GTP hydrolysis in intact rod outer segments and the transmitter cycle in visual excitation

Abstract

THE light-sensitive dark current of vertebrate retinal rods is the main metabolic load on the receptor cells, turning over the cytoplasmic Na⁺ every 1–6 min (refs 1–4) and the cytoplasmic ATP as often as every 20 s (refs 5,6). However, a second process in the rod outer segment (ROS), the transmitter cycle, probably demands metabolic free energy during visual excitation. Photons absorbed in the intracellular rod disks reduce the dark current traversing the nearby plasma membrane by altering the cytoplasmic level of a diffusible intracellular transmitter substance^7–10 that may be free Ca²⁺ (refs 11–14). As tens to thousands of transmitter particles must be released and removed in a single photon response^15–17, the transmitter cycle should cause fast metabolic events in ROSs even when they are isolated from those parts of the receptor cells that sustain the dark current. We report here that light causes ROSs to destroy GTP and form GDP at a rate sufficient to meet the predicted requirement of the transmitter cycle. Briefly, ROSs with intact plasma membranes and normal cytoplasmic nucleotide content were isolated within a few seconds from live frog retinas; aliquots of the ROS suspension were exposed to flashes of light, and the cytoplasmic levels of ROS nucleotides monitored as a function of time. The experimental procedure permits changes in cytoplasmic nucleotides in ROS to be distinguished from those involving nucleotides in the suspending medium or bound to rod disk membranes that are not enclosed within an intact plasma membrane.