Acute effects of unilateral or bilateral superior cervical ganglionectomy on rat pineal N-acetyltransferase activity and melatonin content.

Acute bilateral superior cervical ganglionectomy (SCGX) completely prevents the nocturnal rises in pineal N-acetyltransferase (NAT) activity and melatonin content in male rats kept in light-dark cycles of 14:10. Unilateral SCGX causes the NAT and melatonin levels to be intermediate between those in sham-operated control rats and those in rats from which both ganglia had been removed.     

Effects of oestrogen and progesterone on rat pineal N-acetyl transferase activity and melatonin production

Summary We have extended previous studies on pineal -receptors to include effects of oestradiol or PMSG treatment in the immature female rat. Neither manipulation has any effect on norepinephrine-induced N-acetyl transferase (NAT) activity in vitro. In the adult ovariectomised rat oestrogen/progesterone priming exerts a small sensitising effect to -stimulation with isoproterenol. Progesterone alone, in vitro, inhibits the release of melatonin from pineals of adult ovariectomised rats.The authors are grateful to J. Bradtke, D. de Ziegler and K.B. Ruf for their help, and to the Swiss National Science Foundation and to the Fonds Emil Barrell for financial support.     

Neither prolactin nor growth hormone restore the nocturnal rise in pineal N-acetyltransferase activity or melatonin content in hypophysectomized rats

Hypophysectomy in adult male rats greatly attenuated the nocturnal rise in both pineal N-acetyltransferase (NAT) activity and melatonin content. High nighttime levels of NAT and melatonin were not restored by treating the animals with either prolactin or growth hormone, alone or in combination. Treating intact rats with bromocriptine, which depresses circulating prolactin levels, also was without effect on pineal melatonin synthesis. It appears that neither prolactin nor growth hormone are of major importance in determining pineal melatonin production.     

Neither prolactin nor growth hormone restore the nocturnal rise in pineal N-acetyltransferase activity or melatonin content in hypophysectomized rats

Summary Hypophysectomy in adult male rats greatly attenuated the nocturnal rise in both pineal N-acetyltransferase (NAT) activity and melatonin content. High nighttime levels of NAT and melatonin were not restored by treating the animals with either prolactin or growth hormone, alone or in combination. Treating intact rats with bromocriptine, which depresses circulating prolactin levels, also was without effect on pineal melatonin synthesis. It appears that neither prolactin nor growth hormone are of major importance in determining pineal melatonin production.     

Melatonin biosynthesis in the mammalian pineal gland

Summary Rhythmic production of melatonin by the mammalian pineal occurs in response to noradrenergic stimulation which produces a cascade of biochemical events within the pinealocyte. In the rat, massive changes in NAT activity result from an increase in intracellular c-AMP levels produced by a synergistic interaction whereby an ₁ activation amplifies -adrenergic stimulation. The intracellular events mediating this effect are described. A major aspect of the temporal control of melatonin production is the programmed down-regulation of responses to noradrenergic stimulation once the initial surge of c-AMP is produced. Noradrenergic activation of the gland also influences other enzymic functions, including tryptophan hydroxylase and HIOMT activities, and produces a dramatic increase in intracellular c-GMP levels. Other neurotransmitters and neuropeptides, e.g. VIP, may also influence pineal function and comparisons are, made between the rat, the subject of the bulk of experimental studies, and other species.     

Melatonin decreases the amplitude of the b-wave of the human electroretinogram

In a double-blind placebo crossover study of 13 healthy volunteers, the pineal hormone melatonin (10 mg) was given at 4 pm, and the electroretinogram measured under conditions of dark and light adaptation. A significant diminution of b-wave amplitude was found under both photopic (=5.4 V, p<0.05) and scotopic conditions (=7.4 V, p<0.01). These data indicate that melatonin may transduce the dark signal at the level of the retina as well as the pineal. Acute administration of melatonin decreases sensitivity to light.     

