Determination of pineal melatonin by precolumn derivatization reversed-phase high-performance liquid chromatography and its application to the study of circadian rhythm in rats and mice

Determination of minute amounts of endogenous melatonin in rat and mouse pineal gland was performed using an RP-HPLC system. Melatonin was separated following precolumn derivatization and determined with a fluorescence detector at the emission wavelength of 380 nm with the excitation at 245 nm. The calibration curve of melatonin constructed by adding known amounts of melatonin to the homogenates of mouse pineal gland was linear over the range of 1-500 fmol (injection amount/20 microl). The detection limit of added melatonin was 1 fmol (S/N = 5). Repeatability and day-to-day precision for the melatonin spiked sample of mouse pineal gland was 4.0 and 3.8% (RSD), respectively. Using the present method, circadian changes of melatonin content in rat (Wistar) and mouse (C3H) pineal gland were determined. In addition, a minute amount of melatonin in ddY mouse pineal gland was determined, because pineal melatonin of many inbred mouse strains has been reported to be lower than the detection limit.     

Pineal and plasma melatonin as determined by high-performance liquid chromatography with electrochemical detection.

A rapid and sensitive method for the routine quantitative determination of melatonin in pineal and plasma is described. The assay used reversed-phase high-performance liquid chromatography (RP-HPLC) separation combined with either amperometric (system A) or coulometric (system B) detection. The method gave satisfactory reproducibility and accuracy, and detection limits for melatonin were as low as 8.5 pg (system A) and 1 pg (system B). This high sensitivity, together with the short analysis time (less than 10 min), and the simplicity of sample procedure make the present RP-HPLC method suitable for a wide range of studies concerning melatonin measurements. Melatonin values obtained in this study from both rat pineal and human plasma agree with those reported previously, and clearly determined a circadian pattern.     

5-methoxytryptophol in rat serum and pineal: Detection,quantization, and evidence for daily rhythmicity

A gas chromatography-mass spectrometric method is described which allows the simultaneous detection of melatonin and 5-methoxy-tryptopopthol in rat pineals and sera. The concentrations of 5-methoxytryptophol in the pineal were found to be as great as those of melatonin, and appear to vary with the time of day. Statistical analysis showed a significant correlation between pineal and sera 5-methoxytryptophol levels during the hours of darkness, however, the correlation disappeared during the light phase.     

The profile of melatonin production in tumour-bearing rats

The pineal gland is involved in the regulation of tumour growth through the anticancer activity of melatonin, which presents immunomodulatory, anti-proliferative and anti-oxidant effects. In this study we measured melatonin content directly in the pineal gland, in an attempt to clarify the modulation of pineal melatonin secretory activity during tumour growth. Different groups of Walker 256 carcinosarcoma bearing rats were sacrificed at 12 different time points during 24h (12h:12h light/dark cycle) on different days during the tumour development (on the first, seventh and fourteenth day after tumour inoculation). Melatonin content in the pineal gland was determined by high-performance liquid chromatography with electrochemical detection. During tumour development the amount of melatonin secreted increased from 310.9 ng/mg of protein per day from control animals, to 918.1 ng/mg of protein per day 14 days after tumour implantation, and there were changes in the pineal production profile of melatonin. Cultured pineal glands obtained from tumour-bearing rats turned out to be less responsive to noradrenaline, suggesting the existence, in vivo, of putative factor(s) modulating pineal melatonin production. The results demonstrated that during tumour development there is a modification of pineal melatonin production daily profile, possibly contributing to cachexia, associated to changes in pineal gland response to noradrenaline stimulation.     

Microdialysis of Melatonin in the Rat Pineal Gland: Methodology and Pharmacological Applications

Abstract: The present study describes the development of a new technique to measure melatonin contents in the pineal gland of freely moving rats, by means of on-line microdialysis. The transcerebral cannula was modified, and a sensitive assay of melatonin, using HPLC with fluori-metric detection, was set up. With this system it is possible to monitor the melatonin levels on-line in the pineal gland during day-and nighttime. The nightly increase in melatonin release was recorded. Tetrodotoxin had an inhibitory effect on nighttime levels, whereas even high concentrations did not alter the daytime level. From this we conclude that neuronal activity is necessary to synthesize melatonin and that during daytime no net neuronal activity is present. Melatonin levels could be greatly enhanced by systemic administration of the β-agonist isoprenaline (ISO). Also, local infusion of ISO or 8-bromoadenosine 3',5'-cyclic monophosphate, an analogue of the second messenger cyclic AMP, resulted in increased melatonin levels, demonstrating the presence of β-adrenergic receptors, coupled to a cyclic AMP-based second messenger system, on the pineal gland. Injection of phenylephrine had no effect on daytime levels. Only when administered during ISO-induced stimulation of melatonin release did it enhance this stimulated release. This proved the regulatory role of α₁-receptors on pinealocytes. The method presented is of special interest for investigating the innervation of the pineal gland and the biochemical processes that regulate the biosynthesis of melatonin. Also, for studies on the diurnal rhythms of melatonin release and factors that influence these rhythms in freely moving animals, this model will be of great value.     

