Pineal Microdialysis of the Melatonin in Conscious Sheep: Methodology, Application to a Diurnal Rhythm and Effect of Isoproterenol

This paper describes the development of a new technique to measure melatonin contents in the pineal gland of moving sheep: the microdialysis. A dialysis probe was used to collect extracellular fluid in the sheep pineal gland, but also to inject directly into it different drugs such as isoproterenol at a very low concentration. The probe was implanted the day before the beginning of the experiment in order to obtain low levels of melatonin. This technique makes it possible to measure melatonin in the dialysate and plasma of rams submitted to 8L:16D. No melatonin either in the dialysate or in the plasma was found during the light phase. Shortly after lighting off, the melatonin concentration increased in the dialysate and plasma and remained stable during the dark phase. Melatonin concentrations began to decrease before lighting on and no detectable levels were found during the following light phase. The secretion of melatonin is, at least, under adrenergic regulation. Local infusion of isoproterenol (90  μl at 10⁻⁶ M), an agonist of β adrenergic receptor, through the probe, increased melatonin levels during 2  h, even when infusions were repeated 3 times. This demonstrates the presence of β adrenergic receptors. The technique presented in this paper could be of considerable interest for studying sheep pineal gland and its main secretion, melatonin, for example during diurnal rhythms or for studying its regulation.     

Melatonin Receptors on Ovine Pars Tuberalis: Characterization and Autoradiographicai Localization

The functional significance of the pars tuberalis (PT) of the mammalian adenohypophysis has remained an enigma (1, 2). One view of its function is that it acts as an auxiliary gland to support the endocrine role of the pars distalis (PD) (2), as it has been shown to contain immunocytochemically identifiable thyrotrophs and gonadotrophs (1). Many of the cells of the PT are, however, ultrastructurally unique suggesting an independent function for this tissue. Our recent demonstration that the PT of the rat is a major binding site for the ligand iodomelatonin lends further support to this idea (3). We have utilized the highly specific ligand [¹²⁵l]melatonin, and have demonstrated that it binds exclusively, with very high affinity, to the PT but not the PD of the adult sheep adenohypophysis. These findings support the conclusion that the PT has a distinct role in relation to melatonin action and seasonal reproduction.     

Evidence for a Regulatory Role of Melatonin on Serotonin Release and Uptake in the Pineal Gland

Melatonin has been proposed to exert some regulatory actions within the pineal gland itself. The present study examined the effect of melatonin on the release of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) from rat pineal glands by using an in vitro perifusion system. Melatonin induced a concentration-dependent stimulatory effect on 5-HT secretion from 10^?6 M to 10^?3 M. Maximal effects were obtained with melatonin 10^?3M and concentrations lower than 10^?6 M were without effect. The secretion of 5-HIAA was inhibited by melatonin 10^?3 and 10^?4M, but it was increased when pineals were incubated with 10^?5 and 10^?6 M of melatonin. The indoleamine secretion was also studied on peripherally denervated rat pineal glands. Basal output of 5-HT from these glands was increased when compared with those from control rats. In contrast, the secretion of 5-HIAA was strongly reduced after removal of the sympathetic input to the pineal gland. Melatonin 10^?3 M failed to stimulate 5-HT release from denervated pineal glands, although it inhibited 5-HIAA secretion. In contrast, melatonin 10^?5 M enhanced 5-HT release without altering 5-HIAA output. Fluoxetine, a 5-HT uptake inhibitor, produced similar effects than mM concentrations of melatonin on the indoleamine secretion from control pineal glands, but it had no effect on glands taken from peripherally denervated rats. These data suggest that mM concentrations of the pineal hormone are able to stimulate 5-HT release from the pinealocyte, while mM concentrations of melatonin increase extracellular 5-HT by inhibiting its reuptake in the adrenergic nerve endings. These findings are discussed in relation to the possible role of melatonin regulating the intra- and extracellular availability of 5-HT in the pineal gland and its significance as an autocrine factor.     

