Kinetics and Block of Dopamine Uptake in Synaptosomes from Rat Caudate Nucleus

Abstract

The dopamine (DA) uptake system in mammalian nerve terminals was studied by measuring the unidirectional influx of tritiated DA into synaptosomes prepared from rat caudate nucleus. Two distinct time-dependent components of DA uptake were observed. The principal component was saturable with respect to DA concentration, required both external Na and Cl, and was competitively blocked by micromolar concentrations of the psychotropic agents cocaine, benztropine, nomifensine, amphetamine, and methamphetamine. This principal component of uptake has the properties expected for a carrier-mediated transport system. The second component, which accounted for about 10-30% of the DA uptake at 2 microM DA, was not saturable, and was independent of external Na, Cl, and blockers of the carrier-mediated system. The saturable, Na-dependent component had an apparent Km(DA) of about 0.5 microM. The dependence of DA uptake on external Na was sigmoid [Hill coefficient = 2; Ka(Na) = 45 mM] whereas the dependence on Cl was best described by a rectangular hyperbola [Ka(Cl) = 15 mM]. Depolarizing conditions (elevated external K) reduced the rate of DA influx. The data are consistent with a carrier-mediated DA transport mechanism in which each DA molecule entering the nerve terminal via the carrier is accompanied by two or more Na ions and one Cl ion in a rheogenic process carrying one or more net positive charges into the cell. Net, concentrative accumulation of DA inside nerve terminals may be accomplished by utilizing the Na electrochemical gradient to drive DA against its electrochemical gradient via this carrier system.

State-dependent Conformations of the Translocation Pathway in the Tyrosine Transporter Tyt1, a Novel Neurotransmitter:Sodium Symporter from Fusobacterium nucleatum

“…The Hill coefficient of ϳ2 is consistent with an apparent stoichiometry of 2 Na ϩ :1 tyrosine for coupled uptake. This is in line with much functional data in other NSSs, such as the Hill coefficient of 2 for sodium in dopamine transport by DAT (3,50) as well as the identification of two bound Na ϩ ions in the crystal structure of LeuT (21). In this structure, the carboxyl oxygen of the substrate, leucine, forms an ion pair with one Na ϩ (Na1).…”

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

“…Unlike the standard exchange diffusion model of reverse transport, weak base-mediated reverse transport does not require that the transporter act as a mobile carrier. Under normal conditions cytoplasmic DA is probably much higher than extracellular DA with the transmembrane sodium and/or chloride gradient favoring uptake over release (Krueger, 1990). We suggest that amphetamine decreases the accumulation of vesicular DA, thereby increasing cytoplasmic DA sufficiently to promote net efflux.…”

“…Triethanolamine, due to its hydrophilicity, does not reach intracellular sites and thus does not effectively disrupt intracellular proton gradients (Okhuma and Poole, 198 1). Monensin, a monovalent cationic ionophore frequently used as a protonophore in whole cells, also promotes sodium flux and may therefore potentiate reverse transport by attenuating both the proton gradient required for vesicular accumulation and the sodium gradient used to drive DA uptake by the plasma membrane transporter (Kuhn et al, 1986;Krueger, 1990;Lonart and Zigmond, 1991). Nevertheless, monensin is unlikely to completely disrupt intracellular pH gradients under these conditions, which probably accounts for its reduced toxicity compared with obligatory protonophores such as p-trifluoromethoxyphenylhydrazone carbonyl cyanide (FCCP), which abolish the mitochondria1 proton gradient and uncouple oxidative phosphorylation.…”

Voltammetric characterization of the effect of monoamine uptake inhibitors and releasers on dopamine and serotonin uptake in mouse caudate-putamen and substantia nigra slices

“…When pharmacological agents were used, the change in the current vs. time profile was evaluated as a change in apparent K m ; inhibition of DA or 5-HT uptake was reflected as an increase in apparent K m (John et al, 2006;Jones et al, 1995a). The drugs studied have previously been described to competitively inhibit monoamine transport Davies et al, 1993;Jones et al, 1995b;Krueger, 1990;Missale et al, 1985;Richelson and Pfenning, 1984;Schuldiner et al, 1993;Wu et al, 2001a; but see, e.g., Coyle and Snyder, 1969;Eshleman et al, 1999;McElvain and Schenk, 1992).…”