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Stimulation of bioluminescence by 5-methoxylated indoleamines in the dinoflagellate, Gonyaulax polyedra
Camp. Biochem. Physiol.Vol. 98C, No. 213, pp. 395-391, 1991 Printed in Great Britain
0306~4492/91$3.00 + 0.00 0 1991 Pergamon Pressplc
BY STIMULATION OF BIOLUMINESCENCE 5-METHOXYLATED INDOLEAMINES IN THE DINOFLAGELLATE, GONYAULAX POLYEDRA IVONNEBALZERand R. HARDELAND I. Zoologisches Institut, UniversitHt Gottingen, Berliner Str. 28, D-3400 Gottingen, F.R.G. (Telephone: 0551/39-5415) (Received 4 June 1990)
Abstract-l. Bioluminescence was measured in cells transferred from a light/dark cycle of 12 : 12 hr to constant darkness. 2. Gonyaulux responded strongly and specifically to Smethoxytryptamine and its analogue, N,Ndimethyl-5-methoxytryptamine. The reaction threshold for 5-methoxytryptamine was as low as 2 x IO-* M. At 10e4 M, this indoleamine stimulated bioluminescence by 55-fold. 3. 5-Hydroxylated indoleamines such as serotonin and N-acetyl-serotonin, but also the 5-methoxylated indoleamine, melatonin, and the indole alcohols, 5-hydroxytryptophol and 5-methoxytryptophol, were much less effective or without stimulatory potency. 4. The responsiveness of cells to 5-methoxytryptamine differed considerably in various phases of the circadian cycle. The highest degree of stimulation was attained around the middle of subjective night, whereas almost no stimulation was observed during the second part of subjective day.
INTRODUCTION The role of 5-methoxylated indoleamines is predominantly seen in the transmission of temporal information concerning the light/dark cycle; in the pineal hormone, melatonin, is particular, considered to be a kind of “night hormone” in vertebrates, exerting coordinative effects on the suprachiasmatic nucleus and, eventually, other organs (Reiter, 1980, 1985; Tamarkin, 1985; Arendt, 1986; Laudon et al., 1988; Duncan et al., 1988). Exceptionally, also other indoleamines may play a similar role. In the European hamster, e.g., no rhythm of melatonin or of serotonin N-acetyltransferase activity was detected, despite the occurrence of photoperiodism and a considerable diurnal rhythm in pineal serotonin exhibiting the typical nocturnal trough (Pevet et al., 1989) which usually corresponds to a high rate of melatonin formation; these results strongly suggest a metabolic pathway towards S-methoxytryptamine, which might substitute for melatonin in this species. The occurrence of melatonin is not restricted to vertebrates. This indoleamine has been shown to be present in insects such as Locusta (Vivien-Roels et al., 1984), Mmca (Wetterberg et al., 1987), and Drosophila (Finocchiaro et al., 1988). In planarians, melatonin was reported to exert effects on reproduction (Morita and Best, 1984). Recently, we were able to demonstrate the existence of melatonin in the dinoflagellate, Gonyaulax polyedra, using two entirely different methods, RIA and reverse phase HPLC (Piiggeler et al., 1989). Moreover, 5-methoxytryptamine also appears to be present in Gonyaulax, according to HPLC with electrochemical detection (Poggeler, unpublished data). Also the enzymes involved in the formation of 5-methoxylated
indoleamines, indoleamine (“serotonin”) N-acetyltransferase (NAT) and hydroxyindole O-methyltransferase (HIOMT), have been detected in this dinoflagellate (Balzer et al., 1990). In search of a physiological role of indoleamines in Gonyaulax, we investigated the effects of these substances on bioluminescence. In this paper, we demonstrate that 5-methoxytryptamine is a potent, structurally-specific stimulator of light emission, whereas other related substances including melatonin are almost without effect on this particular parameter. MATERIALSAND METHODS Cultures of Gonyaulax polyedra were grown in a modified f/2 medium (Hoffmann and.Hardelandy 1985), at 20°C in a light/dark cvcle of 12 : 12 hr. At the end of the light period, _ cells were transferred to constant darkness. Bioluminescence was measured in a scintillation spectrometer, at 20°C as previously described (Hardeland, 1980). The indole compounds used in this investigation were purchased from Sigma, Deisenhofen. The substances were dissolved directly before use, first at a concentration of 0.2 M in DMSO, and were then diluted with culture medium to give the desired molarity. Data were evaluated by computing the medians of IO-min intervals. Two different calculations were used for expressing the pharmacological stimulation of light emission, whichever was more appropriate: (a) the ratio of maximally stimulated by basal bioluminescence rates, at a given time point (“stimulation factor”); (b) multiples of the basal bioluminescence level within a minute, over the entire time span of stimulation (expressed as “bioluminescence units”; 1 unit = basal level for 1 min; the basal level was determined in controls between 1 and 3 hr after beginning of darkness). The stimulation factor was calculated preferably for the case of strong, but temporally short, stimulations by high concentrations of amine followed by a decline of light _
395
_
IVONNE BALZER and R. HARDELAND
0.
