progression of the CDC in many cell types (Whitfield,. 1990; Whitfield et al. ..... 22 (ed. P. Greengard and G. A.. Robison), pp. 1-38. New York: Raven Press.
Circadian rhythmicity in the activities of adenyiate cyciase and phosphodiesterase in synchronously dividing and stationary-phase cultures of the achiorophylious ZC mutant of Euglena gracilis
JIAN TONG, ISABELLE A. CARRE and LELAND N. EDMUNDS, JR* Department of Anatomical Sciences, State University of New York, Stony Brook, New York 11794, USA * Author for correspondence
Summary Key factors in the adenosine 3',5'-cyclic monophosphate (cyclic AMP) metabolic pathway are two enzymes responsible for its generation and degradation, namely, adenyiate cyciase (AC) and phosphodiesterase (PDE). In LD: 12,12 (12 h light, 12 h dark), these enzymes were found to undergo bimodal, circadian variation of activity in both dividing and nondividing cultures of the photosynthesis-deficient, achiorophylious ZC mutant of Euglena gracilis Klebs (Z). Maximal AC activity occurred 2 h after the onset of the light interval (CT 02) and at the beginning of darkness (CT 12—14); these times corresponded to the acrophase profile for the rhythmic changes in cyclic AMP content that have been previously reported. The activity of PDE also exhibited a daily oscillation, but with an inverse phase pattern. Both the AC and
Introduction The relationship between the cell division cycle (CDC) and circadian oscillators perhaps has been most intensively investigated in Euglena gracilis, a unicell having a number of well-defined rhythmicities (Edmunds, 1984, 1988). Cyclic AMP (3'5'-cyclic adenosine monophosphate) plays a pivotal role in cellular regulation and has the capacity to control certain rate-limiting steps in the progression of the CDC in many cell types (Whitfield, 1990; Whitfield et al. 1987). Increasing evidence indicates that a cyclic AMP signal is required for DNA synthesis and chromosome replication (Matsumoto et al. 1988; Tamanoi, 1988), and a second cyclic AMP surge during G2 may be correlated with the onset of mitosis (Boynton and Whitfield, 1983). Thus, one would expect a periodic cyclic AMP signal under the control of a cellular pacemaker to generate cell division rhythmicity, as well as rhythms in other physiological or enzymatic activities. In recent studies, we have shown that cultures of the achiorophylious, photosynthesis-deficient ZC mutant of Euglena gracilis Klebs (strain Z), which display freerunning circadian rhythms of cell division in constant darkness (DD) following synchronization by a light-dark cycle (LD), exhibit bimodal circadian variations in their levels of cyclic AMP (Carre et al. 1990). The maximum cyclic AMP levels occur at the beginning of the light period (Circadian Time, CT 0-2), when cells are in Gi, and at the Journal of Cell Science 100, 365-369 (1991) Printed in Great Britain © The Company of Biologists Limited 1991
PDE activity rhythms persisted after the cultures were transferred from LD: 12,12 to constant darkness. The activity of AC was activated significantly in vivo by forskolin at the trough phase (CT 20), while that of PDE was inhibited by 3-isobutyl-l-methylxanthine (IBMX) at its peak phase. These results indicate that the rhythms of both AC and PDE may be the main factors generating the circadian oscillations of cyclic AMP content in Euglena, which appear to be under control of an endogenous pacemaker.
Key words: adenyl cyciase, biological clock, cell cycle, circadian rhythm, cyclic AMP, Euglena gracilis, phosphodiesterase, ZC mutant.
