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

Abstract

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 β-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 β:γ-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.