Short communicationOxygen radicals differentially affect Na+/Cl−-dependent transporters
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, [] 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
[]Norepinephrine and []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), []dopamine uptake into striatal synaptosomes was inhibited by incubation with xanthine oxidase (Fig. 1). In contrast, xanthine oxidase treatment was unexpectedly without effect on []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.
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