Regulation of Melatonin Secretion by Light in the Isolated Pineal Organ of the White Sucker (Catostomus commersoni)

The effects of different lighting conditions and physical parameters of light were investigated in the isolated pineal organ of the white sucker kept under static or superfusion culture. The secretion of pineal melatonin is directly controlled by the photoperiod and completely suppressed under constant illumination. When pineal organs are exposed to unexpected light at night, the secretion of melatonin is significantly reduced within 15 min and reaches basal value after 30–35 min of light treatment. The inhibition of melatonin secretion by unexpected light at night depends on the irradiance, duration, timing of the treatment and the lighting history of the pineal organ.     

Regulation of indoleamine N-Acetyltransferase activity in the retina: Effects of light and dark, protein synthesis inhibitors and cyclic nucleotide analogs

The regulation of indoleamine N-acetyltransferase (NAT) in the posterior eye was investigated in vivo, and in vitro in cultured eye cups. Surgical separation of neural retinal from the retinal pigment epithelium-choroid complex indicated that NAT was localized to neural retina. The activity of retinal NAT fluctuated in vivo in a rhythmic fashion, with peak activity in the dark phase of the light-dark cycle. The rhythm of NAT activity persisted for up to 3 days in constant darkness, with a rhythmic period of approximately 25 h. The rhythm was suppressed by constant light, and could be phase-shifted by exposure to a new light-dark cycle. These observations indicate that retinal NAT activity occurs as a circadian rhythm that is entrained by light and dark. Retinas also responded to light and dark in vitro with changes of NAT activity. A significant increase in retinal NAT activity occurred in eye cups cultured in darkness during the dark phase of the light-dark cycle. This increase was completely suppressed in eye cups cultured at the same time of day in light. The dark-induced increase in NAT was completely blocked by protein synthesis inhibitors, and mimicked in light by cyclic AMP analogs.The similarity of the regulation of NAT activity in retina to that in pineal, and the possible relationship of the retinal NAT rhythm to cyclic metabolism in photoreceptors are discussed.     

The Effect of NO-Donors in Bovine and Rat Pineal Cells: Stimulation of cGMP and cGMP-Independent Inhibition of Melatonin Synthesis

The presence of soluble guanylate cyclase in the pineal and its regulation by adrenergic pathways has been well documented. Recent evidence points to adrenergically stimulated nitric oxide generation as a mechanism for coupling this pathway. To what extent nitric oxide (NO) signalling can influence adrenergically stimulated melatonin synthesis has not been investigated. Cyclic guanosine 3′,5′-monophospate (cGMP) signal transduction in the bovine pineal has also received little attention. We describe in the present report: 1) a dose-dependent elevation of cGMP in response to the nitrovasodilators, sodium nitroprusside (SNP) and 3-morpholino-sydnonimine (SIN-l), 2) a dose-dependent inhibition of melatonin synthesis by SNP and SIN-1, but not by 8-Br-cGMP in both bovine and rat pineal cell cultures, which is not due to cytotoxicity as judged by two different approaches, and 3) immunohistochemical evidence for the presence of nitric oxide synthase (NOS) (EC 1.14.23.-) in the intact bovine pineal gland and in cultured bovine pinealocytes. These data support the view that NOS is a component of the cGMP-generating system in mammalian pinealocytes. Although NO-donor molecules are also potent activators of cGMP accumulation, they may have other important actions in the pineal, namely the inhibition of adrenergic-stimulated melatonin synthesis. As SNP and SIN-1 exerted this inhibitory effect on cells regardless of whether they were stimulated by isoproterenol, forskolin or 8-Br-CAMP it would appear that NO-donors can act ‘downstream’ from the receptor/adenylate cyclase level.     

Local Corticosterone Infusion Enhances Nocturnal Pineal Melatonin Production In Vivo

Melatonin, an important marker of the endogenous rhythmicity in mammals, also plays a role in the body defence against pathogens and injuries. In vitro experiments have shown that either pro- or anti-inflammatory agents, acting directly in the organ, are able to change noradrenaline-induced pineal indoleamine production. Whereas corticosterone potentiates melatonin production, incubation of the gland with tumour necrosis factor-α decreases pineal hormonal production. In the present study, we show that nocturnal melatonin production measured by intra-pineal microdialysis is enhanced in pineals perfused with corticosterone at concentrations similar to those measured in inflamed animals. In vitro experiments suggest that this enhancement may be due to an increase in the activity of the two enzymes that convert serotonin to N -acetylserotonin (NAS) and NAS to melatonin. The present results support the hypothesis that the pineal gland is a sensor of inflammation mediators and that it plays a central role in the control of the inflammatory response.     

