Transport of choline against a concentration gradient by isolated granular pneumocytes

Uptake of 3H-choline was studied in granular pneumocytes isolated from rat lungs by tryptic digestion, attached to Nucleopore membranes coated with rat-tail collagen, and maintained in primary culture for 24 hours. 3-0-Methyl-[14C]-glucose was used as a tracer to calculate H2O space of cells. To stop reaction, cells were quenched in methanol and extracted to give lipid, free-choline, and phosphorylated choline fractions. With 5 uM external choline, cellular accumulation in all three fractions was linear for 30 min and free choline reached a 9-fold accumulation (inside/outside) ratio. With 0.1 mM external choline, the accumulation ratio reached a constant value of approx. 3 after 2 min incubation. With 1.5 min incubation, cell [choline] reached a plateau at approx. 30 uM external choline with a Km for uptake of approx. 8 uM. Efflux from preloaded cells showed two cellular components with a rate constant for the slow component of 0.031/sec. Uptake was inhibited 52% by hemicholinium-3 (10-4 M), 41% by N-ethylmaleimide (10-5M), 30-50% by mitochondrial inhibitors, 15% by ouabain (0.5 mM) and 85% by incubation at 40. Omitting Na+ had no effect on uptake. These results indicate that granular pneumocytes accumulate choline from the medium against a concentration gradient by an energy-dependent process indicating active transport of this substrate