The distribution of amino acids within the rat retina
The distribution of amino acids within the rat retina
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大鼠视网膜内氨基酸的分布
DOI：
10.1111/j.1471-4159.1977.tb03938.x
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发表时间：
1977
影响因子：
4.7
通讯作者：
R. Lolley
中科院分区：
文献类型：
--
作者：
A. Kennedy;M. Neal;R. Lolley
视网膜内的神经元。如在CNS的其它区域中，可利用氨基酸GABA、甘氨酸、牛磺酸。谷氨酸和天冬氨酸2S突触传递物质。(For评论，见格雷厄姆，1974;尼尔，1976）。作为评估它们作为神经递质的可能作用的第一步，我们测量了这些物质的视网膜内分布。已知GABA和牛磺酸相对集中在视网膜内层（KLJRIYAMA等人[. 1968; GRAHAM等人，1970; GRAHAM，1974）和感光层（KENNEDY & VOADEK. 1974; ORR er a/.. 1976年）。但是关于其他假定的氨基酸递质的视网膜内分布的信息很少。在本研究中。我们已经测定了视网膜解剖学上确定的层中几种推定和非推定的"递质"氨基酸的水平，所述层是通过使用先前描述的方法获得的（GRAHAM等，1970年）。将白化病大鼠（Fischer CDJ品系）的光适应眼在杀死动物后摘除，并立即在用液氮冷却的异戊烷中冷冻。使用解剖显微镜。用手从冷冻干燥的切片中分离出以下层：（1）光感受器外节层和内节层，（2）光感受器细胞层，包括外节和突触终末，（3）水平细胞层，在外突触层和双极层中线处切开，（4）无长突细胞层，从双极细胞的中线延伸到内突触层的中部;（5）神经节细胞层，包括视神经纤维层。通过在一定比例的显微切片中对交替切片进行染色来检查样品的纯度和均匀性。合并各层的冷冻干燥样品（30 mg/60 mg），用75 Y乙醇提取氨基酸并蒸干。通过如前所述的双标记丹磺酰化技术（KENNEDY & VOADEN，1974）测定氨基酸。为每种氨基酸的一系列标准品绘制3H/14C（牛磺酸为3H/14S）图。并通过加入精确闪烁计数所需的最小量I4C内标来实现测定的高灵敏度。样品中测定的最低水平氨基酸的3H/14C-标记比率是空白的两倍以上。单个视网膜层和包含所有层的视网膜中的氨基酸浓度总结于表I中。发现冻干的整个视网膜的重量为新鲜组织重量的1702，当转换为新鲜重量当量时，结果与通过分析新鲜解剖的整个视网膜获得的结果一致（表I）。本研究中发现的氨基酸水平与先前报告的大鼠视网膜（NEAL，1976）相似，但牛磺酸除外，牛磺酸的水平约为先前报告水平的两倍。氨基酸在视网膜各层内似乎遵循三种分布模式：（1）
NEURONES within the retina. as in other areas of the CNS may utilize the amino acids GABA, glycine, taurine. glutamate and aspartate 2s synaptic transmitter substances. (For reviews, see GRAHAM, 1974; NEAL, 1976). As a first step in evaluating their possible roles as neurotransmitters, we have measured the intraretinal distribution of these substances. GABA and taurine are known to be relatively concentrated in the inner retina (KLJRIYAMA ef a[.. 1968; GRAHAM et a/., 1970; GRAHAM, 1974) and photoreceptor layer (KENNEDY & VOADEK. 1974; ORR er a/.. 1976) respectively. but there is little information available concerning the intraretinal distribution of other putative amino acid transmitters. In the present study. we have determined the level of several putative and non-putative ‘transmitter’ amino acids in anatomically-defined layers of the retina which were obtained by using methods previously described (GRAHAM et al.. 1970). Light-adapted eyes of albino rats (Fischer CDJ strain) were enucleated after killing the animal and immediately frozen in isopentane cooled with liquid nitrogen. Using a dissecting microscope. the following layers were micd i s s e c t e d by hand from the frozen-dried sections: (1) photoreceptor outer and inner segment layer, (2) photoreceptor cell layer which included outer segments and synaptic terminals, (3) horizontal cell layer, cut at the outer synaptic layer and at the midline of the bipolar layer, (4) amacrine cell layer which extended from the midline of the bipolar cells to the middle of the inner synaptic layers, and (5) ganglion cell layer which included the optic fibre layer. The purity and homogeneity of the samples were checked by staining alternate sections in a proportion of microsections. Frozen-dried samples of the respective layers were pooled (3Ck60mg) and the amino acids were extracted with 75Y, ethanol and evaporated to dryness. The amino acids were assayed by the double-label dansylation technique as described previously (KENNEDY & VOADEN, 1974). Plots of 3H/’4C (3H/’4S for taurine) were set up for a range of standards for each amino acid. and a high sensitivity for the assay was achieved by adding the minimum amount I4C internal standard required for accurate scintillation counting. 3H/’4C-label ratios for the lowest levels of amino acid determined in samples were more than double those of blanks. The concentrations of amino acids in the individual retinal layers and in retina containing all layers are summarized in Table I . The lyophilized whole retina was found to weigh 1702 of the fresh tissue weight and the results, when converted to fresh weight equivalents (Table I), agreed with those obtained by analysis of freshly dissected whole retinae. The amino-acid levels found in the present study are similar to those previously reported for the rat retina (NEAL, 1976) with the exception of taurine which was found to be approximately twice the level previously reported. The amino acids appear to follow three patterns of distribution within the layers of the retina: (1 ) those which