Light-stimulated release of [35S]taurine from frog retinal rod outer segments
Light-stimulated release of [35S]taurine from frog retinal rod outer segments
复制标题
青蛙视网膜杆外节光刺激释放[35S]牛磺酸
DOI：
10.1016/0006-8993(77)91065-4
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发表时间：
1977
期刊：
影响因子：
2.9
通讯作者：
H. Pasantes‐morales
中科院分区：
文献类型：
--
作者：
R. Salceda;A. López;H. Pasantes‐morales
Tau-ne以特别高的浓度存在于脊椎动物视网膜中，其中它占游离氨基酸总库的40-50% 18，19。它在视网膜功能中的作用尚未阐明，尽管它已被假定为抑制性神经递质17。牛磺酸在视网膜内的分布可以通过直接测量显微解剖分离的视网膜层来确定。xs、e6或间接通过跟踪自发或实验诱导的变性后视网膜中发生的牛磺酸水平的变化。eT.，~这些研究已经清楚地证实，视网膜中的大部分牛磺酸集中在光感受器中。此外，牛磺酸似乎是必要的维持光感受器的完整性，因为这些结构退化的猫作为一个结果，减少牛磺酸摄入量xo。这些结果表明，牛磺酸在光感受器中发挥的作用不同于神经递质的作用，因为现有证据表明牛磺酸对神经元活动具有抑制作用15，而光感受器释放的神经递质被认为是兴奋性物质。我们以前已经证明了在光的影响下，鸡视网膜的牛磺酸流出量增加。视网膜结构负责这个lightevoked释放尚未确定。在试图本地化这些网站，在目前的工作中，我们已经研究了[35 S]牛磺酸从离体青蛙视网膜杆外节（ROS）的释放。在该物种中，活性氧可以通过相对温和的方法分离，并且似乎比从其他物种中获得的活性氧保存得更好1，4。通过在含氧的等渗Krebs-碳酸氢盐培养基中轻轻刷视网膜4来分离ROS，所述培养基含有（mM）NaCl，118; KCl，4.7; CaCl 2，2.5; MgSO 4，1.17; KHePO 4，1.2; NaHCO 3，25;葡萄糖，5.6。通过低速离心（900× g，10 μ m，4 ℃）收集ROS并重悬于Krebs培养基中。每次测定使用来自20个视网膜的ROS沉淀（0.5-1 mg蛋白质）。通过在0.1M磷酸盐缓冲液（pH7.0）中的3-/o戊二醛中固定来制备用于电子显微镜的ROS级分。在相同的缓冲液中用2~ o OsO_4后固定。将样品包埋在Epon 812中，并在Jeol 100 B电子显微镜中观察在Reichert超薄切片机UMO 3中获得的薄切片。
Tau~ ne is present in particularly high concentrations in vertebrate retina, where it accounts for 40-50% of the total pool of free amino acids18, 19. Its role in retinal function has not been yet elucidated, although it has been postulated as an inhibitory neurotransmitter 17. The intraretinal distribution of taurine has been determined either by direct measurements in retinal layers separated by microdissectionle. xs, e6 or indirectly by following changes in taurine levels which occur in retina after spontaneous or experimentally-induced degenerationsS. eT.,~ l these studies have clearly established that most of taurine in retina is concentrated in photoreceptors. Moreover, taurine appears to be necessary for maintaining the integrity of photoreceptors, since these structures degenerate in cats as a consequence of a decreased taurine intake xo. These results suggest that taurine plays a role in photoreceptors different from that of a neurotransmitter since available evidence indicate that taurine has an inhibitory effect on neuronal activity 15, whereas the neurotransmitter released by photoreceptors is believed to be an excitatory substance e. We have previously demonstrated an increase in taurine efllux from the chick retina under the influence of light 20. The retinal structures responsible for this lightevoked release have not yet been identified. In an attempt to localize these sites, in the present work we have studied the release of [35S] taurine from isolated frog retinal rod outer segments (ROS). In this species, ROS may be isolated by relatively gentle procedures and appear to be preserved much better than those obtained from other species 1, 4.Frogs were dark adapted for 1-2 h and the retinas dissected under dim red light. ROS were detached by gentle brushing of retinas 4 in an oxygenated, isotonic Krebs-bicarbonate medium containing (raM) NaCl, ll8; KCI, 4.7; CaClz, 2.5; MgSO4, 1.17; KHePO4, 1.2; NaHCOa, 25; glucose, 5.6. ROS were collected by low speed centrifugation (900× g, 10 rain, 4 C) and resuspended in the Krebs medium. The ROS pellet from 20 retinas (0.5-1 mg of protein) was used for each determination. ROS fractions were prepared for electron microscopy by fixation in 3~/o glutaraldehyde in 0.1 M phosphate buffer, pH 7.~. and postfixation in 2~ o OsO4 in t~ e same buffer. Samples were embedded in Epon 812 and thin sections obtained in a Reichert ultramicrotome UMO3 were observed in a Jeol 100B electron microscope.