O-
30
1
1
20
20
1
1
20
1
20
2
Fig. 1. Stimulation of bioluminescence by %methoxytryptamine and N,N-dimethyl-5methoxytryptamine. (a) Control; (b) 10m4M 5methoxytryptamine; (c) 10m6M 5methoxytryptamine; (d) 10m4M NJ-dimethyl-5-methoxytryptamine; (e) 10-j M NJ-dimethyl-5-methoxytryptamine. Substances were added at the beginning of the dark period in the LID cycle. Ordinate: bioluminescence (millions of cpm; data are presented as medians of lo-min intervals); abscissae: hr after transfer to darkness.
emission being presumably due to exhaustion components of the bioluminescence reaction.
of
RESULTS AND DISCUSSION
5_Methoxytryptamine, when administered at the beginning of darkness, stimulated bioluminescence considerably, at high dosages by more than 50-fold (Fig. 1; Table 1). The reaction threshold turned out to be as low as approximately 2 x lo-* M, a concentration resulting in a transient doubling of light emission. Most of the other indoleamines, as well as the indole alcohols tested, were much less effective (Table 2). At the high dose of 10m4M, serotonin provoked a moderate stimulation, which, however, might be due to conversion to 5-methoxytryptamine; this metabolic route should be possible with regard to the occurrence of HIOMT in Gonyaulux (Balzer et al., 1990) and to the fact that serotonin can serve as a substrate (Balzer, unpublished data). The inefficiency of indole alcohols may be not surprising, since these substances presumably represent degradation products. Moreover, it should be emphasized that the N-acetylated indoleamines, N-acetyl-serotonin and melatonin, were practically without effect, even at the high concentration of 10e4 M (Table 2). The only other indoleamine tested which induced responses in the order of magnitude seen with 5methoxytryptamine was its analogue, N,N-dimethyl5-methoxytryptamine (Fig. 1; Table 2). There is,
Table
1. Dosage dependence of 5methoxytryptatnine
Concentration 5x 2x 2x 2x
10-4M W5M lo-‘M lo-‘M IO-‘M lo-’ M IO-‘M IO-* M
response
Stimulation factor 55.0 11.8 8.9 2.6 2.5 2.2 2.0 1.3
to
however, currently no indication for the existence of the N,N-dimethylated compound in Gonyaulax. At a concentration of 10e4 M, 5-methoxytryptamine and N,N-dimethyl-5-methoxytryptamine exerted similarly strong stimulations. However, the effect by the dimethylated compound was observed much earlier (Fig. 1). This is, however, no indication for a higher potency of the latter drug, since it was already almost inefficient at 10m6M( data not shown), by contrast with 5-methoxytryptamine. Therefore, the more rapid response to the dimethylated amine might only be a consequence from a facilitated uptake of this pharmacon. The high sensitivity of the cells towards 5methoxytryptamine along with the considerable structural specificity for the response towards indoleamines suggests the existence of a control mechanism of bioluminescence by 5-methoxytryptamine. Among the biogenic amines tested so far, 5-methoxytryptamine represents the one to which Gonyaulux reacts with the highest sensitivity. In the cases of epinephrine, epinine, and kynuramine, the concentrations required for obtaining responses are 2-3 orders of magnitude higher (cf. Hardeland, 1980; Balzer and Hardeland, 1989a, b). Assuming that bioluminescence might be regulated by 5-methoxytryptamine would lead to the question of the physiological situation in which the amine stimulates light emisison. Since bioluminescence varies considerably within the circadian cycle (Krasnow et al., 1980; Johnson and Hastings, 1986), the efficacy of such a control mechanism might be. related to the diurnal periodicity. In fact, we observed a strong dependence of the response to 5methoxytryptamine on the time of day (Fig. 2). When given in constant darkness during the second part of subjective day, only a very small increase in bioluminescence was detected. The responsiveness was already higher at the beginning and highest around the