Sechenov Institute for Evolutionary Physiology and Biochemistry, Russian Academy of Sciences , St. Petersburg, Russia.
Research Institute of Hygiene, Occupational Pathology and Human Ecology , St. Petersburg, Russia.
J Gen Physiol. 2024 Nov 4;156(11). doi: 10.1085/jgp.202313530. Epub 2024 Oct 22.
The phototransduction cascade enables the photoreceptor to detect light over a wide range of intensities without saturation. The main second messenger of the cascade is cGMP and the primary regulatory mechanism is calcium feedback. However, some experimental data suggest that cAMP may also play a role in regulating the phototransduction cascade, but this would require changes in cAMP on a time scale of seconds. Currently, there is a lack of data on the dynamics of changes in intracellular cAMP levels on this timescale. This is largely due to the specificity of the sensory modality of photoreceptors, which makes it practically impossible to use conventional experimental approaches based on fluorescence methods. In this study, we employed the method of rapid cryofixation of retinal samples after light stimulation and subsequent isolation of outer segment preparations. The study employed highly sensitive metabolomics approaches to measure levels of cAMP. Additionally, PKA activity was measured in the samples using a western blot. The results indicate that when exposed to near-saturating but still moderate light, cAMP levels increase transiently within the first second and then return to pre-stimulus levels. The increase in cAMP activates PKA, resulting in the phosphorylation of PKA-specific substrates in frog retinal outer segments.
光转导级联使光感受器能够在没有饱和的情况下检测到广泛强度范围内的光。级联的主要第二信使是 cGMP,主要的调节机制是钙反馈。然而,一些实验数据表明,cAMP 也可能在调节光转导级联中发挥作用,但这需要 cAMP 在秒级的时间尺度上发生变化。目前,关于在这个时间尺度上细胞内 cAMP 水平变化的动力学数据还很缺乏。这在很大程度上是由于光感受器的感觉模态的特异性,这使得基于荧光方法的常规实验方法在实际上变得不可能。在这项研究中,我们采用了在光刺激后快速冷冻视网膜样本并随后分离外节制剂的方法。该研究采用了高度敏感的代谢组学方法来测量 cAMP 水平。此外,还使用 Western blot 测量了样品中的 PKA 活性。结果表明,当暴露于接近饱和但仍适中的光下时,cAMP 水平在最初的一秒内短暂增加,然后恢复到刺激前的水平。cAMP 的增加激活了 PKA,导致青蛙视网膜外节中 PKA 特异性底物的磷酸化。