INCREASED SURFACTANT INTERNALIZATION BY RAT TYPE-II CELLS CULTURED ON MICROPOROUS MEMBRANES

We evaluated the influence of various substrata in maintaining internalization of biosynthesized natural surfactant by type II pneumocytes in primary culture. Cells were incubated for 2 h with H-3, S-35-labeled surfactant, prepared by perfusing isolated rat lungs with [H-3]choline + [S-35]methionine, and analyzed for trypsin-resistant radiolabel. At 24 h of culture, uptake of [H-3]phosphatidylcholine (PC) was 71.9 +/- 6.1 mug/mg protein and S-35-protein was 12.4 +/- 2.4 mug/mg cell protein for cells cultured on Transwell membranes. Uptake with this substratum was significantly greater than for cells cultured on plastic, Primaria, or Transwell-COL and was maintained for 72 h of culture. [H-3]PC/S-35-protein (mug/mug) for cells on Transwell was 5.8 +/- 0.7 compared with 3.4 +/- 0.1 in isolated surfactant. Results were similar at 72 h of culture. 8-Bromoadenosine 3',5'-cyclic monophosphate or terbutaline significantly stimulated the uptake of both surfactant components for cells on Transwell, but not by cells cultured on plastic. By light and electron microscopy, cells cultured on Transwell maintained characteristics of granular pneumocytes, including cuboidal shape, lamellar bodies, and microvilli. We conclude that the use of Transwell microporous membrane for primary culture of rat type II cells maintains their differentiated characteristics as assessed by the uptake of lung surfactant and its response to beta-adrenergic agonists.