Posttranslational regulation of TPH1 is responsible for the nightly surge of 5-HT output in the rat pineal gland

Serotonin (5-hydroxytryptamine, 5-HT), a precursor for melatonin production, is produced abundantly in the pineal gland of all vertebrate animals. The synthesis of 5-HT in the pineal gland is rate limited by tryptophan hydroxylase 1 (TPH1) whose activity displays a twofold increase at night. Earlier studies from our laboratory demonstrate that pineal 5-HT secretion exhibits dynamic circadian rhythms with elevated levels during the early night, and that the increase is controlled by adrenergic signaling at night. In this study, we report that (a) 5-HT total output from the pineal gland and TPH1 protein levels both display diurnal rhythms with a twofold increase at night; (b) stimulation of cAMP signaling elevates 5-HT output in vivo; (c) 5-HT total output and TPH1 protein content in rat pineal gland are both acutely inhibited by light exposure at night. Consistent with these findings, molecular analysis of TPH1 protein revealed that (a) TPH1 is phosphorylated at the serine 58 in vitro and in the night pineal gland; and (b) phosphorylation of TPH1 at this residue is required for cAMP-enhanced TPH1 protein stability. These data support the model that increased nocturnal 5-HT synthesis in the pineal gland is mediated by the phosphorylation of TPH1 at the serine 58, which elevates the TPH1 protein content and activity at night.     

Daily variation in the content of indoleamines, catecholamines and related compounds in the pineal gland of Syrian hamsters kept under long and short photoperiods

Abstract: This study examined the diurnal changes in the content of 5-hydroxytryptophan (5-HTP), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), N-acetylserotonin (NAS), melatonin, 5-methoxytryptophol (5-ML), noradrenaline (NA), dopamine (DA), and 3,4-dihydroxyphenylacetic acid (DOPAC) in the pineal gland of Syrian hamsters kept under long (14L: 10D) and short (10L: 14D) photoperiods. The nocturnal increase in NAS and melatonin levels was dependent upon the prevailing photoperiod, with a prolonged duration when the night lengthened. In both photoperiods, NAS and melatonin contents increased several hours after the onset of darkness, and, in animals kept in short photoperiod, the levels of both compounds began to decrease before light onset. On the contrary, decreases were noted in 5-HT, 5-HIAA, and 5-ML contents during the night, which was directly proportional to the dark phase. 5-HTP levels did not show a rhythmic variation. Correlations between the mean values of 5-HT-related compounds showing daily rhythms were very high when group means were compared, but they decreased when values from individual animals were considered. In addition, when correlations were calculated on per-animal basis during the night phase, a weak negative correlation was found for 5-HT vs NAS and 5-HT vs melatonin, although the correlation of 5-HT with positively 5-HT-correlated compounds (5-HIAA and 5-ML) continued to be high. These results indicate that the nocturnal increase in the N-acetyl transferase activity is the major factor generating the rhythm of pineal 5-HT content, but that other photoperiod-dependent mechanisms (i.e., 5-HT synthesis or release) seem to be also implicated. On the other hand, this study shows that NA content in the Syrian hamster pineal gland does not exhibit daily variations, although marked nocturnal increases in the levels of DA and DOPAC were evident. These results suggest the existence of parallel daily alterations in pineal catecholamine synthesis and release, and suggest a role for DA in the pineal activation at night.     

Circadian Rhythms of Human Pineal Melatonin, Related Indoles, and Beta Adrenoreceptors: Post-Mortem Evaluation

