Comparison of [3H]WIN 35,428 binding, a marker for dopamine transporter, in embryonic mesencephalic neuronal cultures with striatal membranes of adult rats.
In contrast to striatal membranes of adult rats, where high- (KD1 = 34 nM) and low- (KD2 = 48,400 nM) affinity binding sites for [3H]WIN 35,428 are present, in primary cultures of ventral mesencephalon neurons (CVMNs) only low-affinity binding sites were found (KD = 336,000 nM). The binding of [3H]WIN 35,428 in CVMNs prepared from rat embryos was reversible, saturable, and located in cytosol. Although dopamine (DA) uptake blockers inhibited [3H]DA uptake at nanomolar concentrations in CVMNs, the displacement of [3H]WIN 35,428 binding in CVMNs by DA uptake inhibitors required 100-8,000 times higher concentrations than were needed to displace [3H]WIN 35,428 binding in striatal membranes. Piperazine derivatives, e.g., GBR-12909, GBR-12935, and rimcazole, inhibited [3H]WIN 35,428 binding in CVMNs more effectively than did cocaine, WIN 35,428, mazindol, nomifensine, or benztropin. A positive correlation (r = 0.779; p < 0.001) was found between drug affinities for the striatal membrane sites labeled by [3H]WIN 35,428 and their abilities to inhibit DA uptake in CVMNs, whereas no correlation existed between the IC50 values of drugs that inhibited [3H]WIN 35,428 binding and [3H]DA uptake in CVMNs. The cytosolic [3H]WIN 35,428 binding sites may be a piperazine acceptor and may not be involved in the regulation of the DA transporter.
Valchar
M
M
Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Hanbauer
I
I
D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism*; D002421:Caudate Nucleus / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D003042:Cocaine / Q000031:analogs & derivatives* / Q000378:metabolism; D003342:Corpus Striatum / Q000378:metabolism*; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D004622:Embryo, Mammalian; D005260:Female; D007700:Kinetics; D008297:Male; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008636:Mesencephalon / Q000378:metabolism*; D009419:Nerve Tissue Proteins; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014316:Tritium
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D014316:Tritium; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D003042:Cocaine; D004298:Dopamine