Comparison of [3H]WIN 35,428 Binding, a Marker for Dopamine Transporter, in Embryonic Mesencephalic Neuronal Cultures with Striatal Membranes of Adult Rats

Abstract: In contrast to striatal membranes of adult rats, where high- ( K _D1= 34 n M ) and low- ( K _D2= 48,400 n M ) 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 ( K _D= 336,000 n M ). 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.