COCAINE RECEPTOR--BIOCHEMICAL AND MOLECULAR STUDIES

可卡因受体——生物化学和分子研究

• 批准号: 3752860
• 负责人: M J KUHAR
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3752860
• 关键词: age difference; analog; cocaine; dopamine; drug receptors; gene expression; glycosylation; laboratory rat; membrane transport proteins; mesencephalon; molecular site; molecular weight; neurotransmitter transport; nitric oxide; protein structure function; receptor binding; tissue /cell culture; transfection

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.

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，就其功能而言， 蛋白 这种蛋白质作为可卡因结合位点的特征 作为多巴胺能神经元的一个实体， 了解其功能。 关于多巴胺转运蛋白的一个重要发现是， 似乎是不同的后修正形式，至少与 就其分子量而言。 分子量的差异 似乎至少部分是由于糖基化的变化，尽管 也不可能排除其他异质性来源。 在最近的一项研究中，我们发现，过度发育和衰老， 多巴胺转运蛋白的分子量发生变化。 再次 这种变化的重要部分是由于糖基化的差异。 这些结果表明，如果使用多巴胺能神经元， 不成熟的物种，例如用于移植，转运蛋白不是 与发育完全的成年人相同。 调节多巴胺转运蛋白的机制并不完全 明白 因为新的知识表明， 导致一氧化氮的产生，因为多巴胺能 在纹状体中发现了与突触相邻的突触， 组织，我们测试了一氧化氮是否会抑制多巴胺 摄取。 我们发现一氧化氮会抑制多巴胺的摄取， 剂量和温度依赖的方式。 多巴胺的抑制 摄取发生在较低浓度的一氧化氮发生器比 抑制谷氨酸摄取。 这是第一次展示 一氧化氮会影响神经递质的吸收，并可能产生深远的影响 影响多巴胺转运的调节方式。 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细胞培养物可用于 研究具有某些优势的运输机。 因为很明显， 这些细胞不在大脑中，我们想测试 这些原代培养物中的多巴胺转运蛋白与 至少在成熟的大鼠大脑中，就其药理学而言。 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， 与成年大鼠大脑中的方式没有区别。 我们还展示 溶解的多巴胺转运蛋白可以很容易地通过以下方法进行研究 标准受体结合技术。 这将有助于各种 实验 我们继续在理解自然灾害的性质方面取得实质性进展， 多巴胺转运蛋白，它是如何加工的， 的监管机制。