SECRETORY GRANULE CALCIUM LOSS AFTER ISOLATION OF RAT ALVEOLAR TYPE-II CELLS

Morphological change and lamellar body loss suggests that alveolar type II cells rapidly de- or redifferentiate after several days of primary culture. To determine whether type II cells or lamellar body compositional changes precede these obvious morphological changes, we examined the in situ elemental composition of lamellar bodies and type II cells from intact lung and at different times after isolation using electron probe microanalysis (EPMA). Isolated cells were prepared by standard methods and plated on either tissue culture plastic or kept in suspension with stirrer flasks. Cell pellets obtained at 0, 3, 24, and 48 h after isolation were rapidly frozen, and thin freeze-dried cryosections were prepared and examined cold in a transmission electron microscope equipped for EPMA. Eight to ten type II cells from each of three to four different preparations for each time period were analyzed. A rapid, progressive, and sustained fall in lamellar body calcium and sulfur content occurred by 48 h of primary culture, suggesting rapid alteration in calcium and protein metabolism by type II cells and/or lamellar bodies after isolation. Also, marked changes in type II cell cytoplasmic Na and K occurred in freshly isolated cells, with incomplete normalization by 48 h. Culture on laminin-enriched Matrigel for 1 wk increased both lamellar body calcium or sulfur content, but 100 nM dexamethasone had no effect. Lamellar body calcium accumulation appears to be a very sensitive index of differentiated type II cell function.