Kawamura S, Murakami M
Department of Physiology, Keio University School of Medicine, Tokyo, Japan.
J Gen Physiol. 1989 Oct;94(4):649-68. doi: 10.1085/jgp.94.4.649.
Cyclic GMP is the second messenger in phototransduction and regulates the photoreceptor current. In the present work, we tried to understand the regulation mechanism of cytoplasmic cGMP levels in frog photoreceptors by measuring the photoreceptor current using a truncated rod outer segment (tROS) preparation. Since exogenously applied substance diffuses into tROS from the truncated end, we could examine the biochemical reactions relating to the cGMP metabolism by manipulating the cytoplasmic chemical condition. In tROS, exogenously applied GTP produced a dark current whose amplitude was half-maximal at approximately 0.4 mM GTP. The conductance for this current was suppressed by light in a fashion similar to when it is activated by cGMP. In addition, no current was produced in the absence of Mg2+, which is known to be necessary for the guanylate cyclase activity. These results indicate that guanylate cyclase was present in tROS and synthesized cGMP from exogenously applied GTP. The enzyme activity was distributed throughout the rod outer segment. The amount of synthesized cGMP increased as the cytoplasmic Ca2+ concentration of tROS decreased, which indicated the activation of guanylate cyclase at low Ca2+ concentrations. Half-maximal effect of Ca2+ was observed at approximately 100 nM. tROS contained the proteins involved in the phototransduction mechanism and therefore, we could examine the regulation of the light response waveform by Ca2+. At low Ca2+ concentrations, the time course of the light response was speeded up probably because cGMP recovery was facilitated by activation of the cyclase. Then, if the cytoplasmic Ca2+ concentration of a photoreceptor decreases during light stimulation, the Ca2+ decrease may explain the acceleration of the light response during light adaptation. In tROS, however, we did observe an acceleration during repetitive light flashes when the cytoplasmic Ca2+ concentration increased during the stimulation. This result suggests the presence of an additional light-dependent mechanism that is responsible for the acceleration of the light response during light adaptation.
环磷酸鸟苷（cGMP）是光转导过程中的第二信使，可调节光感受器电流。在本研究中，我们试图通过使用截短的视杆外段（tROS）制剂测量光感受器电流，来了解青蛙光感受器中细胞质cGMP水平的调节机制。由于外源施加的物质从截短端扩散到tROS中，我们可以通过操纵细胞质化学条件来研究与cGMP代谢相关的生化反应。在tROS中，外源施加的鸟苷三磷酸（GTP）产生了一种暗电流，其幅度在约0.4 mM GTP时达到最大值的一半。该电流的电导被光抑制，其方式类似于被cGMP激活时的情况。此外，在没有镁离子（Mg2+）的情况下不产生电流，已知镁离子是鸟苷酸环化酶活性所必需的。这些结果表明鸟苷酸环化酶存在于tROS中，并从外源施加的GTP合成cGMP。该酶活性分布在整个视杆外段。合成的cGMP量随着tROS细胞质钙离子（Ca2+）浓度的降低而增加，这表明在低Ca2+浓度下鸟苷酸环化酶被激活。在约100 nM时观察到Ca2+的半最大效应。tROS包含参与光转导机制的蛋白质，因此，我们可以研究Ca2+对光反应波形的调节。在低Ca2+浓度下，光反应的时间进程加快，可能是因为环化酶的激活促进了cGMP的恢复。那么，如果光感受器的细胞质Ca2+浓度在光刺激期间降低，Ca2+的降低可能解释了光适应期间光反应的加速。然而，在tROS中，当细胞质Ca2+浓度在刺激期间增加时，我们确实观察到在重复光闪烁期间有加速现象。这一结果表明存在一种额外的光依赖机制，该机制负责光适应期间光反应的加速。