Interaction of Forskolin and Melatonin on Cyclic AMP Generation in Pars Tuberalis Cells of Ovine Pituitary

The pineal indoleamine, melatonin, acts on specific secretory cells of the pars tuberalis of the sheep pituitary. Using pars tuberalis cells in primary culture melatonin inhibited forskolin-stimulated cyclic AMP production in both a time - and dose-dependent manner, but the nature of the melatonin response was critically dependent upon the stimulatory concentration of forskolin used. Forskolin alone stimulated dose-dependent cyclic AMP accumulation, which reached an equilibrium state after 15 min. This was maintained for up to 3 h, indicating a lack of desensitization to forskolin. Melatonin (1 μM) inhibited this response by greater than 80% at all doses. However, 100 μM forskolin reduced both the affinity and the number of the melatonin receptors, relative to untreated controls. Consistent with this, melatonin was 100 times less potent at inhibiting forskolin-stimulated cyclic AMP production, when titrated against 100 μM forskolin as compared to 1 μM forskolin. The response to 1 μM forskolin could be potentiated by 10 μM phorbol 12,13 myristate acetate, but not by calcium ionophore (A23187). This provides evidence for the interaction of the phosphatidylinositol pathway with the cyclic AMP system in these cells. Nevertheless melatonin can inhibit both the potentiated and non-potentiated response with equal effect.     

Long-Term Dynamic in vitro System for Investigating Rat Pineal Melatonin Secretion

Several details regarding the control of melatonin (MT) secretion from the pinealocytes are still to be clarified. To obtain more data on the mechanism and kinetics of MT secretion and on the interactions between bioactive materials affecting MT production, a perifusion system has been developed in our laboratory. In this dynamic in vitro system the surviving pinealocytes maintain their full responsiveness for at least 5 days. In order to determine the MT contents of large numbers of samples collected in the perifusion system, a sensitive MT radioimmunoassay was set up utilizing our specific MT antibody. In our perifusion system the basal MT release does not change significantly during the 5-day experiments. Norepinephrine (NE) was used at 1 μM concentration for 30 min as a marker of the responsiveness of pinealocytes, given at the beginning and at the end of the same experiments. No significant difference was found in the MT responses to NE stimulation over 5 days. The kinetics of MT response and the dose-response relationship were investigated after NE exposure at various concentrations (100 nM to 10 mM). NE at 100 nM was found to be ineffective. Between 1 μM and 1 mM concentrations NE increased the MT release in a dose-dependent manner. No significant difference was found between the responses above 1 mM concentration. NE seems to be a specific stimulator of pineal MT production. The MT production reached the maximum value after a relatively long lag (2 to 3 h) when NE application had been stopped, and returned to basal values after 5 to 6 h. This prolonged time-course of MT secretion, in contrast with the fast responses of pituitary cells to releasing hormones, suggests that NE stimulated the synthesis of MT rather than the release of stored hormone. The modulatory effects of light-dark cycle on basal and stimulated MT release of perifused pineals was also investigated: Neither basal nor NE-stimulated (100 nM to 10μM) MT release was influenced significantly by light-dark conditions showing that the light-dark cycle does not have a direct modulatory effect on MT secretion under in vitro circumstances. Based on our observations, this perifusion system should be a useful tool for investigating: 1) hormone interactions on the regulation of pineal MT release, and 2) accurate kinetics of MT response.     

