James O. Schenk

D2 Receptors May Modulate the Function of the Striatal Transporter for Dopamine: Kinetic Evidence from Studies In Vitro and In Vivo

Abstract: Recently it was hypothesized by others that the D2dopamine receptor can regulate the uptake of dopamine. However, the evidence in support of this hypothesis, although compelling, was not based on observations related to direct measures of the kinetic activity of the transporter itself. Here kinetic evidence in support of this hypothesis is shown. The apparent time‐resolved initial velocity of the transport of 1.0 μM dopamine into striatal suspensions, measured using rotating disk electrode voltammetry, was found to increase in the presence of the D2 receptor agonist, quinpirole, at 100 μM. This effect was reversed by sulpiride. In separate studies it was shown that acute and chronic treatments with haloperidol at 0.5 mg/kg, i.p., reduced the reuptake transport of dopamine in vivo following intrastriatal stimulation of its release by K+. Thus, it appears that D2 receptors may influence the functioning of the striatal transporter for dopamine. These results are consistent with a model in which presynaptically released dopamine may feed back onto the function of its transporter to increase the velocity of the clearance of synaptic dopamine following an action potential, suggesting the existence of a mechanism, in addition to release and synthesis modulation, for fine‐tuning dopaminergic chemical signaling.

A multisubstrate mechanism of striatal dopamine uptake and its inhibition by cocaine

A study of Na+ and Cl- as co-substrates in dopamine uptake into striatal suspensions and inhibition of dopamine uptake by cocaine was made by monitoring the initial velocity of the uptake of exogenously added non-radioactively labeled dopamine using a rotating disk electroanalytical technique with 50 msec resolution. Dopamine, in the concentration range of 0.025 to 4.00 microM, was found to be taken up rapidly into the tissue phase of striatal suspensions following the apparent zero order rate law for the first 25 sec. The observed, dopamine concentration-dependent, initial velocity data were first analyzed graphically using the Eadie-Hofstee transformation of the Michaelis-Menten kinetic equation and, subsequently, using all of the velocity data and the results of the graphical analyses, by non-linear curve fitting. Dopamine uptake was found to be first order in dopamine with a Vmax of 582 pmol/sec/g wet weight and a Km of 1.2 microM. The results of experiments in which choline and isethionate were substituted for Na+ and Cl-, respectively, suggested that the uptake process is second order in Na+ and first order in Cl-. Multisubstrate analyses of the initial velocities of uptake over the concentration range of 0.025 to 1.5 microM dopamine suggested that the mechanism of binding of dopamine to the uptake carrier is a partially random, sequential mechanism where dopamine or Na+ binds first with the uptake carrier and Cl- binds last. Cocaine was found to uncompetitively inhibit dopamine uptake and competitively inhibit both Na+ and Cl- binding (apparent Km values: 131 and 51 mM, respectively), suggesting that the mechanism of cocaine inhibition may be to bind to the dopamine occupied uptake carrier complex at the Na+ binding site.

Food Deprivation Decreases mRNA and Activity of the Rat Dopamine Transporter

We have hypothesized that the midbrain dopamine (DA) neurons are a target for insulin action in the central nervous system (CNS). In support of this hypothesis, we have previously demonstrated that direct intracerebroventricular infusion of insulin results in an increase in mRNA levels for the DA reuptake transporter (DAT). In this study, 24- to 36-hour food deprivation was used as a model of decreased CNS insulin levels, to test whether DAT mRNA levels, DAT protein concentration or DAT functional activity would be decreased. DAT mRNA levels, assessed by in situ hybridization, were significantly decreased in the ventral tegmental area/substantia nigra pars compacta (VTA/SNc) (77 ± 7% of controls, p < 0.05) of food-deprived (hypoinsulinemic) rats. Binding of a specific high-affinity DAT ligand (125I-RTI-121) to membranes from brain regions of fasted or free-feeding rats provided an estimate of DAT protein, which was unchanged in both of the major terminal projection fields, the striatum and nucleus accumbens (NAc). In addition, we utilized the rotating disk electrode voltametry technique to assess possible changes in the function of the DAT in fasting (hypoinsulinemic) rats. The Vmax of DA uptake was significantly decreased (87 ± 7% of control, p < 0.05), without a change in the Km of uptake, in striatum from fasted rats. In vitro incubation with a physiological concentration (1 nM) of insulin resulted in an increase of striatal DA uptake to control levels. We conclude that striatal DAT function can be modulated by fasting and nutritional status, with a contribution by insulin.

