Ivonne Balzer

On the primary functions of melatonin in evolution: Mediation of photoperiodic signals in a unicell, photooxidation, and scavenging of free radicals*

Abstract: Melatonin is widely abundant in many eukaryotic taxa, including vari‐ous animal phyla, angiosperms, and unicells. In the bioluminescent dinoflagellate Gonyaulax polyedra, melatonin is produced in concentrations sometimes exceed‐ing those found in the pineal gland, exhibits a circadian rhythm with a pro‐nounced nocturnal maximum, and mimics the short‐day response of asexual encystment. Even more efficient as a cyst inducer is 5‐methoxytryptamine (5MT), which is also periodically formed in Gonyaulax. In this unicell, the photoperiodic signal‐transduction pathway presumably involves melatonin formation, its deace‐tylation to 5MT, 5MT‐dependent transfer of protons from an acidic vacuole, and cytoplasmic acidification. According to this concept, we observe that cyst forma‐tion can be induced by various monoamine oxidase inhibitors and protonophores, that 5MT dramatically stimulates H+‐dependent bioluminescence and leads to a decrease of cytoplasmic pH, as shown by measurements of dicyanohydroquinone fluorescence. Cellular components from Gonyaulax catalyze the photooxidation of melatonin. Its property of being easily destroyed by light in the presence of cel‐lular catalysts may have been the reason that many organisms have developed mechanisms utilizing this indoleamine as a mediator of darkness. Photooxidative reactions of melatonin, as studied with crude Gonyaulax extracts and, more in de‐tail, with protoporphyrin IX as a catalyst, lead to the formation of N1 ‐acetyl‐N ‐formyl‐5‐methoxykynuramine (AFMK) as one of the main products. Photochemical mechanisms involve interactions with a photooxidant cation radi‐cal leading to the formation of a melatonyl cation radical, which subsequently combines with a superoxide anion. Photooxidation of melatonin represents one of several possibilities of a more general, biologically highly important property of this indoleamine to act as an extremely efficient radical scavenger, including its feature of terminating radical reaction chains by a final combination with the su‐peroxide anion. Trapping of free radicals may reflect the primary and evolutionar‐ily most ancient role of melatonin in living beings.

Hemistasia phaeocysticola (Scherffel) comb. nov., Redescription of a Free-living, Marine, Phagotrophic Kinetoplastid Flagellate

Summary We investigated a common, free living, marine, phagotrophic flagellate by light and electron microscopy. It has two heterodynamic flagella, a flexible cell shape and a pronounced apical rostrum. So far it was regarded as dinoflagellate and called Pronoctiluca phaeocysticola (Scherffel) Pavillard. Our investigation revealed, however, that it belongs to the bodonids (Kinetoplastida). The mitochondrium is of the polykinetoplastic type. The two flagella insert in a deep flagellar pocket and have a paraflagellar rod. The cytostome, the cytopharynx and the rostrum are supported by various cytoskeletal elements, the most prominent of which is a reinforced microtubular band. A distinct rod organ(= microtubular prism, nemadesm) has not been found. It has extrusomes of the “lattice tube” type. A distinct glycocalyx and a spiral groove of the cell body are absent. It fits the description of Hemistasia klebsii Griessmann. We regard both as con-specific and introduce the combination Hemistasia phaeocysticola (Scherffel) Elbrachter & Schnepf, comb. nov. The delimitation to other, similar organisms placed in various genera is discussed.

The vertebrate pineal hormone melatonin is produced by the brown alga Pterygophora californica and mimics dark effects on growth rate in the light

Melatonin, a methoxylated indoleamine, plays a role as a mediator of darkness in animals as well as in the unicellular alga Gonyaulax polyedra Stein and was recently detected in higher plants. We report on the first finding of melatonin in a multicellular alga, the brown alga Pterygophora californica Rupr. Melatonin was identified in juvenile sporophytes of P. californica by two independent methods, reverse-phase high-performance liquid chromatography (HPLC) with electrochemical detection, and radioimmunoassay. Another indolic metabolite, 5-methoxytryptophol, was also indentified by HPLC. The rapid decline of growth rate upon the onset of darkness in P. californica is mimicked by melatonin in the light, with increasing efficiency from 5 × 10−5M to 5 × 10−4M, while no effect was obtained at 10−5M.

Effects of Indoleamines and Short Photoperiods on the Encystment of Gonyaulax polyedra

At a temperature of 15 degrees C, Gonyaulax polyedra responds to short days (light less than or equal to 10 h) by transition to the stage of a resting cyst. At 20 degrees C, even an light:dark (LD) cycle of 6:18 is incapable of inducing this process. In otherwise cyst-inducing conditions (15 degrees C; 10 h of light per day), an interruption of the scotophase by 2 h of light (LDLD 8:2:2:12 or 2:2:8:12) prevented encystment. Cyst induction is, therefore, initiated by a photoperiodic mechanism rather than by light deficiency. In Gonyaulax, photoperiodism may be mediated by the action of indoleamines. Melatonin, which exhibits a circadian rhythmicity in this organism, leads to encystment when given 1 h before lights-off in LD 11:13 at 15 degrees C, i.e., under otherwise noninducing conditions. Again, at 20 degrees C, melatonin is inefficient. Some analogues of melatonin, in particular, 5-methoxytryptamine and N,N-dimethyl-5-methoxytryptamine, and, at high concentrations, their respective precursors, serotonin and bufotenin, are capable of inducing cyst formation at 20 degrees C and in LD 12:12, whereas N-acetyl-serotonin does not show this effect.