Prostaglandins of the E Series Stimulate Cyclic AMP Accumulation and N-Acetylation of Serotonin in Chick Pineal Cells

We investigated the effects of prostaglandins on cyclic AMP (cAMP) levels and on the activity of the rate-limiting enzyme of melatonin biosynthesis, arylalkylamine-N-acetyltransferase (NAT). The study was performed on primary cultures of dispersed chick pineal cells. Prostaglandin E, (PGE,) increased cAMP levels 2-fold and this stimulation went up to 4-fold in the presence of a phosphodiesterase inhibitor. The PGE,- evoked increase in cAMP levels did not desensitize over 6 h. The potency order of a series of prostaglandins to increase cAMP levels (PGE₁ PGE₁>PGA2>PGD₂?PGF₂α) agreed with the pharmacological profile of the adenylate cyclase-coupled prostaglandin receptor. Inhibition of endogenous prostaglandin synthesis by two cyclooxygenase inhibitors (indomethacin and aspirin) caused a 30% decrease in cAMP levels. This effect was completely reversed by the addition of exogenous PGE₁ or PGE₂. Indomethacin and aspirin also caused a 50% decrease in NAT activity. Prostaglandins of the E series increased NAT activity up to 2-fold above basal level and restored NAT activity after inhibition by indomethacin or aspirin. These results are the first illustration of a role for prostaglandins in chick pineal cells. The correlations observed between cAMP levels and NAT activity suggest that the regulation of NAT activity by prostaglandins of the E series might be mediated by changes in cAMP concentration.     

Changes in Pineal Indoleamines in Rats after Single Melatonin Injections: Evidence for a Diurnal Sensitivity to Melatonin

We recently determined that melatonin stimulated serotonin (5-HT) secretion from rat pineal glands by increasing 5-HT release from the pinealocytes (μM melatonin concentrations) and by inhibiting 5-HT uptake in the pineal sympathetic nerve endings (mM melatonin concentrations). The present study investigated whether a single melatonin injection could alter the content of indoleamines in the rat pineal gland, as well as its possible dependence on the daytime of administration. Melatonin (150 μg/kg) was i.p. injected at 8 time points (11.00 h, 14.00 h, 17.00 h, 20.00 h, 23.00 h, 02.00 h, 05.00 h and 08.00 h) to rats kept in 12:12 h light:dark cycle (lights on at 07.00 h). Melatonin injections in the afternoon (17:00 h) and late in the nighttime (02.00 h and 05.00 h) decreased pineal 5-HT content 90 min later. The levels of 5-hydroxyindoleacetic acid (5-HIAA) were also decreased 90 min after the melatonin treatment at 14.00 h, 17.00 h and 02.00 h. The effect of melatonin on 5-HT content was a long-lasting effect (still evident after 180 min) only when injected at 02.00 h, whereas 5-HIAA levels were found to be decreased 180 min after melatonin treatment at 14.00 h and 23.00 h. No changes in these compounds were detected 240 min after melatonin treatment. Moreover, melatonin did not change 5-hydroxytryptophan levels at any of the daytime points studied. By contrast, 90 min after the injection of melatonin at 20.00 h, an increased content of pineal N-acetylserotonin was observed. This effect of melatonin could be mediated through a phase alteration of the pineal N-acetyltransferase activity rhythm by acting on the suprachiasmatic clock, althought a direct melatonin effect on the pineal rhythmic function cannot be excluded. The effects of the hormone on 5-HT and 5-HIAA contents agree with previous findings on the inhibitory effect of pharmacological doses of melatonin on pineal 5-HT uptake, which presumably would result in a decreased intraneuronal content of 5-HT and its acid metabolite. These data point to an acute regulatory action of exogenous melatonin on the pineal melatonin synthesis pathway which seems to be limited to two daytime phases: the afternoon-early evening period and the second half of the night.     

