DOWN-REGULATION OF THE HUMAN NOREPINEPHRINE TRANSPORTER IN INTACT 293-HNET CELLS EXPOSED TO DESIPRAMINE

The effects of continuous exposure of cultured cells expressing the hu man norepinephrine transporter (hNET) to the hNET inhibitor desipramin e on hNET expression and function were studied. Exposure of HEK-293 ce lls transfected stably with the hNET cDNA (293-hNET cells) to desipram ine for 3 days reduced the specific binding of [H-3]nisoxetine in memb rane homogenates in a concentration-dependent manner. The magnitude of the reductions in [H-3]nisoxetine binding to hNET was dependent on th e length of time of the exposure to desipramine, reaching 77% after a 21-day exposure, The reduction of [H-3]nisoxetine binding returned to control levels within 72 h after a 3-day exposure to desipramine. Redu ctions in [H-3]nisoxetine binding to hNET were accompanied by time-dep endent and exposure concentration-dependent reductions in hNET protein levels as determined by western blotting. Similar to binding, hNET pr otein levels returned to control levels 72 h after cessation of desipr amine exposure. Northern blotting indicated that exposure of 293-hNET cells to desipramine did not significantly alter hNET mRNA levels. Upt ake of [H-3]norepinephrine by 293-hNET cells was markedly reduced afte r a 3-day exposure to desipramine. However, desipramine exposure had n o effect on uptake of [H-3]glutamate or [H-3]alanine. The present find ings imply that down-regulation of the hNET in 293-hNET cells induced by desipramine results from a selective reduction in hNET protein leve ls, presumably a consequence of either a reduction in the translation of hNET mRNA or from an enhanced degradation of hNET protein.