Interaction of isoflurane with the dopamine transporter.
BACKGROUND
Isoflurane administration is known to increase extracellular dopamine (DA) concentration. Because the dopamine transporter (DAT) is a key regulator of DA, it is likely affected by isoflurane. This study investigates the hypothesis that isoflurane inhibits DA reuptake by causing DAT to be trafficked into the cell.


METHODS
Rhesus monkeys were scanned with positron emission tomography (PET) using [18F]FECNT (a highly specific DAT ligand) while anesthetized with 1% isoflurane. The isoflurane was increased to 2%, and the animals were rescanned. Uptake was analyzed with the tissue reference method using the cerebellum as the reference tissue to determine the binding potential in the putamen. Immunohistochemistry and Western blot analyses were performed in rats to determine if isoflurane administration would change the total amount of DAT. Rats breathed air plus 2% isoflurane for 30 min, and then striatal DAT assays were rapidly performed. immunocytochemistry experiments were performed using human embryonic kidney (HEK) cells stably transfected with human DAT. The cells were exposed to 4% isoflurane for 1 h while the location of DAT was observed with fluorescent confocal microscopy.


RESULTS
The [18F]FECNT binding potential in rhesus monkeys decreased by 63 +/- 6% (SEM, n = 5) when isoflurane was increased from 1 to 2% as compared with no significant change (0.7 +/- 2.5%; SEM, n = 5) when the isoflurane concentration was not changed (P < 0.001). No difference in DAT staining between isoflurane-treated and control rats was apparent from visual inspection, and quantitative Western blot analyses showed no significant change in total DAT protein. After isoflurane treatment, focal puncta of intense fluorescence was visible inside the HEK cells.


CONCLUSIONS
The experiments indicate that DAT is trafficked into the cell by isoflurane without changing the total amount of DAT in the striatum. The PET data are consistent with this finding, provided that intracellular DAT acquires a conformation that has low affinity for [18F]FECNT. Thus, [18F]FECNT appears to be an excellent agent for measuring plasma membrane-expressed DAT and evaluating DAT trafficking.
Votaw
John
J
Department of Radiology, Emory University School of Medicine, Atlanta, Georgia, USA. Votaw@commander.eushc.org
Byas-Smith
Michael
M
Hua
Jian
J
Voll
Ronald
R
Martarello
Laurent
L
Levey
Allan I
AI
Bowman
F DuBois
FD
Goodman
Mark
M
D018685:Anesthetics, Inhalation / Q000493:pharmacokinetics / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D050483:Dopamine Plasma Membrane Transport Proteins; D006439:Hemodynamics / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D007530:Isoflurane / Q000493:pharmacokinetics / Q000494:pharmacology*; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D008253:Macaca mulatta; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000187:drug effects* / Q000378:metabolism*; D018613:Microscopy, Confocal; D008954:Models, Biological; D009419:Nerve Tissue Proteins; D009662:Nortropanes / Q000138:chemical synthesis; D019275:Radiopharmaceuticals / Q000138:chemical synthesis; D051381:Rats; D014055:Tomography, Emission-Computed
C410211:2-carbomethoxy-3-(4-chlorophenyl)-8-(2-fluoroethyl)nortropane; D018685:Anesthetics, Inhalation; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D009662:Nortropanes; D019275:Radiopharmaceuticals; C493214:SLC6A3 protein, human; C493216:Slc6a3 protein, rat; D007530:Isoflurane