Hepatic hydroxylation of melatonin in the rat is induced by phenobarbital and 7,12-dimethylbenzy[a]anthracene — implications for cancer etiology

The protective function of the pineal hormone melatonin in the etiology of cancer and carcinogenic activation is increasingly well-established. Low melatonin levels seem to parallel cancer growth. The question arises as to which factors cause the depression of melatonin levels and what the direct effects are. Melatonin is known to be metabolized in the liver by hydroxylation and subsequent conjugation yielding 6-sulfatoxymelatonin as a main product. Nevertheless, the microsomal monoxygenases catalyzing the first step have been poorly investigated. To further characterize these enzymes, typical inducers of three different sub-classes, namely phenobarbital, 7,12-dimethylbenz[a]anthracene, and 17-estradiol, were administered to female Fischer rats. Circadian urinary excretion patterns of melatonin and 6-sulfatoxymelatonin were determined over a 24-hour period on the third (second) day of induction. Liver homogenates were used to monitor the in vitro conversion of melatonin or 6-hydroxymelatonin to 6-sulfatoxymelatonin. Results of both approaches showed the microsomal monoxygenases catalyzing the 6-hydroxylation of melatonin to be strongly inducible by phenobarbital and to a lesser degree by the polyaromatic hydrocarbon 7,12-dimethylbenz[a]anthracene. The dramatic depletion of circulating melatonin as a result of these induction patterns and its possible implications for oncogenesis are discussed.     

Pineal ‘synaptic’ ribbon numbers and melatonin synthesis of rat are resistant to guanethidine sympathectomy

Chemical sympathectomy is widely used to study the impact of the noradrenergic system on neuronal and neuroendocrine circuits. We tested the effects of intraperitoneal injections of guanethidine, an adrenergic neuron blocking agent, on selected functional parameters of the rat pineal gland which are known to be under sympathetic influence. The reliability of the method was demonstrated by the clear enophthalmus developed by experimental animals. However, neither the numbers of synaptic ribbons nor melatonin synthesis differed between treated and control rats, both parameters exhibiting the nocturnal increase seen in intact animals. These results are in striking contrast to those obtained upon chemical sympathectomy with 6-hydroxydopamine or surgical superior cervical ganglionectomy. We conclude that guanethidine is not capable of sufficiently removing noradrenergic influence from the rat pineal gland, and that this substance is thus inferior to other experimental methods of sympathectomy.     

Orally administered melatonin stimulates the 3α/β-hydroxysteroid oxidoreductase but not the 5α-reductase in the ventral prostate and seminal vesicles of pinealectomized rats

Summary Pinealectomized rats were treated orally with melatonin (MEL) for 14 days. Prostates and seminal vesicles were investigated for the activity of the 4-3-ketosteroid-5-oxidoreductase (5-R) and of the 3/-hydroxysteroid-oxidoreductase (3-HSO). The activity of the 3a-HSO was significantly stimulated (p<0.01, Friedman test) when compared to controls. The activity of the 5-R did not change significantly.This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 34 (Endokrinologie).     

Increased melatonin levels after hemorrhagic shock in male and female C3H/HeN mice

Although hemorrhagic shock leads to significant alterations of several hormones, e.g. ACTH, corticosterone and -endorphin, it is not known whether plasma melatonin levels are affected under this condition and if so, whether the effects are comparable in males and females. Using a radioimmunoassay, it was found that plasma melatonin levels were significantly increased in male and proestrus female C3H/HeN mice immediately after hemorrhagic shock. However, in male mice, by two hours after hemorrhage and resuscitation, plasma melatonin returned to levels comparable to those seen in control and sham-operated animals. Proestrus female mice, on the other hand, showed significantly increased plasma melatonin levels at two hours after surgery when compared to unoperated control animals. Although the significance and biological role of the transient increased plasma melatonin levels after hemorrhagic shock remain to be determined, it appears that the pineal gland and/or an extrapineal source of melatonin, of both male and proestrus female mice responds to severe hypotension by increased release of melatonin.     

Indoleamines and the UV-light-sensitive photoperiodic responses of the melanocyte network: a biological calendar?

The pineal, serotoninergic and pigmented neurons are associated with light-dependent sleep/arousal, serving as a biological clock with a circadian rhythm. This rhythm is maintained by melatonin which serves to recognise the dark phase. The neural network that responds to seasonal variations in day/night length has not been identified. The present study demonstrates that melanocytes in human skin respond to changes in the duration of UV exposure, and can serve as a biological calendar. These responses are mediated by two indoleamines, serotonin and melatonin. Higher melatonin levels correspond to long nights and long days (short UV pulse), while high serotonin levels in the presence of melatonin reflect short nights and long days (long UV exposure). This response recapitulates the sleep/arousal patterns in animals exposed to large variations in day/night cycle that cause changes in coat colour from pure white in winter to complete repigmentation in summer.     

