CYCLIC NUCLEOTIDES IN CONE METABOLISM AND DISEASE

锥体代谢和疾病中的环状核苷酸

基本信息

批准号：
3483955
负责人：
DEBORA B FARBER
金额：
$ 28.3万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
1978
资助国家：
美国
起止时间：
1978-08-01 至 1996-07-31
项目状态：
已结题

项目摘要

The objective is to investigate the metabolism of cyclic nucleotides in normal and diseased visual cells with a special emphasis in the less biochemically characterized cones. In addition, we wish to investigate the mechanism by which elevated cyclic nucleotide levels affect the viability of photoreceptor cells in some retinal degenerations. Our work has suggested that in contrast to the function that cGMP plays in rods, cAMP may be more important than cGMP in cone-dominant retinas since cAMP concentration is higher, cAMP levels are decreased by light, and elevated levels of cAMP seem to be more toxic and cause the specific degeneration of cones. However, to establish that cones and rods utilize different cyclic nucleotides as intracellular regulators it is necessary to study isolated cone photoreceptors because the rest of the retina interferes with its own cAMP metabolism. Using isolated cone cells we will study their cyclic nucleotide content, activities, kinetics and regulation of cyclic nucleotide synthetic and degradative enzymes, and investigate if cyclic nucleotide action is modulated by the phosphorylation of specific proteins. We will determine whether light reduces cyclic nucleotide levels and whether activities of the metabolic enzymes are coupled to bleaching of the visual pigment. The same enzyme systems will also be studied in developing cone-like photoreceptors grown in culture, obtained from dissociated retinas of chick embryos. In addition, we will continue to collect baseline data on cyclic nucleotide metabolism in normal human retina to be used for comparison with results from retinas affected with Retinitis Pigmentosa, macular degeneration or other retinal diseases, whenever these retinas become available. To gain insight into how elevated cyclic nucleotide levels participate in visual cell degeneration, we will study first degeneration of rods in the rd mouse, since we have several probes on hand and clues about what components need to be analyzed. In the future, when more is known about cones and probes are developed, we will focus our attention on cone degeneration. We will use antibodies and molecular biology methods to measure concentration and synthesis of cGMP-phosphodiesterase (PDE), the activity of which is abnormal in rd retina, and mRNAs that code for G-protein and rhodopsin, which are needed for PDE activation. We will also clone cDNAs for PDE and rhodopsin from control and rd retinas. Analyses of the cDNA sequences may reveal anomalies associated with the cause of the rd mouse disease and further our understanding of photoreceptor degeneration.

目的是研究环核苷酸的代谢正常和患病的视觉细胞，特别强调较少的视觉细胞生化特征锥体。此外，我们希望调查环核苷酸水平升高影响活力的机制某些视网膜变性中感光细胞的变化。我们的工作有表明与 cGMP 在视杆细胞中发挥的功能相反，cAMP 在视锥细胞视网膜中，cAMP 可能比 cGMP 更重要浓度越高，cAMP 水平因光而降低，并且升高 cAMP 水平似乎毒性更大，会导致视锥细胞。然而，要确定锥体和杆体利用不同的循环核苷酸作为细胞内调节剂，有必要研究分离的视锥细胞感光器，因为视网膜的其余部分会干扰其自身的 cAMP 代谢。使用分离的视锥细胞，我们将研究它们的循环核苷酸含量、活性、动力学和循环调节核苷酸合成和降解酶，并研究是否循环核苷酸的作用是通过特定的磷酸化来调节的蛋白质。我们将确定光是否会降低环核苷酸水平以及代谢酶的活性是否与漂白有关视觉色素。相同的酶系统也将在在培养物中生长的发育中的锥状光感受器，从鸡胚视网膜的分离。此外，我们还将继续收集正常人环核苷酸代谢的基线数据视网膜用于与受影响的视网膜的结果进行比较视网膜色素变性、黄斑变性或其他视网膜疾病，每当这些视网膜可用时。深入了解如何提升环核苷酸水平参与视觉细胞变性，我们将研究 rd 小鼠视杆细胞的第一次退化，因为我们有几个现有的探针和需要分析哪些组件的线索。在未来，当人们对锥体和探针有更多了解时，我们将将我们的注意力集中在视锥细胞退化上。我们将使用抗体和测量浓度和合成的分子生物学方法 cGMP-磷酸二酯酶（PDE），其活性在rd中异常视网膜，以及编码 G 蛋白和视紫红质的 mRNA，这些都是必需的用于 PDE 激活。我们还将克隆 PDE 和视紫红质的 cDNA 控制和RD视网膜。 cDNA 序列分析可能揭示与 rd 小鼠疾病的病因相关的异常现象以及进一步我们的研究了解光感受器变性。