Short communication
Oxygen radicals differentially affect Na+/Cl-dependent transporters

https://doi.org/10.1016/S0014-2999(99)00487-2Get rights and content

Abstract

Incubation with the oxygen radical-generating enzyme, xanthine oxidase, dramatically reduced striatal dopamine transporter activity, but was unexpectedly without effect on rat hippocampal norepinephrine uptake. To determine whether environmental differences between the striatum and hippocampus contributed to this lack of oxidative effect on norepinephrine transporters, synaptosomal γ-aminobutryic acid (GABA) uptake was assessed in both regions. Xanthine oxidase similarly decreased [3H]GABA uptake in both the striatum and hippocampus, supporting the conclusion that environmental differences did not account for the lack of effect on norepinephrine transport. These data suggest that norepinephrine transporters are less vulnerable than other Na+/Cl-dependent transporters to oxidative inactivation.

Introduction

The norepinephrine, dopamine, serotonin and γ-aminobutyric acid (GABA) transporters are members of the Na+/Cl-dependent transporter family, and help regulate synaptic transmission by influencing release and re-uptake of neurotransmitters from the synaptic cleft (Horn, 1990; Uhl, 1992; Povlock and Amara, 1997). Because of the critical modulatory role of these transporters, pharmacological inhibition of norepinephrine, serotonin (Glod, 1996) and GABA (Bourgeois, 1998) uptake is a useful therapeutic strategy to treat central nervous system disorders such as depression and epilepsy. Due to the potential clinical value derived from altering transporter activity, elucidation of mechanisms by which Na+/Cl-dependent transporter function is altered is warranted.

Previous studies from this laboratory, as well as others, have demonstrated that reactive oxygen species alter the function of some Na+/Cl-dependent transporter proteins. Specifically, it has been determined that dopamine, serotonin, and GABA uptake are inhibited by substances which generate reactive species, such as xanthine oxidase, dopamine, or sodium nitroprusside (Braughler, 1985; Debler et al., 1986; Pögün et al., 1994; Berman et al., 1996; Fleckenstein et al., 1997; Kokoshka et al., 1998). Molecular biological techniques have revealed that dopamine, serotonin, norepinephrine, and GABA transporters all contain two conserved cysteine residues located in the large extracellular loop (Guestella et al., 1990; Blakely et al., 1991; Kilty et al., 1991; Pacholczyk et al., 1991; Shimada et al., 1991), which in the dopamine transporter were determined to be critical for proper function and expression (Wang et al., 1995). Sulfhydryl groups of cysteine residues are readily oxidized (Meucci and Mele, 1997), which makes them likely targets of reactive oxygen species leading to functional alterations in these transporter proteins. In support of this, [3H] dopamine uptake decreases in striatal synaptosomes pre-exposed to sulfhydryl-modifying agents (Cao et al., 1989; Schweri, 1990). Like other Na+/Cl-dependent transporters, the norepinephrine transporter contains cysteinyl residues (Pacholczyk et al., 1991) that may render it vulnerable to oxidative inactivation. Less is known about the consequences of reactive oxygen species on norepinephrine transporter proteins. Since reactive oxygen species have been suggested to influence synaptic transmission by inhibiting several different transporter proteins, the intent of the current study was to determine the effects of xanthine oxidase-generated reactive oxygen species on norepinephrine transporter activity. For comparison, the effects of xanthine oxidase incubation on GABA and dopamine transporters were also assessed. The results indicate that norepinephrine transporters are less susceptible to oxidative inactivation than other Na+/Cl-dependent transporters.

Section snippets

Animals

Male Sprague–Dawley rats (200–300 g; Simonsen Laboratories, Gilroy, CA) were maintained under conditions of controlled temperature and lighting, with food and water provided ad libitum. Rats were sacrificed by decapitation. All procedures were conducted in accordance with approved National Institutes of Health guidelines.

Drugs and chemicals

Pargyline hydrochloride was purchased from Sigma (St. Louis, MO). Desipramine hydrochloride and nipecotic acid were purchased from Research Biochemicals International (Natick,

Results

[3H]Norepinephrine and [3H]dopamine uptake were assessed in hippocampal and striatal synaptosomes, respectively, that had been exposed to the reactive oxygen species generating enzyme xanthine oxidase. As reported previously (Berman et al., 1996; Fleckenstein et al., 1997), [3H]dopamine uptake into striatal synaptosomes was inhibited by incubation with xanthine oxidase (Fig. 1). In contrast, xanthine oxidase treatment was unexpectedly without effect on [3H]norepinephrine uptake into hippocampal

Discussion

Monoamine and GABA transporters are Na+/Cl-dependent and are important elements involved in the control of synaptic transmission. Since pharmacological inhibition of such transporters has lead to the treatment of depression and epilepsy, it is important to identify and understand the mechanism(s) whereby these membrane proteins are regulated. Some of the transporters, such as dopamine-, serotonin-, and GABA-types, which require Na+ and Cl to function, are vulnerable to oxidative inactivation (

Acknowledgements

This research was supported by PHS grants DA 00869, DA 04222, DA 11389 and DA 00378.

References (21)

There are more references available in the full text version of this article.

Cited by (13)

  • Section II. The Dopamine System

    2005, International Review of Neurobiology
    Citation Excerpt :

    In guinea‐pig dorsal striatal slices, H2O2 inhibits synaptic DA release by means of a Ca2+‐dependent mechanism (Chen et al., 2001). In rat HC, ROS inhibit the DA, but not the NE, transporter (Haughey et al., 1999). In rat astroglial cultures, application of DA leads to increased superoxide production accompanied by the oxidation of glutathione (GSH) to GSSH.

  • Glutamate uptake

    2001, Progress in Neurobiology
    Citation Excerpt :

    For example, the NMDA-receptor subtype NR1 has two cysteines (residue numbers 726 and 780) which regulate the receptor function (for review, see Mori and Mishina, 1995). Free radicals differentially affect the members of the Na+/Cl−-dependent neurotransmitter transporter family: Dopamine uptake is inhibited (Fleckenstein et al., 1997; Haughey et al., 1999) while norepinephrine uptake is not affected (Haughey et al., 1999). The SH-reducing agent dithiothreitol (DTT) increases the affinity of the serotonin transporters for imipramine (Tarrant and Williams, 1995).

  • Chronic and acute regulation of Na<sup>+</sup>/Cl<sup>-</sup>-dependent neurotransmitter transporters: Drugs, substrates, presynaptic receptors, and signaling systems

    2001, Pharmacology and Therapeutics
    Citation Excerpt :

    Interestingly, NET was not affected by ROS generators. While NET contains these same cysteine residues, it has been hypothesized that amino acid residues flanking the cysteines confer a protein-folding pattern prohibiting ROS interaction with the cysteine residues, rendering the NET resistant to down-regulation by ROS (Haughey et al., 1999). ROS may play a role in methamphetamine-induced inhibition of SERT and DAT.

View all citing articles on Scopus
View full text