Striatal Transporter for Dopamine: Catechol Structure‐Activity Studies and Susceptibility to Chemical Modification

Abstract: The apparent second‐order association rate constant of dopamine binding to the striatal transporter (∼1 ± 106M−1 s−1) as well as the transporter turnover number (∼1.5 s−1) was estimated using rotating disk electrode voltammetry to monitor apparent zero trans entry of dopamine into striatal suspensions. The substrate specificity of the transporter was also assessed using catechol derivatives. Dopamine and norepinephrine were transported, whereas epinephrine and the acidic metabolites of dopamine were not transported. The metabolite, 3‐meth‐oxytyramine, was transported with a Km seven times greater than and a Vmax close to that for dopamine. 4‐Methoxytyramine was transported more facilely than the 3‐methoxy derivative. N‐Alkylation of the amine side chain of dopamine reduced transport dramatically. 4‐Ethylcatechol and 3,4‐dihydroxybenzylamine were transported with velocities 79 and 91 % less than that for dopamine, respectively. The rigid analogue 6,7‐dihydroxy‐1,2,3,4‐tetrahydronaphthalene was transported with a greater velocity than the 5,7‐dihydroxy derivative. Finally, the apparent Kmvalues for 4‐ethylcatechol, 1‐amino‐2‐phenylethane, tyramine, and m‐tyramine as cosubstrates with dopamine were 1.1, 11, 17, and 2.6 μM, respectively. Pretreatments of striatal suspensions with chloroethylnorapomorphine, N‐ethylmaleimide, Hg2+, 4,5‐dihydroxy‐4,5‐dioxo‐1H‐pyrrolo[2,3‐f]quinoline‐2,7,9‐tricarboxylic acid (a redox modulator of receptors in neuronal as well as other tissues), and neuraminidase reduced the velocity of transport of dopamine, whereas N‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline had no effect. Thus, the dopamine transporter requires an intact catechol with a primary ethylamine side chain for optimal activity relative to shorter side chain derivatives (side chains longer than two carbons were not tested), the 3‐hydroxyl group of dopamine is the more critical hydroxyl group, and the β rotamer of the extended conformation of dopamine is transported preferentially. The catechol appears to mediate the recognition of the substrate, whereas the amine side chain apparently facilitates the conformational change of the transporter that results in movement of dopamine into or across the membrane. The transporter distinguishes between agents known to block dopamine recognition sites on dopamine receptors? appears to possess a reduction/oxidation modulatory site, and requires sulfhydryl groups and external glycosylation for optimal function.

A Multisubstrate Kinetic Mechanism of Dopamine Transport in the Nucleus Accumbens and Its Inhibition by Cocaine

Abstract: Kinetic studies of dopamine transport into suspensions of nucleus accumbens (NAcc) and effects of Na+ and Cl− as cosubstrates were performed using rotating disk electrode voltammetry. To mimic chemical neurotransmission, dopamine was added as a rapid pulse, and transporter‐mediated clearance of dopamine was evaluated kinetically. This paradigm was shown to approximate a zero trans entry transport experiment. Dopamine was taken up with apparent Km and Vmax values of 1.3 µM and 375 pmol/s/g wet weight, respectively. Transport exhibited apparent trans acceleration. Substitution of Na+ with choline or Cl− with isethionate reduced dopamine transport with reaction orders of two and unity, respectively, accompanied by reductions in Vmax with no changes in Km. Apparent KNa and KCl values were 70.0 and 92.1 mM, respectively. Dopamine transport in NAcc was found to follow a partially random, sequential mechanism in which dopamine and Na+ bind randomly to the transporter followed by binding of Cl− before transport. Cocaine inhibited dopamine transport and the influences of the other substrates allosterically with an overall Ki of 0.30 µM. Thus, the general kinetic mechanism of the transport of dopamine in the NAcc is identical to that previously reported by this laboratory for dopamine transport in the striatum. However, the dopamine transporter in the NAcc is more tightly regulated by Na+, possesses a higher kinetic turnover rate, is four times more sensitive to cocaine than the striatal transporter, and exhibits cocaine inhibition independent of [substrate]. These findings suggest that cocaine modulates chemical signaling in NAcc differently than in striatum, providing down‐regulation of function irrespective of [substrate], thereby enhancing dopaminergic signaling more robustly in the NAcc than in the striatum.

