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Signal transduction processes regulating melatonin production in the light-sensitive trout pineal organ were investigated by immunocytochemical and immunochemical demonstration of phosphorylated cyclic AMP-responsive element-binding protein (pCREB) and measurements of cyclic AMP, melatonin, and calcium levels. Melatonin levels were tightly controlled by light and darkness. Elevation of cyclic AMP levels by 8-bromo-cyclic AMP, forskolin, and 3-isobutyl-1-methylxanthine increased the levels of pCREB and melatonin in light- or dark-adapted pineal organs in vitro. Without pharmacological treatment, the levels of pCREB and cyclic AMP remained constant for several hours before and after light onset. Inhibition of cyclic AMP-dependent proteasomal proteolysis by lactacystin, MG 132, and calpain inhibitor I did not prevent the rapid, light-induced suppression of melatonin biosynthesis. However, changes in the intracellular calcium concentration by drugs affecting voltage-gated calcium channels of the L type and intracellular calcium oscillations (cobalt chloride, nifedipine, Bay K 8644) had dramatic effects on the rapid, light-dependent changes in melatonin levels. These effects were not accompanied by changes in cyclic AMP levels. Thus, the rapid, light-dependent changes in melatonin levels in the trout pineal organ are regulated apparently by a novel calcium signaling pathway and do not involve changes in cyclic AMP levels, cyclic AMP-dependent proteasomal proteolysis, or phosphorylation of cyclic AMP-responsive element-binding protein.

Pineal organ of the lamprey, Lampetra japonica, is essential to keep the circadian locomotor activity rhythm as previously reported. In this paper, we tried to show that an endogenous oscillator is located and is working in the pineal organ. When the pineal organs were excised and cultured in a plastic tube with M199 medium at 20 degrees C, melatonin secretion rhythms were clearly observed under both light-dark and continuous dark conditions. The circadian secretion of melatonin continued for more than five cycles under the continuous dark condition. This indicates that the pineal organ has an endogenous oscillator and that the melatonin secretion rhythm is controlled by this oscillator. These findings suggest the possibility that the locomotor activity rhythm of the lamprey is under the control of the oscillator in the pineal organ.

Together with cAMP, calcium ions play an important role in the regulation of melatonin synthesis in the pineal organ of all vertebrate species, irrespective of the conspicuous phylogenetic transformation of the melatonin-producing cell, the pinealocyte. Here we address the question how the intracellular concentration of free calcium ions [Ca2+]i is regulated in directly light-sensitive trout pinealocytes and in rat pinealocytes which have lost the direct light sensitivity and respond to norepinephrine. Isolated pinealocytes identified by the S-antigen immunoreaction were investigated by means of the fura-2 technique, image analysis and patch clamp recordings. Approximately 30% of the trout pinealocytes exhibited spontaneous [Ca2+]i oscillations that were not affected by light or dark adaptation of the cells. Removal of extracellular Ca2+ or application of 10 microM nifedipine caused a reversible breakdown of the [Ca2+]i oscillations. Treatments with 60 mM KCl and nifedipine suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca2+]i in both oscillating and nonoscillating trout pinealocytes. Experiments with thapsigargin (2 microM) revealed the presence of intracellular calcium stores in 80% of the trout pinealocytes, but their role in the regulation of [Ca2+]i remains elusive. Norepinephrine had no apparent effect on [Ca2+]i in any trout pinealocyte. In rat pinealocytes, [Ca2+]i did not show spontaneous oscillations. Norepinephrine evoked a dramatic biphasic rise in [Ca2+]i in more than 95% of the cells via stimulation of alpha1-adrenergic receptors. The response reflects a combination of calcium mobilization from intracellular, thapsigargin-sensitive calcium stores and an increased calcium influx. Voltage-gated calcium channels of the L-type are present in the rat pinealocyte membrane, but they are not involved in the norepinephrine-induced calcium response. These channels, however, mediate the increase in calcium influx which is observed in virtually all rat pinealocytes upon stimulation with acetylcholine or nicotine. The results show that the mechanisms which regulate [Ca2+]i in pinealocytes are complex and differ considerably between poikilothermic and mammalian species.

By means of the fura-2 technique and image analysis the intracellular concentration of free calcium ions [Ca 2+ ] i was examined in isolated rainbow trout pinealocytes identified by S-antigen immunocytochemistry. Approximately 30% of the pinealocytes exhibited spontaneous [Ca 2+ ] i oscillations whose frequency differed from cell to cell. Neither illumination with bright light nor dark adaptation of the cells had an apparent effect on the oscillations. Removal of extracellular Ca 2+ or application of 10 μ M nifedipine caused a reversible breakdown of the [Ca 2+ ] i oscillations. Application of 60 mM KCl elevated [Ca 2+ ] i in 90% of the oscillating and 50% of the non-oscillating pinealocytes. The effect of KCl was blocked by 50 μ M nifedipine. These results suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca 2+ ] i in trout pinealocytes. Experiments with thapsigargin (2 μ M) revealed the presence of intracellular calcium stores in 80% of the trout pinealocytes, but their role for regulation of [Ca 2+ ] i remains elusive. Treatment with norepinephrine (100 pM–50 μ M), previously shown to induce calcium release from intracellular calcium stores in rat pinealocytes, had no apparent effect on [Ca 2+ ] i in any trout pinealocyte. This finding conforms to the concept that noradrenergic mechanisms are not involved in signal transduction in the directly light-sensitive pineal organ of anamniotic vertebrates.

