Spectral and Metabolic Basis of Visual Responses

描述（由申请人提供）：我们将研究在明亮光线照射期间和之后调节视锥细胞和视杆细胞敏感性和反应性恢复的机制，这些细胞可以对巨大强度范围内的光做出反应。为此，我们将对受体电流进行电生理记录，同时对视色素进行微分光光度测量，并对孤立杆状体和视锥状体中视网膜到视黄醇的还原进行微荧光测量。测量将在黑暗中进行漂白前后和外源性视网膜及其类似物的掺入。本文将对以下假设进行验证：(1)不同视锥细胞类型的视蛋白和视网膜上的假体基团之间的关键非共价相互作用通过不同的机制调节光激活视锥蛋白（Meta II）的衰变。这些机制有助于影响在强光照射期间和之后发生的暗电流和闪光反应的快速恢复。我们将关联Meta II衰变、视网膜生成和漂白后受体反应恢复的影响，以及随后将各种天然和模拟类维生素a纳入完整细胞的视觉色素中，以确定视网膜上调节反应恢复的相互作用的特定位点。(2)色素再生过程中锥体结合位点的非共价占用有助于在黑暗适应过程中迅速恢复敏感性。这种占用在杆中有相反的效果。我们将确定相对于暴露于类维生素a的视锥细胞和视杆细胞中色素再生的敏感性恢复的时间过程，这些类维生素a要么只是占据视蛋白结合位点，要么与视蛋白结合并随后形成视觉色素。我们将使用不同的类视黄酮类似物来确定类视黄酮必须具有的基本特征，以加速/阻碍这些细胞在黑暗适应期间的敏感性恢复。(3) NADPH使全反式视网膜还原为全反式视黄醇，视蛋白释放视黄醇，限制了视色素的再生和视敏感性的恢复。我们将在透析前后用含有NADPH或其代谢前体（葡萄糖-6磷酸，ATP等）的溶液测量漂白棒外段局部灵敏度的恢复，以确定是否必须发生视网膜还原为视黄醇才能恢复暗电流和闪光灵敏度，并观察这种反应恢复是否会受到OS中NADPH供应的限制。摘要：在明亮的光线照射期间和之后，视杆细胞和视锥细胞的敏感性和反应性的恢复对我们感知光环境变化的能力很重要。我们建议进行生理和生化实验，以加深我们对这种能力背后的细胞机制的理解。我们的最终目标是了解正常和疾病相关条件下的视网膜功能。

DESCRIPTION (provided by applicant): We will investigate the mechanisms that regulate recovery of sensitivity and responsiveness of cones and rods during and following exposure to bright light which allow these cells to respond to light over an immense intensity range. To do this we will make electrophysiological recordings of receptor current together with microspectrophotometric measurements of visual pigment and microfluorometric measurements of retinal to retinol reduction in solitary rods and cones. Measurements will be made in darkness before and following bleaching and following exogenous incorporation of retinal and its analogs. The following hypotheses will be tested: (1) Critical non-covalent interactions between prosthetic groups on retinal and opsin in different cone types regulate the decay of light-activated cone opsin (Meta II) by different mechanisms. These mechanisms assist to affect the rapid recovery of dark current and flash responses that occur during and following bright light exposure. We shall correlate effects on Meta II decay, retinal production, and receptor response recovery following bleaching and subsequent incorporation of a variety of native and analog retinoids into the visual pigment of intact cells to determine specific sites of interaction that on retinal that regulate response recovery. (2) Non-covalent occupancy of the cone binding site during pigment regeneration assists in the very rapid recovery of sensitivity that occurs during dark-adaptation. Such occupancy has the opposite effects in rods. We shall determine the time course of sensitivity recovery relative to that of pigment regeneration in cones and rods that have been exposed to retinoids that either simply occupy the opsin binding site or bind to opsin and subsequently form a visual pigment. We will use different retinoid analogs to determine the essential features that a retinoid must have to accelerate/impede sensitivity recovery during dark adaptation in these cells. (3) The reduction of all-trans retinal to all-trans retinol by NADPH, and subsequent retinol release from opsin limit the regeneration of visual pigment and recovery of visual sensitivity in vertebrate rods. We shall measure the recovery of sensitivity locally within outer segments of bleached rods before and following dialysis with solutions containing NADPH or its metabolic precursors (glucose-6 phosphate, ATP, etc.) to determine whether the reduction of retinal to retinol must occur to allow recovery of dark current and flash sensitivity, and to see if this response recovery can be limited by the supply of NADPH in the OS. Lay Summary: Recovery of sensitivity and responsiveness of rods and cones during and following bright light exposure is important for our ability to sense changes in our light environment. We propose physiological and biochemical experiments designed to increase our understanding of the cellular mechanisms that underlie this ability. Our ultimate goal is to understand retinal function under normal and disease related conditions.