middle of subjective night. These results are, on the one hand, fully compatible with the assumption of a regulation by the indoleamine within the frame of diurnal cyclicity; on the other hand, these temporal variations reflect, to a certain extent, oscillations in the availability of the elements of the bioluminescence reaction, i.e. luciferin, luciferinbinding protein (LBP), and luciferase (Johnson and Hastings, 1986; Morse er al., 1989). However, the rhythm of responsiveness to 5-methoxytryptamine can be only partially explained by a cycle of bioluminescence capacity due to varying concentrations of the above-mentioned components. This becomes particularly obvious by the weak reaction of cells in the second part of subjective day, which was
Table 2. Effects of various indole compounds on bioluminescence Compound Serotonin N-Acetylserotonin Melatonin 5-Methoxytryptamine N,N-Dimethyl-5-methoxytryptamine 5-Hydroxytryptophol 5-Methoxytryptophol
Stimulation (units) 231 0 3 4573 2088 20 199
Substances were added at the beginning of darkness, at a concentration of 0.1 mM.
Gonyaulax bi oluminescence
397
membranes: pharmacological characteristics and regional distribution. Endocrinology 122, 1825-1833. Finocchiaro L., Callebert J., Launay J. M. and Jallon J. M. (1988) Melatonin biosynthesis in Drosophila: its nature and its effects. J. Neurochem. SO, 382-387. Hardeland R. (1980) Effects of catecholamines on bioluminescence in Gonyaulax polyedra (Dinoflagellata), Comp. Biochem. Physiol. 66C, 53-58.
Hoffman B. and Hardeland R. (1985) Membrane fluidization by propranolol, tetracaine and I-aminoadamantane in the dinoflagellate, Gonyaulaxpolyedra. Camp. Biochem. Physiol. 81C, 39-43.
12
15
18
21
0
3
6
9
Fig. 2. Circadian rhythm of responsiveness to 2 x lo-’ M 5-methoxytryptamine, as compared to the periodicity of spontaneous bioluminescence. Bars: stimulation, expressed as bioluminescence units; line: spontaneous bioluminescence. At the beginning of the dark period, cells were transferred to constant darkness. Substances were added at various phases of the circadian cycle. Left ordinate: bioluminescence units; right ordinate: millions of cpm (medians of 10 min intervals); abscissa: hr of circadian time (CT 12: beginning of dark period = beginning of subjective night; CT 0: beginning of subjective day). much less than expected with regard to the capacity of the bioluminescent system. Therefore, in addition to the rhythmicity which regulates the formation of luciferin, LBP, and luciferase, a second periodic regulation mechanism should exist, controlling the sensitivity of the cell towards 5-methoxytryptamine. REFERENCES Arendt J. (1986) Role of the pineal gland in seasonal reproduction function in mammals. Oxford Rev. Reprod. Biol. 8, 266-320.
Balzer I. and Hardeland R. (1989a) Structural specificity of catecholamine action in the dinoflagellate, Gonyaulax polyedra. Cotnp. Biochem. Physiol. 92C, 297-300.
Balzer I. and Hardeland R. (1989b) Action of kynuramine in a dinoflagellate: stimulation of bioluminescence in Gonyaulax
polyedra.
Comp.
Biochem.
Physiol.
94C,
129-132. Balzer I., Pijggeler B. and Hardeland R. (1990) S-Methoxylated indoleamines in the dinoflagellate Gonyaulax polye of bioluminescence, and dra: presence, stimulation circadian variations in responsiveness. Eur. J. Cell Biol. 51 (Suppl. 30), 52. Duncan M. J., Takahashi J. S. and Dubovich J. S. (1988) 2-1251 Iodomelatonin binding sites in hamster brain
Johnson C. H. and Hastings J. W. (1986) The elusive mechanism of the circadian clock. Am. Scient. 74, 29-36. Krasnow R., Dunlap J. C., Taylor W., Hastings J. W., Vetterling W. and Gooch V. (1980) Circadian spontaneous bioluminescent glow and flashing of Gonyaulax polyedra. J. camp. Physiol. B. 138, 19-26. Laudon M.. Nir I. and Zisauel N. (1988) Melatonin receptors in discrete brain areas of the- male rat. Impact of aging on density and circadian rhythmicity. Neuroendocrinology 48, 577-583.
