Regulation and Repair of the Alveolar-Capillary Barrier in Acute Lung Injury

Considerable progress has been made in understanding the basic mechanisms that regulate fluid and protein exchange across the endothelial and epithelial barriers of the lung under both normal and pathological conditions. Clinically relevant lung injury occurs most commonly from severe viral and bacterial infections, aspiration syndromes, and severe shock. The mechanisms of lung injury have been identified in both experimental and clinical studies. Recovery from lung injury requires the reestablishment of an intact endothelial barrier and a functional alveolar epithelial barrier capable of secreting surfactant and removing alveolar edema fluid. Repair mechanisms include the participation of endogenous progenitor cells in strategically located niches in the lung. Novel treatment strategies include the possibility of cell-based therapy that may reduce the severity of lung injury and enhance lung repair

Vacuolar ATPase Regulates Surfactant Secretion in Rat Alveolar Type II Cells by Modulating Lamellar Body Calcium

Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase) is the enzyme responsible for pumping H+ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase) dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1), an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca2+ chelator, BAPTA-AM, the protein kinase C (PKC) inhibitor, staurosporine, and the Ca2+/calmodulin-dependent protein kinase II (CaMKII), KN-62. Baf A1 induced Ca2+ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca2+ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca2+ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion

Role of the N-Terminal Seven Residues of Surfactant Protein B (SP-B)

Breathing is enabled by lung surfactant, a mixture of proteins and lipids that forms a surface-active layer and reduces surface tension at the air-water interface in lungs. Surfactant protein B (SP-B) is an essential component of lung surfactant. In this study we probe the mechanism underlying the important functional contributions made by the N-terminal 7 residues of SP-B, a region sometimes called the “insertion sequence”. These studies employed a construct of SP-B, SP-B (1–25,63–78), also called Super Mini-B, which is a 41-residue peptide with internal disulfide bonds comprising the N-terminal 7-residue insertion sequence and the N- and C-terminal helices of SP-B. Circular dichroism, solution NMR, and solid state 2H NMR were used to study the structure of SP-B (1–25,63–78) and its interactions with phospholipid bilayers. Comparison of results for SP-B (8–25,63–78) and SP-B (1–25,63–78) demonstrates that the presence of the 7-residue insertion sequence induces substantial disorder near the centre of the lipid bilayer, but without a major disruption of the overall mechanical orientation of the bilayers. This observation suggests the insertion sequence is unlikely to penetrate deeply into the bilayer. The 7-residue insertion sequence substantially increases the solution NMR linewidths, most likely due to an increase in global dynamics