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The differentiated phenotype of the alveolar type II cell is rapidly altered in vitro. To evaluate factors that might influence this process, we isolated and plated rat type II cells in serum-supplemented media to promote adherence and then maintained the cells in a simple nutrient medium in the absence (S− cells) or presence (S+ cells) of serum for 5 to 7 d. The type II S− cells remained metabolically active. Despite protein synthesis that was 50% that of S+ cells, S− cells continued to synthesize a broad spectrum of proteins and to express several features of type II cell differentiation. They synthesized an apical integral membrane glycoprotein, Maclura pomifera agglutinin (MPA)-gp200, and a cytokeratin, No. 19, while S+ cells did not. When supplemented with linoleic acid, S− cells contained lamellar and multivesicular bodies, incorporated cell surface MPA into these structures, and secreted their phosphatidylcholine (PC) in response to mastoparan. Despite the relative synthesis of higher levels of total and saturated PC in S− cells supplemented with linoleic acid, phosphatidylglycerol remained diminished. A surfactant protein (SP-A) was present in S− cells, but synthesis was not detected. These studies demonstrate that serum accelerates the loss of type II cell differentiation in vitro and that the expression of type II cell markers of differentiation is not inherently linked.