Regulation of cGMP levels by guanylate cyclase in truncated frog rod outer segments.
Regulation of cGMP levels by guanylate cyclase in truncated frog rod outer segments.
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DOI：
10.1085/jgp.94.4.649
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发表时间：
1989-10
期刊：
影响因子：
--
通讯作者：
Murakami M
中科院分区：
文献类型：
--
作者：
Kawamura S;Murakami M
环GMP是光传导中的第二信使，调节光感受器电流。在本工作中，我们试图了解细胞质cGMP水平的调节机制，在青蛙的光感受器通过测量光感受器电流使用截断杆外节（tROS）的准备。由于外源施加的物质从截短的末端扩散到tROS中，我们可以通过操纵细胞质化学条件来检查与cGMP代谢相关的生化反应。在tROS中，外源性应用GTP产生暗电流，其幅度在约0.4 mM GTP时为半最大值。该电流的电导被光抑制，其方式与cGMP激活时类似。此外，在不存在Mg 2+的情况下不产生电流，已知Mg 2+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，并从外源应用的GTP合成cGMP。酶活性分布在整个杆外节。cGMP的合成量随着tROS胞浆内Ca ~（2+）浓度的降低而增加，表明鸟苷酸环化酶在低Ca ~（2+）浓度时被激活。在约100 nM时观察到Ca 2+的半数最大效应。tROS含有参与光转导机制的蛋白质，因此，我们可以研究Ca 2+对光响应波形的调节。在低Ca 2+浓度下，光响应的时间过程加快，可能是因为环化酶的激活促进了cGMP的恢复。然后，如果在光刺激期间光感受器的细胞质Ca 2+浓度降低，则Ca 2+降低可以解释在光适应期间光响应的加速。然而，在tROS中，当细胞质Ca 2+浓度在刺激期间增加时，我们确实观察到重复闪光期间的加速。这一结果表明存在一种额外的光依赖性机制，该机制负责光适应期间光反应的加速。
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.