Circadian rhythms and environmental lighting regulate a number of endocrine and behavioral functions. Arguably, the best understood endocrine rhythm is that of the pineal gland, which secretes the hormone melatonin almost entirely at night. Melatonin is made from serotonin, in two steps. The first of these, is mediated by the enzyme serotonin N-acetyl transferase (SNAT). This is the key enzyme in the synthesis of melatonin, for its activity changes markedly between day and night and it is regulated in several ways. Unlike cells from rat pineal, dispersed cells from chick pineal remain rhythmic in their synthesis of melatonin, and responsive to light, in culture. In these cells, synthesis and activity of SNAT reflect cAMP and calcium stimuli, as well as "directly" constituting the "hands of the clock". Last year, we showed that the melatonin rhythm in chick pineal cells is driven by an endogenous clock that changes SNAT gene expression. We also found that much of the other regulation of SNAT enzyme activity (e.g. by forkolin and a second action of light) could not be accounted for by changes in mRNA, suggesting that it occurs posttranscriptionally. Work on SNAT regulation has continued this year (in collaboration with David Klein's lab) as we examined the regulation of SNAT protein levels directly. Enzyme activity is quite labile and protein levels can decline rapidly. Agents that increase enzyme activity, such as forskolin, increase SNAT protein levels and agents that decrease enzyme activity, such as light or norepinephrine, decrease protein levels. These results confirm the previous distinction between acute and clock-mediated regulation of SNAT and indicate that the clock acts primarily pretranslationally while other agents, act post-translationally. A new insight, gained this year, is that regulation of degradation appears to be important in these acute effects. Agents that interfere with the action of proteosomal proteases can increase, or prevent the decrease in, SNAT protein levels and activity. Perhaps acute changes in protein levels and activity reflect regulated changes in degradation rates. In addition, we have initiated a collaboration with Mark Rollag to explore the possibility that melanopsin (a novel photopigment he discovered in frog skin, whose homologous message we have found to be present in chick pineal cells) is the unknown photopigment that mediates photo-entrainment. We plan on using transfections to compare the effects of specific perturbations of this and other photopigment candidates. Finally, in attempting to identify the unknown signal transduction pathway from the photpigment to the clock, we have undertaken to determine whether the ERK, or related, pathways play a role in photoentrainment, after obtaining promising preliminary results at the pharmacologic level.