TEMPORAL SEGREGATION OF SURFACTANT SECRETION AND LAMELLAR BODY BIOGENESIS IN PRIMARY CULTURES OF RAT ALVEOLAR TYPE-II CELLS

Pulmonary alveolar type II cells synthesize and secrete the phospholipids of surfactant. However, type II cells isolated from adult rat lungs rapidly lose their characteristic morphology and differentiated functions (such as surfactant-specific phospholipid and protein biosynthesis) when maintained on tissue culture plastic. In this study, phospholipid secretion and its regulation by type II cells grown on tissue culture plastic were examined up to 8 days after isolation. Type II cells were preincubated with [H-3]choline for varying 24-h periods during culture prior to examining phosphatidylcholine ([H-3]PtdCho) secretion. Type II cells cultured for 4 days and incubated with [H-3]choline 24 h before the secretion experiment failed to show significant basal and tetradecanoyl phorbol acetate (TPA, 100 nM)-stimulated [H-3]PtdCho secretion (basal, 0.29 +/- 0.01%; TPA, 0.48 +/- 0.04%). In contrast, type II cells incubated with [H-3]choline for the first 24 h during culture and then cultured for 3 more days showed significant [H-3]PtdCho secretion (basal, 1.27 +/- 0.19%; TPA, 6.24 +/- 0.82%). Subcellular fractionation of type II cells revealed that [H-3]choline was incorporated into phosphatidylcholines in a lamellar body-enriched fraction during the first 24 h of culture but that the assimilation of phosphatidylcholine into the lamellar body fraction progressively declined with increasing time in culture. Radiolabel incorporated into the lamellar body fraction labeled during the first 24 h of culture was detectable for up to 8 days in culture. The [H-3]PtdCho incorporated into the lamellar body during the first 24 h of culture was lost gradually over 8 days, suggesting the continuous secretion or turnover of the lamellar bodies during culture. Cells incubated with [H-3]choline for the first 24 h of culture and cultured for 4 days were examined for their response to different secretagogues. TPA (100 nM) and calcium ionophore (ionomycin, 400 nM) stimulated [H-3]PtdCho secretion from these type II cells but 10-mu-M adenosine triphosphate, 100-mu-M terbutaline, and 2 mM 8-bromo cyclic adenosine monophosphate failed to induce significant [H-3]PtdCho secretion. Surfactant protein A (1-mu-g/ml) completely inhibited the TPA-induced [H-3]PtdCho secretion from type II cells cultured for 4 days. From these results, we conclude that: (1) type II cells retain the capacity to secrete significant levels of surfactant for at least 4 days in culture, (2) the lamellar body pool labeled in freshly isolated cells remains competent for secretion for several days and turns over slowly, (3) the accumulation of newly synthesized phospholipids in the lamellar body declines with increasing time in culture, (4) there is selective loss of purinergic and beta-adrenergic dependent secretion with increasing time in culture, and (5) the antagonistic action of surfactant protein A upon stimulated secretion is retained for several days in culture.