SODIUM-DEPENDENT INFLUX OF ORTHOPHOSPHATE IN MAMMALIAN NON-MYELINATED NERVE

The rate of uptake of radiophosphate was measured in desheathed vagus nerves of rabbits mounted in an apparatus where the incubating solution flowed along the preparation. Replacement of the Na+ of the Locke by either choline or Tris slowed the rate of uptake to about 10% of its value in Na+; with K+ it was slowed to 20%; and with Li+ to 50%. Measurements of the rate of uptake at different extracellular phosphate concentrations showed that in choline-Locke the influx of phosphate was proportional to the extracellular phosphate concentration, while in Locke the rate of inward flow showed a tendency to saturation with increasing phosphate concentrations. Extracts of nerves after 45 min exposure to labeled solutions showed for different phosphate concentrations a slowing of the labeling of ATP, ADP and creatine-phosphate (CrP) and inorganic phosphate (P1) when Locke was replaced by choline-Locke. A similar slowing was found when, at 0.2 mM phosphate, the labeling of these compounds was measured at different times: application of choline-Locke reduced the rate of incorporation of 32P to about 15% of the rate in Locke. In preparations that were loaded with radiophosphate and then washed with inactive solution, and the effluent fractionated, over 90% of the radioactivity was found in the inorganic phosphate fraction. The efflux of 32P showed an initial exponential phase with a time constant of 20-30 min and a much slower one. The slow efflux had a rate constant of 0.0014 min-1 in 0.2 mM external phosphate. Increasing the external phosphate concentration increased the efflux. Prolonged incubation in choline-Locke reduced the efflux. The influx of phosphate was calculated for different external phosphate concentrations using the rate of uptake of radiophosphate measured 45 min after the application of isotope and the corresponding efflux rate coefficients and the intracellular specific activities of the labeled compounds. A correction was also introduced for diffusion, using the limited biophase model. The Na+-sensitive influx, i.e., the difference between influx in Locke and influx in choline-Locke, showed saturation kinetics. A lineweaver-Burk plot for Na+-sensitive uncorrected fluxes gave a Vmax of 16.7 .mu.mole/kg wet weight min and an apparent Km of 0.42 mM; for the corrected fluxes these values were 18.2 and 0.36. A large part of phosphate influx and some of the phosphate efflux is apparently mediated by a specific Na+-dependent phosphate transport system. This system seems to be present also in other types of nervous tissues and probably in many types of animal cells.