has been morphologically localized in clathrincoated pits in type II cells by microscopic studies (39,42). We reported previously the involvement of clathrin-coated pits in SP-A internalization by type II cells in culture (8). In the present study, we have extended our observations about clathrin-mediated SP-A uptake by using two functionally different clathrin inhibitors, amantadine and PAO.…”

Measurement of stretch-induced loss of alveolar epithelial barrier integrity with a novel in vitro method

“…One untreated, unstretched well and one untreated well stretched at 37% ⌬SA from each isolation served as untreated controls. Treatment of alveolar epithelial cells with PAO using a similar concentration and duration has been shown to decrease endocytotic activity without adversely affecting cellular viability (2). In a limited manner, cells were also treated with a higher concentration of PAO (10 M), but these results were no different than those at 5 M (data not shown).…”

Surfactant protein-A plays an important role in lung surfactant clearance: evidence using the surfactant protein-A gene-targeted mouse

“…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).…”

“…Although actin plays a role in clathrin-dependent pathways, some actin-mediated pathways such as phagocytosis, macropinocytosis, and bulk membrane retrieval are clathrin independent. In type II cells, we determined that the turnover of specialized membrane patches containing the lamellar body membrane ABCA3 was sensitive to the actin inhibitor cytochalasin D but not affected by clathrin inhibitors (4).…”