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.