COCAINE RECEPTOR--BIOCHEMICAL AND MOLECULAR STUDIES

这个项目的目标是阐明多巴胺的性质。 转运蛋白，一个关键的可卡因受体，就其功能和作为一种 蛋白。该蛋白作为可卡因结合位点的特性 作为多巴胺能神经元的一个实体，在 了解它的功能。 关于多巴胺转运体的一个重要发现是 看起来是不同的翻译后修饰形式，至少 考虑到它的分子质量。分子量上的差异 似乎至少部分是由于糖基化的变化，尽管 也不可能排除异质性的其他来源。 在最近的一项研究中，我们发现在发育和衰老过程中， 多巴胺转运体的分子量会发生变化。再说一次，一个 这一变化的重要部分是由于糖基化的差异。 这些结果表明，如果将多巴胺能神经元从 未成熟的物种，例如用于移植，运输者不是 与完全发育成人的情况相同。 调节多巴胺转运体的机制还不完全 明白了。因为新的知识表明谷氨酸能突触 导致一氧化氮的生成，而且因为多巴胺能 在纹状体中发现了与谷氨酸能突触相邻的突触 组织，我们测试了一氧化氮是否会抑制多巴胺 领悟。我们发现，一氧化氮在一段时间内抑制了多巴胺的摄取， 剂量和温度依赖的方式。多巴胺的抑制作用 在较低浓度的一氧化氮生成器中发生摄取 谷氨酸摄取的抑制。这是第一次演示 一氧化氮影响神经递质的摄取，并可能对 对多巴胺运输调控方式的影响。下一个目标 是为了证明这种调节发生在体内。 胚胎大鼠中脑细胞的原代培养可用于 研究具有一定优势的运输机。因为很明显， 这些细胞不在大脑内，我们想测试一下 这些原代培养物中的多巴胺转运体类似于 至少在成熟的大鼠大脑中，就它们的药理而言。我们发现 可卡因类似物在这些培养物中抑制多巴胺的摄取 这种方式与成年大鼠的大脑没有什么不同。我们还展示了 溶解的多巴胺转运体可以很容易地通过 标准的受体结合技术。这将促进各种 实验。 我们继续在理解问题的性质方面取得实质性进展 多巴胺转运蛋白，它是如何处理的和可能的 它的监管机制。

The goal of this project is to elucidate the nature of the dopamine transporter, a key cocaine receptor, in terms of its function and as a protein. The characterization of this protein as a cocaine binding site and as an entity of the dopaminergic neuron is essential in understanding its function. One of the key findings about the dopamine transporter is that there appear to be different post-translationally modified forms, at least with regard to its molecular weight. The differences in molecular weight appear to be due at least in part to changes in glycosylation, although it is not possible to rule out other sources of heterogeneity as well. In a recent study, we have found that over development and aging, the molecular weight of the dopamine transporter changes. Again, a significant part of this change is due to differences in glycosylation. These results indicate that if dopaminergic neurons are used from immature species, for example for transplant, the transporter is not the same as that in the fully developed adult. The mechanisms that regulate the dopamine transporter are not fully understood. Because of the new knowledge that glutamatergic synapses result in the elaboration of nitric oxide, and because dopaminergic synapses have been found adjacent to glutamatergic synapses in striatal tissue, we tested whether or not nitric oxide would inhibit dopamine uptake. We found that nitric oxide inhibits dopamine uptake in a time, dose and temperature dependent fashion. The inhibition of dopamine uptake occurs at lower concentrations of nitric oxide generator than does the inhibition of glutamate uptake. These are the first demonstrations that nitric oxide affects neurotransmitter uptake and could have profound implications in the way dopamine transport is regulated. The next goal is to show that this regulation occurs in vivo. Primary cell cultures of embryonic rat mid-brain cells can be used to study the transporter with certain advantages. Since it is clear that these cells are not within the brain, we wanted to test whether or not the dopamine transporter in these primary cultures was similar to that in mature rat brain at least, in regard to their pharmacology. We found that cocaine analogs inhibited dopamine uptake in these cultures in a fashion that did not differ from that in adult rat brain. We also showed that the solubilized dopamine transporter can readily be studied by standard receptor binding techniques. This will facilitate a variety of experiments. We continue to make substantial progress in understanding the nature of the dopamine transporter protein, how it is processed and possible mechanisms for its regulation.