COCAINE RECEPTOR--BIOCHEMICAL AND MOLECULAR STUDIES

这个项目的目标是阐明多巴胺的性质

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.