onset of darkness (CT 12-14), corresponding to the onset of mitosis. These variations, however, appear to be under the control of a circadian oscillator rather than to be cell cycledependent, since they persist, independently of the CDC, after the cultures have reached the stationary phase of growth. The intracellular level of cyclic AMP is determined by a dynamic balance between synthetic and degradative processes. The two key enzymes responsible for the generation and degradation of cyclic AMP, adenyiate cyciase (AC) and phosphodiesterase (PDE), were first described in detail about 30 years ago (Sutherland et al. 1962; Sutherland and Rail, 1958). Since then, there has been considerable work on both the characterization of the enzymes and the significance of their activities in physiological regulation. In fact, there is no evidence to date suggesting that there are other pathways for cyclic AMP metabolism; thus, the determination of the cyclic AMP content of cell preparations may be attributable primarily to the activities of AC and PDE under certain experimental conditions (Perkins, 1973). In most cases, the change in cyclic AMP level that occurs during cell activation is produced by stimulation of AC, which is associated with the plasma membrane. There are examples, however, as in Dictyostelium discoideum (Wang et al. 1988) and in mammalian cells (Lemmer et al. 1987), where the cyclic AMP level also can be altered by a change in PDE activity. Adenyiate cyciase has been found in 365
many organisms, including Brevibacterium liquifaciens (Hirata and Hayaishi, 1967), Saccharomyces fragilis (Sy and Richter, 1972), Neurospora crassa (Flawia and Torres, 1972), as well as Euglena gracilis (Keirns et al. 1973). In one study, Euglena was found to have both AC and PDE in high concentration (Keirns et al. 1973). The activity of AC was mainly particulate, being associated with membrane components that sedimented at moderate centrifugal force, while PDE activity was found principally in the 78 000 g supernatant fraction of broken cell preparations, indicating its soluble fraction origin. Since cyclic AMP levels - known to undergo circadian variation in Euglena gracilis - are controlled via the activities of AC and PDE, an understanding of the regulation of these enzymes would be the first step in tracing the pathway upstream to the circadian oscillator. We report in this paper similar circadian changes of AC and PDE activity in this organism. In addition, we give results from the chemical stimulation and inhibition in vivo of the activities of the two enzymes in an initial attempt to determine how these oscillations in activity might be generated. Materials and methods
cyclic AMP, 0.1 mM GTP, 20 mM phosphoenol pyruvate, and 6 units of pyruvate kinase. The activity of PDE was measured by separating the [3H]adenosine formed from [3H]3',5'-AMP and [3H]5'-AMP by precipitation with an anion exchange resin, and then counting with a liquid scintillation spectrometer (Packard Tri-Carb, model 3320) (Ho and Hoskins, 1989; Thompson et al. 1979). The incubation mixture for the PDE assay (final concentrations) consisted of 25 pM of 3H-labeled cyclic AMP (2.2xl0 6 ctsmin~ 1 ), 5mM MgCl2, 40mM Tris (pH8.0), 3.75mM mercaptothanol, 0.15/(M cyclic AMP, and 10/d of snake (Ophiophagus hannah) venom (lmgml" 1 ). Incubation time for both enzymes was 10 min at 31°C. Enzyme activities were asssayed either in duplicate or in triplicate and were expressed aspmol cyclic AMP formed or hydrolyzed per min per 10 cells.
Stimulation and inhibition of enzyme activities Forskolin was used as an activator of AC and IBMX (3-isobutyl-lmethylxanthine) as an inhibitor of PDE. For in vitro experiments, extracts were prepared from synchronously dividing cultures at CT 12 and CT 20 (corresponding to the peaks and troughs of the two enzyme activities), and forskolin ( 1 0 ~ 5 - 1 0 ~ 2 M ) or IBMX ( 5 X 1 0 ~ 6 - 1 0 ~ 2 M ) was added to them. After a 30-min preincubation of the extracts at 31°C, the reaction was initiated by the addition of the assay medium. For in vivo experiments, forskolin and IBMX were added to intact cells (instead of cell extracts) 1 h before the preparation of cell extracts and subsequent measurement of enzyme activities.
Cell culture The achlorophyllous ZC mutant of Euglena gracilis Klebs was derived from the wild-type Z strain by the action of 25 ;JM diuron [3-(3,4)-dichlorophenyl-l,l-dimethylurea (DCMU)] in a 33-mM lactate medium (pH3.5) under illumination and anoxia (Calvayrac and Ledoigt, 1976; Carrti et al. 1988). Axenic, aerated, magnetically stirred, 4-1 batch cultures of ZC cells were grown at 16.5±(0.5)°C on a modified Cramer and Myers' medium supplemented with vitamins Bi and B 12 and containing ethanol (0.1 %, v/v) as the sole carbon source (Edmunds and Funch, 1969). Cysteine and methionine (10~ 5 M), which improve the coupling between the CDC and the underlying circadian oscillator (Edmunds et al. 1976), were also added. Illumination (3000 lx) was provided by clock-programmed, cool-white fluorescent lamps. Cell numbers were monitored every 2h by a miniaturized fraction collector and a Coulter Electronic Particle Counter (Edmunds, 1964). Since cells were entrained by a strong Zeitgeber (LD: 12,12), extracts were prepared from two out-of-phase cultures during a 12-h time span, and samples were taken every 2h from either dividing cultures or those having reached the stationary phase. For experiments under DD (free-running conditions), however, measurements were carried out on single cultures over 40-h time spans in order to avoid the problem caused by possible variations in the free-running period (T) of twin cultures.
