Isolation and metabolism of granular pneumocytes from rat lungs.

Abstract

A population of rat lung cells enriched in granular pneumonocytes (type II cells) was prepared by modification of the method of Kikkawa (Lab. Invest.30, 76, 1974). Lungs were minced, mechanically agitated and incubated with a fluorochemical emulsion and 1% trypsin. The isolated cells were centrifuged in a discontinuous Ficoll gradient. Granular pneumonocytes were identified by the presence of cytoplasmic inclusions on Papanicolaou and acid phosphatase staining and a strongly positive alkaline phosphatase reaction. The yield at the interface between 1.058 and 1.100 density Ficoll was (4.0 ± 1.5) × 106 (mean ± SD) cells per g of lung tissue consisting of 72.7 ± 9.0% granular pneumonocytes and 20.3 ± 8.4% alveolar macrophages. O2 uptake by the cells at room temperature was 46.9 ± 5.8 nmol/h per 106 cells (mean ± SE; n=5). Respiration was inhibited by oligomycin and subsequently stimulated by an uncoupler of oxidative phosphorylation. During incubation with [U-14C] glucose, the cells produced14CO2, lactate and pyruvate at rates of 17.0 ± 2.9, 17.1 ± 1.6 and 8.7 ± 0.8 nmol/h/106 cells, respectively. These results show that a cell population enriched in granular pneumonocytes can be isolated by lung trypsinization. The cells in this preparation show respiration that is coupled to oxidative phosphorylation and intact glycolytic pathways.