Distinct, Developmentally Regulated Brain mRNAs Direct the Synthesis of Neurotransmitter Transporters
Distinct, Developmentally Regulated Brain mRNAs Direct the Synthesis of Neurotransmitter Transporters
复制标题
独特的、发育调节的大脑 mRNA 指导神经递质转运蛋白的合成
作者：
R. Blakely;Janet A. Clark;T. Pacholczyk;S. Amara
摘要：非洲爪蟾卵母细胞表达系统被用来定义神经递质转运蛋白mRNA的发育和结构特性和编码载体的药理学特性，而不依赖于脑组织制备的复杂性。将新生和成年大鼠脑区的Poly（A）+ RNA显微注射到爪蟾卵母细胞中，48小时后测定Na+依赖性神经递质转运蛋白的表达。对注射幼年大鼠组织RNA的卵母细胞进行的转运研究表明，mRNA丰度的区域和转运蛋白特异性出生后增加是脑高亲和力氨基酸摄取系统观察到的发育变化的主要因素。L-谷氨酸（Glu）和γ-氨基丁酸（GABA）摄取系统在出生后第3天的前脑mRNA中均可检测到，并在接下来的2周前脑发育过程中逐渐富集。与此相反，脑干谷氨酸和GABA转运富集是60-70%的成人值的第3天，超过成人水平的第10天。发育过程中L-谷氨酸脱羧酶mRNA丰度的平行测定证明了对指导递质合成和再摄取的mRNA的不同调控影响。在前脑发育的任何时间点均无法检测到甘氨酸摄取，并且在脑干发育的前3周内，甘氨酸摄取逐渐在出生后升高至成人水平。使用Glu、GABA、多巴胺和胆碱转运的良好表征的抑制剂（分别为D-天冬氨酸、哌啶甲酸、诺米芬辛和半胆碱-3）进行的摄取研究表明，由成年大鼠脑mRNA编码的卵母细胞转运蛋白保留了体外脑制备物所表现出的拮抗剂敏感性。此外，观察到对Glu转运拮抗剂二氢红藻氨酸（1 mM）的不同区域敏感性，支持了先前关于区域特异性Glu转运蛋白亚型的报告。为了确定脑转运蛋白mRNA之间存在的结构多样性，在卵母细胞注射之前，在线性（10-31%）蔗糖密度梯度上对poly（A）+ RNA进行大小分级。这些实验揭示了能够独立地指导Glu、GABA和甘氨酸转运蛋白合成的两种mRNA大小类别（2.4-3.0 kb、4.0-4.5 kb）。在小脑以外的区域中，Glu和GABA转运蛋白活性迁移为单个但不同的4.0-4.5 kb峰。相比之下，Glu和GABA转运蛋白在2.5-3.0 kb处显示出活性主峰，具有大小分级的小脑mRNA。脑干甘氨酸摄取表现出广泛的沉降曲线，峰明显在2.4和4.0 kb。两者合计，这些发现表明以前未得到重视的复杂性，在mRNA的结构和调节，其基础上的氨基酸神经递质摄取系统在啮齿动物中枢神经系统的表达。
Abstract: The Xenopus laevis oocyte expression system was utilized to define developmental and structural properties of neurotransmitter transporter mRNAs and the pharmacological characteristics of encoded carriers independent of the complexities of brain tissue preparations. Poly(A)+ RNA from dissected brain regions of neonatal and adult rats was microinjected into Xenopus oocytes and the expression of Na+‐dependent neurotransmitter transporters determined 48 h later. Transport studies conducted with oocytes injected with RNAs derived from juvenile rat tissues indicate a region‐ and transporter‐specific, postnatal increase in mRNA abundance as a major factor in the developmental changes observed for brain high‐affinity amino acid uptake systems. Both L‐glutamic acid (Glu) and γ‐aminobutyric acid (GABA) uptake systems were detectable by day 3 in postnatal forebrain mRNA and became progressively enriched during the next 2 weeks of forebrain development. In contrast, brainstem Glu and GABA transporter enrichment was 60–70% of adult values by day 3 and exceeded adult levels by day 10. Parallel determinations of L‐glutamic acid decarboxylase mRNA abundance during development argue for distinct regulatory influences on mRNAs directing transmitter synthesis and reuptake. Glycine uptake could not be detected at any point of forebrain development and exhibited a gradual postnatal rise to adult levels over the first 3 postnatal weeks of brainstem development. Uptake studies conducted with well‐characterized inhibitors of Glu, GABA, dopamine, and choline transport (D‐aspartate, nipecotic acid, nomifensine, and hemicholinium‐3, respectively) revealed that oocyte transporters encoded by adult rat brain mRNAs retained antagonist sensitivities exhibited by in vitro brain preparations. In addition, a differential regional sensitivity to the Glu transport antagonist dihydrokainate (1 mM) was observed, lending support to previous reports of region‐specific Glu transporter subtypes. To determine the structural diversity present among brain transporter mRNAs, poly(A)+ RNA was size‐fractionated on linear (10–31%) sucrose density gradients prior to oocyte injection. These experiments revealed two mRNA size classes (2.4–3.0 kb, 4.0–4.5 kb) independently capable of directing the synthesis of Glu, GABA, and glycine transporters. In regions other than the cerebellum, Glu and GABA transporter activities migrated as single, yet distinct, peaks of 4.0–4.5 kb. In contrast, both Glu and GABA transporters exhibited major peaks of activity at 2.5–3.0 kb with size‐fractionated cerebellar mRNA. Brainstem glycine uptake exhibited a broad sedimentation profile, with peaks apparent at 2.4 and 4.0 kb. Taken together, these findings indicate previously unappreciated complexity in mRNA structure and regulation which underlies the expression of amino acid neurotransmitter uptake systems in the rodent CNS.
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影响因子：
56.9
作者：
GUASTELLA, J;NELSON, N;KANNER, BI
通讯作者：
KANNER, BI
大鼠脑和脊髓中甘氨酸受体及其信使 RNA 的异质性。
DOI：
10.1126/science.2845580
发表时间：
1988
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Akagi,H;Miledi,R
通讯作者：
Miledi,R
DOI：
10.1073/pnas.86.15.5748
发表时间：
1989-08-01
影响因子：
11.1
作者：
HEDIGER, MA;TURK, E;WRIGHT, EM
通讯作者：
WRIGHT, EM
快速印迹：固定细胞中的 DNA 和 RNA。
影响因子：
--
作者：
Costanzi,C;Gillespie,D
通讯作者：
Gillespie,D
影响因子：
5.3
作者：
Otis, TS;Wu, YC;Trussell, LO
通讯作者：
Trussell, LO