Characterization of [3H]dopamine uptake sites and [3H]cocaine recognition sites in primary cultures of mesencephalic neurons during in vitro development.
[3H]Dopamine uptake and [3H]cocaine binding sites were studied in primary cultures of ventral mesencephalon from 14-day-old rat embryos. Specific binding sites for [3H]cocaine and [3H]mazindol were detected only in intact cell cultures of ventral mesencephalon, and were absent in sonicated, washed membranes prepared from these cell cultures. [3H]Cocaine was not taken up by the cells through an active transport process because [3H]cocaine binding occurred also at 4 degrees C. Moreover, the possibility of [3H]cocaine entering the cells by passive diffusion and ion trapping was also excluded because extensive washing failed to remove [3H]cocaine from the cells. [3H]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, [3H]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 [3H]dopamine. When cells were permeabilized with streptolysin O (0.2 U/ml; 5 min) after [3H]dopamine was taken up, [3H]dopamine was retained by the cells and did not leak into the incubation medium, indicating that [3H]dopamine was stored in sites that could not pass through the perforated membranes. In contrast, [3H]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, [3H]cocaine binding in intact mesencephalic cell cultures was Na+ independent.(ABSTRACT TRUNCATED AT 250 WORDS)
Grilli
M
M
Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
Wright
A G
AG
Hanbauer
I
I
D000818:Animals; D001426:Bacterial Proteins; D001665:Binding Sites; D002470:Cell Survival; D002478:Cells, Cultured; D003042:Cocaine / Q000378:metabolism*; D004298:Dopamine / Q000493:pharmacokinetics*; D004622:Embryo, Mammalian / Q000378:metabolism; D004625:Embryo, Nonmammalian; D008636:Mesencephalon / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D013301:Streptolysins / Q000494:pharmacology; D014316:Tritium
D001426:Bacterial Proteins; D013301:Streptolysins; C025964:streptolysin O; D014316:Tritium; D003042:Cocaine; D004298:Dopamine