Tryptophan hydroxylase is modulated by L-type calcium channels in the rat pineal gland

Calcium is an important second messenger in the rat pineal gland, as well as cAMP. They both contribute to melatonin synthesis mediated by the three main enzymes of the melatonin synthesis pathway: tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase. The cytosolic calcium is elevated in pinealocytes following alpha(1)-adrenergic stimulation, through IP(3)-and membrane calcium channels activation. Nifedipine, an L-type calcium channel blocker, reduces melatonin synthesis in rat pineal glands in vitro. With the purpose of investigating the mechanisms involved in melatonin synthesis regulation by the L-type calcium channel, we studied the effects of nifedipine on noradrenergic stimulated cultured rat pineal glands. Tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase activities were quantified by radiometric assays and 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin contents were quantified by HPLC with electrochemical detection. The data showed that calcium influx blockaded by nifedipine caused a decrease in tryptophan hydroxylase activity, but did not change either arylalkylamine N-acetyltransferase or hydroxyindole-O-methyltransferase activities. Moreover, there was a reduction of 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin intracellular content, as well as a reduction of serotonin and melatonin secretion. Thus, it seems that the calcium influx through L-type high voltage-activated calcium channels is essential for the full activation of tryptophan hydroxylase leading to melatonin synthesis in the pineal gland.     

Determination of melatonin in rat pineal,plasma and retina by high-performance liquid chromatography with electrochemical detection

A sensitive method for the routine measurement of endogenous melatonin (MEL) in pineal, retina and plasma rat tissues has been developed using reversed-phase high-performance liquid chromatography with electrochemical detection. Quantification limit for MEL was 0.2 ng/mg protein in pineal, 15 pg/ml in plasma and 2.0 pg/mg protein in retina. To improve both MEL quantification and the reproducibility of the assay, an internal standard was used when an extraction in organic solvent was required, in contrast with other available chromatographic methods. MEL values and the circadian profile obtained in this study from both rat pineal and plasma agree with those reported previously. This method allows MEL detection in mammal retina, particularly in rat, where MEL levels are very low.     

Melatonin and the synthesis of vasopressin in pinealectomized male rats

The pineal hormone, melatonin, is known to modify, under different experimental conditions, neurohypophysial hormone secretion in the rat. The aim of this study was to investigate the effect of melatonin on the vasopressin biosynthesis rate in the hypothalamus of either pinealectomized or sham-operated rats, using the colchicine method. To estimate whether colchicine affects the function of the neurohypophysis in these animals, the neurohypophysial and plasma vasopressin levels were also measured. The vasopressin synthesis rate was increased after pineal removal, when compared with sham-operated animals, and melatonin strongly inhibited the rise in the hormone synthesis due to pinealectomy. After pineal removal plasma vasopressin concentration was significantly elevated, and melatonin attenuated this effect. On the contrary, the neurohypophysial vasopressin content was significantly decreased after pinealectomy, but it was not further modified by melatonin.Thus, melatonin suppresses the synthesis and secretion of vasopressin in pinealectomized rats. The present results confirm our previous reports as to the inhibitory impact of the pineal on both vasopressin synthesis and release and suggest that melatonin may mediate the effect of the pineal gland on vasopressinergic neuron activity.     

Circadian rhythms of melatonin release from chicken pineal in vitro: modified melatonin radioimmunoassay

An improved and simplified radioimmunoassay for measuring pineal, serum, and in vitro cultured medium melatonin is described. Using 2-[125I]iodomelatonin as radiolabeled ligand and a polyclonal rabbit antimelatonin antiserum, melatonin concentrations were determined in all three types of samples by a 2-day direct equilibrium double-antibody assay method without prior extraction. Serial dilutions of pineal homogenates, serum, and cultured medium all gave parallel displacement curves. Cross-reactivity of the antisera with other indoles was negligible. Intraassay coefficients of variation (n = 3) were 5.09, 3.32, and 5.05% at 7.81, 62.5, and 500 pg/tube, respectively, and the interassay coefficients of variation (n = 20) were 12.18% at 62.5 pg/tube. A characteristic diurnal rhythm of melatonin was observed using this direct assay for measuring daytime and nighttime chicken pineal and serum samples. An in vitro incubation of chicken pineal glands with a lighting cycle of 12-hr light:12-hr dark showed that the diurnal rhythm of melatonin secretion into the cultured medium was maintained. The direct assay method described in this report for measuring chicken melatonin using 2-[125I]iodomelatonin as radiolabeled ligand coupled with the in vitro cultured chicken pineal gland clearly offers great potential for studying the chicken pineal circadian oscillator and its underlying mechanism.     

