INFLUENCE OF STRAIN ON [H-3] THYMIDINE INCORPORATION, SURFACTANT-RELATED PHOSPHOLIPID-SYNTHESIS, AND CAMP LEVELS IN FETAL TYPE-II ALVEOLAR CELLS

Episodic fetal breathing movements occur in utero; however, their purpose is not known. While some evidence indicates these movements produce distention of the fetal lung, the effects of eliminating the movements on lung development are not clear. Using preparations of the surfactant-producing fetal rabbit lung type II cells, we have examined the hypothesis that distention is involved in maturation of these cells as determined by replication rate and phospholipid synthesis. Differentiating type II cells were isolated from fetal rabbit lungs on gestational day 24. After allowing 24 h for the cells to attach, distention (10%) was applied using a Flexercell apparatus at a rate of 3 s of strain and 57 s of rest for 24 or 48 h. Cells were concurrently incubated with [H-3]thymidine. Autoradiography showed that [H-3]thymidine-labeled nuclei of fetal type II cells and fibroblasts could be identified. Strain significantly increased incorporation of the radioisotope into DNA. In the remaining studies, cells were grown to confluence prior to use. Cell viability after straining (3 or 50 cycles per minute [cpm] for 48 h) was assessed by incubating with diacetyl fluorescein/ethidium bromide and viewing by fluorescence microscopy. Straining did not alter cellular viability. Lactate dehydrogenase was also assayed in the culture medium. Straining did not cause release of lactate dehydrogenase. Ultrastructural examination showed typical cellular appearance in the strained cells. Extracellular lamellar body material was also observed. The incorporation of [H-3]choline (3 or 50 cpm) into cellular phospholipids was measured under static conditions or under distention. [H-3]choline-labeling of phosphatidylcholine and disaturated phosphatidylcholine was significantly increased by distention at either 3 or 50 cpm. Pre-exposure of the cells to fetal lung fibroblast-conditioned medium, which induces differentiation, reduced the effect of straining on [H-3]choline incorporation. Strain at 50 cpm for 24 h significantly increased the intracellular levels of cAMP in cells incubated under control conditions or after exposure to conditioned medium. In contrast, straining at 3 cpm only altered cAMP levels in those cells previously exposed to the conditioned medium. These results suggest that in vitro distention of fetal type II alveolar cells potentially alters replication rate and, depending on the degree of differentiation, may also affect synthesis of surfactant-related phospholipids.