Human pineal glands obtained from 77 post-mortem sources from various age groups and times of death were used to examine the 24-hour cycle of serotonin (5-HT), melatonin, N-acetylserotonin (NAS), and beta adrenoceptor density. Pineal glands were divided sagitally and a single half was used to measure 5-HT, NAS, and melatonin concentrations, while the remaining half from the same gland was employed to assess changes in the density of beta adrenoceptors on partially purified membranes. The results show that density of pineal beta adrenoceptors was relatively constant between midnight and 18.00 h and became significantly higher between 18.00 and 20.00 h as measured by ligand saturation binding experiments using (125-1) iodocyanopindolol. The receptor affinity of all of the samples assayed remained in relatively narrow range near 58 pM and only changes in the relative receptor density were apparent. The up-regulation of receptors coincided with an increase in the concentration of 5-HT that began to rise between 16.00 and 20.00 h and became maximal between 20.00 and midnight. NAS, the immediate precursor of melatonin, was also at maximal levels between 20.00 h and midnight. Both 5-HT and NAS began declining after midnight and this change corresponded to the maximal pineal gland concentration of melatonin between midnight and 4.00 h. It is therefore suggested that the up-regulation of beta adrenoceptors noted during the late afternoon and early evening hours corresponds to the increased synthesis of 5-HT and the subsequent conversion to NAS. These events are followed by the highest accumulation of melatonin after midnight and represent the synthesis of melatonin from its precursor NAS in a sequential pattern.     

Tryptophan administration inhibits nocturnal N-acetyltransferase activity and melatonin content in the rat pineal gland. Evidence that serotonin modulates melatonin production via a receptor-mediated mechanism.

In the rat pineal gland, the activity of serotonin N-acetyltransferase (NAT) and the concentration of melatonin are normally high at night; conversely, the concentration of serotonin (5-HT), the precursor of melatonin, is low. Since tryptophan administration increases the concentration of pineal 5-HT at night, we examined its effect of melatonin production. Nighttime tryptophan loading led to substantial increases in pineal 5-hydroxytryptophan, 5-hydroxyindole acetic acid (5-HIAA), and 5-HT but a highly significant reduction in NAT activity in comparison to saline-injected controls. In contrast to other measured indoles, melatonin levels also were significantly diminished by tryptophan loading. Nocturnally high pineal norepinephrine levels were unaltered by tryptophan administration. The idea that high concentrations of 5-HT could lead to substrate inhibition of NAT activity was not supported by kinetic analysis of control NAT levels versus tryptophan-inhibited NAT activity under varied substrate concentrations. Hypotheses to explain these results include the possibility that tryptophan inhibition of melatonin synthesis is mediated by the release of 5-HT from the pinealocyte and its subsequent autocrine action on melatonin production.     

Daily rhythm in pineal phosphodiesterase (PDE) activity reflects adrenergic/3',5'-cyclic adenosine 5'-monophosphate induction of the PDE4B2 variant

The pineal gland is a photoneuroendocrine transducer that influences circadian and circannual dynamics of many physiological functions via the daily rhythm in melatonin production and release. Melatonin synthesis is stimulated at night by a photoneural system through which pineal adenylate cyclase is adrenergically activated, resulting in an elevation of cAMP. cAMP enhances melatonin synthesis through actions on several elements of the biosynthetic pathway. cAMP degradation also appears to increase at night due to an increase in phosphodiesterase (PDE) activity, which peaks in the middle of the night. Here, it was found that this nocturnal increase in PDE activity results from an increase in the abundance of PDE4B2 mRNA (approximately 5-fold; doubling time, approximately 2 h). The resulting level is notably higher (>6-fold) than in all other tissues examined, none of which exhibit a robust daily rhythm. The increase in PDE4B2 mRNA is followed by increases in PDE4B2 protein and PDE4 enzyme activity. Results from in vivo and in vitro studies indicate that these changes are due to activation of adrenergic receptors and a cAMP-dependent protein kinase A mechanism. Inhibition of PDE4 activity during the late phase of adrenergic stimulation enhances cAMP and melatonin levels. The evidence that PDE4B2 plays a negative feedback role in adrenergic/cAMP signaling in the pineal gland provides the first proof that cAMP control of PDE4B2 is a physiologically relevant control mechanism in cAMP signaling.     

