The uptake of [3H]dopamine by homogenates of rat corpus striatum: Effects of cations
Abstract
The uptake of [3H]dopamine was studied with a synaptosomal preparation of the corpus striatum. The accumulation of dopamine was found to be temperature-dependent and very rapid, but linear over time for at least 5 min. at 37°C with characteristics of saturable kinetics. The optimum concentrations for Na+ and K+ were 150–160 m and 2.5–4.8 m, respectively, while uptake was progressively inhibited at concentrations of K+ greater than 5 m. Rubidium was capable of substituting for potassium whereas cesium was a much less effective replacement. The uptake of DA was blocked by the antibiotics, valinomycin and gramicidin-D which bind K+ or both Na+ and K+, respectively, and thereby might interfere with the transport of cations across neuronal membranes. Similarly, ouabain which blocks the active transport of Na+ markedly antagonized the accumulation of DA into striatal homogenates. In contrast, tetrodotoxin which does not prevent the active transport of Na+, had no effect. Uptake appeared not to require Ca++ and it was not inhibited by increasing total osmolarity to 400 mos. In general, the cationic requirements for DA-uptake in striatal tissue and its responses to several inhibition of ionic transport, do not appear to be greatly different from those reported for NE with synaptosomes prepared from whole brain.
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What can be learned from studies of multisubstrate mechanisms of neuronal dopamine transport?
2003, European Journal of PharmacologyThe dopamine transporter (DAT) is a Na+- and Cl−-dependent transporter and, with respect to its three apparent substrates, both partially random sequential as well as ordered mechanisms have been reported. Here we describe some of the features of DAT, such as the coupling of energy to concentrate dopamine and the properties of slippage and leakage. Further, in considering the regulation of transport velocities by DAT few have considered issues related to substrate regulation of DAT activity. Specifically, what effect do changes in the constants (K) for the participation of Na+ and Cl− have on dopamine transport velocity? It is shown that DAT may possess properties of slippage, an argument is made that leakage may be important in neuronal systems containing DAT, and the influence of changing values of K for the participation of Na+ and Cl− in transport is shown to produce large effects on DAT activity depending on the multisubstrate kinetic mechanism.
Methamphetamine-induced alterations in dopamine transporter function
1998, Brain ResearchRepeated methamphetamine (METH) administration has been shown to produce differing neurochemical as well as behavioral effects in rats. This study was designed to examine the effects of acute and chronic METH exposure on uptake and release of [3H]dopamine (DA) in cultured midbrain dopamine neurons to determine if persistent neuronal adaptations ensue. In addition, we have assessed DA D2 receptor function to determine if chronic METH alters this receptor. Fetal midbrain cultures were exposed to METH (1, 10 μM) for 5 days and dopaminergic function examined 1 or 7 days after drug removal. The ability of METH to release [3H]DA was compared to other releasing agents as well as several potent uptake inhibitors. Chronic exposure to a release-promoting concentration of METH resulted in either no change or a reduction in [3H]DA release upon subsequent METH challenge. Pretreatment with METH was also found to cause a decrease in the Bmax for [3H]raclopride binding, suggesting that persistently elevated DA levels cause a downregulation of DA D2 receptors. Examination of transporter kinetics utilizing initial velocity of uptake revealed that METH treatment caused a significant decrease in affinity (Km) for the substrate (DA), while not altering the maximal velocity of uptake (Vmax). Binding studies with [125I]RTI-55 revealed that there was no alteration in either the Bmax or Kd for this ligand, suggesting that the changes induced by METH treatment are due to alterations in Km and not in the number of DA transport sites. The results from these studies indicate that METH treatment produces a modification in transporter function which may be associated with both the altered uptake and release of [3H]DA. These changes have broad implications for the regulation of transporter activity not only because of the relevance to pre-synaptic mechanisms controlling neurotransmission, but also to the importance of the neuronal adaptation that occurs in response to chronic METH exposure.
Peroxidation of Synaptosomes Alters the Dopamine Uptake Complex but Spares the Exocytotic Release of Dopamine
1995, NeurodegenerationSynaptosomes, prepared from the striata of mice, and incubated for 1 h in a Krebs-Ringer medium with the peroxidative combination of ascorbic acid (0.1 mM)/Fe2+(1 μM), lose their ability to take up [3H] dopamine. This effect is associated with a decrease in binding of the dopamine uptake inhibitor [3H] GBR 12783. The free radical scavenger trolox C (0.1 mM) and theGinkgo bilobaextract EGb 761 (10 μg/ml) prevent both effects. Although submitted to these peroxidative conditions after loading with [3H] DA, superfused synaptosomes retain their ability to release [3H] DA when depolarized by high potassium concentrations (40 mM). This release is higher than that observed when synaptosomes are incubated without ascorbic acid/Fe2+1, and does not seem to depend upon peroxidation, since it is also observed when incubation is performed in the presence of the free radical scavengers EGb 761 (10 μg/ml) and trolox C (0.1 mM).
Specific binding of [<sup>3</sup>H]GBR 12783 to the dopamine neuronal carrier included in polarized membranes
1993, European Journal of Pharmacology: Molecular PharmacologyWe have compared the properties of the binding to the neuronal dopamine carrier located either in polarized membranes of synaptosomes or in non polarized, classical membranes. Non-polarized membranes were prepared by sonication of the partially purified synaptosomal fraction obtained from rat striatum which was used as the source of polarized membranes. Binding experiments were carried out at 37°C in Krebs Ringer related media. [3H]GBR 12783 (1-[2-(diphenylmethoxy)ethyl]4-(3-phenyl-2-[1-3H]propenyl) piperazine) specifically bound with a nanomolar affinity to a homogenous population of site (maximal binding site concentration: 8–10 pmol/mg protein). Pure uptake inhibitors, but not substrates, competed for the [3H]GBR 12783 binding site located in polarized membranes of synaptosomes at concentrations effective against dopamine neuronal transport. Except for [3H]GBR 12783, the replacement of Cl− by isethionate− did not result in significant change in the ability of pure uptake inhibitors to compete for the specific binding site. A reduction in the Na+ concentration from 135 to 10 mM induced a significant decrease in the inhibitory potency of GBR 12783, mazindol, nomifensine and methylphenidate. This decrease was likely to result from the presence of K+, Mg2+ and Ca2+, whose inhibitory effects were modified and/or increased by decreasing the Na+ concentration. These data indicate that the membrane polarity is not clearly involved in the binding of pure uptake inhibitors to the dopamine neuronal carrier; furthermore they underline the critical role of Na+ and K+ transmembrane gradients in both the recognition of the carrier by dopamine and its inward transport.
Inhibitory effect of xylamine on the uptake of [<sup>3</sup>H]dopamine into isolated striatal synaptosomes of the rats
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