Transport of choline by plasma membrane vesicles from lung-derived epithelial cells

A549 cells, a lung epithelium-derived cell line, were used as a model system to study choline transport by granular pneumocytes. Intact cells accumulated free choline against a concentration gradient by a low-affinity transport system with kinetic characteristics similar to that previously described for granular pneumocytes (Am. J. Respir. Cell Mol. Biol. 1: 455, 1989). Membrane vesicles prepared from these cells showed a 10-fold enrichment in plasma membrane marker enzymes with a vesicular H2O space of 5.7 +/- 0.05 (SE) microliters/mg protein. Vesicles showed a time- and concentration-dependent uptake of free [3H]choline in Na(+)-free medium. With 5 microM choline, choline uptake reached an apparent steady-state concentration gradient (inside/outside) of 50. 3H that was membrane associated ("bound" choline) represented approximately 5% of total uptake. In the presence of an initial gradient of NaCl, choline uptake showed an overshoot with a plateau value similar to Na(+)-free conditions; a similar effect was observed for plasma membrane vesicles from rat lung type 2 epithelial cells. The steady-state uptake of choline was inhibited at low pH (6.5) and by the presence of valinomycin or carbonyl cyanide p-tri-fluoromethoxyphenylhydrazone and was abolished when both were present. These results show that plasma membrane vesicles from A549 cells accumulate choline by binding to the membranes and by Na(+)-dependent and -independent transport mechanisms, the latter apparently reflecting a transmembrane proton gradient.