Responses of the Circadian System in the Aplysia Eye to Inhibitors of Protein Synthesis

Protein synthesis seems to be a general requirement for circadian timing. Defining the time period when inhibition of protein synthesis changes the phase of the biological clock may help identify proteins that are involved in the molecular mechanism of circadian timing. Rothman and Strumwasser (1976), Jacklet (1977), and Lotshaw and Jacklet (1986) gen erated phase response curves (PRCs) for relatively long pulses (6 hr) of anisomycin and puromycin administered to Aplysia eyes. Using somewhat different conditions, we generated a 4-hr anisomycin PRC from Aplysia eyes and found that our anisomycin PRC was similar to that previously described by Lotshaw and Jacklet (1986). We studied recovery of protein synthesis after 1-hr and 6-hr anisomycin treatments and found recovery to be very slow; from 8 to 12 hr appeared to be required for full recovery after anisomycin. Slow recovery occurred when eyes were treated either in buffered artificial seawater or in enriched culture media. Because of the slow recovery after anisomycin, it is difficult to infer accurately from the anisomycin PRC when protein synthesis is important. To identify an inhibitor whose effect reverses quickly, we studied recovery from inhibi tion of protein synthesis after emetine, L-O-methylthreonine, and cycloheximide. Both eme tine and L-O-methylthreonine seemed to reverse no faster than anisomycin, but cycloheximide reversed faster than all the other inhibitors. Cycloheximide (10 mM, 1 hr) produced 89% inhibition of [³H] leucine incorporation, and within 3 hr after removal of cycloheximide, the recovery was 85%. A PRC was obtained using 1-hr treatments of cycloheximide (10 mM). Cycloheximide did not significantly phase-shift from circadian time (CT) 8 to CT 20, and cycloheximide delayed (by about 1 hr or less) the circadian rhythm from CT 20 to CT 8. The cycloheximide PRC was not due to different kinetics of recovery at different phases, as evidenced by similar recovery times when recovery from inhibition by cycloheximide was measured at two phases (a phase when cycloheximide produced no phase shift and a phase when cycloheximide delayed the rhythm).