Light regulation of adenosine 3′, 5′ cyclic monophosphate levels in vertebrate photoreceptors

Abstract

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. This disproportionality was further studied by mixing dark and totally bleached material in various proportions. With as little as 2% of the material bleached the cyclase activity is the same as in fully bleached material. The inhibitory action of the bleached material is destroyed by heating but is not affected by dialysis or treatment with tryspin. If the photoreceptor material is fractionated by sedimentation at 60 000 × g and 200 000 × g, the rhodopsin and adenylyl cyclase are primarily in the 60 000 × g pellet. The inhibitor is present in both pellets but not in the high speed supernatant. However, the high speed supernatant is essential for action of the inhibitor on unbleached material. Bleached (but not dark) photoreceptor material was found to inhibit cyclase in brain, liver and melanoma. Recent work carried out after presentation of the above findings has indicated that although the activity of cAMP phosphodiesterase is not affected by light under the usual assay conditions, in the presence of ATP light produces a marked stimulation of this activity. This stimulation of phosphodiesterase exhibits many of the characteristics previously attributed to the inhibition of cyclase and accounts completely or in large part for the apparent light dark differences in cyclase activity. At present the evidence points to phosphodiesterase as the main locus of light regulation of cAMP concentrations in photoreceptors, though a role for the light regulation of cyclase activity is not excluded.