Jesús M. Míguez

The role of the intracellular and extracellular serotonin in the regulation of melatonin production in rat pinealocytes

Miguez JM, Simonneaux V, Pevet P. The role of the intracellular and extracellular serotonin in the regulation of melatonin production in rat pinealocytes. J. Pineal Res. 1997; 23:63–71.

Daily and Photoperiodic Melatonin Binding Changes in the Suprachiasmatic Nuclei, Paraventricular Thalamic Nuclei, and Pars Tuberalis of the Female Siberian Hamster (Phodopus sungorus)

Using quantitative autoradiography, 2-125I-melatonin binding was investigated throughout the light:dark cycle in the suprachiasmatic nuclei (SCN), paraventricular nuclei (PVT), and pars tuberalis (PT) of adult female Siberian hamsters kept for 10 weeks in either long or short photoperiods (LP or SP, respectively). Plasma melatonin concentrations were measured by radioimmunoassay, and the sexual status of the animals was established by visual inspection of vaginal smears and by weighing uteri after sacrifice. The SCN displayed neither daily nor photoperiod-dependent variations in specific binding. Melatonin receptors in these nuclei would be regulated neither by plasma melatonin nor by the light:dark cycle or sexual steroids. By contrast, melatonin receptor density in the PT displayed both strong daily (maximal values during the first half of the light period and minimal values during the night) and photoperiod-dependent (maximal values in LP) variations. These variations depended on changes in the maximal binding (Bmax ) without differences in the dissociation constant (Kd ). Daily melatonin receptor densities in the PT of LP- and SP-exposed animals might be regulated by the dark:light transition but not by melatonin. Daily profiles of 2- 125I-melatonin-specific binding in the PT were independent of photoperiod. Factors underlying the photoperiod-related variations presently are unknown. Concerning the PVT, weak variations in specific binding were detected in SP only when time points were grouped according to the light or dark periods. It is not yet possible to conclude whether they have any physiological relevance. These results show clearly that the regulation of melatonin receptors varies among structures (SCN, PVT, and PT) in the Siberian hamster and is also totally different from that found in the rat.

Diurnal changes in the content of indoleamines, catecholamines, and methoxyindoles in the pineal gland of the Djungarian hamster (Phodopus sungorus): effect of photoperiod.

Abstract: Previous studies in Syrian hamster have shown that the correlations between the daily fluctuations in the contents of pineal indoleamines and methoxyindoles are influenced by the photoperiod, and that dopamine may play a role in the regulation of pineal function. The present study investigated the 24 hour changes in the content of 5‐hydroxytryptophan (5‐HTP), serotonin (5‐HT), 5‐hydroxyindoleacetic acid (5‐HIAA), N‐acetylserotonin (NAS), melatonin, 5‐methoxytryptophol (5‐MT), noradrenaline (NA), dopamine (DA), and 3,4‐dihydroxyphenylacetic acid (DOPAC) in the pineal gland of female Djungarian hamsters exposed to long (LP; LD 16: 8) or short (SP; LD 10: 14) photoperiods for 10 weeks. Pronounced nocturnal increases of N‐acetylserotonin and melatonin content were observed irrespective of the photoperiod regime. The content of 5‐HT was markedly decreased during the first hour of the night in LP, which contrasted with the lack of changes in NAS and melatonin content at this time. In SP, an increased 5‐HTP content and a less obvious decrease in 5‐HT content was observed during the night, although melatonin and NAS content were even higher than in LP. Similar daily patterns as for 5‐HT were observed in the 5‐HT oxidative metabolites (5‐HIAA and 5‐MT). When considering values throughout the day, a poor correlation for 5‐HT vs. NAS and melatonin content was observed, which was particularly evident during the nighttime. These data indicate that the daily variation of pineal indoles may not only be dependent on changes in the N‐acetyltransferase activity but also in other mechanisms regulating pinealocyte 5‐HT availability. As previously reported in other species of hamster, pineal NA content did not show daily variations in LP conditions, although a nocturnal increase was detected in SP. In contrast, pronounced nocturnal increases were observed in the content of DA and its acid metabolite, DOPAC, irrespective of the photoperiod. These data indicate the existence of an increased dopaminergic turnover during the night and further support a role for DA in the regulation of melatonin synthesis and in the synchronization of the pineal functions.

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.

