Protein Synthesis Requirement of the Aplysia Circadian Clock Tested by Active and Inactive Derivatives of the Inhibitor Anisomycin

The proper timing of physiological events is crucial for an harmonious relationship between an organism and its environment and equally important for establishing temporal order among the many physiological processes that maintain the organism. One of the important physiological timers is the circadian clock (Pittendrigh, 1976), which is endogenous to the organism and may be reset by an appropriate ‘time giving’ environmental stimulus. Timing by the clock confers the definite advantage of allowing the organism to anticipate environmental change. It is not surprising that circadian clocks are a basic property of all eucaryotes.

The question of whether the proper functioning of the circadian clock is dependent upon protein synthesis and therefore can be perturbed by protein synthesis inhibitors has been debated for more than a decade. A study (Feldman, 1967) showed the period length of the phototaxis rhythm of the flagellate Euglena was increased by the continuous application of cycloheximide. On the other hand, the effects of inhibitors on the clock of the dinoflagellate, Gonyaulax, were equivocal (Sweeney, 1969) and studies on the circadian rhythm of photosynthesis in the alga, Acetabularia (Sweeney, Tuffli & Rubin, 1967) suggested that the clock timing mechanism was independent of protein synthesis.

Support for the concept of required protein synthesis has come from recent experiments using protein synthesis inhibitors. In Acetabularia, a pulse of anisomycin or cycloheximide at low concentrations caused phase-dependent phase shifts of the O₂ evolution rhythm (Karakashian & Schweiger, 1976a, b). Phase-dependent phase shifts of the Aplysia eye rhythm were obtained with pulses of anisomycin (Jacklet, 1977) and similar responses occurred with puromycin (Rothman & Strumwasser, 1976). Even in Gonyaulax it is now known that inhibitors cause phase shifts of the rhythm (Dunlop et al. 1979).

Protein synthesis inhibitors are used to determine if new protein is required or how rapidly new protein must be synthesized for a function to continue. However, the inhibiting agent must be free of side effects on other cellular functions to conclude with certainty that the effects observed with the inhibitor are due specifically to inhibition of protein synthesis. This problem of specificity may be approached by using derivatives of inhibitor molecules that are slightly different in structure from the parent molecule and lacking in potency to inhibit protein synthesis.

A highly effective inhibitor of eurcaryotic protein synthesis is anisomycin (Grollman, 1967). It had proved to be remarkably free of side effects on neuronal functions, such as action potentials and synaptic potentials, when it was tested on the neuronal tissue of Aplysia (Schwartz, Castillucci & Kandel, 1971 ; Jacklet, 1977, 1979). To determine the specificity of the inhibitor effects on the circadian clock of the Aplysia eye derivatives of anisomycin were tested. Only those compounds that inhibited protein synthesis also caused phase shifts of the circadian clock.

Aplysia californica (100-300 g) were obtained from Pacific Bio-Marine (Venice, CA) and kept in Instant Ocean aquariums exposed to light-dark cycles of 13—11 h maintained at 16-17 °C. Eyes with attached optic nerves were dissected from the animals 2-3 h after dawn and placed in 50 ml of culture medium, maintained thereafter at 17 °C in constant darkness. The optic nerve was drawn into a J-shaped tubing electrode (Jacklet, 1974) in the culture chamber allowing the compound action potentials (CAP) to be amplified and continuously recorded on a Grass polygraph. The frequency of CAP were counted automatically with an Ortec system and printed out each 20 min on a teletype. The culture medium contained 90% artificial sea water (ASW) and 10% nutrient mixture, which included amino acids, vitamins, dextrose, penicillin and streptomycin (Gibco) as used previously (Jacklet, 1974, 1977). Anisomycin or one of the derivatives (Pfizer) was dissolved in ASW and added to the culture medium at specific phases of the circadian rhythm in a pulse of six hours. After the pulse treatment, the inhibitor solution was removed, the chamber was washed with 150 ml of ASW and fresh culture medium was added all under dim red light. Exposure to the red light and changing the culture medium did not alter the rhythm. Another protein synthesis inhibitor, pactamycin, a gift from the Upjohn Company, was also tested. Pactamycin was first dissolved in acetone but otherwise handled the same as anisomycin.