Locally synthesized angiotensin modulates pineal melatonin generation

We aimed to study the mechanisms and the significance of the influence exerted by the renin-angiotensin system (RAS) on the pineal melatonin production. Pineal melatonin and other indoles were determined by HPLC with electrochemical detection after angiotensin AT1-receptor blockade with Losartan in vivo or in cultured glands. N-acetyltransferase (NAT) activity was radiometricaly measured. To test the in vivo relevance of the local RAS, pineal melatonin and its indole precursors were determined in transgenic rats with inhibited production of angiotensinogen exclusively in astrocytes, TGR(ASrAOGEN). Tryptophan hydroxylase (TPH) and NAT mRNA levels were determined by real-time RT-PCR. Pineal melatonin content was significantly decreased by AT1-receptor blockade in vivo, in cultured glands and in TGR(ASrAOGEN) (35%, 32.4% and 17.5% from control, respectively). Losartan produced a significant decrease of pineal 5-hydroxytryptophan, serotonin, 5-hydroxyindole acetic acid and N-acetylserotonin in pineal cultures. Also, the pineal content of the precursor indoles in TGR(ASrAOGEN) rats was significantly lowered. The reduction of 5-hydroxytryptophan levels by 33-75% in both in vivo and in vitro studies suggests a decreased activity of TPH. Moreover, the TPH mRNA levels in TGR(ASrAOGEN) rats were significantly lower than control rats. On the other hand, NAT activity was unaffected by Losartan in pineal culture and its expression was not significantly different from control in TGR(ASrAOGEN) rats. Our results demonstrate that a local pineal RAS exerts a tonic modulation of indole synthesis by influencing the activity of TPH via AT1-receptors.     

Pineal function during ethanol intoxication, dependence, and withdrawal

Pineal melatonin and serotonin content were determined during one to four days of continuous intoxication, and during the alcohol withdrawal syndrome. The nocturnal rise in pineal melatonin was blunted in continuously intoxicated animals, however this was found to be unrelated to duration of treatment. The initial dependent-intoxicated phase of the alcohol withdrawal syndrome produced a reduction of nocturnal pineal melatonin content with a concomitant elevation in pineal serotonin. The overt withdrawal phase of the alcohol withdrawal syndrome had no effect on pineal melatonin or serotonin content. This data suggests that ethanol may perturb pineal melatonin synthesis either directly, or indirectly by altered receptor function. Contrary to our expectations the pineal may not be a useful model to probe the physiology of increased noradrenergic neurotransmission produced by ethanol withdrawal.     

松果体接触脑脊液神经元的实验研究

为证实大鼠接触脑脊液神经元内视紫红质的存在及光照对其的影响,将大鼠处死后立即取出松果体,通过免疫组织化学方法用荧光显微镜检测视紫红质的存在;用电生理方法来证实光照对松果体上的视紫红质可产生作用。在松果体接触脑脊液神经元及松果体内部有视紫红质反应阳性细胞存在;光照松果体后,可使松果体的神经元诱发放电频率明显增加。并且,与光照前松果体自发放电相比有显著差异。哺乳动物松果体接触脑脊液神经元内存在有视蛋白;松果体除了经典的途径调节褪黑素的释放外,可能还有其他途径:光照松果体,可诱导松果体放电或放电频率增加,从而影响褪黑素的释放。     

Studies on the development of growth hormone and prolactin cells in the rat pituitary gland by in situ hybridization

Summary An antiserum raised against N-amino-3-propyl melatonin bound to a protein carrier was used to visualize melatonin by immunohistochemistry and to measure melatonin concentration by radioimmunoassay in the pineal gland of intact mink females killed throughout the 24 h cycle and females killed after a bilateral ablation of the cervical superior ganglion. Melatonin immunoreactivity revealed by immunofluorescence or by the peroxidase-antiperoxidase complex was observed in the cytoplasm of presumed pinealocytes of all the females. Circadian changes in pineal melatonin content were not visualized by immunohistochemistry; furthermore, immunoreactivity was also present in the pineal gland of the ganglionectomized females. However, the melatonin content measured by radioimmunoassay was significantly higher in the pineal gland from intact females killed during the night compared with that of intact females killed during the day or of ganglionectomized females. The discrepancy between the results obtained using the two methods may arise because immunohistochemistry can detect very small amounts of melatonin.     