Melatonin, Serotonin, 5-Hydroxyindole-3-Acetic Acid and N-Acetyltransferase in the Pineal of the European Hamster (Cricetus cricetus) Kept Under Natural Environmental Conditions: Lack of a Day/Night Rhythm in Melatonin Formation in Spring and Early Summer

In female European hamsters killed in spring and early summer, pineal melatonin content exhibited no day/night rhythm. Absolute levels measured were relatively low, being on the order of daytime levels detected in other hamster species. An absence of day/night changes in the activity of N-acetyltransferase was also observed. However, a marked rhythm in pineal serotonin (5-HT) was found, an abrupt large increase being observed at the beginning of the light period. The day/night rhythm of pineal 5-HIAA content is similar to that of 5-HT. This absence of rhythm in pineal melatonin formation might mean that in the European hamster it is not melatonin but another substance that is of importance in photoperiodism. An absence of melatonin rhythm, however, could also be simply a peculiar pattern of melatonin production observed at a given period of the year. In this case, melatonin would be able to transduce photoperiodic information in the European hamster, as in other photoperiodic species.     

Serotonin content and melatonin production in the pineal gland of the male Djungarian hamster (Phodopus sungorus)

Diurnal variations in serum melatonin levels and pineal concentrations of serotonin (5-HT), N-acetylserotonin (NAS), 5-hydroxyindole acetic acid (5-HIAA), and melatonin were estimated in adult male Djungarian hamsters kept under long-day (LD 16:8) or short-day (LD 8:16) photoperiods. The nocturnal increase in melatonin production was accompanied by a marked drop in pineal serotonin concentrations. Serotonin levels, however, decreased approximately 4 hr before pineal melatonin increased. Correlations of the mean values for pineal serotonin and pineal melatonin indicated significant correlations at both LD 16:8 (r = -0.92, P less than 0.001) and LD 8:16 (r = -0.85, P less than 0.001). The mean levels of pineal serotonin and serum melatonin were correlated as well (LD 16:8, r = -0.91, P less than 0.001; LD 8:16, r = -0.81, P less than 0.01). The levels of pineal serotonin declined at approximately the same time as serum melatonin levels increased. These data suggest that the drop in pineal serotonin is primarily a consequence of melatonin production (as reflected by increasing serum concentrations). Consequently, pineal concentrations of melatonin may not be the best estimate of actual melatonin production, but a measure of how much melatonin is accumulated within the pineal due to high synthesis rates while the release of the hormone from the gland is limited.     

Indoleamines and 5-methoxyindoles in trout pineal organ in vivo: daily changes and influence of photoperiod

This study describes the diel rhythms in several indoleamines, melatonin, and related 5-methoxyindoles in the pineal organ of rainbow trout in vivo. In addition, the effect of different photoperiod conditions was evaluated. Melatonin levels displayed clear daily rhythms in the pineal organ of rainbow trout kept experimentally under long (LD 16:08), neutral (LD 12:12), and short (LD 08:16) photoperiods. Duration of melatonin signal was dependent on the night length of prevailing photoperiod, while peak amplitude was higher when lengthening the photoperiod. Significant daily rhythms in 5-HT content, the precursor of melatonin synthesis, were found in neutral and short photoperiod with increases of the amine content just after the light-dark interphase and decreases in the middle of the night, which were more important under short photoperiod. In contrast, no significant 24-h cyclic variation was found in pineal 5-HT content under long photoperiod. Daily profiles in the content of the main 5-HT oxidative metabolite, the 5-hydroxyindoleacetic acid (5-HIAA), outlined those of the amine precursor. The chronograms of both aminergic compounds contrast with those of 5-hydroxytryptophan content, which displayed a net tendency to increase at night. This study also provides evidence for the existence of daily cyclic changes in the content of 5-methoxytryptamine (5-MT), 5-methoxyindoleacetic acid (5-MIAA), and 5-methoxytryptophol (5-MTOL) in trout pineal organ, which were also dependent on photoperiod. The 24-h profiles in 5-MT content correlated well with those of 5-HT, showing a peak at the first hour of darkness in all photoperiodic conditions, and a decay at midnight only in both neutral and long photoperiods. Similarly, the content of 5-MTOL also displayed high values during the day-night transition in trout kept under neutral and long photoperiods, followed by a slow decay all along the night. Finally, levels of 5-MIAA increased in all photoperiods when lights were turned off, being this nocturnal increase maximal in fish kept under LD 16:08. These results suggest that light-dark cycle modulates daily rhythms in pineal indoles and non-melatonin 5-methoxyindoles by acting mainly through the melatonin synthesis activity, which limits the availability of 5-HT for the oxidative and direct methylation pathways. In addition, it seems that a nocturnally increased synthesis of 5-HT might be a requirement for the optimal formation of melatonin and other 5-methoxyindoles in the pineal organ when trout remain under short photoperiods.     

