Isolation of lung lamellar bodies and their conversion to tubular myelin figures in vitro

Abstract

Lamellar bodies of type II alveolar epithelial cells are the intracellular storage sites of lung surfactant, while tubular myelin figures are an extracellular surfactant form found in the alveolar fluid. A refined procedure was used to isolate intact lamellar bodies from rat lung homogenates in a fraction of high purity and yield. The stability of isolated lamellar bodies under various conditions was determined by electron microscopy. Lamellar bodies were completely disrupted after incubation at 37, 24, and O°C for 0.6, 2, and 12 hr, respectively, in 0.33 M sucrose, 0.01 M HEPES (pH 7.4). In addition, they were completely disrupted after incubation for 1 hr in 0.33 M sucrose, 1 mM EGTA at 0°C or 0.154 M NaCl or 0.10 M sodium phosphate (pH 7.4) at 24°C. Incubation of isolated lamellar body fractions in medium containing 5 mM Ca⁺⁺ or Mg⁺⁺ at 37°C for 1 hr resulted in the appearance of tubular myelin figures. A procedure is also presented for the isolation of tubular myelin figures from rat lung lavage fluid.