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 0 degrees 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 degrees C or 0.154 M NaCl or 0.10 M sodium phosphate (pH 7.4) at 24 degrees C. Incubation of isolated lamellar body fractions in medium containing 5 mM Ca++ or Mg++ at 37 degrees 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.
Lamellar bodies, an intracellular source of lung alveolar surfactant, were isolated from rat lung homogenates and studied in the Langmuir-Adam surface balance. By layering intact lamellar bodies on the surface of a more dense sucrose subphase, we studied the factors affecting film formation from surface tension-vs-time data and determined surface tensionsurface area isotherms by compression and expansion of the resulting films. We found that films with properties representative of the alveolar surfactant are formed in the presence of Ca2+ or Mg2+ alone, or either plus Na+; that film formation is incomplete with Na+ alone or on ion-free subphases; and that Ca2+-induced film formation is blocked by chelation with EGTA but is unaffected by diisopropylfluorophosphate. The results suggest that divalent cations induce film formation by interactions at sites within the lamellar bodies and may be responsible for the binding of membrane lipids to membrane proteins in lung surfactant.Despite morphological (1, 2) and cytochemical (3,4) evidence suggesting that lung alveolar surfactant is derived from the lamellar bodies of the type II epithelial cell, it has not been shown that intact lamellar bodies are capable of forming films with the extraordinary surface properties attributed to surfactant. For example, bubbles (5) or films (6) from lung extracts exhibit surface tensions approaching 0 mN/m and large surface tension-surface area hysteresis. And recent results from wettability experiments performed directly on the alveolar surface indicate that surface tensions as low as 9 mN/m may be reached in situ (7). Low surface tension in the lung is thought to facilitate opening of small airways and alveoli on inflation (8) and to stabilize lung units during deflation (9), and surfactant abnormalities have been identified in both the infant (10) and the adult (11) respiratory distress syndrome.Because surface-active lipids float on physiological saline and intact lamellar bodies (d = 1.06-1.07 g/ml) do not, previous observations of surface activity on saline surfaces have been limited to the lamellar body contents obtained by ethanol extraction (12, 13), sonication (13), or dispersion in salt solutions (12-16). Because these procedures resulted in the loss of integrity of the lamellar bodies, we layered intact lamellar bodies on a more dense sucrose subphase and monitored surface activity directly as a function of subphase composition.In this report, we present evidence that suggests that lamellar bodies require divalent cations in order to produce films having surface tension-surface area relationships characteristic of the extracellular surfactant.MATERIALS AND METHODS Lamellar Bodies. Lamellar bodies were obtained from rat lung by a refined homogenization and sucrose density gradient centrifugation procedure (17). Lamellar
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