Food deprivation and refeeding effects on pineal indoles metabolism and melatonin synthesis in the rainbow trout Oncorhynchus mykiss

The effects of food deprivation and refeeding on daily rhythms of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and melatonin contents, as well as on arylalkylamine N-acetyltransferase (AANAT) activity were evaluated in the pineal organ of rainbow trout. In addition, changes in circulating melatonin and cortisol levels were tested at one single point at day and night. Immature rainbow trout were distributed in 3 experimental groups: fish fed, fish fasted (7 days), and fish fasted for 7 days and refed for 5 days. All fish were sampled from each treatment group at different times of the day-night cycle. Pineal melatonin levels and AANAT activity showed daily variations in either fed, fasted and refed trout, displaying highest values at night. Fasted trout showed reduced melatonin content throughout the 24-h cycle, which was associated with decreased AANAT activity. Rhythms of pineal 5-HT and 5-HIAA levels were evident in all groups and were negatively correlated to melatonin in fed fish groups, but not in fasted fish. A higher content of 5-HT and 5-HIAA was observed in fasted fish during the night with no apparent changes during daytime for 5-HT and increased 5-HIAA levels. Furthermore, decreased circulating levels of melatonin were observed at midday, but not at night, in food deprived trout. Refeeding for 5 days generally counteracted the effects of food deprivation. Cortisol levels in plasma were reduced after food deprivation and remained low in refed fish. The results show that food deprivation impairs daily rhythms of melatonin content in trout pineal organ by affecting the activity of melatonin synthesizing enzymes rather than by a deficiency in substrate availability.     

Long-term in vivo pineal microdialysis

This study describes the development of a new technique for long-term measurement of daily 5-hydroxytryptamine (5-HT) and melatonin contents in the pineal gland of freely moving rats. The technique features a number of novel improvements over previous protocols. It allows visualization of the pineal gland for accurate targeting of the guide cannula, which minimizes bleeding; incurs no direct injury to the surrounding brain tissues; and causes no interference with the sympathetic innervation from the superior cervical ganglia. Robust releases of melatonin and indole precursors were continuously monitored quantitatively and reproducibly for more than 2 wk in the same animal. In addition, effects of pharmacological agents on in vivo pineal circadian rhythms can be studied reproducibly over time, and gene expression profiles can be correlated with physiological consequences in single animals. Using these approaches, it is found that beta-adrenergic activation leads to decreased release of 5-HT, and that increased cAMP signaling in vivo results in activation of N-acetyltransferase gene induction and melatonin production. These studies will enhance the understanding of signaling pathways that regulate pineal 5-HT and melatonin synthesis and secretion.     

Day/night serotonin levels in the pineal gland of male BALB/c mice with melatonin deficiency

Studies from another laboratory have shown that several strains of laboratory mice have a genetic defect for melatonin synthesis. In non-deficient species, melatonin synthesis undergoes a typical, beta-adrenergically regulated day/night rhythm with low melatonin levels during daytime and high levels at night, the precursor serotonin showing an inverse behaviour. This study examines whether a day/night rhythm of pineal serotonin levels exists in melatonin-deficient male BALB/c mice. Mice kept under a lighting schedule of 12 h light (lights on at 07.00 h) and 12 h dark were killed at 13.00 and 01.00 h, respectively. Serotonin amounted to 12-15 ng/pineal and did not show regular day/night differences. Administration of the beta-adrenergic agonist, isoproterenol, which is known to affect melatonin synthesis in a number of species, was without effect on pineal serotonin levels. Melatonin and two of the melatonin-forming enzymes, serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) were below the detection limit in the pineal. It is concluded that in melatonin-deficient BALB/c mice, pineal serotonin synthesis is apparently intact. In BALB/c mice, serotonin synthesis and release do not appear to be directly or indirectly regulated by beta-adrenergic mechanisms.     

