Drug interactions with the dopamine transporter in cryopreserved human caudate

Although several model systems have been developed to characterize the func tion of the dopamine transporter (DAT), there is a relative lack of data re garding dopamine (DA) uptake by human caudate, as contrasted to binding stu dies. Cryopreserved human brain tissue can be used for functional as well a s radioligand binding studies of neuronal proteins. The drug-induced inhibi tion of [I-125]RTI-55 binding to, and [H-3] DA uptake by, cryopreserved hum an caudate preparations has now been characterized. Using human caudate mem branes, a single site for [I-125] RTI-55 binding was observed in associatio n and saturation experiments. The relative potencies of 22 drugs for inhibi tion of [I-125] RTI-55 binding to membranes prepared from cryopreserved hum an caudate, fresh rat striatum, and HEK293 cells expressing the recombinant human DAT (HEK-hDAT) were highly correlated. The affinity of DA for the DA T, as measured by [H-3] DA uptake experiments, was higher in both the cryop reserved human caudate and freshly prepared rat striatum than in HEK-hDAT c ells. Although affinities were similar in rat and human brain tissue prepar ations, the maximal uptake rate in the cryopreserved human caudate was appr oximately 1 to 4% and 7% of the rate in fresh and cryopreserved rat striata l preparations, respectively. The relative potencies of 22 drugs for inhibi tion of [H-3] DA uptake were similar for tissue prepared from cryopreserved human caudate, nonfrozen rat striatum, and intact HEK-hDAT cells. These da ta suggest that cryopreserved human caudate can be used to characterize dru g interactions with the DAT, and that HEK-hDAT cells provide a comparable s ystem for modeling the initial interaction of drugs with native hDAT.