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Characterization of [ 3 H]Dopamine Uptake Sites and [ 3 H]Cocaine Recognition Sites in Primary Cultures of Mesencephalic Neurons During In Vitro Development
Author(s) -
Grilli Mariagrazia,
Wright A. Gilbert,
Hanbauer Ingeborg
Publication year - 1991
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1991.tb03473.x
Subject(s) - dopamine , dopamine transporter , mazindol , biology , dopamine plasma membrane transport proteins , microbiology and biotechnology , cytosol , streptolysin , chemistry , biochemistry , biophysics , dopaminergic , endocrinology , enzyme , bacterial protein , gene
[ 3 H]Dopamine uptake and [ 3 H]cocaine binding sites were studied in primary cultures of ventral mesencephalon from 14‐day‐old rat embryos. Specific binding sites for [ 3 H]cocaine and [ 3 H]mazindol were detected only in intact cell cultures of ventral mesencephalon, and were absent in sonicated, washed membranes prepared from these cell cultures. [ 3 H]Cocaine was not taken up by the cells through an active transport process because [ 3 H]cocaine binding occurred also at 4°C. Moreover, the possibility of [ 3 H]cocaine entering the cells by passive diffusion and ion trapping was also excluded because extensive washing failed to remove [ 3 H]cocaine from the cells. [ 3 H]Cocaine binding was reduced to 6% of control when cells were permeabilized with streptolysin O (0.2 U/ml, 5 min). Taken together, these results suggest that in cultured mesencephalic neurons, [ 3 H]cocaine may enter the cell by passive diffusion and then be sequestered by a cytosolic compartment that is lost in the process of permeabilization or sonication and washing of membrane preparations. Permeabilization of cultured neurons failed to alter the storage of [ 3 H]dopamine. When cells were permeabilized with streptolysin O (0.2 U/ml; 5 min) after [ 3 H]dopamine was taken up, [ 3 H]dopamine was retained by the cells and did not leak into the incubation medium, indicating that [ 3 H]dopamine was stored in sites that could not pass through the perforated membranes. In contrast, [ 3 H]dopamine uptake into already permeabilized cells was reduced by 33%, suggesting that a cytosolic protein that had leaked out may play a functional role in the uptake process. In contrast to striatal membrane preparations of adult rats, [ 3 H]cocaine binding in intact mesencephalic cell cultures was Na + independent. The expression of [ 3 H]dopamine uptake and [ 3 H]cocaine binding sites appeared to be developmentally linked to neuritic outgrowth, supporting the view that cocaine binding sites may be closely associated with the dopamine transporter.