Cations Affect [3H]Mazindol and [3H]WIN 35,428 Binding to the Human Dopamine Transporter in a Similar Fashion

Abstract: The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn²⁺, Mg²⁺, Hg²⁺, Li⁺, K⁺, and Na⁺ were assessed on the binding of [³H]mazindol and [³H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn²⁺ (30–100 µ M ) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [³H]mazindol; Mg²⁺ (0.1–100 µ M ) had no effect; Hg²⁺ at ∼3 µ M stimulated binding to membranes, with a relatively smaller effect for [³H]mazindol than [³H]WIN 35,428 at 0°C, and at 30–100 µ M inhibited both intact cell and membrane binding; Li⁺ and K⁺ substitution (30–100 m M ) inhibited binding to membranes more severely than to intact cells; and Na⁺ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn²⁺ at 0 and 21°C and Hg²⁺ at 0°C.     

Relationship Between [3H]Mazindol Binding to Dopamine Uptake Sites and [3H]Dopamine Uptake in Rat Striatum During Aging

Certain drugs exhibit a remarkable correlation between their ability to inhibit synaptosomal uptake of dopamine and the binding of [3H]mazindol to striatal membranes. To investigate the role of mazindol binding sites in the dopamine uptake process and the fate of these sites (labeling dopaminergic neurons) during aging, we have examined the properties of mazindol binding and dopamine uptake in individual young and old rats. There was a 48% decrease (p = 0.0001) in the Bmax of mazindol binding and a 23% decrease (p = 0.0166) in the Vmax of dopamine uptake with no apparent change in their affinities with age. Regression analysis of the relationship between Bmax and Vmax exhibited a significant correlation in old (p = 0.0156) but not young rats (p = 0.1398). These data suggest that the number of mazindol binding sites decreases with age and that the number of sites on the dopamine transporter complex far exceeds the number required to elicit maximal dopamine uptake.     

Characterization of [3H]Mazindol Binding in Rat Brain: Sodium-Sensitive Binding Correlates with the Anorectic Potencies of Phenylethylamines

Saturable low-affinity binding sites for [3H]mazindol have been demonstrated in crude synaptosomal membranes from rat brain using both a centrifugation and a filtion assay. Studies on the regional distribution of these binding sites revealed that the hypothalamus and brainstem had the highest density of sites. Kinetic analysis of the binding of [3H]mazindol to hypothalamic membranes demonstrated a single class of noninteracting binding sites with an apparent affinity constant (KD) of 10.2 +/- 0.7 microM and maximal number of binding sites (Bmax) of 786 +/- 94 pmol/mg of protein. Specific [3H]mazindol binding was rapidly reversible, temperature sensitive, labile to pretreatment with proteolytic enzymes, and inhibited by physiological concentrations of sodium. In most peripheral tissues, such as the liver and kidney, very low levels of binding were observed; however, the adrenal gland had a relatively high density of sites. The potency of a series of anorectic drugs in inhibiting specific [3H]mazindol binding to hypothalamic membranes was highly correlated with their anorectic potencies in rats, but not with their motor stimulatory effects. These results suggest the presence of a specific drug recognition site in the hypothalamus that may mediate the anorectic activity of mazindol and related phenylethylamines.     

Effect of Unilateral Perinatal Hypoxic-Ischemic Brain Injury in the Rat on Dopamine D1 and D2 Receptors and Uptake Sites: A Quantitative Autoradiographic Study

The effect of a unilateral perinatal hypoxic-ischemic brain injury on dopamine D1 and D2 receptors and uptake sites was investigated in rats by using in vitro quantitative binding autoradiography, 2-3 weeks after the insult. We observed significant decreases in the Bmax and KD for [3H]SCH 23390-labeled D1 and in the Bmax for [3H]spiperone-labeled D2 receptors in the lesioned caudate-putamen in rats with moderate brain injury (visible loss in hemispheric volume ipsilateral to the injury) compared with the nonlesioned contralateral caudate-putamen or with control rats. Changes in [3H]SCH 23390 and [3H]spiperone binding predominated in the dorsolateral part of the lesioned caudate-putamen. Pronounced reduction in [3H]SCH 23390 binding was also observed in the substantia nigra pars reticulata on the side of the lesion. In contrast, we did not observe any significant change in Bmax or KD for [3H]mazindol-labeled dopamine uptake sites. Similarly, no significant changes in the levels of dopamine or its metabolites were found on the side of the lesion. The observed reductions in striatal dopamine D1 and D2 receptors are a reflection of striatal cell loss induced by the hypoxic-ischemic injury. The absence of changes in [3H]mazindol binding or dopamine levels in the lesioned caudate-putamen indicates that the dopaminergic presynaptic structures are preserved.     

