Membrane fusion between lamellar bodies and plasma membrane is an obligatory distal step in lung surfactant secretion in alveolar type II (T2) cells. We have previously postulated that annexin A7 (A7) can facilitate such membrane fusion in vitro and can increase surfactant secretion in permeabilized T2 cells. We now report that surfactant secretagogues increase in vivo phosphorylation of A7 in T2 cells that can possibly facilitate stimulated secretion of lung surfactant. Isolated T2 cells were cultured overnight and treated with 80 nM phorbol myristate acetate (PMA) for varying periods. The cells were extracted in lysis buffer and A7 was immunoprecipitated with anti‐annexin A7 antibodies that were raised against recombinant rat A7. Western blot analysis of immunoprecipitated proteins with anti‐ A7, anti‐phosphoserine and anti‐phosphothreonine antibodies showed that PMA‐treatment increased A7 phosphorylation in T2 cells in a time‐dependent manner at both serine and threonine residues. The phosphorylation reached maximum within 5–10 min and was elevated during the study period (30min). Treatment of T2 cells for 10 min with other secretagogues, ATP, terbutaline or calcium ionophore A23187 also showed increased phosphorylation of A7 suggesting that it was related to increased surfactant secretion. In vitro phosphorylation of recombinant A7 with a protein kinase C preparation showed increased membrane fusion activity of annexin A7. These observations suggest that secretagogue activation of protein kinase causes phosphorylation of A7 for increased membrane fusion activity to facilitate increased surfactant secretion.

Annexin A7 (A7) increases lung surfactant secretion in permeabilized alveolar type II cells, possibly by increasing fusion between lamellar bodies (LB) and plasma membrane as shown in vitro. Stimulation of type II cells with ATP increases the phospholipase C-mediated hydrolysis of phosphatidylinositol bisphosphate (PIP2), elevates cell diacylglycerol (DAG), and augments surfactant secretion. For this study, we hypothesized that the presence of DAG or PIP2 would alter protein-membrane interaction and the Ca2+-dependent fusion activity of A7. The Ca2+-dependent increase in A7 fluorescence was higher in the presence of 5% PIP2 containing phospholipid vesicles (PLV) in comparison to that with PLV without PIP2, indicating that PIP2 altered the protein-membrane interaction. Fluorescence quenching studies also indicated change in molecular organization since 5% dansyl phosphatidylethanolamine-PLV quenched the A7 fluorescence in presence of PIP2-PLV or 5% DAG-PLV, but not in presence of PLV without PIP2 or DAG. These results suggest that PIP2 hydrolysis to DAG does not further affect the mode of protein-membrane interaction. Membrane fusion was measured by Ca2+-dependent change in transfer efficiency of resonance energy as reflected in increased fluorescence. Incubation of isolated lung LB with DAG and 10μM Ca2+ caused LB enrichment with DAG. The A7-mediated fusion of DAG-enriched LB was higher in comparison to that of untreated LB. Thus, ATP treatment of type II cells could increase the DAG content of LB and facilitate A7-mediated membrane fusion activity during surfactant secretion. Supported by HL 49959