Lesions in suprachiasmatic nuclei simulate effects of pinealectomy on prolactin release in ovariectomized and sulpiride-treated female rats

Summary Previous studies indicate that the pineal gland alters prolactin secretion, and it was suggested that at least part of the effect of the pineal hormone melatonin on prolactin release may be mediated by the hypothalamic structures. In this study, pinealectomy and lesions of the suprachiasmatic nuclei were found to alter serum levels of prolactin in the same direction, an effect that was counteracted by daily afternoon melatonin administration. Melatonin, but not other pineal indoles, also prevented sulpiride-induced prolactin secretion in pinealectomized or suprachiasmatic nuclei-lesioned and ovariectomized rats, which suggested that the pineal gland can modulate prolactin secretion by acting through a dopamine mechanism independent of hypothalamic suprachiasmatic structures.The authors thank Ms Karen Shashok for revising the English style. This work was supported in part by a grant GG85-0168 from the Comisión Asesora de Investigación Cientifica y Ténica. The NIAMDD, through the National Pituitary Agency, supplied the radioimmunoassay materials for prolactin determinations.     

Exogenous melatonin accelerates re-entrainment: Attenuation of the circadian rhythm of metabolic rate in the canary,Serinus canaria

Administration of melatonin in the drinking water (200 g/ml in 1% ethanol) decreased the time of re-entrainment of the circadian rhythm of the metabolic rate (measured as oxygen uptake) of domestic canaries (Serinus canaria) after 10-h delay phase shifts of the light-dark (LD) cycle by 1.3 days on average. Associated with faster re-entrainment, the amplitude of the metabolic rhythm was attenuated by 46% on, average on the first day after the shift as compared with about 25% in the controls. After re-entrainment, the amplitude of the metabolic rhythm during melatonin administration was about 23% lower than in the controls. The minimum resting metabolic rate increased by ca 5% on average during treatment with melatonin. The results are consistent with the hypothesis that constant high plasma levels of melatonin act on higher levels of the circadian oscillatory system rather than by directly affecting peripheral or central photoreceptors.     

Development of melatonin rhythm in the pineal gland and eyes of chick embryo

A melatonin rhythm was observed in the pineals of 18-day-old chick embryos incubated under a light-dark regime of 186 h. A low pineal melatonin content was found during the light phase of the day. Concentrations started to increase 2 h after dark onset and reached maximum levels after 4 h of darkness. The amplitude of the pineal melatonin rhythm increased considerably after 2 days and night-time concentrations in 20-day-old embryos were more than 5 times higher than in 18-day-old ones. Significant day/night differences in melatonin production were found both in pineals and eyes. Exposure of eggs to 1 h of light during the dark period decreased the high melatonin concentrations in the eyes but not in the pineals of the 20-day-old chick embryo. The results suggest that in this precocial bird at least part of the circadian system may already operate during embryonic life.     

Effects of oestrogen and progesterone on rat pineal N-acetyl transferase activity and melatonin production

We have extended previous studies on pineal beta-receptors to include effects of oestradiol or PMSG treatment in the immature female rat. Neither manipulation has any effect on norepinephrine-induced N-acetyl transferase (NAT) activity in vitro. In the adult ovariectomised rat oestrogen/progesterone priming exerts a small sensitising effect to beta-stimulation with isoproterenol. Progesterone alone, in vitro, inhibits the release of melatonin from pineals of adult ovariectomised rats.     

The presence and function of melatonin and structurally related indoleamines in a dinoflagellate,and a hypothesis on the evolutionary significance of these tryptophan metabolites in unicellulars

The bioluminescent dinoflagellateGonyaulax polyedra contains various indoleamines, in particular, melatonin and 5-methoxytryptamine, as well as enzymes of their biosynthetic pathway. Melatonin exhibits a high-amplitude circadian rhythm characterized by a dramatic increase shortly after the onset of darkness. The maximum of melatonin is followed by a peak of 5-methoxytryptamine. These 5-methoxylated indoleamines seem to be involved in the mediation of the information darkness.G. polyedra shows a short-day response, which consists in the formation of asexual cysts. Light break experiments demonstrate the photoperiodic nature of this reaction. Cells become sensitive to short days only upon exposure to a lowered temperature (<16°C). Melatonin mimics the short-day effect, but only at decreased temperature. 5-Methoxytryptamine is even a better inducer of cyst formation, acting also at 20°C and in any lighting schedule, including LL. Cyst induction is associated with stimulation of bioluminescence and cytoplasmic acidification. A model on the intracellular pathway of photoperiodic information transduction assumes increased deacetylation of melatonin under cyst-inducing conditions, binding of 5-methoxytryptamine to the membrane of an acidic vacuole, proton transfer to the cytoplasm, and decreased intracellular pH as the stimulus for encystment. Melatonin shows the property of a scavenger of superoxide anions. This reaction, which is efficiently catalyzed by hemin, leads to the formation of a substituted kynuramine (AFMK). Destruction of melatonin by light-induced superoxide anions in the presence of cellular hemin may represent a property which, during evolution, has made this molecule suitable as an indicator of darkness. On the other hand, AFMK, which is formed under illumination, might have become a mediator of the information light. Photoperiodism inGonyaulax shows surprising parallels to that in mammals, but allows the analysis of this phenomenon at an entirely cellular level.     

