Differential effects of forskolin on tyrosine hydroxylase gene transcription in identified brainstem catecholaminergic neuronal subtypes in organotypic culture.
Differential effects of forskolin on tyrosine hydroxylase gene transcription in identified brainstem catecholaminergic neuronal subtypes in organotypic culture.
复制标题
毛喉素对器官型培养中已确定的脑干儿茶酚胺能神经元亚型中酪氨酸羟化酶基因转录的差异影响。
DOI：
10.1111/j.1460-9568.2005.03913.x
复制
发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Gainer,Harold
中科院分区：
文献类型：
--
作者：
Rusnak,Milan;Gainer,Harold
在体外研究了脑干去甲肾上腺素能核、蓝斑（LC）、A2和A1中酪氨酸羟化酶（TH）（儿茶酚胺合成的限速酶）基因表达的调控。本研究中采用的几种新型实验方法包括：（i）建立切片-外植模型，其中这些脑干核维持了去甲肾上腺素能神经元的高存活率、器官型拓扑结构以及除TH之外的两种识别标记物的共表达，即去甲肾上腺素转运蛋白（NET）和囊泡单胺转运蛋白 2（VMAT 2）;（ii）使用标记的内含子探针定量分析这些核中的TH转录以测量TH杂合RNA（hnRNA），和（iii）在培养基中使用河豚毒素以消除这些核中的自发神经活动，从而提供基础状态作为在各种药理学扰动下研究TH转录的起始点。在TTX的存在下，腺苷酸环化酶刺激剂，毛喉素，产生了155%的增加LC，增加130%的A1，和增加220%的A2 TH hnRNA相比，控制核。在LC和A1中，PKA抑制剂H89（5 µm）消除了毛喉素的这种作用，但MAP激酶途径（MEK）抑制剂PD 98059（75 µm）没有消除毛喉素的这种作用。 相反，毛喉素在A2神经元中产生的TH转录的稳健增加被PD 98059完全抑制，并且仅被H89部分抑制，表明诱导的TH转录在特定的中枢去甲肾上腺素能神经元亚型中由不同的激酶途径介导。
The regulation of gene expression of tyrosine hydroxylase (TH), the rate‐limiting enzyme in catecholamine synthesis, was studied in brainstem noradrenergic nuclei, locus coeruleus (LC), A2 and A1,in vitro. Several novel experimental approaches employed in this study included: (i) the development of a slice‐explant model in which these brainstem nuclei maintained a high survival of the noradrenergic neurons, an organotypic topology and the coexpression of two identifying markers in addition to TH, i.e. norepinephrine transporter (NET) and vesicular monoamine transporter 2 (VMAT2); (ii) quantitative analysis of TH transcription in these nuclei was made using a labelled intronic probe to measure TH heteronuclear RNA (hnRNA) and (iii) the use of tetrodotoxin in the media to eliminate spontaneous neural activity in these nuclei, thereby providing a basal state as the starting point for the study of TH transcription under various pharmacological perturbations. In the presence of TTX, the adenylcyclase stimulator, forskolin, produced a 155% increase in LC, a 130% increase in A1, and a 220% increase in A2 in TH hnRNA as compared to control nuclei. This effect of forskolin was abolished in the LC and A1 by the PKA inhibitor, H89 (5 µm), but not by the MAP kinase pathway (MEK) inhibitor, PD98059 (75 µm). In contrast, the robust increase in TH transcription produced by forskolin in A2 neurons, was completely inhibited by PD98059, and only partially inhibited by H89, showing that induced TH transcription is mediated by different kinase pathways in specific central noradrenergic neuronal subtypes.