[³H]WIN 35,065–2: A Ligand for Cocaine Receptors in Striatum

Abstract

[3H]WIN 35,065-2 binding to striatal membranes was characterized, primarily by centrifugation assay. Like [3H]cocaine, [3H]WIN 35,065-2 binds to both high- and low-affinity sites. [3H]WIN 35,065-2, however, exhibits consistently higher affinities than [3H]cocaine. Saturation experiments indicate a low-affinity binding site with an apparent KD of approximately 160 nM and a Bmax of 135 fmol/mg of tissue. A high-affinity site has also been identified with an apparent KD of 5.6 nM and a Bmax of 5.2 fmol/mg of tissue. The specific-to-nonspecific binding ratios with [3H]WIN 35,065-2 were higher than with [3H]cocaine in both centrifugation and filtration assays. Pharmacological characterization suggests that [3H]WIN 35,065-2 binds to the dopamine transporter. Mazindol, GBR 12909, nomifensine, and (-)-cocaine are potent inhibitors of [3H]WIN 35,065-2 binding. In contrast, the norepinephrine transporter ligand desipramine is a weak inhibitor, and the serotonin transporter ligand citalopram does not inhibit binding. The effect of sodium on binding was examined under conditions in which (a) the low-affinity site was primarily (87%) occupied and (b) approximately 50% of both sites were occupied. The results indicate that both sites are sodium dependent. Injection of 6-hydroxydopamine into the striatum results in a significant loss of both high- and low-affinity sites, a finding suggesting that both sites are on dopaminergic nerve terminals. Taken together, these data are consistent with the presence of multiple cocaine binding sites associated with the dopamine transporter.

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

“…Several reports in the literature using striatal membrane preparations of rodents or primates indicate that the binding of [3H]cocaine and congeners is associated with the DA transporter (Kennedy and Hanbauer, 1983;Madras et al, 1989a.h;Ritz et al, 1990~). Recently, the cDNA that encodes the DA transporter has been isolated and expressed in Xenopus oocytes and COS cells (Shimada et al, 1991).…”

Inhibition of DAT function attenuates manganese accumulation in the globus pallidus

“…This suggests a potential role for the DAT and dopamine metabolism in the transport of Mn. However, both cocaine and reserpine are known to affect other neurotransmitters, most notably serotonin and norepinephrine (Ritz et al, 1990). Therefore, it is unclear if this decrease in ventral pallidum Mn concentrations is directly related to the DAT being inhibited or is related to the inhibition of the serotonin transporter and/or the norepinephrine transporter.…”

Amphetamine and Other Weak Bases Act to Promote Reverse Transport of Dopamine in Ventral Midbrain Neurons

“…However, in part because it has not been possible previously to differentiate blockade of amphetamine uptake from inhibition of DA release, the mechanism of transporter blockade has been controversial. Some blockers, including GBR 12909, might act extracellularly at the substrate binding site (Andersen, 1989) whereas others, such as WIN 35,065-2, may bind to multiple sites and could have allosteric effects (Ritz et al, 1990). In the exchange diffusion model, high levels of amphetamine would increase the frequency of translocation of the binding site, increasing the rate of DA reverse transport and in effect promoting exchange of DA for amphetamine (Liang and Rutledge, 1982;Butcher et al, 1988).…”

“…In this model, if uptake blockers bind to the substrate site at the extracellular face and are not themselves transported (Andersen, 1989), they would inhibit DA release by preventing intracellular translocation of the binding site. If allosteric binding sites contribute (Ritz et al, 1990), the blockers might inhibit the reverse transport of DA previously bound at the intracellular face; however, the net effect would still be to inhibit amphetamine uptake, thereby decreasing exchange of amphetamine for DA.…”