[3H]GBR 12935 Binding to Dopamine Uptake Sites in the Human Brain

Binding of 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine ([3H]GBR 12935) was studied in membrane preparations of several human brain regions. In putamen, the substituted piperazine derivates cis- and trans-flupenthixol displaced 90% of the total [3H]GBR 12935 binding. Computer-assisted analysis of the competition curves revealed a high-affinity site (30%; KiH = 54 nM) and a low-affinity site (60%; KiL = 4.5 microM). The dopamine uptake blockers mazindol and nomifensine only displaced 30% of the total [3H]GBR 12935 binding in a monophasic way. Binding of [3H]GBR 12935 to the dopamine uptake sites, i.e., that displaced by dopamine uptake blockers, corresponded to part of the binding having low affinity for flupenthixol and was only detected in putamen, nucleus caudatus, nucleus accumbens, and substantia nigra. Even after masking the high-affinity binding site for flupenthixol by including 1 microM cis-flupenthixol in the binding assays, no dopamine uptake sites could be detected in globus pallidus, amygdala, thalamus, hippocampus, and cerebral cortex. Binding of [3H]GBR 12935 to dopamine uptake sites was lost in the nucleus caudatus ipsilateral to ventral midbrain infarctions, confirming their location on nigrostriatal nerve endings. Gross unilateral lesions of the striato- and pallidonigral pathways did not affect the number of dopamine uptake sites in the ipsilateral substantia nigra, suggesting that they may reside on the soma or dendrites of nigral neurons.     

人参皂苷生物合成相关新基因GBR6的cDNA克隆及序列分析

从人参根组织中分离总RNA,使用RT—PCR扩增出人参皂苷生物合成相关基因GBR6开放阅读框(ORF)cDNA片断,并将其定向克隆入原核高效表达载体pQE30,阳性克隆经BamHⅠ和PstⅠ双酶切鉴定、测序验证与已知的GBR6ORF序列完全一致．因而成功构建了pQE30-GBR60RF融合原核表达载体,为下一步进行GBR6功能表达分析奠定了基础．     

Studies,using in vivo microdialysis,on the effect of the dopamine uptake inhibitor GBR 12909 on 3,4-methylenedioxymethamphetamine ('ecstasy')-induced dopamine release and free radical formation in the mouse striatum

The present study examined the mechanisms by which 3,4-methylenedioxymethamphetamine (MDMA) produces long-term neurotoxicity of striatal dopamine neurones in mice and the protective action of the dopamine uptake inhibitor GBR 12909. MDMA (30 mg/kg, i.p.), given three times at 3-h intervals, produced a rapid increase in striatal dopamine release measured by in vivo microdialysis (maximum increase to 380 +/- 64% of baseline). This increase was enhanced to 576 +/- 109% of baseline by GBR 12909 (10 mg/kg, i.p.) administered 30 min before each dose of MDMA, supporting the contention that MDMA enters the terminal by diffusion and not via the dopamine uptake site. This, in addition to the fact that perfusion of the probe with a low Ca(2+) medium inhibited the MDMA-induced increase in extracellular dopamine, indicates that the neurotransmitter may be released by a Ca(2+) -dependent mechanism not related to the dopamine transporter. MDMA (30 mg/kg x 3) increased the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA) from salicylic acid perfused through a probe implanted in the striatum, indicating that MDMA increased free radical formation. GBR 12909 pre-treatment attenuated the MDMA-induced increase in 2,3-DHBA formation by approximately 50%, but had no significant intrinsic radical trapping activity. MDMA administration increased lipid peroxidation in striatal synaptosomes, an effect reduced by approximately 60% by GBR 12909 pre-treatment. GBR 12909 did not modify the MDMA-induced changes in body temperature. These data suggest that MDMA-induced toxicity of dopamine neurones in mice results from free radical formation which in turn induces an oxidative stress process. The data also indicate that the free radical formation is probably not associated with the MDMA-induced dopamine release and that MDMA does not induce dopamine release via an action at the dopamine transporter.     

1-Methyl-4-phenylpyridinium accumulates in cerebellar granule neurons via organic cation transporter 3

1-Methyl-4-phenylpyridinium (MPP+), the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, induces apoptosis in cerebellar granule neurons (CGNs). We have tested the hypothesis that organic cation transporter (OCT) 3 mediates the accumulation and, hence, the toxicity of MPP+ in CGNs. CGNs in primary culture express OCT3 but do not express mRNA for OCT1, OCT2 or the dopamine transporter. Cerebellar astrocytes are negative for OCT3 protein by immunocytochemistry. [3H]MPP+ accumulation by CGNs exhibits first-order kinetics, and a Kt value of 5.3 +/- 1.2 micro m and a Tmax of 0.32 +/- 0.02 pmol per min per 106 cells. [3H]MPP+ accumulation is inhibited by corticosterone, beta-estradiol and decynium 22 with Ki values of 0.25 micro m, 0.17 micro m and 4.0 nm respectively. [3H]MPP+ accumulation is also inhibited by desipramine, dopamine, serotonin and norepinephrine, but is not affected by carnitine (10 mm), mazindol (9 micro m) or GBR 12909 (1 micro m). MPP+-induced caspase-3-like activation and cell death are prevented by pretreatment with 5 micro mbeta-estradiol. In contrast, the neurotoxic effects of rotenone are unaffected by beta-estradiol. Interestingly, GBR 12909 protects CGNs from both MPP+ and rotenone toxicity. In summary, CGNs accumulate MPP+ in manner that is consistent with uptake via OCT3 and the presence of this protein in CGNs explains their sensitivity to MPP+ toxicity.