Requirement for protein synthesis in the regulation of a circadian rhythm by serotonin

Serotonin (5-HT, 5-hydroxytryptamine) regulates the phase of a circadian pacemaker located within the eye of Aplysia. We are attempting to define the cellular and biochemical events involved in the regulatory pathway through which serotonin acts. Previously, we have shown that an activation of adenylate cyclase and an increase in cAMP are events in the 5-HT phase-shifting pathway. In this paper, we examine the role of protein synthesis in mediating the effect of 5-HT and cAMP on the phase of the circadian rhythm. Exposure of eyes to anisomycin, an inhibitor of protein synthesis, completely blocked the advance shift in phase produced by 5-HT. Although anisomycin by itself can produce phase shifts, it did not affect the rhythm at the phases where the blocking experiments were performed. The specificity of action of anisomycin was investigated in two ways. First, deacetylanisomycin, an analogue of anisomycin that is inactive in inhibiting protein synthesis, did not affect the shift in phase produced by 5-HT. Second, anisomycin did not inhibit two other effects of 5-HT on the eye that also appear to be mediated by cAMP: an inhibition of spontaneous optic nerve activity and an increase in the photosensitivity of the eye. The step in the 5-HT phase-shifting pathway that is sensitive to anisomycin appears to occur after the cAMP step because anisomycin also inhibits the ability of 8-benzylthio-cAMP to shift the phase of the rhythm. We have also examined whether 5-HT directly regulates the synthesis of any proteins in the eye. Using two-dimensional gel electrophoresis, we have found that 5-HT appears to increase the synthesis of a protein with an apparent molecular weight of 67,000. Our results indicate that protein synthesis is necessary for 5-HT to shift the phase of the rhythm and that 5-HT appears to regulate the expression of at least one protein in the eye.