Preparation of cell extracts for AC and PDE assays Cells were pelleted at 4°C by centrifugation for lOmin at 7700g. The cells were resuspended in 2 ml of 50 mM Tris (pH7.8) at a concentration of 3 xlO 6 cells ml" 1 . Each sample was sonicated for 3 intervals of 20 s (MSE sonicator set on high power at amplitude 4). The crude sonicate was then centrifuged for 10 min at 39 000 g. The resulting supernatant fraction was kept for the assay of PDE activity, and the pellet was resuspended in 3 ml Tris (50 mM, pH7.8) for the AC assay. The enzyme assays were performed either on fresh extracts or on extracts that had been frozen in liquid nitrogen and stored at -70°C (which showed no loss in activity).
Assays of AC and PDE activities The AC assay was performed as described by Alvarez and Daniels (1990), in which a one-step method was used for the separation of cyclic AMP from other nucleotides on polypropylene columns of neutral aluminum oxide. The final concentrations of the components of the incubation medium were 0.5 mM (O.lfjCi) [of32 P]ATP, lmM MgSO.,, 40 mM Tris-HCl buffer (pH7.4), 2mM
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Results Average AC and PDE activities in dividing and nondividing cells The average AC and PDE activities were determined for cultures in both the exponentially dividing phase and the stationary phase of growth. In dividing cultures, 24.9±4.0 pmol min" 1 of cyclic AMP were formed by AC and 8.9±2.0 pmol min" 1 of cyclic AMP were hydrolyzed by PDE per 106 cells - a three-fold difference between the two average activities. In nondividing cultures, however, the difference was much less: an average of 20.8± 1.8 pmol min" 1 for AC and 13.1±2.2pmolmin" 1 for PDE per 106 cells. Oscillations of AC and PDE activity in dividing cultures Under the experimental conditions, bimodal, 24-h oscillations of AC and PDE activities were observed (Fig. 1A,C). For AC, peaks were found at CT 02 and at CT 12-14 (that is, the beginning of the light and the dark intervals) and troughs at CT 08 and CT 20. For PDE, an inverse phase relationship to the patterns of the AC oscillation was found, with the two peaks' appearing at CT 06-08 and CT 18-20 and the two minima at CT 02-04 and CT 12-14. The range of the oscillations in AC activity for dividing cultures was 12.1-41.8 pmol cyclic AMP min" 1 10 6 cells" 1 and that for PDE was 2.5-22.5 pmol cyclic AMP min" 1 10 6 cells" 1 . Oscillations of AC and PDE activity in nondividing cultures After the cells had stopped dividing in the stationary phase, the bimodal circadian changes of AC and PDE activity still persisted with approximately the same patterns (Fig. 1B,D) as those in dividing cultures (Fig. 1A,C). The ranges for the AC and PDE oscillations were 10.7-32.2 and 2.9-29.6 pmol cyclic AMP min" 1 10 6 cells" 1 . Although there were some differences in the ranges of the oscillations between dividing and nondividing cultures (for example, that of PDE appeared
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Fig. 1. Bimodal circadian rhythms of AC (A,B) and PDE (C,D) activity in dividing (A,C) and nondividing (B,D) cultures of the achlorophyllous ZC mutant of Euglena gracilis Klebs (strain Z), grown in LD: 12,12 at 16.5°C on a mineral medium supplemented with ethanol (0.1%). For experiments A and C, the results shown are averages of triplicate assays from two different cultures, and for experiments B and D, the data are averages of triplicate from one culture only.
to be greater than in dividing cells), they probably were not significant. Persistence of the AC and PDE rhythms in constant conditions To test for the endogenicity of the enzyme rhythms, cultures of the ZC mutant Euglena gracilis were transferred from LD: 12,12 to DD shortly before they attained the stationary phase. After 2 to 3 circadian cycles in DD had elapsed, enzyme activities were assayed during a 40-h time span. The bimodal rhythms of AC and PDE observed in LD were found to persist with a free-running period of approximately 24 h (Fig. 2), similar to the x of 25 h observed in DD for the cell division rhythm. The ranges of activity oscillations in these nondividing cultures were 7.2-36.7pmol cyclic AMPmin" 1 10 6 cells" 1 for AC and 2.9-26.6 pmol cyclic AMPmin" 1 10 6 cells" 1 for PDE. In vitro and in vivo modulation of AC and PDE activities To determine whether the changes in the activity of AC are generated by changes in the amount of enzyme, rather than by its degree of activation, total (fully stimulated) AC activity was measured after treatment of cells with forskolin. The addition of increasing concentrations of forskolin (0.01-10 /.IM) to cell extracts caused the activity of AC to increase until saturation was reached. If added to
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Fig. 2. Free-running circadian rhythms in AC activity (A) and PDE activity (B) measured over a 40-h time span in stationary cultures of the ZC mutant of Euglena gracilis maintained in constant darkness (DD). Cultures, synchronized by LD: 12,12, were transferred to DD shortly before the cells had ceased dividing, and the onset of the last burst of cell division was used as a phase reference point for CT 12. Hatched bars indicate subjective night. In order to facilitate the comparision with curves in Fig. 1, the free-running periods have been normalized to 24 h.
