Hanneman W H, Legare M E, Barhoumi R, Burghardt R C, Safe S, Tiffany-Castiglioni E
Department of Veterinary Anatomy, Texas A & M University, College Station 77843, USA.
Toxicology. 1996 Aug 1;112(1):19-28. doi: 10.1016/0300-483x(96)03346-x.
This study examined the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and related compounds on the uptake of intracellular calcium ([Ca2+]i) in primary cultures of rat hippocampal neuronal cells. [Ca2+]i levels were detected and quantified by interactive laser cytometry with microscopic image analysis. Cells were noninvasively labeled with fluo-3/AM and all experiments were conducted on cultured rat hippocampal neurons 14 days in culture. Treatment of cell cultures with 2,3,7,8-TCDD (10-100 nM) resulted in a rapid concentration-dependent increase in [Ca2+]i associated with a decrease in mitochondrial membrane potential and activation of alpha-protein kinase C (alpha-PKC). In contrast, 1,2,3,4-TCDD, a weak Ah receptor agonist, had no effect on [Ca2+]i at concentrations as high as 10 microM and similar results were also observed for 2,2',5,5'-tetrachlorobiphenyl. Maximal [Ca2+]i was observed within 30 s after addition of 2,3,7,8-TCDD and remained elevated (at higher concentrations) above resting levels for the duration of the experiment. This rapid increase in [Ca2+]i was blocked by addition of EDTA (2 mM) to the external medium or by pretreatment of the cells with the calcium channel antagonist nifedipine (10 microM). However, pretreatment of the cells with 100 microM cycloheximide failed to block calcium uptake in neuronal cells. These data indicate that rat hippocampal neuronal cells are responsive to 2,3,7,8-TCDD; however, the mechanism is not associated with altered gene transcription and may involve cellular targets.
本研究检测了2,3,7,8-四氯二苯并对二噁英（2,3,7,8-TCDD）及相关化合物对大鼠海马神经元细胞原代培养物中细胞内钙摄取（[Ca2+]i）的影响。通过交互式激光细胞术结合显微图像分析检测并定量[Ca2+]i水平。细胞用Fluo-3/AM进行非侵入性标记，所有实验均在培养14天的大鼠海马神经元上进行。用2,3,7,8-TCDD（10 - 100 nM）处理细胞培养物会导致[Ca2+]i迅速呈浓度依赖性增加，同时伴有线粒体膜电位降低和α-蛋白激酶C（α-PKC）激活。相比之下，1,2,3,4-TCDD作为一种弱芳烃受体激动剂，在高达10 μM的浓度下对[Ca2+]i没有影响，2,2',5,5'-四氯联苯也观察到类似结果。添加2,3,7,8-TCDD后30秒内观察到最大[Ca2+]i，在实验期间（较高浓度下）一直高于静息水平。向细胞外培养基中添加EDTA（2 mM）或用钙通道拮抗剂硝苯地平（10 μM）预处理细胞可阻断[Ca2+]i的这种快速增加。然而，用100 μM放线菌酮预处理细胞未能阻断神经元细胞中的钙摄取。这些数据表明大鼠海马神经元细胞对2,3,7,8-TCDD有反应；然而，其机制与基因转录改变无关，可能涉及细胞靶点。
