Abstract
Since secretagogues have been shown to increase the internalization of surfactant phospholipid and protein by lung cells, we postulated that their action occurred through a mechanism involving increased surfactant protein A (SP-A) receptor density. Therefore, we evaluated the influence of secretagogues on the binding of iodinated SP-A to alveolar type II cells. Type II cells were isolated from rat lung and maintained in primary culture for 18 h on Transwell membranes. Upon exposure to 8-bromo-cyclic AMP (cAMP, 0.1 mM), phorbol 12-myristate 13-acetate (PMA, 10 nM), terbutaline (0.1 mM), or ATP (1 mM), the binding of SP-A increased 1.5-2-fold. This stimulation was cell substrate-dependent since type II cells plated on plastic dishes did not show this effect. A time course of the stimulation of SP-A binding due to secretagogues showed that both cAMP and PMA increased SP-A binding by 2-fold after 20 min. With cAMP, binding remained elevated for 2 h, while binding in the presence of PMA had returned to control values. The effects of submaximal concentrations of cAMP and PMA on binding were additive. Inhibition of cellular protein synthesis with cycloheximide did not alter the increase of SP-A binding stimulated by the secretagogues. Type II cells pretreated with PMA responded to subsequent treatment with cAMP by increasing SP-A binding, while these cells were refractory to subsequent treatment with PMA. Both constitutive and regulated binding of SP-A to type II cells were sensitive to trypsin. The binding of SP-A to type II cells showed saturation at a concentration of 1 microg/ml SP-A under control and secretagogue-stimulated conditions, with both total and calcium-dependent binding showing a 2-fold increase upon secretagogue exposure. The data are consistent with the hypothesis that secretagogues stimulate surfactant uptake, at least in part, through recruitment of SP-A receptors to the type II cell surface, resulting in an increase in the number of SP-A binding sites.
Highlights
Pulmonary surfactant is a complex mixture of phospholipids and proteins secreted by the type II alveolar epithelial cells
Secretagogues have augmented the clearance of lipid from rabbit lungs (Pettenazzo et al, 1989) and the uptake of surfactant protein and dipalmitoylphosphatidylcholine into both isolated, perfused rat lungs (Fisher et al, 1985, 1989, 1991) and type II cells cultured on microporous membranes (Chinoy et al, 1993)
Type II cells plated on plastic dishes bound 5.7 Ϯ 0.7 ng of surfactant protein A (SP-A)/mg of cell protein (n ϭ 8), whereas the binding of SP-A to cells plated on Transwell membranes was 27.7 Ϯ 2.7 ng of SP-A/mg of protein (n ϭ 13), approximately five times higher than that for cells on plastic (p Ͻ 0.01)
Summary
Pulmonary surfactant is a complex mixture of phospholipids and proteins secreted by the type II alveolar epithelial cells. Secretagogues have augmented the clearance of lipid from rabbit lungs (Pettenazzo et al, 1989) and the uptake of surfactant protein and dipalmitoylphosphatidylcholine into both isolated, perfused rat lungs (Fisher et al, 1985, 1989, 1991) and type II cells cultured on microporous membranes (Chinoy et al, 1993). SP-A functions to inhibit surfactant phospholipid secretion by type II cells (Dobbs et al, 1987; Kuroki et al, 1988). Strayer et al (1996) have shown that antibodies to the 30-kDa protein inhibit the binding of SP-A and mimic the functional ability of SP-A to down-regulate phospholipid secretion stimulated by secretagogues in isolated type II cells. Since secretagogues stimulated the uptake of biosynthesized SP-A and phosphatidylcholine (Chinoy et al, 1993), in this report we have examined the possibility that secretagogues induce SP-A receptor activity and have found that secretagogues enhance SP-A binding to type II cells
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