whole cells at CT 20, during the trough of the AC rhythm (Figs 1A,B, 2A), 10 ,UM forskolin elevated AC activity from 14 to 30pmol cyclic AMPmin" 1 10 6 cells" 1 (Fig. 3), a factor of 2.14, in contrast to only a 10 % stimulation at CT 12 (the peak phase). In vitro PDE activity was inhibited by 62% by IBMX at concentrations of 5xlO" 5 to 5 X 1 0 ~ 2 M . The addition of 50,UM IBMX to intact cells at CT 20 (corresponding to the peak of the PDE rhythm; see FigslC,D, 2B) depressed PDE activity by about 51% (Fig. 3), while at CT 12 (the trough phase), IBMX had only a slight (8 %) inhibitory effect.
Discussion Previous studies on circadian rhythms in the AC-cyclic AMP-PDE system have been limited to mammalian cells. Diurnal rhythms have been reported for AC activity in rat forebrain (Lemmer et al. 1987) and heart tissue (Lemmer and Witte, 1989) and for PDE activity in the pineal gland (Epplen et al. 1982; Minneman and Iverson, 1976) and in rat ventricles (Lemmer et al. 1985). In the study on rat heart, the AC rhythm was bimodal (acrophases at CT 04 and CT 22), whereas PDE daily activity changed unimodally (the trough at CT 20). In the present study on the achlorophyllous ZC mutant Rhythms of AC and PDE activity in Euglena
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Fig. 3. In vivo activation of AC activity by forskolin (IOJIM), or inhibition of PDE activity by 3-isobutyl-l-methylxanthine (IBMX, 50/IM) in the ZC mutant of Euglena gracllls (Z). The action of the effectors was tested at CT 12 (stippled bars) and at CT 20 (hatched bars), corresponding, respectively, to the peak and trough values of the AC rhythm and to the trough and peak values of the PDE rhythm (see Figs 1 and 2).
of Euglena gracilis, the activities of both AC and PDE exhibited bimodal circadian rhythmicity (Figs 1, 2). The maxima in AC activity corresponded to the onset of the light interval (CT 0-2) and to the onset of darkness (CT 12-14). Furthermore, the first AC activity peak occurred when most cells were in the Gi phase of the CDC, and the second peak corresponded to the onset of mitosis (Edmunds, 1964; Carre and Edmunds, unpublished data). This acrophase profile is consistent with our previous finding of a bimodal circadian rhythm in cyclic AMP content (Carr6 et al. 1990). On the other hand, it is the inverse of that for PDE, which exhibited peaks at CT 06 and CT 14. From these results it can be concluded that the rhythms of both AC and PDE are responsible for generating the circadian oscillations in cyclic AMP content in Euglena gracilis. In order to determine if the rhythms in enzyme activity were dependent on the imposed LD cycle, or upon transit of the cells through the different stages of their CDC, or both, assays were performed on cultures that had been transferred to DD and that had entered the stationary phase of growth (Fig. 2). In these nondividing cultures under constant conditions in which environmental timing cues were absent, free-running rhythms of both AC and PDE activity persisted with the phasing identical to those in LD-synchronized, dividing cultures (Fig. 1A,C). Thus, the circadian oscillations of AC and PDE activity can be uncoupled from the cell division rhythm and seem to be under control of an endogenous pacemaker. It is interesting to compare the activity levels of the enzymes in different cultures. Peak PDE activity at CT 06, in stationary cultures under LD (Fig. ID) was 6-8pmol higher than that in dividing cells (Fig. 1C), and, in the second peak at about CT 20, the difference was as much as 7-14 pmol. In contrast, the level of AC activity did not exhibit much difference at peak times in nondividing cultures as compared to dividing cells, and only a slightly lower activity at the trough time of CT 20 (Fig. 1A,B). The mean value of AC activity, however, was greater than that for PDE in both dividing and nondividing cells (approximately three-fold or two-fold, respectively). These results suggest that cyclic AMP generated by AC might not be completely destroyed by PDE. For example, intracellular cyclic AMP in Saccharomyces cereuisiae may be exported 368