Serotonin modulates melatonin synthesis as an autocrine neurotransmitter in the pineal gland

The pineal gland secretes melatonin principally at night. Regulated by norepinephrine released from sympathetic nerve terminals, adrenergic receptors on pinealocytes activate aralkylamine N-acetyltransferase that converts 5-hydroxytryptamine (5-HT, serotonin) to N-acetylserotonin, the precursor of melatonin. Previous studies from our group and others reveal significant constitutive secretion of 5-HT from pinealocytes. Here, using mass spectrometry, we demonstrated that the 5-HT is secreted primarily via a decynium-22–sensitive equilibrative plasma membrane monoamine transporter instead of by typical exocytotic quantal secretion. Activation of the endogenous 5-HT receptors on pinealocytes evoked an intracellular Ca²⁺ rise that was blocked by RS-102221, an antagonist of 5-HT_2C receptors. Applied 5-HT did not evoke melatonin secretion by itself, but it did potentiate melatonin secretion evoked by submaximal norepinephrine. In addition, RS-102221 reduced the norepinephrine-induced melatonin secretion in strips of pineal gland, even when no exogenous 5-HT was added, suggesting that the 5-HT that is constitutively released from pinealocytes accumulates enough in the tissue to act as an autocrine feedback signal sensitizing melatonin release.

The principal role of pinealocytes of the pineal gland (PG) is to synthesize and secrete the hormone melatonin, driven by the neurotransmitter norepinephrine (NE) released at night from sympathetic nerve terminals. Acting through α₁- and β₁-adrenergic receptors on pinealocytes, the adrenergic signal generates Ca²⁺ and cyclic AMP (cAMP) to activate up to 150-fold (in rat) a key synthetic enzyme aralkylamine N-acetyltransferase (AANAT) (1). This signaling pathway is shown in Fig. 1. AANAT in turn converts serotonin (5-HT) from a constitutive pool to generate N-acetylserotonin (NAS), from which hydroxyindole-O-methyltransferase generates melatonin (1). The secretion of melatonin and NAS can be increased 100-fold at night (2). In addition to 5-HT, many other neurotransmitters are present elsewhere in the PG, including acetylcholine (ACh), glutamate, GABA, and neuropeptides (3). Their roles in modulating the activity of pinealocytes and melatonin synthesis remain to be established. Here we focus on modulatory effects of 5-HT.Open in a separate windowFig. 1.Synthesis and release of 5-HT and melatonin in rat pinealocytes. The enzymatic pathway from L-tryptophan to 5-HT and melatonin uses four enzymes. AANAT is regulated by adrenergic signaling. Two possibilities are shown (red dotted arrows) for the mechanism of release of 5-HT from pinealocytes: through vesicular exocytosis or through monoamine transporters running in reverse (orange). Released serotonin activates serotonin receptors (blue) on the plasma membrane of pinealocytes. We are suggesting that the receptors up-regulate AANAT acting as an auto- or paracrine mechanism to promote melatonin synthesis. AADC, aromatic amino acid decarboxylase; HIOMT, hydroxyindole-O-methyltransferase; TPH, tryptophan hydroxylase.Serotonergic signaling plays diverse roles in the CNS and peripheral tissues. It is critically involved in mood control, the sleep–wake cycle, breathing, locomotion, and more (4, 5) and acts through 14 distinct, mostly G protein–coupled 5-HT receptors. In the mammalian PG, 5-HT content shows a circadian rhythm, higher during daytime and somewhat lower at night while melatonin is being produced (6, 7). Compared to melatonin release, the 5-HT release from the gland shows only modest (±twofold) circadian variation, continuing in appreciable amounts throughout (8–10). The large pineal pool of 5-HT is continually turning over through constitutive synthesis and secretion (11). Exogenous NE increases 5-HT production and secretion in PGs partly by elevating tryptophan hydroxylase activity (10, 12–17).Secretion of 5-HT from the PG is larger than the elevated nocturnal release of melatonin (9, 10), but the physiological significance of this secretion has not been clear. Released 5-HT is suggested to prime the cAMP-dependent activation of AANAT for melatonin synthesis (17–20). We tested the hypothesis that 5-HT secretion acts as an autocrine signal to promote melatonin synthesis and secretion (Fig. 1) by answering the following questions: What is 1) the secretory mechanism, 2) the type of 5-HT receptor used, and 3) the effect of 5-HT on melatonin secretion? Using optical, electrical, and mass spectrometric (MS) measurements, we found that 5-HT is released from pinealocytes via a nonconventional mechanism and sensitizes NE-induced melatonin synthesis and secretion by activating 5-HT_2C receptors present in the PG. We show that 5-HT qualifies as an autocrine transmitter in the PG.     