Morita M. and Best J. B. (1984) Effects of photoperiods and melatonin on planarian asexual reproduction. J. exp. 2001. 231, 273-282.
Morse D., Milos P. M., Roux E. and Hastings J. W. (1989) Circadian regulation of bioluminescence in Gonyaulax involves translational control. Proc. natn Acud. Sci. U.S.A. 86, 172-176.
P&vet P., Vivien-Roels B., Masson-P&vet M, Steinlechner B., Skene D. and Canguilhem B. (1989) Melatonin, serotonin, 5-hydroxy-indole-3-acetic acid and N-acetyltransferase in the pineal of the European hamster (Cricetus cricetus) kept under natural conditions: lack of day/night rhythm in melatonin formation in spring and early summer. Congr. cur. Chronobiol. (G.E.R.B.), Be&con 1989, p. 167 Piiaeeler B.. Balzer I.. Fischer J.. Behrmann G. and HardeGid R. (1989) A role of melatdnin in dinoflagellates? Acta endocr. Copen. 120 (Suppl. 1) 97. Reiter R. J. (1980) The pineal and its hormones in the control of reproduction in mammals. Endocr. Rev. 1, 109-131. Reiter R. J. (1985) Action spectra, dose-response relationships, and temporal aspects of light’s effects on the pineal gland. Ann. NIY. Acah. Sci. 453, 215-230. _ Tamarkin L.. Baird C. J. and Almeida 0. F. X. (1985) Melatonin.’ A coordinative signal for mammalian reproduction? Science 227, 714720. Vivien-Roels B., P&et P., Beck 0. and Fevre-Montagne M. (1984) Identification of melatonin in the compound eye of an insect, the locust (Locusta migratoriu), by radioimmunoassay and gas chromatography-mass spectrometry. Neurosci. L&t. 49, 153-157. Wetterberg L., Hayes D. K. and Halberg F. (1987) Circadian rhythms of melatonin in the brain of the face fly, Musca autumnalis De Geer. Chronobiologia 14, 377-38 1.
0306~4492/91$3.00 + 0.00 0 1991 Pergamon Pressplc
BY STIMULATION OF BIOLUMINESCENCE 5-METHOXYLATED INDOLEAMINES IN THE DINOFLAGELLATE, GONYAULAX POLYEDRA IVONNEBALZERand R. HARDELAND I. Zoologisches Institut, UniversitHt Gottingen, Berliner Str. 28, D-3400 Gottingen, F.R.G. (Telephone: 0551/39-5415) (Received 4 June 1990)
Abstract-l. Bioluminescence was measured in cells transferred from a light/dark cycle of 12 : 12 hr to constant darkness. 2. Gonyaulux responded strongly and specifically to Smethoxytryptamine and its analogue, N,Ndimethyl-5-methoxytryptamine. The reaction threshold for 5-methoxytryptamine was as low as 2 x IO-* M. At 10e4 M, this indoleamine stimulated bioluminescence by 55-fold. 3. 5-Hydroxylated indoleamines such as serotonin and N-acetyl-serotonin, but also the 5-methoxylated indoleamine, melatonin, and the indole alcohols, 5-hydroxytryptophol and 5-methoxytryptophol, were much less effective or without stimulatory potency. 4. The responsiveness of cells to 5-methoxytryptamine differed considerably in various phases of the circadian cycle. The highest degree of stimulation was attained around the middle of subjective night, whereas almost no stimulation was observed during the second part of subjective day.
