Woodruff Michael L, Sampath A P, Matthews Hugh R, Krasnoperova N V, Lem J, Fain Gordon L
Department of Physiological Science, University of California, Los Angeles, CA 90095-1606, USA.
J Physiol. 2002 Aug 1;542(Pt 3):843-54. doi: 10.1113/jphysiol.2001.013987.
A 10 microm spot of argon laser light was focused onto the outer segments of intact mouse rods loaded with fluo-3, fluo-4 or fluo-5F, to estimate dark, resting free Ca(2+) concentration (Ca(2+)) and changes in Ca(2+) upon illumination. Dye concentration was adjusted to preserve the normal physiology of the rod, and the laser intensity was selected to minimise bleaching of the fluorescent dye. Wild-type mouse rods illuminated continuously with laser light showed a progressive decrease in fluorescence well fitted by two exponentials with mean time constants of 154 and 540 ms. Rods from transducin alpha-subunit knock-out (Tralpha-/-) animals showed no light-dependent decline in fluorescence but exhibited an initial rapid component of fluorescence increase which could be fitted with a single exponential (tau~1-4 ms). This fluorescence increase was triggered by rhodopsin bleaching, since its amplitude was reduced by pre-exposure to bright bleaching light and its time constant decreased with increasing laser intensity. The rapid component was however unaffected by incorporation of the calcium chelator BAPTA and seemed therefore not to reflect an actual increase in Ca(2+). A similar rapid increase in fluorescence was also seen in the rods of wild-type mice just preceding the fall in fluorescence produced by the light-dependent decrease in Ca(2+). Dissociation constants were measured in vitro for fluo-3, fluo-4 and fluo-5F with and without 1 mM Mg(2+) from 20 to 37 degrees C. All three dyes showed a strong temperature dependence, with the dissociation constant changing by a factor of 3-4 over this range. Values at 37 degrees C were used to estimate absolute levels of rod Ca(2+). All three dyes gave similar values for Ca(2+) in wild-type rods of 250 +/- 20 nM in darkness and 23 +/- 2 nM after exposure to saturating light. There was no significant difference in dark Ca(2+) between wild-type and Tralpha-/- animals.
将氩激光的一个10微米光斑聚焦到装载有Fluo-3、Fluo-4或Fluo-5F的完整小鼠视杆细胞的外段上，以估计黑暗中静止的游离Ca(2+)浓度（Ca(2+)）以及光照时Ca(2+)的变化。调整染料浓度以维持视杆细胞的正常生理功能，并选择激光强度以尽量减少荧光染料的漂白。持续用激光照射的野生型小鼠视杆细胞显示荧光逐渐下降，用两个指数函数拟合效果良好，平均时间常数分别为154和540毫秒。转导蛋白α亚基敲除（Tralpha-/-）动物的视杆细胞在光照下荧光没有依赖性下降，但显示出荧光增加的初始快速成分，可用单个指数函数拟合（τ~1-4毫秒）。这种荧光增加是由视紫红质漂白引发的，因为其幅度因预先暴露于强光漂白而降低，且其时间常数随激光强度增加而减小。然而，快速成分不受钙螯合剂BAPTA掺入的影响，因此似乎不反映Ca(2+)的实际增加。在野生型小鼠视杆细胞中，在Ca(2+)因光照依赖性下降而导致荧光下降之前，也观察到类似的荧光快速增加。在20至37摄氏度下，在有和没有1 mM Mg(2+)的情况下，体外测量了Fluo-3、Fluo-4和Fluo-5F的解离常数。所有三种染料都表现出强烈的温度依赖性，在此温度范围内解离常数变化3-4倍。使用37摄氏度时的值来估计视杆细胞Ca(2+)的绝对水平。在黑暗中，所有三种染料在野生型视杆细胞中给出的Ca(2+)值相似，为250±20 nM，在暴露于饱和光后为23±2 nM。野生型和Tralpha-/-动物在黑暗中的Ca(2+)没有显著差异。
