CHARACTERIZATION OF NA-DEPENDENT PHOSPHATE-UPTAKE IN CULTURED FETAL-RAT CORTICAL-NEURONS()

Our laboratory has recently cloned and expressed a brain- acid neuron- specific Na+-dependent inorganic phosphate (P-i) cotransporter that is constitutively expressed in neurons of the rat cerebral cortex, hippo campus, and cerebellum. We have now characterized Na+-dependent P-32(i ) cotransport in cultured fetal rat cortical neurons, where >90% of sa turable P-i uptake is Na+-dependent. Saturable, Na+-dependent P-32(i) uptake was first observed in primary cultures of cortical neurons at 7 days in vitro (DIV) and was maximal at 12 DIV. Na+-dependent P-i tran sport was optimal at physiological temperature (37 degrees C) and pH ( 7.0-7.5), with apparent K-m values for P-i and Na+ of 54 +/- 12.7 mu M and 35 +/- 4.2 mM, respectively. A reduction in extracellular Ca2+ ma rkedly reduced (>60%) Na+-dependent P-i uptake, with a threshold for m aximal P-i import of 1-2.5 mM CaCl2. Primary cultures of fetal cortica l neurons incubated in medium where equimolar concentrations of cholin e were substituted for Na+ had lower levels of ATP and ADP and higher levers of AMP than did those incubated in the presence of Na+. Further more, a substantial fraction of the P-32(i) cotransported with Na+ was concentrated in the adenine nucleotides. inhibitors of oxidative meta bolism, such as rotenone, oligomycin, or dinitrophenol, dramatically d ecreased Na+-dependent P-i import rates. These data establish the pres ence of a Na+-dependent P-i cotransport system in neurons of the CNS, demonstrate the Ca2+-dependent nature of P-32(i) uptake, and suggest t hat the neuronal Na+-dependent P-i cotransporter may import P-i requir ed for the production of high-energy compounds vital to neuronal metab olism.