Characterization of Na(+)-dependent phosphate uptake in cultured fetal rat cortical neurons.
Our laboratory has recently cloned and expressed a brain- and neuron-specific Na(+)-dependent inorganic phosphate (P(i)) cotransporter that is constitutively expressed in neurons of the rat cerebral cortex, hippocampus, and cerebellum. We have now characterized Na(+)-dependent 32Pi cotransport in cultured fetal rat cortical neurons, where > 90% of saturable P(i) uptake is Na(+)-dependent. Saturable, Na(+)-dependent 32Pi 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) transport was optimal at physiological temperature (37 degrees C) and pH (7.0-7.5), with apparent Km values for P(i) and Na+ cf 54 +/- 12.7 microM and 35 +/- 4.2 mM, respectively. A reduction in extracellular Ca2+ markedly reduced (> 60%) Na(+)-dependent P(i) uptake, with a threshold for maximal P(i) import of 1-2.5 mM CaCl2. Primary cultures of fetal cortical neurons incubated in medium where equimolar concentrations of choline were substituted for Na+ had lower levels of ATP and ADP and higher levels of AMP than did those incubated in the presence of Na+. Furthermore, a substantial fraction of the 32Pi cotransported with Na+ was concentrated in the adenine nucleotides. Inhibitors of oxidative metabolism, such as rotenone, oligomycin, or dinitrophenol, dramatically decreased Na(+)-dependent P(i) import rates. These data establish the presence of a Na(+)-dependent P(i) cotransport system in neurons of the CNS, demonstrate the Ca(2+)-dependent nature of 32Pi uptake, and suggest that the neuronal Na(+)-dependent P(i) cotransporter may import P(i) required for the production of high-energy compounds vital to neuronal metabolism.
Glinn
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Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA.
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Paul
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D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002412:Cations / Q000494:pharmacology; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D005333:Fetus / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D051381:Rats / Q000196:embryology; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters
D000963:Antimetabolites; D002352:Carrier Proteins; D002412:Cations; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D000255:Adenosine Triphosphate; D002118:Calcium