Elevated daytime rat pineal and serum melatonin levels induced by isoproterenol are depressed by swimming

Isoproterenol (1 mg/kg) was subcutaneously injected into adult male rats during the day to stimulate pineal N-acetyltransferase (NAT) activity and pineal and serum melatonin levels. Two hours after isoproterenol administration when levels of each of these variables had increased significantly, the experimental animals swam for 10 min in 22 degrees C water. At 15 min after swimming onset, pineal and serum melatonin levels were highly significantly depressed compared to those in control animals that did not swim. The high NAT level was not influenced by swimming. In a second study, isoproterenol injected rats swam for either 1, 3, 6 or 10 min and were sampled 15 min after the onset of swimming. The reduction in the elevated pineal melatonin in these animals was correlated with the length of the swim, i.e., as the duration of swim increased the percent reduction in pineal melatonin also increased. Neither pineal NAT nor hydroxyindole-O-methyltransferase (HIOMT) activities were influenced by swimming. The results suggest that elevated pineal and serum melatonin induced by isoproterenol can be depressed with no effect on the activity of the enzymes which convert serotonin to melatonin.     

Negative correlation of age and the levels of pineal melatonin, pineal N-acetylserotonin, and serum melatonin in male rats

Pineal concentrations of N-acetylserotonin and melatonin and serum levels of melatonin were studied in 3-wk-old (prepubertal), 8-wk-old (adult), and 17-mo-old (senile) male rats. They were adapted to a photoperiod of 12 h light/12 h darkness for a minimum of 1 wk and killed at mid-light and mid-dark. Melatonin and N-acetylserotonin were determined by radioimmunoassay. The concentrations of pineal N-acetylserotonin and melatonin were high in the dark period and low in the light period. Statistical analysis indicated that pineal N-acetylserotonin and melatonin levels per 100 gm body weight declined with age. Similarly, serum melatonin demonstrated diurnal changes in all the age groups studied. In addition, there was a significant reduction in the levels of serum melatonin with age. The parallel patterns of decrease in pineal and serum melatonin levels with age suggest a decline in pineal secretion of melatonin in the older animals.     

Effects of the blockade of high voltage-activated calcium channels on in vitro pineal melatonin synthesis

The presence of high voltage-activated calcium channels in the rat pineal gland is well known. However, their role in pineal metabolism is not completely understood and is even controversial. Better to understand this matter, we investigated the effects of L-, N- or P/Q-type calcium channel blockers (nifedipine, omega-conotoxin GVIA, omega-agatoxin IVA, respectively) on melatonin content and arylalkylamine-N-acetyltransferase activity of denervated rat pineal glands kept for 48 h in culture and stimulated with norepinephrine. Melatonin was measured by high performance liquid chromatography with electrochemical detection and arylalkylamine-N-acetyltransferase activity was quantified by radiometric assay. Pre-incubation with any of these high voltage-activated calcium channel blockers reduced the melatonin production induced by norepinephrine although arylalkylamine-N-acetyltransferase activity was reduced only by the N-type calcium channel antagonist, omega-conotoxin GVIA. The results indicate that calcium influx through L-, N- or P/Q-type of high voltage-activated calcium channels is necessary for the full expression of the metabolic process leading to melatonin synthesis in the rat pineal glands. However, the mechanisms involved in this process are different for the L- or P/Q- and N-type calcium channels.     

Regulation by Light and Darkness of Melatonin Secretion from the Superfused Masu Salmon (Oncorhynchus masou) Pineal Organ

The pineal organ of masu salmon Oncorhynchus masou was maintained in a flow-through, whole-organ culture (superfusion) system and melatonin secretory profiles were determined at 15 °C under light-dark cycles of 12:12 h (LD 12:12) or the same in combination with constant darkness (DD) for 72 h. Under LD 12:12, superfused pineal organs showed a rhythmic melatonin secretion with high and low rates during the dark phase and the light phase, respectively. When the pineal organs maintained under LD 12:12 for 24 h were transferred to DD, melatonin secretion was consistently activated and no endogenous component was evident. When the pineal organs maintained under DD for 48 h were transferred to LD 12:12, melatonin secretion was reduced only during the light phase. These results indicate that melatonin secretion from the superfused pineal organ of masu salmon is regulated not by an intra-pineal circadian oscillator but by the environmental LD cycles, via local photoreceptors.     

