title: Down-regulation of the human norepinephrine transporter in intact 293-hNET cells exposed to desipramine.
abstract: The effects of continuous exposure of cultured cells expressing the human norepinephrine transporter (hNET) to the hNET inhibitor desipramine on hNET expression and function were studied. Exposure of HEK-293 cells transfected stably with the hNET cDNA (293-hNET cells) to desipramine for 3 days reduced the specific binding of [3H]nisoxetine in membrane homogenates in a concentration-dependent manner. The magnitude of the reductions in [3H]nisoxetine binding to hNET was dependent on the length of time of the exposure to desipramine, reaching 77% after a 21-day exposure. The reduction of [3H]nisoxetine binding returned to control levels within 72 h after a 3-day exposure to desipramine. Reductions in [3H]nisoxetine binding to hNET were accompanied by time-dependent and exposure concentration-dependent reductions in hNET protein levels as determined by western blotting. Similar to binding, hNET protein levels returned to control levels 72 h after cessation of desipramine exposure. Northern blotting indicated that exposure of 293-hNET cells to desipramine did not significantly alter hNET mRNA levels. Uptake of [3H]norepinephrine by 293-hNET cells was markedly reduced after a 3-day exposure to desipramine. However, desipramine exposure had no effect on uptake of [3H]glutamate or [3H]alanine. The present findings imply that down-regulation of the hNET in 293-hNET cells induced by desipramine results from a selective reduction in hNET protein levels, presumably a consequence of either a reduction in the translation of hNET mRNA or from an enhanced degradation of hNET protein.
authors.affiliation: Department of Psychiatry and Human Behavior, University of Mississippi Medical Center, Jackson 39216, USA.
authors.forename: M Y
authors.initials: MY
authors.lastname: Zhu
authors.forename: R D
authors.initials: RD
authors.lastname: Blakely
authors.forename: S
authors.initials: S
authors.lastname: Apparsundaram
authors.forename: G A
authors.initials: GA
authors.lastname: Ordway
mesh_terms: D018759:Adrenergic Uptake Inhibitors / Q000494:pharmacology*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003891:Desipramine / Q000494:pharmacology*; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005473:Fluoxetine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D009638:Norepinephrine / Q000493:pharmacokinetics; D050484:Norepinephrine Plasma Membrane Transport Proteins; D012333:RNA, Messenger / Q000378:metabolism; D027981:Symporters
chemical_list: D018759:Adrenergic Uptake Inhibitors; D002352:Carrier Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D012333:RNA, Messenger; C493227:SLC6A2 protein, human; D027981:Symporters; D005473:Fluoxetine; C011386:nisoxetine; D003891:Desipramine; D009638:Norepinephrine