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rapidly and in large quantities into the cell environment to serve in intercellular communication (Smith et al. 1990). Although the differences between the activities of the two enzymes in Euglena might reflect their varying functional capacity in the regulation of cyclic AMP metabolism in cell populations at different growth stages; we cannot conclude from our results that PDE played a greater relative role than AC in the generation of cyclic AMP oscillations, as has been suggested recently for mammalian cells (Lemmer, 1989). Forskolin is a highly specific activator of AC and can fully stimulate AC activity directly, independently of G proteins (Harper, 1988). Thus, the total amount of AC (as opposed to the activated amount) can be measured in cell preparations. Our results (Fig. 3) showed that forskolin stimulation of AC does not result in uniform effects throughout the 24-h day but varies considerably with circadian time. The degree of AC potentiation by an in vivo forskolin pulse appears to be inversely dependent on the basal level of AC: at its trough phase (CT 20), AC activity was significantly stimulated by as much as 114% (bringing it to exactly the same level as that found at the peak phase), while at the peak phase (CT 12), only a 10% stimulation was observed. The same result has been demonstrated by Lemmer et al. (1986) in rat heart: cyclic AMP level was significantly increased by the addition of forskolin (5mgkg" x ) at CT 12 in LD: 12,12, but the effect disappeared at CT 20 under the same experimental conditions. Thus, since forskolin appears to stimulate AC activity at different circadian times to the same maximal value regardless of its initial basal level, we conclude that the oscillatory activity of the enzyme derives from modulatory cellular effectors emanating from the circadian pacemaker rather than by changes in the amount of the enzyme protein itself. While previous studies have emphasized the role of AC, along with cyclic AMP, in signal transduction and neoplastic transformation (Whitfield, 1990; Hunt and Martin, 1980), less attention has been paid to the role of PDE. PDE is an important component of the signal system because it is responsible for the destruction of cyclic AMP after a pulse of synthesis and, thereby, permits the cell to recover from a refractory state induced by cyclic AMP. It is known that the hydrolysis of cyclic AMP by cell extracts is catalyzed by at least five types of different PDE isoenzymes: calmodulin-stimulated PDE, cyclic GMP-stimulated PDE, cyclic GMP-specific PDE, low Km PDE, and nonspecific PDE (Beavo, 1988). IBMX is a nonspecific PDE inhibitor, which can affect the activity of calmodulinstimulated PDE and cyclic GMP-inhibited PDE 20-25 times as much as that of cyclic GMP-stimulated PDE in mammalian cells (Beavo, 1988). Thus, the inhibitory effects of IBMX on PDE observed in Euglena extracts may be the result of its action on several PDE species. Furthermore, the differing effects of IBMX on PDE activity at CT 12 and CT 20 (Fig. 3) showed that the degree of inhibition was circadian-time-dependent and that IBMX had little or no effect on PDE at CT 12 (corresponding to the time when the activity was at a minimum). At CT 20 (when PDE activity was at its peak), IBMX brought the activity down to the same level as that observed at CT 12. This suggests that the relative amounts of the different types of PDE vary with circadian time and that a specific PDE (or a specific subset of enzymes that are inhibited by IBMX) is responsible for the oscillation. Forskolin is known to be able to reset the rhythm of compound action potential in the eye of the sea hare,
Aplysia, through the mediation of cyclic AMP, producing both advance and delay phase shifts (Eskin and Takahashi, 1983). IBMX has been reported to cause an increase in T of the conidiation rhythm of the wild-type bread mold, Neurospora and its 'clock' period mutants frq-1, -2 and -3 (Feldman, 1975). This agent also could potentiate the effect of 5-hydroxytryptamine on the rhythm in the Aplysia eye (Eskin et al. 1982). Although these results might be taken to suggest that AC and PDE are elements of the circadian oscillator itself, in our system cyclic AMP pulses do not appear to induce steady-state phase shifts in the cell division rhythm (Carr6 and Edmunds, unpublished). Consequently, it is unlikely that AC or PDE would phase shift division rhythmicity or constitute clock 'gears'. Since both AC and PDE are themselves oscillatory and are responsible for the generation of the circadian rhythm of cyclic AMP content in Euglena, it will be interesting to determine how they, in turn, are regulated. Results of a preliminary up-stream analysis indicate that effectors such as calcium, calmodulin, and cyclic GMP may play a role in modulating their activities.
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This work was supported by National Science Foundation grants DCB-8901944 and DCB-9105752 and National Institutes of Health grant RR05736 to L.N.E.
LEMMER, B., BISSINGER, H. AND LANG, P. H. (1986). Effect of forskolin
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(Received 28 May 1991 - Accepted 1 July 1991)
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