Non-suppressibility by room light of pineal N-acetyltransferase activity and melatonin levels in two diurnally active rodents, the Mexican ground squirrel (Spermophilus mexicanus) and the eastern chipmunk (Tamias striatus)

The rhythms in pineal N-acetyltransferase (NAT) activity and melatonin levels were studied in the diurnally active Mexican ground squirrel and Eastern chipmunk. In the ground squirrel, both NAT activity and melatonin levels exhibited a marked nocturnal rise; these increases were not prevented by the exposure of these animals to a light irradiance of 200 microW/cm2 throughout the night. In the Eastern chipmunk, darkness at night was also associated with a marked rise in both the activity of the acetylating enzyme as well as the levels of melatonin. Again, these rhythms were not suppressed by the exposure of these animals to a light irradiance of 200 microW/cm2 for either 1 night or for 7 nights; exposure of chipmunks to light for 7 consecutive days did, however, reduce the rise in melatonin normally associated with darkness. The non-suppressibility of pineal NAT and melatonin by a 200 microW/cm2 light irradiance may relate either to the activity pattern of these animals, i.e., diurnal, or to their previous lighting history.     

Adenosine effects on the rat pineal gland in vitro: Cyclic adenosine monophosphate levels, N-acetyltransferase, and thyroxine type II 5''-deiodinase activities and melatonin production

The presence of adenosine and its receptors in the pineal gland suggests that they could have a regulatory influence on pineal physiology. Rat pineal glands cultured in the presence of the adenosine analog 5'-N-ethylcarboxamido adenosine (NECA) promoted a significant increase in pineal adenosine 3'5'-monophosphate (cAMP) content, similar to that caused by the beta-adrenergic receptor agonist isoproterenol (ISO). A potentiation of the cAMP response occurred when the glands were simultaneously stimulated with both NECA and ISO. Whereas high cAMP levels induced by ISO were associated with an increased N-acetyltransferase (NAT) activity and rise in the melatonin production and release, the NECA-induced rise in cAMP concentration failed to promote an increase in the activity of either NAT or another pineal enzyme thyroxine 5'-deiodinase type II; likewise, pineal melatonin levels did not rise.     