Secretoneurin: a new neuropeptide in the rodent pineal gland

Abstract.The pineal gland of mammals is richly innervated by nerve fibres containing various peptides of central or sympathetic origin. In this study, we have investigated the presence, regulation and effect of secretoneurin (SN), a 33-amino-acid peptide discovered recently in the central and sympathetic nervous systems. The rat pineal gland contains about 34 fmol SN/pineal, although only 20% of the secretoneurin immunoreactivity (SN-IR) is the free peptide. SN-IR is localised in only a few nerve fibres found in the parenchyma of the gland and along the pineal stalk. In the rat pineal, SN-IR shows no midday/midnight differences but is significantly decreased (55%–65%) after superior cervical ganglionectomy or exposure to constant light. These observations suggest that, in the rat pineal, SN-IR originates partly from central areas and partly from the superior cervical ganglia. The pineal of the Syrian hamster contains much more SN-IR: 120 fmol SN-IR/pineal (about 20% being the free peptide). SN-IR is found both in nerve fibres and pineal cells. Some of the fibres run along the pineal stalk, indicating that they are of central origin, although a sympathetic origin cannot be ruled out. Pineal SN-IR is below detectable levels in the Siberian hamster. In cultured rat pinealocytes, SN significantly inhibits serotonin release and, to a lower extent, melatonin release.

Peptidergic Modulation of Serotonin Release from Cultured Rat Pinealocytes

This paper describes the effects of β‐adrenergic and peptidergic inputs on serotonin (5‐HT) synthesis, outflow and metabolism into melatonin in cultured dissociated rat pinealocytes. The spontaneous outflow of 5‐HT from pinealocytes was high as demonstrated by the elevated levels of extracellular 5‐HT accumulated in the medium (about 5u2003ng/h/70,000 pineal cells). The β‐adrenergic agonist isoproterenol (ISO) used at concentrations up to 10−6 M induced a moderate (+20–40%) increase in intra‐ and extracellular 5‐HT levels together with a large release of melatonin. At a higher ISO stimulation (10−5 M), the intra‐ and extracellular levels of 5‐HT were significantly (−25–30%) reduced whereas melatonin secretion was dramatically increased. This is interpreted as a large 5‐HT mobilization for melatonin synthesis and release, consequently reducing both the intracellular pool and outflow of 5‐HT. The peptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) up to 10−7 M induced always a moderate (+20–30%) increase in intra‐ and extracellular levels of 5‐HT. However, the use ofu2003nM concentrations of VIP or PACAP together with 10−6 M ISO induced a decrease in 5‐HT outflow (−25–30%) and a dramatic increase in melatonin secretion as did 10−5 M ISO alone. Neuropeptide Y (NPY) is another pineal peptide which induced a stimulation of 5‐HT outflow (+30–40%) although its effect on melatonin release was marginal. The above results are discussed in term of the multineuronal regulation of the synthetic and secretory activities of the rat pineal gland.

Melatonin receptors in the suprachiasmatic nuclei and pars tuberalis of testosterone induced photoresponsive rats

The sexual axis of rats can be rendered photoresponsive by testosterone implants. We have studied in these conditions whether rat pars tuberalis (PT) melatonin receptor density would be decreased after exposure to short photoperiod (SP) like as observed in long day seasonal breeders. The answer is no, but we observed that testosterone induced a photoperiod independent decrease in PT melatonin receptor density. These results show that testosterone-induced photosensitivity in rat is not linked to an SP-induced decrease in PT melatonin receptor density. However, testosterone regulates PT melatonin receptors independently of the photoperiod.

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 (150u2003μg/kg) was i.p. injected at 8 time points (11.00u2003h, 14.00u2003h, 17.00u2003h, 20.00u2003h, 23.00u2003h, 02.00u2003h, 05.00u2003h and 08.00u2003h) to rats kept in 12:12u2003h light:dark cycle (lights on at 07.00u2003h). Melatonin injections in the afternoon (17:00u2003h) and late in the nighttime (02.00u2003h and 05.00u2003h) decreased pineal 5‐HT content 90u2003min later. The levels of 5‐hydroxyindoleacetic acid (5‐HIAA) were also decreased 90u2003min after the melatonin treatment at 14.00u2003h, 17.00u2003h and 02.00u2003h. The effect of melatonin on 5‐HT content was a long‐lasting effect (still evident after 180 min) only when injected at 02.00u2003h, whereas 5‐HIAA levels were found to be decreased 180u2003min after melatonin treatment at 14.00u2003h and 23.00u2003h. No changes in these compounds were detected 240u2003min after melatonin treatment. Moreover, melatonin did not change 5‐hydroxytryptophan levels at any of the daytime points studied. By contrast, 90u2003min after the injection of melatonin at 20.00u2003h, 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.