Ratto G M, Payne R, Owen W G, Tsien R Y
Department of Biophysics and Medical Physics, University of California, Berkeley 94720.
J Neurosci. 1988 Sep;8(9):3240-6. doi: 10.1523/JNEUROSCI.08-09-03240.1988.
The use of fluorescent indicators such as fura-2 (Grynkiewicz et al., 1985) to measure the cytosolic free calcium activity in retinal rods is complicated by the rods' sensitivity both to the fluorescence and to the light that excites it. By stimulating fluorescence from large numbers of rods in whole, loaded retinas and averaging repeated measurements, however, we have been able to monitor changes in free [Ca2+]i during exposure to nonsaturating lights under physiological conditions. Retinas, isolated from the bullfrog Rana catesbeiana, were loaded with fura-2 by incubation and mounted, receptor-side up, in a perfusion chamber placed on the stage of a specially designed apparatus. A step of light delivered from above, whose wavelength alternated between 340 and 380 nm every 110 msec, excited fluorescence from 24 mm2 of retina and evoked a light response (the aspartate-isolated pIII component of the electroretinogram--ERG). By comparing the fluorescence intensities excited by the 2 wavelengths (corrected for background and dark-noise), the free [Ca2+]i of the rod outer segment was determined. In darkness, the [Ca2+]i of the outer segment was found to be approximately 220 nM. A bright light caused it to fall exponentially to approximately 140 nM, with a time constant of approximately 1.6 sec. The value of [Ca2+]i at the onset of illumination was independent of stimulus intensity over a 2 log-unit range, and in all cases the fall was monotonic. After terminating the illumination, [Ca2+]i rose again to its time-zero value.(ABSTRACT TRUNCATED AT 250 WORDS)
使用诸如fura-2（格林凯维茨等人，1985年）之类的荧光指示剂来测量视网膜视杆细胞中的胞质游离钙活性，会因视杆细胞对荧光及其激发光的敏感性而变得复杂。然而，通过刺激完整加载视网膜中大量视杆细胞的荧光并对重复测量进行平均，我们能够在生理条件下监测非饱和光照射期间游离[Ca2+]i的变化。从牛蛙北美牛蛙分离出的视网膜，通过孵育加载fura-2，并将其受体面朝上安装在放置在专门设计仪器载物台上的灌注室中。从上方发出的光脉冲，其波长每110毫秒在340和380纳米之间交替，激发来自24平方毫米视网膜的荧光，并诱发光反应（视网膜电图——ERG的天冬氨酸分离的pIII成分）。通过比较两种波长激发的荧光强度（校正背景和暗噪声），确定视杆细胞外段的游离[Ca2+]i。在黑暗中，外段的[Ca2+]i约为220纳摩尔。强光使其呈指数下降至约140纳摩尔，时间常数约为1.6秒。光照开始时[Ca2+]i的值在2个对数单位范围内与刺激强度无关，并且在所有情况下下降都是单调的。终止光照后，[Ca2+]i再次上升至其初始值。（摘要截断于250字）