Derivatives of anisomycin were obtained from Pfizer Inc, through the courtesy of Dr J. Beereboom. The parent compound, anisomycin, and four derivatives that are inactive in inhibiting protein synthesis (Grollman, 1967) were tested. The structural formulas are shown in Fig. 1 below.

The incorporation of tritiated leucine (New England Nuclear, NET-135H, specific activity 40-60 Ci/m-mol) into eye protein insoluble in trichloroacetic acid (TCA) was determined. Eyes from the same animal were compared. One was preincubated in ASW and buffer while the other was preincubated in ASW and buffer plus anisomycin or a derivative. After 15 min of preincubation eyes were placed in separate droplets containing labelled leucine. These droplets were prepared by drying down 20 µl of labelled leucine under vacuum and then dissolving it in 100 µl of ASW containing 30 mm Hepes buffer (pH 7.8). Eyes were incubated for 3 h in DD at 17 °C, then rinsed several times in ASW, rapidly frozen and homogenized. The homogenate was precipitated with 10% TCA, kept on ice 1 h, heated to 90 °C for 15 min, cooled and filtered on Gelman metricel membrane filters. Radioactivity was counted in a beckman LS-230 using 5 ml of Econofluor (NEN) scintillation fluid. Counting efficiency was 58%.

In culture medium and complete darkness at 17 °C the period of the rhythm is 26.1 ±0.5 h (Jacklet, 1979) and on the first day in culture the CAP frequency increases to half its maximum rate of firing at subjective dawn (the solar time when dawn would have been observed by the eye had it not been dissected from the animal), which is indicated by convention as circadian time o (CT o). On subsequent cycles of the rhythm the time when the frequency increased to half the maximum rate was used as a phase reference for CT o and 26 h cycle was normalized to 24 circadian hours. The phase of a 6 h pulse of inhibitor was designated in circadian hours at the midpoint of the pulse (i.e. a pulse designated at CT 3 was actually started at CT o and ended at CT 6). The period of the rhythm was measured in hours between successive maximum rate increases or between successive centroids (Benson & Jacklet, 1977 a).

Previous studies of the anisomycin treatment of the circadian rhythm in CAP frequency recorded from the Aplysia eye have shown that the inhibitor has no detectable side-effects on the mechanism for CAP generation (Jacklet, 1977, 1979). Only the circadian clock control of the periodic firing was affected. Pulses of anisomycin phase shifted the rhythm to an extent dependent upon the phase of the rhythm at which the inhibitor was applied, which resulted in a characteristic phase-response curve for anisomycin (Jacklet, 1977, 1979)-Derivatives were therefore tested for effects at a number of different phases of the rhythm. The results were then compared with a paired control eye treated with anisomycin or a paired control eye treated with normal culture medium. Fig. 2 contrasts the effects of anisomycin to those of deacetyl-anisomycin after each compound was delivered to one of a pair of eyes from the same animal. Before addition of the inhibitor (↓) the eyes were firing at a similar rate and the rhythms were precisely in phase. During the treatment (centered at CT o) the CAP activity of the deacetylanisomycin treated eye continued as expected for an untreated eye and the eye treated with anisomycin performed as expected (Jacklet, 1977, 1979) for a rhythm treated at that phase; the firing frequency was stabilized. Following the pulse treatment the deacetlyanisomycin eye behaved like an untreated eye ; the period of the rhythm was not changed and the CAP rate was normal. In contrast, the rhythm of the anisomycin eye was delayed in phase by 8 h but regained the normal period and amplitude a few cycles after the treatment as expected (Jacklet, 1977, 1979). These results show that rhythms treated with deacetlyanisomycin are virtually unperturbed compared to the large phase delay affected by anisomycin. In order to test the derivatives more precisely, the deacetylanisomycin treated eyes were compared to a paired control eye treated by changing the normal culture medium only. One result is shown in Fig. 3. No consistent alteration of the deacetylanisomycin treated eyes could be detected; the periods were normal, the phase and amplitude were not changed beyond that expected for a pair of normal eyes (Jacklet, 1974, 1979)-Other experiments on pairs of eyes treated similiarly to those illustrated in Fig. 2 and 3 are listed in Table 1. Normal/deacetylanisomycin pairs were tested at two phase points (CT 23-5 and CT 0-5) at which maximum phase changes were obtained for anisomycin pulses. No phase shifting by deacetylanisomycin was observed and the period of the rhythm was normal before and after the pulse. Deacetylanisomycin/anisomycin pairs were tested at phases where large delays were expected for anisomycin (CT 0, CT 0.5 and CT 2) and a phase at which no phase shifting by anisomycin was expected (CT 4). In each case the deacetylanisomycin treated eyes were not phase shifted or otherwise perturbed. Three other eyes were treated with derivatives (CPD’ s III, V, and VII) similarly and compared to a normal control (Table 1). These derivatives also did not change the rhythm.

