Aspartate Transport In Glial Cells

Studies described in the previous section have demonstrated unequivocally that cloned NaC3s are capable of mediating the transport of N-acetyl-L-aspartate in a Na+-coupled manner. The functional characteristics of NaC3-mediated N-acetyl-L-aspartate transport show marked similarities to those of N-acetyl-L-aspartate transport in astroglial cells. However, to implicate NaC3 definitively as the transporter responsible of N-acetyl-L-aspartate transport in astroglial cells, the following questions need to be addressed. Is NaC3 expressed functionally in glial cells? Is there a direct interaction between N-acetyl-L-aspartate and the known substrates of NaC3 for uptake in glial cells? What is the role, if any, of NaC2, also a Na+-coupled transporter for dicarboxylates and tricarboxylates, in N-acetyl-L-aspartate transport in glial cells?

We have sought to answer these questions in a systematic manner using primary cultures of astrocytes from rat brain.18 These studies have shown that astrocytes in culture take up succinate by a Na+-dependent process. The uptake process is saturable with a Michaelis constant of ~35 ^M. Succinate uptake in these cells is inhibitable by N-acetyl-L-aspartate with an inhibition constant of ~140 ^M. Citrate is also capable of inhibiting succinate uptake but with an inhibition constant of ~900 ^M. The relative affinities of succinate and citrate for the uptake process suggest that NaC3 is likely to be responsible for the process. Reverse transcriptase-polymerase chain reaction (RT-PCR) has confirmed that these cells do indeed express NaC3 mRNA. We then used these cells for characterizing the uptake of N-acetyl-L-aspartate. The cells are able to take up radiolabeled N-acetyl-L-aspartate avidly. The uptake process is saturable with a Michaelis constant of —110 ^M. Succinate is able to inhibit the uptake in a strictly competitive manner. The dependence of N-acetyl-L-aspartate uptake on Na+ shows a sigmoidal relatiolship with a Hill coefficient of 3. Thus, the Na+ : N-acetyl-L-aspartate stoichiometry is 3 : 1. Furthermore, the uptake is inhibitable by a-ketoglutarate and fumarate. NaC2 is not expressed in these cells as assessed by RT-PCR and by the lack of high-affinity citrate uptake. These studies provide direct answers to the questions raised above and demonstrate unequivocally that NaC3 is indeed responsible for N-acetyl-L-aspartate uptake in astrocytes.

0 0

Post a comment