Expression and functional characterization of the extraneuronal monoamine transporter in normal human astrocytes.

Abstract

In this study we examined the functional expression of the extraneuronal monoamine transporter (EMT) in normal human astrocytes (NHA). RT-PCR with EMT-specific primers demonstrated the presence of EMT mRNA in NHA. The RT-PCR products were subjected to restriction-site analysis using three different enzymes (HinfI, SacI and BclI). The restriction patterns with the three enzymes were identical and were exactly as expected from the known restriction map of human EMT cDNA. DNA sequencing was performed for the RT-PCR products from NHA. Sequence analysis demonstrated that the sequences of RT-PCR products were identical to that of EMT. The extract of NHA was immunoblotted with anti-EMT polyclonal antibody raised against EMT polypeptides. Western blotting indicated that anti-EMT polyclonal antibody recognized a band of 63 kDa. Immunocytochemical staining using anti-EMT polyclonal antibody in NHA revealed that the plasma membrane, as well as intracellular, perinuclear compartments, presumably endoplasmic reticulum or Golgi membranes, showed a considerable level of immunoreactivity. We examined the time course of temperature-dependent [3H]MPP+ uptake in NHA for 60 min. Temperature-dependent [3H]MPP+ uptake increased in a time-dependent manner for the initial 45 min and almost reached a plateau level (8.70 +/- 0.59 pmol/mg protein) at 60 min. In the presence of 3 micro m decynium22 (D22) (the most potent EMT inhibitor), temperature-dependent [3H]MPP+ uptake was strongly reduced by 61% (3.39 +/- 0.76 pmol/mg protein at 60 min). D22-sensitive [3H]MPP+ uptake was saturable over a MPP+ concentration of 6.25-200 micro m. Km for this process was 78.01 +/- 7.64 micro m and Vmax was 295.4 +/- 12.8 pmol/mg protein/min. D22-sensitive [3H]MPP+ uptake was reduced when the astrocyte membrane potential was depolarized by increasing the concentration of K+ in the uptake buffer or by adding Ba2+ to the uptake buffer. These results provide evidence that the MPP+ transport activity in NHA is potential-sensitive. Moreover, D22-sensitive [3H]MPP+ uptake was independent of extracellular Na+. D22-sensitive [3H]MPP+ uptake was inhibited by D22, various organic cations, steroids and monoamine neurotransmitters. Our results showed that the EMT is functionally expressed in NHA and may also play a key role in the disposition of cationic drugs, neurosteroids, the neurotoxin MPP+ and monoamine neurotransmitters in the brain.