| Abstract: | The metabolism of photoreceptor cGMP and the relationship of its light-sensitive regulation to rhodopsin photoisomerization and to the photoreceptor electrical response was examined in isolated, intact rabbit retinas. The dynamics of cGMP metabolism were assessed by measuring the rate of 18O incorporation from 18O-water into the alpha-phosphoryls of the guanine nucleotides. The photoreceptor electrical response was determined by measuring the aspartate-isolated mass receptor potential. Basal cGMP flux in dark-adapted retinas was 33 pmol cGMP X mg protein-1 X s-1 which translates into a metabolic rate in the rod outer segment (ROS) of 1.7 mM/min in ATP equivalents. Photic stimulation increased this flux as much as 4.5-fold. With continuous illumination, increasing intensity caused increments in cGMP metabolic flux to a maximum of 4.5-fold, with corresponding increases in the electrical response over the same 3-log unit intensity range. Tight coupling between activation of guanylate cyclase and phosphodiesterase was indicated by either no changes in cGMP steady state concentrations or relatively small fluctuations represented by increases of 50% at lower light intensities and a 12% decrease at one of the highest intensities. A stoichiometry of about 10,000 molecules of cGMP generated and hydrolyzed per photon absorbed was calculated for the lowest light intensity when the increment in cGMP metabolic flux per photon was maximal. Flashing light caused an increase in flux in proportion to frequency up to 1 Hz and a nearly proportional increase in the voltage time integral of the electrical response up to 0.5 Hz. This indicates that the temporal resolution, or "on"/"off" rate, of the cGMP metabolic response was as fast or faster than the temporal resolution of the electrical response. The concentration of cGMP remained relatively stable in spite of the marked acceleration of cGMP flux that occurred over the 32-fold range of frequencies tested. Taken together these results show that the light-accelerated rate of cGMP synthesis tightly coupled to hydrolysis becomes a primary energy-utilizing system in the photoreceptor and represents a response that fulfills certain of the fundamental criteria required of a metabolic event playing an essential role in phototransduction. |
