Invited Symposium: Molecular and Cellular Analysis of Dopamine and Serotonin Transporters
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Introduction
Nitric oxide (NO) is an intercellular messenger molecule that is synthesized from L-arginine. NO is implicated in many physiological and pathological processes such as learning and memory, glutamate neurotoxicity drug dependence, tolerance and withdrawal (Dawson et al., 1991 a, b; Garthwaite, 1991; Moncada, 1991; Moncada et al., 1991). Glutamate (Glu) is an excitatory amino acid neurotransmitter in the brain and implicated in learning and memory. Glutamate, through NMDA receptors, cause in an increase in intracellular Ca2+, thereby stimulating nitric oxide synthase and NO production. Dopamine (DA) is another important neurotransmitter involved in mechanisms related to addiction. Amyl nitrate, an abused nitrate, is a NO generator.
Reuptake is a major mechanism for inactivation of released Glu and DA. NO, inhibits 3H-Glu and 3H-DA uptake in synaptosomes prepared from hippocampus and striatum respectively (Pogun et al., 1994a, b). If this effect can be demonstrated in-vivo, it can present an important transsynaptic regulation mechanism, which could have important impact on events ranging from learning and memory to neurotoxicity and addiction. In the present study, we investigated the effect of in-vivo NO synthase (NOS) inhibition on glutamate and dopamine uptake, determined ex-vivo.
Materials and Methods
Male Sprague Dawley rats were used in this study. Rats injected with NG Nitro-L-arginine (L-NNA, 50 mg/kg, i.p) or saline and decapitated at various times post-injection (5, 10, 15, 30, 60, 120 min). 3H-Glu and 3H-DA uptake and stable metabolites of NO, which are accepted to reflect NO levels, were determined in synaptosomes prepared from the hippocampus and striatum respectively. Synaptosomes were prepared in 0.32M sucrose, and a P2 pellet was obtained by centrifugation. The tissue was resuspended in sucrose. Uptake assays were carried out in modified Krebs-Ringer phosphate buffer. Synaptosomes were incubated with either 3H-Glu or 3H-DA at 30oC for 3 min. The assay was terminated by addition of ice-cold sucrose and the tissue was collected by rapid filtration. Radioactivity was measured by liquid scintillation counter. NO2+NO3 determinations were carried out as previously described: Nitrate levels were determined spectrophotometrically, based on the reduction of nitrate to nitrite by nitrate reductase (Bories and Bories, 1995); nitrite levels were obtained by the Griess reaction (Green et al, 1982).
Results
Glutamate
Our results show a significant facilitation (11.09 %, p < 0.05) of uptake (compared to controls) at 15 min post-injection, when the NO levels are 7,5 % (0.05) lower than the controls. However, while lower NO levels persist up to 120 minutes, the facilitation of Glu transport does not accompany this decline (Figure 1). Glu uptake was lower in NNA treated rats than controls at 5 min post-injection.
Fig. 1: Effect of NNA on 3H-Glu Uptake and NO2 + NO3 Levels. Numbers on the bars indicate n numbers.
Dopamine
A similar pattern for DA uptake and NO2+NO3 levels were observed. We found a significant increase of DA uptake (13 %, p < 0.05) and a decrease of NO2+NO3 levels (8 %, p < 0.01) at 15th min (Figure 2). In addition, a significant increase of DA uptake at 10th min while NO levels were not altered, and a decrease at 120th min were also observed.
Fig. 2: Effect of NNA on 3H-DA Uptake and NO2 + NO3 Levels. Numbers on the bars indicate n numbers.
Discussion and Conclusion
Previously, it has been shown that NO inhibits Glu and DA transport in synaptosomes. In this present study our results suggest that NOS inhibition and the subsequent lowering of brain NO levels result in a transient facilitation of 3H-Glu and 3H-DA uptake in rat brain. This effect is significant particularly at 15th min post-injection. However, an increase was found on DA uptake at 10th min without any accompanying change in NO levels. Conversely, the decline of NO levels at 30th, 60th and 120th minutes do not seem to effect Glu and DA uptake. Furthermore, a significant decrease of DA transport was observed at 120th min post-injection in spite of the low NO levels.
In a previous study, Cupello at al. showed that NG Nitro-L-arginine inhibits GABA uptake. This effect, if it operates on the glutamate and dopamine transporters similarly, may explain the return of Glu and DA uptake to control levels while NO levels are still low. Other compensatory mechanisms may be regulating Glu and DA uptake as well.
We are currently studying the effects of 7-Nitroindasole (a specific neuronal NOS inhibitor) and NG Nitro-D-arginine (a less active enantiomer of L-NNA) on Glu and DA uptake. A dose response study at 15th min is also in our future plans.
References
Bories, P. N., and Bories, C. (1995). Nitrate determination in biological fluids by an enzymatic one-step assay with nitrate reductase. Clin Chem 41, 904-7.
Cupello, A., Mainardi, P., Robello, M., Thellung, S. (1997) Effect of Nitric Oxide Donors on GABA Uptake by Rat Brain Synaptosomes. Neurochem. Res., 22(12): 1517-1521.
Dawson, V. L., Dawson, T. M., Bartley, D. A., Uhl, G. R., Snyder, S. H. (1991) Mechanisms of nitric oxide mediated neurotoxicity in primary brain cultures. J. Neurosci., 13: 2651-2661.
Dawson, V. L., Dawson, T. M., London, E. D., Bredt, D. S., Snyder, S. H. (1991) Nitric oxide mediated glutamate neurotoxicity in primary cortical cultures. Proc. Natl. Acad. Sci. USA, 88: 6368-6371
Garthwaite, J. (1991) Glutamate, nitric oxide and cell-cell signaling in the nervous system. Trends Neurosci., 14: 60-67.
Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L., Wishnok, J. S., and Tannenbaum, S. R. (1982). Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 126, 131-8.
Moncada, S. (1991) The L-arginine-nitric oxide pathway. Acta Physiol. Scand., 145:201-227
Moncada, S., Palmer, R. M., Higgs, E. A. (1991) Nitric oxide: physiology, pathology, pathophysiology and pharmacology. Pharmacol. Rev., 43: 109-142.
Pogun, S., Baumann, M. H., Kuhar, M. J. (1994) Nitric Oxide Inhibits 3H-Dopamine uptake. Brain Res., 641: 83-91.
Pogun, S., Dawson, W., Kuhar, M. J. (1994) Nitric Oxide Inhibits 3H-Glutamate Transport in Rat Synaptosomes. Synapse, 18: 21-26.
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