Castration Reduces the Nocturnal Rise of Pineal Melatonin Levels in the Male Rat by Impairing its Noradrenergic Input

The effects of castration and testosterone treatment on pineal day-night rhythms were studied in male rats. Bilateral gonadectomy was performed at 21 days of age. Testosterone propionate was given subcutaneously to castrated animals in a dose of 10 μg/100 g body weight during two consecutive days before sacrifice. Animals were killed 40 days after gonadectomy at four different times of a 12:12 h light-dark cycle (1600, 2400, 0400 and 0800h). Tyrosine hydroxylase activity was measured in individual pineals by means of high-performance liquid chromatography determination of L-DOPA formed. Pineal levels of norepinephrine, dopamine, 5-hydroxytryptamine and 5-hydroxyindole acetic acid were determined by high-performance liquid chromatography with amperometric detection, while pineal melatonin content was measured by radioimmunoassay. Castration abolished the day-night rhythms of pineal tyrosine hydroxylase activity and norepinephrine content, both by elevating their daytime levels and by blocking their nocturnal rise. In addition, gonadectomy drastically modified pineal indoleamine metabolism by increasing daytime levels of both 5-hydroxytryptamine and 5-hydroxyindole acetic acid, and by reducing the nocturnal elevation of pineal melatonin content. Testosterone treatment was unable to prevent the effect of orchidectomy on pineal rhythms of tyrosine hydroxylase activity, 5-hydroxytryptamine or 5-hydroxyindole acetic acid content, however it partially restored the day-night pineal rhythms of both norepinephrine and melatonin content. These results are indicative of a possible participation of reproductive hormones in the control of pineal rhythmic activity in the male rat. Apparently, since gonadectomy abolished the nocturnal rise of both pineal tyrosine hydroxylase activity and norepinephrine content, the primary site of action of reproductive hormones could be at the level of the superior cervical ganglion.     

Photoreceptor Cells of the Pike Pineal Organ as Cellular Circadian Oscillators

In the pike pineal, the rhythm of melatonin (MEL) secretion is driven by a population of cellular circadian oscillators, synchronized by the 24 h light/dark (LD) cycle. Because the pineal photoreceptor contains both the input and output pathways of the clock, this cell is likely to be a cellular circadian system by itself. To support this idea, we have dissociated and cultured pike pineal cells as well as purified photoreceptors. In culture, the pineal cells reassociated in follicles, surrounded by collagen fibres. At the electron microscopic level, they appeared well preserved. Total cells consisted mainly of photoreceptors and glia. Purified cells corresponded exclusively to photoreceptors. Under LD, MEL production was rhythmic. Under constant darkness (DD), the rhythm was well sustained for at least six 24 h cycles (τ= 24/27 h) with 1 × 10⁶ total cells/well or below; with 2 × 10⁶ total cells/well, a strong damping occurred towards high levels as soon as after the second cycle. At the density of 0.5 × 10⁶ cells/well, purified photoreceptors produced less MEL than an equivalent amount of total cells. However, the pattern of the oscillations was similar to that observed with 2 × 10⁶ total cells, i.e. a damping occurred rapidly. Decreasing the density to 0.125 × 10⁶ photoreceptors/well resulted in a loss of homogeneity among replicates. The production of melatonin by single photoreceptors was monitored by means of the reverse haemolytic plaque assay. Both under LD and under DD, the number of photoreceptors releasing melatonin was higher during the (subjective) dark than during the (subjective) light. The results provide strong support to the idea that the pike pineal photoreceptor is a cellular circadian system. Expression of the oscillations seemed to depend on several factors, including cell to cell contacts between photoreceptors. There is indication that also MEL and glia might be involved.     

Melatonin: Generation and Modulation of Avian Circadian Rhythms

The pineal organ and its hormone melatonin are significant components of avian circadian pacemaking systems. In songbirds, pinealectomy results in the abolition or destabilization of overt circadian rhythms such as the rhythm of locomotor activity, feeding, or body temperature. A stable rhythmicity can be restored either by reimplanting a pineal organ, by periodic injections or infusions of melatonin, or by applying melatonin rhythmically through the drinking water. Several results suggest that the pineal melatonin rhythm acts on at least one other oscillator within the circadian pacemaking system, presumably the SCN, which in turn, feeds back to the pineal. As described by the “Neuroendocrine Loop” and “Internal Resonance” models, overall pacemaker output thus depends on the relative strengths of the oscillations in the pineal and the SCN. Investigations on migratory birds have shown that the amplitude of the 24-h plasma melatonin rhythm is reduced during the migratory seasons compared with the nonmigratory seasons. According to the models mentioned above, such a reduced melatonin amplitude should result in a reduction in the degree of self-sustainment of the pacemaker as a whole. This, in turn, should facilitate adjustment to the altered Zeitgeber conditions encountered by these birds as a result of their own migratory flights. A seasonal reduction in melatonin amplitude also occurs in some high-latitude birds during midsummer and midwinter. Under such conditions a less self-sustained circadian pacemaker may enhance entrainability to weak zeitgeber conditions. These examples suggest that the properties of the circadian system may be adjusted to match the changing requirements for synchronization, and that this is achieved by altering the melatonin amplitude.     

