The regulation of adenylyl cyclase activity (measured as the production of labeled cyclic-AMP (cAMP) from labeled ATP) in frog rod outer segment photoreceptor membranes has been studied. The activity was found to be more than 20 times higher in fully dark-adapted than in bleached material. A number of experimental parameters must be carefully regulated to obtain this large ratio of dark to light activities. The disrupted rod outer segments must not be washed, since a soluble factor is essential for inhibition of activity by light. The photoreceptor membranes should be assayed for adenylyl cyclase immediately after preparation since the specific activity and light-dark differences are substantially reduced on storage at 4°C. Mechanical trauma (homogenization or sonication) causes some decrease in dark activity and a lessening of sensitivity to illumination. Preparation and assay of the dark material must be carried out using infrared light since there is a marked disproportionality between photic bleaching of rhodopsin and loss of cyclase activity.

Early studies of vertebrate rod outer segment cyclic nucleotide metabolism indicated that illumination of photoreceptor membranes causes a marked reduction in apparent cyclic AMP production. Current studies confirm these observations and explain light effects in terms of an unanticipated, ATP-dependent activation of phosphodiesterase. Here we present recent progress in our understanding of the components and mechanisms of this light dependent activation. We also describe the characteristics and substrate preference (cyclic GMP) of the photoreceptor phosphodiesterase, and a suggested model for the role of cyclic nucleotides in photoreceptor physiology.

Regulation of cyclic nucleotide concentrations in rod outer segments (Rana pipiens) has been further examined. The present studies show that illumination markedly diminishes the concentration of cyclic nucleotides in suspensions of photoreceptor membranes, but the locus of regulation is cyclic nucleotide phosphodiesterase (EC 3.1.4.c) (light-stimulated) and not adenylate cyclase. There is a marked disproportionality between bleaching of rhodopsin and stimulation of phosphodiesterase. Bleaching only 0.6% of the rhodopsin produces half the stimulation produced by bleaching 100% of the rhodopsin. The process of activation of phosphodiesterase by light is in two steps, a light-dependent step followed by an ATP-dependent step. Illumination (in the absence of ATP) produces a trypsin-resistant, heat-labile, macromolecular stimulator. In the presence of 0.75 mM ATP (GTP or ITP) this stimulator produces a greater than 5-fold increases in the V(max) of photoreceptor phosphodiesterase without changing the K(m). At physiological substrate concentrations (10(-7) M) the rate of hydrolysis of cyclic GMP is 23 times greater than that of cyclic AMP. The light-produced stimulator appears unique to the photoreceptor membranes and does not activate phosphodiesterase in other tissues.