The pineal and melatonin: Regulators of circadian function in lower vertebrates

Summary The pineal has been identified as a major circadian pacemaker within the circadian system of a number of lower vertebrates although other pacemaking sites have been implicated as well. The rhythmic synthesis and secretion of the pineal hormone, melatonin, is suggested as the mechanism by which the pineal controls circadian oscillators located elsewhere. Both light and temperature cycles can entrain the pineal melatonin rhythm. The pineal, therefore, acts as a photo and thermoendocrine transducer which functions to synchronize internal cycle with cycles in the environment. A model is presented which portrays the pineal as a major component of a multioscillator circadian system and which suggests how these multiple circadian clocks are coupled to each other and to cycles of light and temperature in the external world.     

Effect of removal of the Harderian glands on pineal melatonin concentrations in the Syrian hamster

Summary Peak melatonin levels which are normally present in male Syrian hamsters at 8 h after the onset of darkness in animals maintained under a lightdark cycle of 1410, were significantly decreased following the removal of the Harderian glands.Supported by NSF grant PCM 77-05734.     

Expression of the mutant gene for L-gulono-γ-lactone oxidase in scurvy-prone rats

A mutant strain of Wistar rats with L-gulono--lactone oxidase deficiency has recently been established. To investigate this deficiency by DNA and RNA blot hybridization analyses, a fragment of a previously cloned cDNA encoding rat L-gulono--lactone oxidase was used as a probe. When genomic DNA of the mutant rat was digested with several restriction enzymes, the probe hybridized to fragments of the same sizes as those produced from DNA of normal rats. Poly(A)⁺RNA from the liver of the mutant rat was found to contain an L-gulono--lactone oxidase-specific mRNA of a normal size at a comparable level to that of normal rats. An in vitro translation experiment revealed that the mRNA programmed the synthesis of an enzyme protein which had the same molecular weight as that of the translational product of the normal mRNA, although the amount synthesized was markedly reduced as compared with that synthesized with the normal mRNA. In accordance with this observation, a very low but definite degree of L-gulono--lactone oxidase activity was detected in the microsomes of the mutant rat by a newly developed, highly sensitive method.Acknowledgments. The authors thank Dr Susumu Makino, Shionogi Research Laboratories, Shionogi & Co., Ltd, Japan, for his kind donation of normal (ODS- +/+) and ODS (ODS-od/od) rats. This work was supported in part by Grant-in-Aid (59570103) for Scientific Research from the Ministry of Education, Science and Culture of Japan.     

Expression of membrane and nuclear melatonin receptor mRNA and protein in the mouse immune system

The neurohormone melatonin plays a fundamental role in neuroimmunomodulation of several mammalian species, including mice. This effect is supported by the existence of specific melatonin-binding sites in murine immunocompetent organs. Moreover, using melatonin receptor analogues, several effects of the neurohormone on mice physiology through its membrane and nuclear receptors have been described. The expression of these receptors has never been studied, despite indirect evidence showing the presence of melatonin receptor in the murine immune system. At present, the MT₁ and MT₂ membrane receptors, and nuclear receptors belonging to the RZR/ROR family have been related to the immunomodulator effect of melatonin. Here, we show the presence of membrane and nuclear melatonin-binding sites in mouse thymus and spleen, using the specific melatonin membrane (S 20098) and nuclear (CGP 52608) receptor agonist. To confirm the presence of melatonin receptors, we analyzed the presence of membrane and nuclear receptor mRNA and protein by RT-PCR, Southern blot, and Western blot. Thus, we show that MT₁ and ROR receptor mRNA and protein are expressed in both thymus and spleen, while MT₂ receptor mRNA is only detected in the thymus. This expression of melatonin receptors strongly supports the idea of an immunomodulatory role of melatonin through its receptors.Received 2 June 2003; received after revision 6 August 2003; accepted 14 August 2003