Kinetic Evaluation of the Commonality Between the Site(s) of Action of Cocaine and Some Other Structurally Similar and Dissimilar Inhibitors of the Striatal Transporter for Dopamine

Abstract: The inhibition by cocaine of the apparent initial rate of the transport of striatal dopamine was compared with inhibitions produced by cocaethylene, benztropine, GBR-12909, mazindol, and nomifensine. Rotating disk electrode voltammetry was used to measure the kinetically resolved, inwardly directed transport of dopamine in striatal suspensions. Evidence is presented that the primary site of action of cocaine may be at the external face of the transporter. Experiments to determine whether or not the other inhibitors bind to the same site as cocaine were conducted by comparing the inhibitions observed for each of the inhibitors alone with that observed when paired with cocaine. The resulting changes in the velocity of the transport of dopamine induced by the inhibitors were then fit to one of the previously developed models of inhibition by pairs of inhibitors affecting the kinetics of actively transporting systems: a single-site model, a two-site model in which the two binding sites for the inhibitors interact, and a two-site model in which the two binding sites for the two inhibitors act independently. Cocaine inhibited the transport of dopamine competitively with its structural analogues, cocaethylene and benztropine. The structurally dissimilar inhibitor, GBR-12909, was found also to be competitive with cocaine. In contrast, mazindol and nomifensine were found to bind to separate interactive sites when individually paired with cocaine. These results suggest that mazindol and nomifensine may interact with the kinetically active transporter for dopamine in a manner different from that of cocaine. Mazindol was tested and found to inhibit competitively the inward transport of dopamine into striatal suspensions. In contrast, our previous published findings show cocaine to be an uncompetitive inhibitor of the transport of striatal dopamine. These results suggest that cocaine inhibits inward transport of dopamine by reducing the intramembrane turnover of the transporter, whereas mazindol alters the kinetics of the recognition of dopamine by the transporter. Finally, the potential effects of these binding modes of inhibitors on synaptic chemical communication in dopaminergic systems were analyzed. The results of these analyses suggest that different effects on the extracellular concentrations of dopamine can result from the different patterns of inhibition, suggesting that different modulatory influences on pre- and postsynaptic receptor occupation can result from inhibition of the transport of dopamine.     

Relationships Between the Catechol Substrate Binding Site and Amphetamine, Cocaine, and Mazindol Binding Sites in a Kinetic Model of the Striatal Transporter of Dopamine In Vitro

Abstract: Experiments were conducted to determine how (−)-cocaine and S (+)-amphetamine binding sites relate to each other and to the catechol substrate site on the striatal dopamine transporter (sDAT). In controls, m -tyramine and S (+)-amphetamine caused release of dopamine from intracellular stores at concentrations ≥12-fold those observed to inhibit inwardly directed sDAT activity for dopamine. In preparations from animals pretreated with reserpine, m -tyramine and S (+)-amphetamine caused release of preloaded dopamine at concentrations similar to those that inhibit inwardly directed sDAT activity. S (+)-Amphetamine and m -tyramine inhibited sDAT activity for dopamine by competing for a common binding site with dopamine and each other, suggesting that phenethylamines are substrate analogues at the plasmalemmal sDAT. (−)-Cocaine inhibited sDAT at a site separate from that for substrate analogues. This site is mutually interactive with the substrate site ( K _int = 583 n M ). Mazindol competitively inhibited sDAT at the substrate analogue binding site. The results with (−)-cocaine suggest that the (−)-cocaine binding site on sDAT is distinct from that of hydroxyphenethylamine substrates, reinforcing the notion that an antagonist for (−)-cocaine binding may be developed to block (−)-cocaine binding with minimal effects on dopamine transporter activity. However, a strategy of how to antagonize drugs of abuse acting as substrate analogues is still elusive.     

Differing Neurotoxic Potencies of Methamphetamine, Mazindol, and Cocaine in Mesencephalic Cultures

Abstract: The potent reinforcing effects of methamphetamine and cocaine are thought to be mediated by their interactions with CNS dopamine neurons. Both stimulants share the ability to block dopamine uptake potently, and methamphetamine can release cytoplasmic dopamine as well. There is also abundant evidence demonstrating the neurotoxic effects of methamphetamine. There are, however, limited studies that attempt to discern the neurotoxic mechanisms of these agents. The purpose of the present study was to characterize and compare the chronic in vitro effects of methamphetamine, cocaine, and the dopamine uptake blocker, mazindol, on cultured fetal mesencephalic dopamine neurons. Our studies examined biochemical mechanisms to evaluate the contribution of reuptake blockade versus release of dopamine. Using a dispersed cell preparation of fetal mesencephalon, cultures were treated for 5 days with the three uptake blockers. Dopamine function was assessed by measuring high-affinity [³H]dopamine uptake and by examining cultures for the presence of tyrosine hydroxylase-immunopositive neurons. Nonspecific neurotoxicity was assessed by staining for neuron-specific enolase and measuring lactate dehydrogenase activity. The results indicate that repeated administration of high concentrations of methamphetamine (10^?4 and 10^?3M) caused a generalized neurotoxicity whereas the effects of 10^?5M methamphetamine appeared to be specific to dopamine cells. Likewise, treatment of the cultures with mazindol (10^?6M) resulted in reduced dopamine uptake while not significantly affecting neuron-specific enolase or tyrosine hydroxylase immunostaining. On the other hand, repeated exposure to cocaine (10^?5 and 10^?4M) did not alter dopaminergic function in these cultures. The different mechanisms of action of these stimulants may explain the differences in neurotoxic potency of these compounds. The results demonstrate that a tissue culture model of fetal mesencephalic dopamine neurons provides a useful tool for the study of dopamine uptake systems and neuronal function.