title: Drug interactions with the dopamine transporter in cryopreserved human caudate.
abstract: Although several model systems have been developed to characterize the function of the dopamine transporter (DAT), there is a relative lack of data regarding dopamine (DA) uptake by human caudate, as contrasted to binding studies. Cryopreserved human brain tissue can be used for functional as well as radioligand binding studies of neuronal proteins. The drug-induced inhibition of [125I]RTI-55 binding to, and [3H]DA uptake by, cryopreserved human caudate preparations has now been characterized. Using human caudate membranes, a single site for [125I]RTI-55 binding was observed in association and saturation experiments. The relative potencies of 22 drugs for inhibition of [125I]RTI-55 binding to membranes prepared from cryopreserved human caudate, fresh rat striatum, and HEK293 cells expressing the recombinant human DAT (HEK-hDAT) were highly correlated. The affinity of DA for the DAT, as measured by [3H]DA uptake experiments, was higher in both the cryopreserved human caudate and freshly prepared rat striatum than in HEK-hDAT cells. Although affinities were similar in rat and human brain tissue preparations, the maximal uptake rate in the cryopreserved human caudate was approximately 1 to 4% and 7% of the rate in fresh and cryopreserved rat striatal preparations, respectively. The relative potencies of 22 drugs for inhibition of [3H]DA uptake were similar for tissue prepared from cryopreserved human caudate, nonfrozen rat striatum, and intact HEK-hDAT cells. These data suggest that cryopreserved human caudate can be used to characterize drug interactions with the DAT, and that HEK-hDAT cells provide a comparable system for modeling the initial interaction of drugs with native hDAT.
authors.affiliation: Research Service, Veterans Affairs Medical Center, Portland, Oregon, 97201, USA. eshleman@ohsu.edu
authors.forename: A J
authors.initials: AJ
authors.lastname: Eshleman
authors.forename: K
authors.initials: K
authors.lastname: Wolfrum
authors.forename: D C
authors.initials: DC
authors.lastname: Mash
authors.forename: K
authors.initials: K
authors.lastname: Christensen
authors.forename: A
authors.initials: A
authors.lastname: Janowsky
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000187:drug effects* / Q000378:metabolism*; D002421:Caudate Nucleus / Q000187:drug effects* / Q000378:metabolism*; D003042:Cocaine / Q000031:analogs & derivatives / Q000494:pharmacology; D015925:Cryopreservation; D004298:Dopamine / Q000378:metabolism; D018491:Dopamine Agonists / Q000494:pharmacology; D018492:Dopamine Antagonists / Q000494:pharmacology; D050483:Dopamine Plasma Membrane Transport Proteins; D006801:Humans; D007700:Kinetics; D008297:Male; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D009419:Nerve Tissue Proteins; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D018491:Dopamine Agonists; D018492:Dopamine Antagonists; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493214:SLC6A3 protein, human; C493216:Slc6a3 protein, rat; C069723:2beta-carbomethoxy-3beta-(4-iodophenyl)tropane; D003042:Cocaine; D004298:Dopamine