SUMMARY The rodent pineal organ transduces a photoneural input into a hormonal output. This photoneuroendocrine transduction leads to highly elevated levels of the hormone melatonin at nightime which serves as a message for darkness. The melatonin rhythm depends on transcriptional, translational and posttranslational regulation of the arylalkylamine- N -acetyltransferase, the key enzyme of melatonin biosynthesis. These regulatory mechanisms are fundamentally linked to two second messenger systems, namely the C AMP- and the Ca 2+ -signal transduction pathways. Our data gained by molecular biology, immunohistochemistry and single-cell imaging demonstrate a time-and substance-specific activation of these signaling pathways and provide a framework for the understanding of the complex signal transduction cascades in the rodent pineal gland which in concert not only regulate the basic profile but also finetune the circa-dian rhythm in melatonin synthesis. Melatonin after Four Decades, edited by James Olcese.Kluwer Academic / Plenum Publishers, New York, 2000.

Immunocytochemical double-labeling methods are important tools in cell and neurobiology. Here we describe a method which is based on double immunofluorescence and allows specific detection of two different antigens located in the same cell compartment by two primary antibodies raised in the same species. As an example, we present the double-immunolabeling method for the S-antigen (SAg), a photoreceptor-specific protein, and the indoleamine serotonin (5HT) in dissociated trout and rat pineal cells immobilized on coverslipped and in frozen sections of the trout pineal organ. As a first step, the preparations on the slides or coverslips were sequentially incubated with the first primary antibody (rabbit anti-SAg), the fluorescein-labeled (anti-rabbit) secondary antibody, and then with normal rabbit serum. Meanwhile, the second primary antibody (rabbit anti-5HT) was coupled to a Cy3-labeled secondary (anti-rabbit) antibody in a reaction tube and excess binding sites were quenched with normal rabbit serum. This complex was applied to the specimens after completion of the first (SAg) immunoreaction on the slide. For control experiments, the first (anti-SAg) or the second (anti-5HT) primary antibody were omitted. Most of the rat and trout pinealocytes were double immunolabeled for SAg and 5HT. In the trout, few cells contained SAg or 5HT immunoreaction only. This underlines the selectivity of each immunoreaction. The results show that the method can be used for the analysis of whole cells and tissue sections by means of conventional fluorescence and confocal laser scanning microscopy.

The present in vitro study correlates melatonin production of explanted trout pineal organs with intracellular calcium concentrations of isolated and immunocytochemically identified pinealocytes. Melatonin production of pineal organs maintained in flow-through organ culture is closely related to the incident light, showing high levels in the scotopic and mesopic range of illumination and low levels in the photopic range. Diminishing extracellular calcium (Ca2+) and enhancing magnesium (Mg2+) as well as application of Co2+ (3 mM) reduces melatonin release in the light- and dark-adapted state and causes a reversible breakdown of spontaneous oscillations in the intracellular concentration of free calcium ions [Ca2+]i that are observed in approximately 20% of the isolated S-antigen-immunoreactive pinealocytes. Intracellular calcium oscillations are also reversibly blocked by application of 10 μM nifedipine. Depolarization of pinealocytes by treatment with 60 mM KCl elevates [Ca2+]i in 90% of the oscillating and 50% of the non-oscillating cells. The effect of KCl is blocked by 50 μM nifedipine and 3 mM CoCl2. Furthermore, application of CoCl2 reduces the basal [Ca2+]i in non-oscillating pinealocytes. These results suggest that voltagegated L-type calcium channels play a major role in the regulation of [Ca2+]i in trout pinealocytes. It appears that Co2+ and low calcium/high Mg2+ buffer reduces melatonin release through an action on the intracellular calcium concentration of trout pinealocytes. All these data show that the trout pineal organ synthesizes and releases melatonin in relation to the irradiance of the incident light and depends on the presence of extracellular calcium which enters the cell via voltage-gated calcium channels. Treatment of cultured pineal organs with norepinephrine (1 μM) or the β-adrenergic agonist isoproterenol has no significant effect on the light-dependent melatonin release. Furthermore, application of norepinephrine in varying concentrations (100 pM - 50 μM) that were previously shown to induce calcium release from intracellular calcium stores in rat pinealocytes has no effect on [Ca2+]i of oscillating or non-oscillating trout pinealocytes. These data suggest that in the trout pineal organ a norepinephrine sensitive pinealocyte of the mammalian type is not involved in the transduction of light signals into a melatonin response.

The present in vitro study correlates measurements of the melatonin production from trout pineal organs with those of the intracellular calcium concentration in pinealocytes. Melatonin production increases with decreasing irradiance and shows maximal values in darkness. Some pinealocytes exhibit spontaneous calcium oscillations, although most of them have a stable basal calcium concentration. Diminishing extracellular calcium and enhancing magnesium reduces melatonin release in the light-and dark-adapted state. The application of Co2+ decreases melatonin secretion in the mesopic and scotopic range, reversibly blocks spontaneous calcium oscillations, reduces the basal intracellular calcium concentration in non-oscillating pinealocytes, and inhibits the KCl-induced rise in intracellular calcium. Application of glutamate, norepinephrine, isoproterenol, or dopamine has no significant effect on melatonin secretion. Norepinephrine does not influence the calcium concentration in any of the trout pinealocytes. Treatment with the GABAA-receptor agonist muscimol causes a slight reduction of melatonin release in the mesopic and scotopic range of illumination, without affecting intracellular calcium concentrations. Thus, Co2+ and low calcium/high magnesium buffer reduce melatonin release through an action on the calcium concentration in trout pinealocytes and not through a blockade of synaptic transmission. All the data show that the trout pineal organ synthesizes and releases melatonin in relation to the irradiance of the incident light and that neuronal inputs have a minor, if any, influence on melatonin synthesis.