Melatonin modulates spike coding in the rat suprachiasmatic nucleus

The effects of the application of melatonin in vitro on the electrophysiological activity of suprachiasmatic neurones were characterised using novel measures of coding based on the analysis of interspike intervals. Perfusion of 1 nM melatonin in vitro (n = 53) had no consistent effect on mean spike frequency (Wilcoxon's sign rank, z = -0.01, P = 0.989), but increased the irregularity of firing (Student's paired t-test, t = -3.02, P = 0.004), as measured by the log interval entropy, and spike patterning (z = -3.43, P < 0.001), as measured by the mutual information between adjacent log intervals. Intracellular recordings in vitro in current clamp mode showed that 1 nM melatonin significantly hyperpolarised (n = 11, z = -2.35, P = 0.019) those cells that showed 'rebound' spikes upon termination of a hyperpolarising current pulse. Grouping all cells together (n = 27), melatonin application decreased the duration of the afterhyperpolarisation (z = -2.49, P = 0.013) and increased the amplitude of the depolarising afterpotential (z = -2.71, P = 0.007). The effects of melatonin seen in vitro from extracellular recordings on interspike interval coding were consistent with the changes in spike shape seen from intracellular recordings. A melatonin-induced increase in the size of the depolarising afterpotential of suprachiasmatic cells might underlie the increased irregularity of spike firing seen during the subjective night time. The method of analysis demonstrated a difference in spike firing that is not revealed by frequency alone and is consistent with the presence of a melatonin-induced depolarising current.     

Genetic,temporal and developmental differences between melatonin rhythm generating systems in the teleost fish pineal organ and retina

Complete melatonin rhythm generating systems, including photodetector, circadian clock and melatonin synthesis machinery, are located within individual photoreceptor cells in two sites in Teleost fish: the pineal organ and retina. In both, light regulates daily variations in melatonin secretion by controlling the activity of arylalkylamine N-acetyltransferase (AANAT). However, in each species examined to date, marked differences exist between the two organs which may involve the genes encoding the photopigments, genes encoding AANAT, the times of day at which AANAT activity and melatonin production peak and the developmental schedule. We review the fish pineal and retinal melatonin rhythm generating systems and consider the evolutional pressures and other factors which led to these differences.     

Melatonin Inhibits the Activation of Cyclic AMP-Dependent Protein Kinase in Cultured Pars Tuberalis Cells from Ovine Pituitary

The effect of melatonin upon the activation of the intracellular effector enzyme, cyclic AMP (cAMP)-dependent protein kinase (PKA), was investigated in primary cultures of ovine pars tuberalis cells. Incubation of these cells with forskolin caused a rapid and dose-dependent activation of PKA (ED₅₀ 10^～6M). When cells were incubated with forskolin and melatonin simultaneously, the activation of PKA by forskolin was dramatically inhibited. This inhibitory effect of melatonin was dose-dependent (ED₅₀ 10^?10M). Furthermore, treatment with melatonin rapidly deactivated PKA in cells prestimulated with forskolin. When pars tuberalis cell extracts were incubated with 8N₃-[³²P]cAMP, an analogue of cAMP used for photoaffinity labelling of native PKA, specific binding was observed in three bands with M_r of 54, 52 and 48 kd, representing the regulatory subunits of PKA II (in phosphorylated and dephosphorylated forms) and PKA I, respectively. These results indicate that melatonin is a potent inhibitory regulator of cAMP-mediated signal transduction in the ovine pars tuberalis, and suggest that the cellular effects of melatonin in this tissue are mediated by the dephosphorylation of specific substrate proteins.