Previous investigations have shown that the binding of a selective hydrophilic positron emission tomography radiotracer for the dopamine transporter (DAT) (2beta-carbomethoxy-3beta-(4-chlorophenyl)-8-(2-18F-fluoroethyl)nortropane) decreased in monkey striatum during deep isoflurane anesthesia. Immunohistochemistry experiments suggested but did not prove that isoflurane induced a decrease in cell surface DAT. The present investigation was undertaken to demonstrate quantitatively the isoflurane-induced internalization of DAT using a rapid and relatively uncomplicated biochemical technique in human embryonic kidney (HEK-293) cells stably expressing the human DAT (h-DAT) protein. Biotinylation followed by Western blot analysis was used to determine the extent of change in cell surface expression of the DAT under control conditions and in the presence of a clinically relevant concentration of isoflurane. Isoflurane treatment for 30 min resulted in a highly significant decrease in the amount of h-DAT on the cell surface (21 +/- 15% of control; P < 0.01) (mean +/- SD; n = 4). These data are consistent with the hypothesis that isoflurane results in internalization of DAT from the cell membrane and further validate our qualitative results reported previously. In addition, the current results confirm the hypothesis that biotinylation can be used to quantitate the extent of disappearance of DAT from the cell surface making dose-response studies and comparisons of DAT internalization with other general anesthetics practical.
Copyright 2004 Wiley-Liss, Inc.