INTRODUCTION The role of 5-methoxylated indoleamines is predominantly seen in the transmission of temporal information concerning the light/dark cycle; in the pineal hormone, melatonin, is particular, considered to be a kind of “night hormone” in vertebrates, exerting coordinative effects on the suprachiasmatic nucleus and, eventually, other organs (Reiter, 1980, 1985; Tamarkin, 1985; Arendt, 1986; Laudon et al., 1988; Duncan et al., 1988). Exceptionally, also other indoleamines may play a similar role. In the European hamster, e.g., no rhythm of melatonin or of serotonin N-acetyltransferase activity was detected, despite the occurrence of photoperiodism and a considerable diurnal rhythm in pineal serotonin exhibiting the typical nocturnal trough (Pevet et al., 1989) which usually corresponds to a high rate of melatonin formation; these results strongly suggest a metabolic pathway towards S-methoxytryptamine, which might substitute for melatonin in this species. The occurrence of melatonin is not restricted to vertebrates. This indoleamine has been shown to be present in insects such as Locusta (Vivien-Roels et al., 1984), Mmca (Wetterberg et al., 1987), and Drosophila (Finocchiaro et al., 1988). In planarians, melatonin was reported to exert effects on reproduction (Morita and Best, 1984). Recently, we were able to demonstrate the existence of melatonin in the dinoflagellate, Gonyaulax polyedra, using two entirely different methods, RIA and reverse phase HPLC (Piiggeler et al., 1989). Moreover, 5-methoxytryptamine also appears to be present in Gonyaulax, according to HPLC with electrochemical detection (Poggeler, unpublished data). Also the enzymes involved in the formation of 5-methoxylated
indoleamines, indoleamine (“serotonin”) N-acetyltransferase (NAT) and hydroxyindole O-methyltransferase (HIOMT), have been detected in this dinoflagellate (Balzer et al., 1990). In search of a physiological role of indoleamines in Gonyaulax, we investigated the effects of these substances on bioluminescence. In this paper, we demonstrate that 5-methoxytryptamine is a potent, structurally-specific stimulator of light emission, whereas other related substances including melatonin are almost without effect on this particular parameter. MATERIALSAND METHODS Cultures of Gonyaulax polyedra were grown in a modified f/2 medium (Hoffmann and.Hardelandy 1985), at 20°C in a light/dark cvcle of 12 : 12 hr. At the end of the light period, _ cells were transferred to constant darkness. Bioluminescence was measured in a scintillation spectrometer, at 20°C as previously described (Hardeland, 1980). The indole compounds used in this investigation were purchased from Sigma, Deisenhofen. The substances were dissolved directly before use, first at a concentration of 0.2 M in DMSO, and were then diluted with culture medium to give the desired molarity. Data were evaluated by computing the medians of IO-min intervals. Two different calculations were used for expressing the pharmacological stimulation of light emission, whichever was more appropriate: (a) the ratio of maximally stimulated by basal bioluminescence rates, at a given time point (“stimulation factor”); (b) multiples of the basal bioluminescence level within a minute, over the entire time span of stimulation (expressed as “bioluminescence units”; 1 unit = basal level for 1 min; the basal level was determined in controls between 1 and 3 hr after beginning of darkness). The stimulation factor was calculated preferably for the case of strong, but temporally short, stimulations by high concentrations of amine followed by a decline of light _
395
_
IVONNE BALZER and R. HARDELAND
0.
O-
30
1
1
20
20
1
1
20
1
20
2
Fig. 1. Stimulation of bioluminescence by %methoxytryptamine and N,N-dimethyl-5methoxytryptamine. (a) Control; (b) 10m4M 5methoxytryptamine; (c) 10m6M 5methoxytryptamine; (d) 10m4M NJ-dimethyl-5-methoxytryptamine; (e) 10-j M NJ-dimethyl-5-methoxytryptamine. Substances were added at the beginning of the dark period in the LID cycle. Ordinate: bioluminescence (millions of cpm; data are presented as medians of lo-min intervals); abscissae: hr after transfer to darkness.
emission being presumably due to exhaustion components of the bioluminescence reaction.