Alpha-adrenergic control of serotonin release from rat pineal glands

This study was designed to determine if norepinephrine (NE) stimulated release of pineal serotonin (5-HT) is receptor mediated and to identify the receptor(s) most influential in the release process. Efflux of 3H-5-HT from rat pineals in vitro was significantly increased within 5 min after NE was added to perifusion buffer in concentrations ranging from 0.1 to 3 microM. 3H-5-HT was not simply displaced from pinealocytes by NE, since buffer containing 3 microM 5-HT had no effect on 3H-5-HT efflux. Furthermore, NE enhanced 3H-5-HT secretion by a stereospecific process, since d-NE was significantly less effective than l-NE. The alpha 1-adrenoreceptor agonists phenylephrine and cirazoline simulated the effects of NE, significantly increasing 3H-5-HT efflux in single and sequential stimulations. Furthermore, the alpha 1-adrenergic receptor antagonist prazosin reduced NE-stimulated release of 3H-5-HT. In contrast, the alpha 2- and beta-adrenoreceptor agonists naphazoline or clonidine and isoproterenol, respectively, were without effect. In addition, the alpha 2- and the beta-receptor antagonists rauwolscine and timolol, respectively, had no effect on NE-stimulated release of pineal 5-HT. In conclusion, the data show that NE stimulates 5-HT release within minutes from rat pineal glands in vitro. Unlike the pineal mechanism controlling melatonin synthesis, which has a longer latency and requires beta-adrenergic receptor activation, 5-HT release is regulated by activation of alpha 1-receptors. Thus, the pineal may be useful model for studying how separate intracellular processes are controlled by common neural stimuli.     

Unusual responses of nocturnal pineal melatonin synthesis and secretion to swimming: Attempts to define mechanisms

Abstract: The effect of swimming at night on rat pineal melatonin synthesis was compared with that of light exposure at night. Rats were forced to swim at 0030 hr (lights out at 2000 hr) and sacrificed by decapitation 15 and 30 min later, immediately after swimming. Other groups of animals were exposed to white light (650μW/cm²) for 15 and 30 min at same time. Swimming caused a rapid and highly significant drop in the melatonin content in the pineal gland; however, the activity of N-acetyltransferase (NAT), the supposed rate limiting enzyme in the melatonin production, was not changed. Despite the drop in pineal melatonin levels, serum concentrations of the indole remained elevated in the rats that swam. In contrast, melatonin levels in the pineal and serum of light exposed rats fell precipitously, accompanied by a significant suppression of NAT activity. Since we anticipated that the strenuous exercise associated with swimming may induce release of artrial natriuretic peptide (ANP) from the heart, which in turn could cause the release of pineal melatonin, in a second study we injected physiological saline intravenously to stretch the cardiac muscle and release ANP. Three milliliters of normal saline was injected during the day into the jugular vein of anesthetized rats that were pretreated with isoproterenol to stimulate pineal melatonin production. Animals were killed 15 min after the saline injection, and pineal NAT activity and pineal melatonin levels were measured. The saline injections caused no alteration in the elevated levels of either NAT or melatonin. These data suggest that the disparity in pineal NAT activity (which was high) and pineal melatonin (which was low), in animals swum at night, may not be caused by ANP which is released during strenuous exercise such as swimming.     

Phase-shifting effects of light on the circadian rhythms of 5-methoxytryptophol and melatonin in the chick pineal gland

In the chick pineal gland, 5-methoxytryptophol and melatonin concentrations fluctuate in a rhythmic manner. These rhythms are circadian in nature persisting in constant darkness and have opposite phases. Acute exposure of chicks to white light (30 lux for 5, 10, 20, and 30 min) at night increased the amount of pineal 5-methoxytryptophol and decreased pineal melatonin content. A 6 hr pulse of light (100 lux) applied early in the subjective night (CT12-CT18) caused a delay in the phase of the circadian rhythms of 5-methoxytryptophol and melatonin by 3.7 and 4.5 h, respectively, compared to untreated controls. When the 6 hr light pulse was given during the late subjective night (C18 CT24) it advanced the phase of the 5-methoxytryptophol and melatonin rhythms by 8.1 and 11.9 h, respectively. In the chick pineal the phase-advancing effects of light on the circadian rhythms of 5-methoxytryptophol and melatonin were more pronounced than the phase-delaying effects. Our results provide the first evidence that light is capable of phase shifting the 5-methoxytryptophol rhythm in a manner similar to its action on the melatonin rhythm.     