Influence of temperature on biological rhythms

The various effects that temperature can exert on biological periodicities are reviewed. Particular consideration is given to the remarkable capability of many rhythms either to respond in a temperature dependent manner, especially in nonsteady-state conditions, or to behave almost independently of temperature (‘temperature compensated’) in the steady state. Therefore, organisms are able both to use temperature changes as synchronization cues and to measure time at different temperatures. Moreover, changes of temperature can induce transients, after-effects, and eventually alterations in the phase response behaviour, sometimes even the rhythmicity itself. Treatment with low temperatures can, at least in some cases, hold the circadian clock, and considerably reduce the sensitivity of rhythms towards certain drugs.

Concomitance of circadian and circa‐4‐hour ultradian rhythms in Euglena gracilis

Abstract Stationary cultures of Euglena were investigated in LD 12:12, LD 0.25:1.75, and in LL, at a temperature of 23 °C. In these conditions, protein synthesis, cell shape and/or phototaxis exhibited circadian rhythmicity, whereas tyrosine aminotransferase activity showed a circa‐4‐h periodicity. Simultaneous measurements of cell shape and tyrosine aminotransferase, carried out in LL, at either 27.5 or 18 °C, demonstrated concomitance of circadian and ultradian rhythms in the same cell cultures.

Temperature compensation in an ultradian rhythm of tyrosine aminotransferase activity inEuglena gracilis Klebs

Tyrosine aminotransferase activity ofEuglena oscillates with an ultradian period of approximately 4–5 h. The oscillation frequency in the time series was determined by cosine fitting. Experiments which were performed between 16 and 31.5°C revealed temperature compensation.

Circadian rhythmicity in the stimulation of bioluminescence by biogenic amines and MAO inhibitors in Gonyaulax polyedra

In the dinoflagellateGonyaulax polyedra bioluminescence was investigated in constant darkness. Light emission was stimulated considerably and specifically by the biogenic smines epinephrine, 5-methoxytryptamine, and kynuramine. Various analogues and motabolites of these substances, such as norepinephrine, isoproterenol, phenylephrine, synephrine, metanephrine isoproterenol, phenylephrine, synephrine, metanephrine dopamine, 3,4-dihydroxymandelic and 3-methoxy hydroxymandelic acids, serotonin, N-acetylserotonin, melatonin, 5-hydroxytryptophol, 5-methoxytryptophol, kynurenine, 4-hydroxyquinoline, 3-hydroxyanthrani ic, and quinolinic acids were much less effective. Strong enhancement of bioluminescence, in the range of those obtained with the three stimulatory biogenic amines was also observed after administration of several compeunds acting as MAO inhibitors in mammalian systems, in particular, pargyline, amitriptyline,p-benzoquinone, tranylcypromine, harmaline, and noreleagnine. The responsiveness of cells towards epinephrine, 5-methoxytryptamine, kynuramine, amitriptyline,p-benzoquinone, and noreleagnine varied considerably within the circadian cycle, with the highest stimulations obtained during subjective night. These rhythms can be only partially explained by periodic bioluminescence capacity, and seem to comprise a cyclicity in the sensitivity of cells to the compounds mentioned.

Stimulation of bioluminescence by 5-methoxylated indoleamines in the dinoflagellate, Gonyaulax polyedra

Abstract 1. Bioluminescence was measured in cells transferred from a light/dark cycle of 12: 12hr to constant darkness. 2. Gonyaulax responded strongly and specifically to 5-methoxytryptamine and its analogue, N,N-dimethyl-5-methoxytryptamine. The reaction threshold for 5-methoxytryptamine was as low as 2 × 10−8 M. At 10−4M, 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.

Action of kynuramine in a dinoflagellate: Stimulation of bioluminescence in Gonyaulax polyedra

1. Bioluminescence of Gonyaulax polyedra was measured at the beginning of night within a light/dark cycle of 12:12 hours. 2. Kynuramine stimulated light emission in a dose-dependent manner within the first hour after administration, the reaction threshold being close to 10−5 M; a stimulation factor of ca. 40-fold was obtained at 10−4 M. 3. The early action was followed by late effects; with increasing primary stimulation, the circadian maximum of bioluminescence was progressively reduced; moderate long-lasting stimulations were observed at intermediate concentrations of 2 or 3 × 10−5 M during the subsequent circadian minimum. 4. Various other tryptophan metabolites such as kynurenine, 4-hydroxyquinoline, 3-hydroxyanthranilic, quinolinic, quinaldic and xanthurenic acids exerted only minor effects on bioluminescence. 5. Pargyline and amitriptyline, which are known as inhibitors of mammalian MAO, strongly stimulated light emission.