Abstract
The alveolar type II cell which synthesizes and secretes surfactant also plays a major role in the reuptake of surfactant lipids. In a recent in vivo study we found that the subfractions of natural surfactant that contained the surfactant protein with molecular weights of 26,000-36,000 (SP-26-36) were preferentially taken up into lamellar bodies of type II cells to a greater extent than were fractions that did not contain SP-26-36. Because the subfractions of natural surfactant in that study differed in other properties than the presence or absence of SP-26-36, the current study was undertaken to determine whether purified SP-26-36 enhanced the uptake of surfactant-like lipids by freshly isolated type II cells. SP-26-36 increased the uptake of label in radioactive surfactant-like lipids by up to 10-fold, and the effect of SP-26-36 was dependent on time, protein concentration, and temperature. The enhancement was inhibited by heat-treating the protein, by a polyclonal antibody against SP-26-36, and by metabolic inhibitors. The distribution of radioactivity in cell-associated phospholipids differed if cells were incubated with or without SP-26-36. If SP-26-36 was present during the incubation, greater than 96% of the radioactivity remained associated with phosphatidylcholine. In the absence of SP-26-36, only 85% of the radioactivity remained associated with phosphatidylcholine and 7% of the label appeared in phosphatidylglycerol. We hypothesize that SP-26-36 may act as a ligand to direct surfactant lipids to type II cells, perhaps to different metabolic pathways, and to regulate recycling and surfactant homeostasis.
Highlights
From the $CardiovascularResearch Institute and the Departments of §Pediatrics and TMedicine, Universityof California San Francisco, San Francisco, California94143
In arecent in vivo study we found highly ordered lattice-like structure called tubular myelin, that the subfractions of natural surfactant that con- which in turn maybe able to generate a surface film [2]
Small unilamellar liposomes were prepared in the medium used for the cell incubation as described below by the SP-26-36Analysis
Summary
11 Career Investigator of the American Heart Association. The abbreviations used are: DPPC, dipalmitoylphosphatidylcholine; SP-26-36, surfactant protein of M, 26,000-36,000; OGP, octyl glucopyranoside;SDS, sodium dodecylsulfate; PAGE, polyacrylamide gel electrophoresis; PC, phosphatidylcholine; NBD-PC, l-palmitoyl2-(6-((7-nitro-2-1,3-benzoxadiazol-4-yl)amino)caproylp)hosphatidylcholine; MEM, minimal essential medium; HEPES, 4-(2-hydroxthe method of Dobbs et al [16]. + Isolation of SP-26-36 and SP-5-16-Rat lungs were lavaged eight the centrifuge tubes in the absence of cells was subtracted from the times with a solution containing 154 mM NaCl, 5 mM HEPES, pH cell samples. This correction factor averaged 13 f 2% Insoluble proteins were removed by centrif- Fluorescence Microscopy-Cells that had been incubated with ugation a t 100,000 X g for 60 min (type 40 rotor, Beckman Ultracen- liposomes containing the fluorescent lipid NBD-PC were washed as trifuge). Polar Lipids (Birmingham,AL) and were routinely checked for purity by two-dimensional thin-layer chromatography by the method of
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