The alveolar type II cell performs many important functions within the lung, including regulation of surfactant metabolism, ion transport, and alveolar repair. Because type II cells comprise only 15% of all lung cells, it is difficult to attribute specific functions to type II cells from studies of whole lungs or mixed cell cultures. At the present time, there is no passaged line that exhibits the full range of known type II cell functions. For these reasons, investigators have used isolated type II cells to study alveolar cell biology, biochemistry, and molecular biology. This review addresses many of the issues involved in isolating and culturing type II cells, including the choice of a method of isolating cells, the importance of using specific markers of the differentiated type II cell phenotype, and the problems of maintaining these differentiated phenotypic characteristics in tissue culture.
Deep inflation of the lung stimulates surfactant secretion by unknown mechanisms. The hypothesis that mechanical distension directly stimulates type II cells to secrete surfactant was tested by stretching type II cells cultured on silastic membranes. The intracellular Ca2+ concentration was measured in single cells, before and after stretching. A single stretch of alveolar type II cells caused a transient (less than 60 seconds) increase in cytosolic Ca2+ followed by a sustained (15 to 30 minutes) stimulation of surfactant secretion. Both Ca2+ mobilization and exocytosis exhibited dose-dependence to the magnitude of the stretch-stimulus. Thus, mechanical factors can trigger complex cellular events in nonneuron, nonmuscle cells and may be involved in regulating normal lung functions.
We report here the identification and characterization of a novel gene, T1 alpha, expressed in high abundance in adult rat lung, fetal lung, and early fetal brain. T1 alpha was identified by a monoclonal antibody previously shown to be specific for an antigen expressed by alveolar epithelial type I cells. The cDNA for T1 alpha is 1.85 kb and identifies a single mRNA species of the same size on Northern blots of adult rat lung. The longest open reading frame of the cDNA is 498 bases which would encode a protein of approximately 18 kDa. The protein has a putative membrane spanning domain near the C-terminus but lacks consensus sequences for N-glycosylation. Northern blots and RT-PCR show high expression of T1 alpha in adult lung, with marginally detectable expression in adult brain, intestine, and kidney. RT-PCR analysis shows expression of T1 alpha in freshly isolated type I cells (50-60% purity) but not in highly purified type II cells or other lung cells. We believe therefore that T1 alpha is primarily if not uniquely expressed in alveolar type I cells in the adult rat. Polyclonal antisera against a 16-amino-acid peptide identified in the deduced sequence reacts with the apical membranes of adult type I cells in lung tissue sections but does not label other cell types. The above antiserum as well as the original monoclonal antibody recognize a single approximately 18-kDa protein derived from bacterial expression of a construct containing the T1 alpha open reading frame. By RT-PCR T1 alpha is detected in rat lung from Day 13.5 onward, but is detected by in situ hybridization earlier in lung, brain and neural derivatives, and foregut. Expression is down-regulated in all but lung tissues as development proceeds.
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