Seasonal variations in pineal 5-methoxytryptophol (5-ML) concentrations and in the daily pattern of pineal 5-ML and melatonin in the desert rodent Jaculus orientalis: Effect of prolonged illumination during the night

Seasonal variations in daytime pineal 5-methoxytryptophol (5-ML) and in the daily pattern of both pineal 5-ML and melatonin concentrations were measured by radioimmunoassay in male and female jerboas, Jaculus orientalis. Pineal 5-ML content was found to be low in winter and spring and showed a short but marked increase in summer. A clear daily rhythm was present in pineal 5-ML in September, with high concentrations during daytime and low concentrations during nighttime. In May there was a considerable drop in the daytime values and a marked decrease in the amplitude of the rhythm, while in December the daily rhythm completely disappeared. On the contrary, a clear daily rhythm was observed for pineal melatonin in September, December, and May with high values during nighttime and low values during daytime; no differences in the amplitude of the rhythm could be observed. Illumination during early night prevented both the nocturnal decrease of 5-ML and the increase of melatonin in September; in May illumination had no clear effect on 5-ML, while it prevented the normal increase of melatonin. These results suggest a possible desynchronization between the regulation of 5-ML and melatonin synthesis and release, and stress the complexity of the mechanisms involved in the environmental synchronization of seasonal functions.     

Pineal ultrastructure and indole profiles spanning the summer rise in arginine vasotocin immunoactivity

A correlative radioimmunological-biochemical-ultrastructural study of the rat pineal gland was undertaken during the summer months when pineal arginine vasotocin (AVT) immunoactivity increases up to 200-fold. RIA confirmed a rapid rise in AVT activity during mid-August regardless of the time of day sampled. Pineal indoles were separated by HPLC and measured using electrochemical detection. Serotonin (5-HT) and 5-hydroxyindoleacetic acid levels were consistently elevated in daytime samples, and there was a significant trend for increased day and nighttime levels of 5-HT from July to September. Mid-dark levels of melatonin also exhibited a significant increase over the sample period. Nighttime levels of N-acetylserotonin mirrored fluctuations in 5-HT in the preceding photoperiod. Ultrastructural components implicated in peptide/protein and/or indole biosynthesis were quantified by stereological morphometry. The greatest amounts of rough endoplasmic reticulum stacks, lipid droplets, and annulate lamellae-like bodies coincided with peak AVT activity. Dense-cored vesicles and synaptic ribbons were consistently more frequent during the dark period. The number of dense-cored vesicles and nucleolar size tended to be greatest before and after the peak in AVT immunoactivity. These observations are consistent with the hypotheses that endoplasmic reticulum and lipid are functionally related to the synthesis and/or storage of peptide/protein factors and that numerical changes in synaptic ribbons and dense-cored vesicles are more closely related to day/night differences in indole metabolism.     

Lower Tryptophan:Phenylalanine Ratios in Culture Media Increase Medium: Pineal Melatonin Ratios in Early Dark but not Late Light Phase

Pineals from male Long-Evans rats (60-65 days old; adapted to a 0700-1900 photoperiod) were cultured for 6 h either in light (1200-1800) or in dark (1800-2400). The objective was to ascertain the effects of tryptophan (trp) and phenylalanine (phe) levels and ratios in the culture medium on melatonin levels in the pineals and their respective media. Total culture (pineal + medium) melatonin levels, determined by RIA, were similar under all conditions. However, in cultures during the early dark phase (1800-2400) lower trp:phe ratios in the medium led to lower pineal:medium ratios of melatonin content. In cultures during the late light phase (1200-1800) the trp:phe ratio had little impact on the pineal:medium distribution of melatonin. Trp:phe ratio rather than absolute level of either amino acid appeared responsible for this effect. Functionally this means that during early dark phase, but not late light phase, movement of melatonin from cultured pineal to medium is progressively facilitated by lower trp:phe ratios. It remains to be determined to what extent darkness per se and/or endogenous pineal rhythmic mechanisms have a permissive role in the action of trp:phe ratio on pineal melatonin release. A melatonin compartmentalization/release effect of these or other amino acids, or their ratios, has not been reported previously and may possibly contribute to mechanisms for melatonin's transport or release at night.