Inconsistent suppression of nocturnal pineal melatonin synthesis and serum melatonin levels in rats exposed to pulsed DC magnetic fields

The purpose of these experiments was to determine whether the exposure of rats at night to pulsed DC magnetic fields (MF) would influence the nocturnal production and secretion of melatonin, as indicated by pineal N-acetyltransferase (NAT) activity (the rate limiting enzyme in melatonin production) and pineal and serum melatonin levels. By using a computer-driven exposure system, 15 experiments were conducted. MF exposure onset was always during the night, with the duration of exposure varying from 15 to 120 min. A variety of field strengths, ranging from 50 to 500 μT (0.5 to 5.0 G) were used with the bulk of the studies being conducted using a 100 μT (1.0 G) field. During the interval of DC MF exposure, the field was turned on and off at 1-s intervals with a rise/fall time constant of 5 ms. Because the studies were performed during the night, all procedures were carried out under weak red light (intensity of <5 μW/cm²). At the conclusion of each study, a blood sample and the pineal gland were collected for analysis of serum melatonin titers and pineal NAT and melatonin levels. The outcome of individual studies varied. Of the 23 cases in which pineal NAT activity, pineal melatonin, and serum melatonin levels were measured, the following results were obtained; in 5 cases (21.7%) pineal NAT activity was depressed, in 2 cases (8.7%) studies pineal melatonin levels were lowered, and in 10 cases (43.5%) serum melatonin concentrations were reduced. Never was there a measured rise in any of the end points that were considered in this study. The magnitudes of the reductions were not correlated with field strength (i.e., no dose-response relationships were apparent), and likewise the reductions could not be correlated with the season of the year (experiments conducted at 12-month intervals under identical exposure conditions yielded different results). Duration of exposure also seemed not to be a factor in the degree of melatonin suppression. The inconsistency of the results does not permit the conclusion that pineal melatonin production or release are routinely influenced by pulsed DC MF exposure. In the current series of studies, a suppression of serum melatonin sometimes occurred in the absence of any apparent change in the synthesis of this indoleamine within the pineal gland (no alteration in either pineal NAT activity or pineal melatonin levels). Because melatonin is a direct free radical scavenger, the drop in serum melatonin could theoretically be explained by an increased uptake of melatonin by tissues that were experiencing augmented levels of free radicals as a consequence of MF exposure. This hypothetical possibly requires additional experimental documentation. Bioelectromagnetics 19:318–329, 1998. © 1998 Wiley-Liss, Inc.     

Pineal and serum melatonin at midday and midnight following pinealectomy or castration in male rats.

Melatonin has been extractkd by chloroform from rat serum and quantified by the Rana pipiens tadpole bioassay. Like pineal melatonin, serum melatonin was high at mid-dark and low at mid-light. This finding suggests a diurnal rhythm of serum melatonin in the rat. Serum samples obtained at mid-dark had melatonin levels ranging from 0.02 to 0.05 ng per ml of serum. When rats were pinealectomized, melatonin activity in the serum was abolished. Thus, rat serum melatonin may originate primarily from the pineal. There was no significant change in pineal melatonin content nor in serum melatonin concentrations 7 or 12 days following orchidectomy.     

Pineal melatonin rhythms in female Turkish hamsters: effects of photoperiod and hibernation

Daily rhythms of pineal and serum melatonin content were characterized for adult female Turkish hamsters (Mesocricetus brandti) exposed to long days (16L:8D, 22 degrees C) or after transfer to short days (10L:14D, 22 degrees C). The nocturnal peak of pineal melatonin content was found to be approximately 3 b greater in duration on short than on long days. Changes in levels of serum melatonin closely paralleled those of pineal melatonin. Thus, an effect of photoperiod on synthesis and secretion of pineal melatonin was demonstrated. In a separate experiment, female hamsters were induced to hibernate by exposure to a short-day, cold environment (10L:14D, 6 degrees C). During the 4 to 5-mo hibernation season, Turkish hamsters are known to display 4 to 8-day hours of torpor (body temperature = 7-9 degrees C) alternating with 1 to 3-day intervals of euthermia (body temperature = 35-37 degrees C). Little evidence of nocturnal synthesis or secretion of pineal melatonin was detected in females sampled during torpor. However, animals sampled during the first day after arousal from a torpor bout displayed melatonin rhythms no different in phase or amplitude from those seen in females held at 22 degrees C. Thus, despite the absence of pineal melatonin output during torpor, the pineal gland of hibernating Turkish hamsters produces an appropriately phased, rhythmic melatonin signal during intervals of euthermia.