of
RESULTS AND DISCUSSION
5_Methoxytryptamine, when administered at the beginning of darkness, stimulated bioluminescence considerably, at high dosages by more than 50-fold (Fig. 1; Table 1). The reaction threshold turned out to be as low as approximately 2 x lo-* M, a concentration resulting in a transient doubling of light emission. Most of the other indoleamines, as well as the indole alcohols tested, were much less effective (Table 2). At the high dose of 10m4M, serotonin provoked a moderate stimulation, which, however, might be due to conversion to 5-methoxytryptamine; this metabolic route should be possible with regard to the occurrence of HIOMT in Gonyaulux (Balzer et al., 1990) and to the fact that serotonin can serve as a substrate (Balzer, unpublished data). The inefficiency of indole alcohols may be not surprising, since these substances presumably represent degradation products. Moreover, it should be emphasized that the N-acetylated indoleamines, N-acetyl-serotonin and melatonin, were practically without effect, even at the high concentration of 10e4 M (Table 2). The only other indoleamine tested which induced responses in the order of magnitude seen with 5methoxytryptamine was its analogue, N,N-dimethyl5-methoxytryptamine (Fig. 1; Table 2). There is,
Table
1. Dosage dependence of 5methoxytryptatnine
Concentration 5x 2x 2x 2x
10-4M W5M lo-‘M lo-‘M IO-‘M lo-’ M IO-‘M IO-* M
response
Stimulation factor 55.0 11.8 8.9 2.6 2.5 2.2 2.0 1.3
to
however, currently no indication for the existence of the N,N-dimethylated compound in Gonyaulax. At a concentration of 10e4 M, 5-methoxytryptamine and N,N-dimethyl-5-methoxytryptamine exerted similarly strong stimulations. However, the effect by the dimethylated compound was observed much earlier (Fig. 1). This is, however, no indication for a higher potency of the latter drug, since it was already almost inefficient at 10m6M( data not shown), by contrast with 5-methoxytryptamine. Therefore, the more rapid response to the dimethylated amine might only be a consequence from a facilitated uptake of this pharmacon. The high sensitivity of the cells towards 5methoxytryptamine along with the considerable structural specificity for the response towards indoleamines suggests the existence of a control mechanism of bioluminescence by 5-methoxytryptamine. Among the biogenic amines tested so far, 5-methoxytryptamine represents the one to which Gonyaulux reacts with the highest sensitivity. In the cases of epinephrine, epinine, and kynuramine, the concentrations required for obtaining responses are 2-3 orders of magnitude higher (cf. Hardeland, 1980; Balzer and Hardeland, 1989a, b). Assuming that bioluminescence might be regulated by 5-methoxytryptamine would lead to the question of the physiological situation in which the amine stimulates light emisison. Since bioluminescence varies considerably within the circadian cycle (Krasnow et al., 1980; Johnson and Hastings, 1986), the efficacy of such a control mechanism might be. related to the diurnal periodicity. In fact, we observed a strong dependence of the response to 5methoxytryptamine on the time of day (Fig. 2). When given in constant darkness during the second part of subjective day, only a very small increase in bioluminescence was detected. The responsiveness was already higher at the beginning and highest around the middle of subjective night. These results are, on the one hand, fully compatible with the assumption of a regulation by the indoleamine within the frame of diurnal cyclicity; on the other hand, these temporal variations reflect, to a certain extent, oscillations in the availability of the elements of the bioluminescence reaction, i.e. luciferin, luciferinbinding protein (LBP), and luciferase (Johnson and Hastings, 1986; Morse er al., 1989). However, the rhythm of responsiveness to 5-methoxytryptamine can be only partially explained by a cycle of bioluminescence capacity due to varying concentrations of the above-mentioned components. This becomes particularly obvious by the weak reaction of cells in the second part of subjective day, which was
Table 2. Effects of various indole compounds on bioluminescence Compound Serotonin N-Acetylserotonin Melatonin 5-Methoxytryptamine N,N-Dimethyl-5-methoxytryptamine 5-Hydroxytryptophol 5-Methoxytryptophol
Stimulation (units) 231 0 3 4573 2088 20 199
Substances were added at the beginning of darkness, at a concentration of 0.1 mM.
Gonyaulax bi oluminescence
397
membranes: pharmacological characteristics and regional distribution. Endocrinology 122, 1825-1833. Finocchiaro L., Callebert J., Launay J. M. and Jallon J. M. (1988) Melatonin biosynthesis in Drosophila: its nature and its effects. J. Neurochem. SO, 382-387. Hardeland R. (1980) Effects of catecholamines on bioluminescence in Gonyaulax polyedra (Dinoflagellata), Comp. Biochem. Physiol. 66C, 53-58.
Hoffman B. and Hardeland R. (1985) Membrane fluidization by propranolol, tetracaine and I-aminoadamantane in the dinoflagellate, Gonyaulaxpolyedra. Camp. Biochem. Physiol. 81C, 39-43.
