Visualization of cyclic nucleotide binding sites in the vertebrate retina by fluorescence microscopy

Cyclic nucleotides play a major role in cell signaling, especially in the nervous system. They act as cytoplasmic messengers in a wide range of physiological responses, but the spatial distribution of their sites of action within cells and tissues is not well-known. In the vertebrate retina, there is a class of well-characterized cGMP binding sites which control the permeability of cation channels in the rod outer segments (ROS), while cAMP is involved in several other systems in the inner retina. Biochemical studies of the cGMP-activated permeability in ROS have not distinguished between the subcellular compartments of disk and plasma membrane. By a new method using fluorescein-conjugated cyclic nucleotides, we have found strong cyclic GMP binding to the plasma membrane of the ROS, both on frozen sections of retina and in freshly isolated, leaky ROS. We also found a high density of cGMP binding sites on structures resembling the inner segment calycal processes. Little specific binding could be detected on the disk membranes or on any other retinal layer. In contrast, fluorescent cAMP did not label ROS, but gave a striking pattern of labeling on several deeper layers of the retina. These results suggest that the ROS plasma membrane has a much higher density of cGMP-controlled cation channels than the disk membranes, and point to other retinal layers where cAMP is likely to shape cellular responses. This method opens up novel morphological approaches to the study of cyclic nucleotide regulation.