Functional expression of the norepinephrine transporter in cultured rat astrocytes

Abstract

We assessed the functional expression of the norepinephrine (NE) transporter (NET) in cultured rat cortical astrocytes. Specific [ 3 H]NE uptake increased in a time-dependent manner, and this uptake involves temperature-and Na + -sensitive mechanisms. The Na + -dependent [ 3 H]NE uptake was saturable, and the K m for the process was 539.3 ± 55.4 nM and the V max was 1.41 ± 0.03 pmol/mg protein/min. Ouabain, a Na + -K + ATPase inhibitor, significantly inhibited Na + -dependent [ 3 H]NE uptake. The selective NE uptake inhibitor nisoxetine, the tricyclic antidepressants desipramine and imipramine, and the serotonin and NE reuptake inhibitor (SNRI) milnacipran very potently inhibited Na + -dependent [ 3 H]NE uptake. On the other hand, GBR-12935 (a selective dopamine uptake inhibitor), fluvoxamine (a selective serotonin reuptake inhibitor), venlafaxine (a SNRI) and cocaine had weaker inhibitory activities. RT-PCR demonstrated that astrocytes expressed mRNA for the cloned NET protein, which was characterized as neuronal NET. Western blots indicated that anti-NET polyclonal antibody recognized a major band of 80 kDa in astrocytes. These data indicate that the neuronal NET is functionally expressed in cultured rat astrocytes. Glial cells may exert significant control of noradrenergic activity by inactivating NE that escapes neuronal re-uptake in sites distant from terminals, and are thus cellular targets for antidepressant drugs that inhibit NE uptake.

Corticosterone-Sensitive Monoamine Transport in the Rat Dorsomedial Hypothalamus: Potential Role for Organic Cation Transporter 3 in Stress-Induced Modulation of Monoaminergic Neurotransmission

“…The following primers were used: rSERT (Inazu et al, 2001) [forward, 5Ј-GTA CCA CCG AAA CGG GTG CA-3Ј; reverse, 5Ј-TGG TGG ATC TGC AGG ACA TG-3Ј; nucleotides 401-700; T ann ϭ 60°C; cycling parameters: 2 min at 95°C; 35 times (45 s at 95°C; 45 s at 60°C; 30 s at 72°C); 10 min at 72°C], rDAT (Takeda et al, 2002) [forward, 5Ј-TCC CTG ACA AGC TTC TCC-3Ј; reverse, 5Ј-GCC AGG ACA ATG CCA AGA-3Ј; nucleotides 1077-1381; T ann ϭ 56°C; cycling parameters: 2 min at 95°C, 35 times (1 min at 95°C; 2 min at 56°C; 4 min at 72°C)]; rNET (Inazu et al, 2003b) [forward, 5Ј-CAT CAA CTG TGT TAC CAG TTT TAT T-3Ј; reverse, 5Ј-AAA CAT GGC CAG AAG AAA GGT ACC-3Ј; nucleotides 1044 -1354; T ann ϭ 60°C; cycling parameters: 2 min at 95°C, 35 times (1 min at 95°C; 2 min at 60°C; 4 min at 72°C)], rVMAT1&2 (Hayashi et al, 1999) [VMAT1 forward, 5Ј-AGA CAG CAA CTC TTC TCT GC-3Ј; reverse, 5Ј-CTA TCC CTT GCA AGC AGT TGT-3Ј; nucleotides 426 -640; T ann ϭ 55°C; VMAT2 forward, 5Ј-ACT CTA CGG AAA TCC AGA CC-3Ј; reverse, 5Ј-AAT TCC CTG AAG GGA CCT GG-3Ј; nucleotides 267-685; T ann ϭ 53°C; cycling parameters: 2 min at 94°C; 30 times (30 s at 94°C; 30 s at T ann ; 1 min at 72°C)]. PCR products were separated by electrophoresis in 1.5% agarose gels stained with ethidium bromide.…”

Gliogenesis and Glial Pathology in Depression

“…These transporter systems regulate both the availability and reuptake of neurotransmitters at the synapse [34]. Studies in primary astrocyte cultures and in vivo studies demonstrate that these glia are able to take up glutamate [35][36][37][38], GABA [39][40][41][42], serotonin [43,44], dopamine [45], norepinephrine [45,46] and histamine [47] and they contain the enzymes (i.e., monoamine oxidase (MAO), or catechol-O-methyl transferase (COMT) [48][49][50], required for further regulation of the extracellular concentrations of these neurotransmitters. Moreover, the ability of cultured astrocytes to take up serotonin can be blocked by fluoxetine or paroxetine [43,51], suggesting that glia along with neurons may participate in the therapeutic effects of these compounds in depression.…”

Chronic atomoxetine treatment during adolescence decreases impulsive choice, but not impulsive action, in adult rats and alters markers of synaptic plasticity in the orbitofrontal cortex

“…Data shown are the mean±SEM also be found in glial cells throughout the brain (Inazu et al 2003;Rajkowska and Miguel-Hidalgo 2007;Takeda et al 2002). Many chronic drug manipulations have been found to increase levels of the transcription factor (TF) ΔFosB in both frontal and striatal brain regions, and this TF can regulate numerous genes associated with neuroplastic processes ).…”