12
15
18
21
0
3
6
9
Fig. 2. Circadian rhythm of responsiveness to 2 x lo-’ M 5-methoxytryptamine, as compared to the periodicity of spontaneous bioluminescence. Bars: stimulation, expressed as bioluminescence units; line: spontaneous bioluminescence. At the beginning of the dark period, cells were transferred to constant darkness. Substances were added at various phases of the circadian cycle. Left ordinate: bioluminescence units; right ordinate: millions of cpm (medians of 10 min intervals); abscissa: hr of circadian time (CT 12: beginning of dark period = beginning of subjective night; CT 0: beginning of subjective day). much less than expected with regard to the capacity of the bioluminescent system. Therefore, in addition to the rhythmicity which regulates the formation of luciferin, LBP, and luciferase, a second periodic regulation mechanism should exist, controlling the sensitivity of the cell towards 5-methoxytryptamine. REFERENCES Arendt J. (1986) Role of the pineal gland in seasonal reproduction function in mammals. Oxford Rev. Reprod. Biol. 8, 266-320.
Balzer I. and Hardeland R. (1989a) Structural specificity of catecholamine action in the dinoflagellate, Gonyaulax polyedra. Cotnp. Biochem. Physiol. 92C, 297-300.
Balzer I. and Hardeland R. (1989b) Action of kynuramine in a dinoflagellate: stimulation of bioluminescence in Gonyaulax
polyedra.
Comp.
Biochem.
Physiol.
94C,
129-132. Balzer I., Pijggeler B. and Hardeland R. (1990) S-Methoxylated indoleamines in the dinoflagellate Gonyaulax polye of bioluminescence, and dra: presence, stimulation circadian variations in responsiveness. Eur. J. Cell Biol. 51 (Suppl. 30), 52. Duncan M. J., Takahashi J. S. and Dubovich J. S. (1988) 2-1251 Iodomelatonin binding sites in hamster brain
Johnson C. H. and Hastings J. W. (1986) The elusive mechanism of the circadian clock. Am. Scient. 74, 29-36. Krasnow R., Dunlap J. C., Taylor W., Hastings J. W., Vetterling W. and Gooch V. (1980) Circadian spontaneous bioluminescent glow and flashing of Gonyaulax polyedra. J. camp. Physiol. B. 138, 19-26. Laudon M.. Nir I. and Zisauel N. (1988) Melatonin receptors in discrete brain areas of the- male rat. Impact of aging on density and circadian rhythmicity. Neuroendocrinology 48, 577-583.
Morita M. and Best J. B. (1984) Effects of photoperiods and melatonin on planarian asexual reproduction. J. exp. 2001. 231, 273-282.
Morse D., Milos P. M., Roux E. and Hastings J. W. (1989) Circadian regulation of bioluminescence in Gonyaulax involves translational control. Proc. natn Acud. Sci. U.S.A. 86, 172-176.
P&vet P., Vivien-Roels B., Masson-P&vet M, Steinlechner B., Skene D. and Canguilhem B. (1989) Melatonin, serotonin, 5-hydroxy-indole-3-acetic acid and N-acetyltransferase in the pineal of the European hamster (Cricetus cricetus) kept under natural conditions: lack of day/night rhythm in melatonin formation in spring and early summer. Congr. cur. Chronobiol. (G.E.R.B.), Be&con 1989, p. 167 Piiaeeler B.. Balzer I.. Fischer J.. Behrmann G. and HardeGid R. (1989) A role of melatdnin in dinoflagellates? Acta endocr. Copen. 120 (Suppl. 1) 97. Reiter R. J. (1980) The pineal and its hormones in the control of reproduction in mammals. Endocr. Rev. 1, 109-131. Reiter R. J. (1985) Action spectra, dose-response relationships, and temporal aspects of light’s effects on the pineal gland. Ann. NIY. Acah. Sci. 453, 215-230. _ Tamarkin L.. Baird C. J. and Almeida 0. F. X. (1985) Melatonin.’ A coordinative signal for mammalian reproduction? Science 227, 714720. Vivien-Roels B., P&et P., Beck 0. and Fevre-Montagne M. (1984) Identification of melatonin in the compound eye of an insect, the locust (Locusta migratoriu), by radioimmunoassay and gas chromatography-mass spectrometry. Neurosci. L&t. 49, 153-157. Wetterberg L., Hayes D. K. and Halberg F. (1987) Circadian rhythms of melatonin in the brain of the face fly, Musca autumnalis De Geer. Chronobiologia 14, 377-38 1.