The functioning neuronal transporter for dopamine: kinetic mechanisms and effects of amphetamines, cocaine and methylphenidate.

The dopamine transporter (DAT) is a transmembrane spanning protein that catalyzes the transport of dopamine across the neuronal membrane to concentrate the neurotransmitter inside the cell. Although the uptake of dopamine has been studied since the 1960s, more recent advances in knowledge of the protein itself and in making kinetically resolved measurements of its action have led to more insights into its mechanism and pharmacology. The literature of the kinetics of transporters and kinetic measurements of DAT activity is reviewed to provide an overview of the multisubstrate mechanism of DAT activity, its pharmacology with regard to amphetamine, cocaine and methylphenidate, and correlations of DAT activity with some behavioral outputs.

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.

Neurotensin binding to dopamine.

Rotating disk electrode voltammetry at glassy carbon electrodes and ultraviolet/visible spectroscopy were used to demonstrate that dopamine binds to neurotensin with a dissociation constant of 7.5 × 10‐8. By measuring the binding constants of various neurotensin analogs, it was found that the ‐Arg8‐Arg9‐portion of the neurotensin sequence is critical for binding dopamine. Neurotensin also formed a complex with 4‐ethylcatechol, 4‐methylcatechol, 3‐methoxytyramine, and norepinephrine. Although changes in the side chain did not alter the binding constant, methoxylation of the catechol moiety significantly increased the dissociation constant. These data along with additional studies of dopamine interactions with arginine derivatives suggest that the guanidino groups of arginine and the catechol hydroxyls of dopamine are responsible for mediating the observed binding. It is hypothesized that the capacity of neurotensin to bind directly to dopamine may be partly responsible for its previously observed antagonism of dopamine‐induced locomotor activity.

Effects of methylphenidate analogues on phenethylamine substrates for the striatal dopamine transporter: potential as amphetamine antagonists?

Abstract : Methylphenidate (MPD) was found to inhibit competitively the striatal dopamine transporter (DAT) and bind at sites on the DAT in common with both cocaine (a non‐substrate site ligand) and amphetamine (a substrate site ligand). Some methylphenidate analogues modified on the aromatic ring and/or at the nitrogen were tested to determine whether the profile of inhibition could be altered. None was found to stimulate the release of dopamine in the time frame (≤60 s) of the experiments conducted, and each of the analogues tested was found to noncompetitively inhibit the transport of dopamine. It was found that halogenating the aromatic ring with chlorine (threo‐3,4‐dichloromethylphenidate hydrochloride ; compound 1) increased the affinity of MPD to inhibit the transport of dopamine. A derivative of MPD with simultaneous, single methyl group substitutions on the phenyl ring and at the nitrogen (threo‐N‐methyl‐4‐methylphenidate hydrochloride ; compound 2) bound at a site in common with MPD. A benzyl group positioned at the nitrogen (threo‐N‐benzylmethylphenidate hydrochloride ; compound 3) imparted properties to the inhibitor in which binding at substrate and non‐substrate sites could be distinguished. This analogue bound at a mutually interacting site with that of methylphenidate and had a Kint value of 4.29 μM. Furthermore, the N‐substituted analogues (compounds 2 and 3), although clearly inhibitors of dopamine transport, were found to attenuate dramatically the inhibition of dopamine transport by amphetamine, suggesting that the development of an antagonist for substrate analogue drugs of abuse may be possible.

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 (Kint = 583 nM). 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.