Another protein synthesis inhibitor, pactamycin, known to have a potent effect on protein synthesis in Aplysia central neurones (Schwartz, Castellucci, & Kandel, 1971) was also tested. It proved to be of only slight value in testing the rhythm since the rhythm did not recover from the treatment as shown in Fig. 4. The eye continued to be active after the treatment as evidenced by the continued CAP frequency but the rhythmic alterations in frequency that are the manifestations of clock control are absent. Such a result is hard to interpret. The clock may be stopped, uncoupled from the CAP generation mechanism or otherwise impaired.

Pairs of eyes from the same animal were compared for their ability to incorporate tritiated leucine into protein after one of the pair was treated with anisomycin or a derivative at 10^-6 M concentrations. The results in Table 2 show that for six pairs of eyes the average leucine incorporation was 40017 cpm for normal controls and 9523 cpm for anisomycin treated eyes for a ratio of 0.25, or about 75 % inhibition of protein synthesis by anisomycin. Deacetylanisomycin caused no significant inhibition of protein synthesis. Other neural tissue (buccal and pleural ganglia) were also tested and the results were similar ; no inhibition of protein synthesis by deacetylanisomycin or CPD III as contrasted to 75-85 % inhibition by anisomycin. The levels of incorporation of leucine for control tissues were 0.12 p-mol/h for eyes, 0.32 p-mol/h for buccal ganglia and 1.25 p-mol/h for pleural ganglia. Giant neurone R₂ of the abdominal ganglia of Aplysia incorporated 5 p-mol/h as a comparison (Schwartz et al. 1971). The variability of leucine incorporation was rather high even for paired eyes from the same animal. The reasons for it remain largely unexplained and a similar high variability was noted in studies of central neurones by Schwartz et al.1971 ).

Protein synthesis is strongly inhibited (90%) by anisomycin at 10^-6 M in HeLa cells, intact rabbit reticulocyte (Grollman, 1967) and in Aplysia central neurones (Schwartz, Castellucci & Kandel, 1971). The inhibition is reversible with synthesis being quickly restored to 2/3 of the original value after 1 h of treatment (Grollman, 1967). Derivative of anisomycin tested by Grollman and used in the present study had no more than 1 % of the inhibitory activity of the parent molecule even though the changes in structure of the derivative molecules involved the removal of only a single acetyl group in CPD II (deacetylansiomycin) or the addition of an acetyl group (CPD III). The finding that none of the inactive derivatives caused any phase shifting of the rhythm or produced any alterations of the normal periodicity considerably strengthens the argument (Jacklet, 1978) that it is indeed the inhibition of protein synthesis which causes perturbation of the clock by anisomycin.

