Figure 5

Stability of the sigma-1 receptors in CHO-K1 cells. Endogenous sigma-1 receptors were [¹²⁵I]-IACoc photolabeled in live CHO-K1 cells by a brief photolysis (see Materials and Methods for details). Labeled cells were washed free of excess ligand and plated on culture dishes for various periods of time with or without inhibitors of proteolysis. A. Specificity of I¹²⁵-azidococaine labeling in live cells was tested by preincubation of cells with 5 μM haloperidol (hal), prior to [¹²⁵I]-IACoc labeling (plus conditions). B. Autoradiogram of CHO-K1 cell lysate collected at different time points after plating of photolabeled cells: time 0 – (lane # 1); after 24 h – (lanes # 2,3,4); after 48 h – (lanes # 5,6,7); after 72 h – (lanes # 8,9,10). The proteosomal inhibitor, lactacystine, 2 μM (lanes #3,6,9) was added; the lysosomal inhibitor, chloroquine, 100 nM (lanes # 4,7,10) was added. C. Quantified data from the situ photolabeled sigma-1 receptor time-course from 3 independent experiments are represented in part B. These data indicate that in CHO-K1 cells the turnover of the ligand occupied sigma-1 receptor is slow with a half-life at least 72 hours.