Characterization of [3H]Dopamine Uptake Sites and [3H]Cocaine Recognition Sites in Primary Cultures of Mesencephalic Neurons During In Vitro Development
Characterization of [3H]Dopamine Uptake Sites and [3H]Cocaine Recognition Sites in Primary Cultures of Mesencephalic Neurons During In Vitro Development
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体外发育过程中中脑神经元原代培养物中[3H]多巴胺摄取位点和[3H]可卡因识别位点的表征
DOI：
10.1111/j.1471-4159.1991.tb03473.x
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发表时间：
1991
影响因子：
4.7
通讯作者：
I. Hanbauer
中科院分区：
文献类型：
--
作者：
M. Grilli;A. Wright;I. Hanbauer
摘要：研究了14日龄大鼠胚胎腹侧中脑原代培养细胞的[~3H]多巴胺摄取和[~3H]可卡因结合部位。仅在中脑腹侧完整细胞培养物中检测到[~3H]可卡因和[~3H]马吲哚的特异性结合部位，而在这些细胞培养物制备的超声洗涤膜中未检测到。细胞不通过主动转运过程摄取[~3H]可卡因，因为在4°C时也发生了[~3H]可卡因结合。此外，由于广泛的清洗未能将[~3H]可卡因从细胞中去除，因此也排除了[~3H]可卡因通过被动扩散和离子捕获进入细胞的可能性。当细胞被链溶素O(0.2U/ml，5min)通透时，[~3H]可卡因结合量减少到对照的6%。综上所述，这些结果表明，在培养的中脑神经元中，[~3H]可卡因可能通过被动扩散进入细胞，然后被细胞质隔室隔离，该隔室在渗透或超声和清洗膜制剂的过程中丢失。培养神经元的通透性不能改变[~3H]多巴胺的储存。当细胞摄取[~3H]多巴胺后用链溶素O(0.2U/ml，5min)通透细胞时，[~3H]多巴胺被细胞保留，不会渗入孵育液中，表明[~3H]多巴胺储存在不能通过穿孔膜的部位。相比之下，已经通透的细胞对[~3H]多巴胺的摄取减少了33%，这表明泄漏出来的胞浆蛋白可能在摄取过程中发挥了作用。与成年大鼠纹状体膜标本相比，在完整的中脑细胞培养中，[~3H]可卡因的结合是不依赖于Na+的。[3 H]多巴胺摄取和[3 H]可卡因结合部位的表达似乎与神经炎的生长发育有关，支持可卡因结合部位可能与多巴胺转运体密切相关的观点。
Abstract: [3H]Dopamine uptake and [3H]cocaine binding sites were studied in primary cultures of ventral mesencephalon from 14‐day‐old rat embryos. Specific binding sites for [3H]cocaine and [3H]mazindol were detected only in intact cell cultures of ventral mesencephalon, and were absent in sonicated, washed membranes prepared from these cell cultures. [3H]Cocaine was not taken up by the cells through an active transport process because [3H]cocaine binding occurred also at 4°C. Moreover, the possibility of [3H]cocaine entering the cells by passive diffusion and ion trapping was also excluded because extensive washing failed to remove [3H]cocaine from the cells. [3H]Cocaine binding was reduced to 6% of control when cells were permeabilized with streptolysin O (0.2 U/ml, 5 min). Taken together, these results suggest that in cultured mesencephalic neurons, [3H]cocaine may enter the cell by passive diffusion and then be sequestered by a cytosolic compartment that is lost in the process of permeabilization or sonication and washing of membrane preparations. Permeabilization of cultured neurons failed to alter the storage of [3H]dopamine. When cells were permeabilized with streptolysin O (0.2 U/ml; 5 min) after [3H]dopamine was taken up, [3H]dopamine was retained by the cells and did not leak into the incubation medium, indicating that [3H]dopamine was stored in sites that could not pass through the perforated membranes. In contrast, [3H]dopamine uptake into already permeabilized cells was reduced by 33%, suggesting that a cytosolic protein that had leaked out may play a functional role in the uptake process. In contrast to striatal membrane preparations of adult rats, [3H]cocaine binding in intact mesencephalic cell cultures was Na+ independent. The expression of [3H]dopamine uptake and [3H]cocaine binding sites appeared to be developmentally linked to neuritic outgrowth, supporting the view that cocaine binding sites may be closely associated with the dopamine transporter.
[3H]马吲哚结合与神经元多巴胺和去甲肾上腺素摄取位点相关。
影响因子：
3.6
作者：
Javitch,JA;Blaustein,RO;Snyder,SH
通讯作者：
Snyder,SH
DOI：
10.1016/0006-291x(85)91071-x
发表时间：
1985-01-01
影响因子：
3.1
作者：
CHIBA, K;TREVOR, AJ;CASTAGNOLI, N
通讯作者：
CASTAGNOLI, N