The level of protein synthesis inhibition observed in this study (75 % at 10^-6 M) by anisomycin are quite comparable to that observed previously (90% at 2 · 10^-5 M, Jacklet, 1977) and to the levels found by Schwartz et al. (1971) (90% at 1.8 x 10^-6 M and 95% at 18 · 10^-6 M) for central neurones. The derivatives caused no detectable inhibition in the eye when tested at 10^-8 M. This result is expected from the work of Grollman (1967) where a 1000 x increase in concentration of the derivatives was needed to obtain the inhibition produced by anisomycin.

The incorporation of tritiated leucine into eye protein in this study was good but variable, with standard deviations about one-third of the mean of 43000 cpm for 12 control eyes. However, considerable variability was noted by others as well. The level of about 12000 cpm in SDS gels obtained by Rothman & Strumwasser (1976) was comparable to counts obtained in this study and their radioactive carbon counts averaged about 2900 cpm with a standard deviation of about one-quarter of the mean. In Aplysia central neurones variability was also seen with an average of 79 p-mol ± 24 (one-third of mean) for 39 abdominal ganglia studied by Schwartz et al. (1971). The source of this disconcerting variability remains obscure.

Other protein synthesis inhibitors such as puromycin and cycloheximide cause phase-dependent phase shifts of the eye circadian clock (Rothman & Strumwasser, 1976). The phase-response curve for puromycin from that study is very similar to the one for anisomycin (Jacklet, 1977, 1979) with the exception that the phase delays are limited to 6 h as compared with 15 h with anisomycin. The concentration of puromycin used to generate the phase response curve was rather high (2.3 x 10^-6* M) and some side-effects on the eye were evident. An explanation of the weak phase shifting effects of puromycin is that protein synthesis was also weakly inhibited. It required 20µg/ml for 50% inhibition, according to Rothman & Strumwasser (1976), but in Aplysia central neurones Schwartz et al. (1971) found no inhibition at 80µg/ml. Puromycin, then, would not be expected to cause phase shifts at low concentrations since protein synthesis is poorly inhibited. Cycloheximide is even less effective both as a phase shifting agent and as a protein synthesis inhibitor in Aplysia’. Rothman & Strumwasser (1976) found 500 µg/ml of the chemical inhibited protein synthesis by 50% and caused phase delays at the same concentration while Schwartz et al. (1971) found no inhibition of synthesis at 60 µg/ml. Thus at levels where synthesis inhibition is 50% or so, phase shifts in the rhythm are seen, but where inhibition is slight, phase shifts are small or lacking.

Among the inhibitors, other than anisomycin, found to be effective at low concentrations on Aplysia central neurones were pactamycin and sparsomycin. Pactamycin tested on the eye at 2 x 10^-6 M was effective in altering the eye circadian rhythm without undue influence on the CAP or the short-term pacemaker activity. It altered the rhythm profoundly since it did not recover to its normal cyclic activity after the removal of the inhibitor. These results are not readily interpretable. It would be of considerable interest to know if the protein synthesis ability recovered. If it did not, the results would be consistent with the necessity of protein synthesis for proper clock timing.

The mode of action of anisomycin is to specifically block peptide bond formation and thus prevent the transfer reaction of protein synthesis. It acts on the same step as puromycin (Vasquez, 1974), but its action can be distinguished from that of puromycin since polyribosomes remain intact and nascent peptides remain attached during inhibition by anisomycin (Grollman, 1967). It binds specifically to the 60 S subunit of eucaryotic ribosomes (Vasquez, 1974) and may be acting on peptidyl transferase. Knowing the site of action of the inhibitor is useful for a first approximation of the necessary pathway for the circadian clock timing but it does not help much in the specific localization of the clock. Pertubations of the pathway leading to the timing mechanism may have effects on the clock that can not presently be unequivocally interpreted in terms of specifying the mechanism or exact site of the clock (Tyson et al., 1976). That must await further penetrating inquiry.

Support by NSF grant BNS 11154. I thank Celine Rolerson for excellent technical assistance and gratefully acknowledge the Pfizer and Upjohn chemical companies for inhibitors and Dr Peter Marfey for discussion.