Alloxan-Induced Diabetes and the Pineal Gland: Differential Effects on the Levels of Pineal N-Acetylserotonin, Pineal Melatonin, and Serum Melatonin

Effects of alloxan treatment on the levels of pineal melatonin, pineal N-acetylserotonin, and serum melatonin were investigated. Male rats were housed under a photoperiod of 12 h light: 12 h darkness and a temperature of 23 +/- 3 degrees C. Three weeks after alloxan (170 mg/kg) or carrier injection (s.c.), the animals were killed at mid-light (1200 h) and mid-dark (2400 h). Pineal and serum indoles were extracted and quantified by radioimmunoassays. It was found that pineal levels of N-acetylserotonin in the diabetic rats were significantly higher (P less than 0.05) than those of the controls. Conversely, pineal and serum levels of melatonin in the control rats were significantly higher (P less than 0.05) than those of the alloxan-induced diabetics. Our results suggest that alloxan-induced diabetes may decrease pineal melatonin synthesis in rats by reducing the activity of hydroxyindole-O-methyltransferase, resulting in a decrease in pineal melatonin secretion.     

Fluctuation of Blood Melatonin Concentrations With Age: Result of Changes in Pineal Melatonin Secretion, Body Growth, and Aging

Melatonin in the systemic circulation of rats fluctuates with age, and the causes for such changes were investigated. Male rats (aged 7 days, 16 days, 18 days, 20 days, 30 days, 48 days, 60 days, and greater than 17 months) were adapted under a lighting regime of 12L:12D for at least 7 days. Pineals and blood samples from the trunk or confluens sinuum were collected in the dark period. Melatonin in tissues was extracted, identified, and determined by gas chromatography-mass spectrometry (GC-MS) and/or radioimmunoassay. Tissue melatonin levels obtained by radioimmunoassay correlated closely with those quantified by GC-MS. Thus, the melatonin radioimmunoassay used is a reliable assay method for melatonin in the plasma and pineal of the rat. Plasma melatonin in the confluens sinuum of rats exhibited episodic release superimposed on a basal release pattern. It was suggested that there are two pools of melatonin in the pineal gland, a readily releasable pool and a bound pool. The mean plasma levels of melatonin in the confluens sinuum of rats increased with age with the highest level recorded at 60 days old and declined to a lower level at greater than 17 months old. The above age-related changes, being similar to the alterations in pineal melatonin levels with growth and aging, suggest that, under our experimental conditions, levels of pineal melatonin increase or decrease with its secretory rate. In developing rats, the age-related increase in the rate of secretion of pineal melatonin as reflected by increases in melatonin levels in the confluens sinuum or pineal melatonin content before adulthood is different from the changes in melatonin levels in the systemic circulation which showed an early developmental rise, followed by an active period and then a prepubertal decline. However, when the body weight was taken into consideration, changes in the levels of pineal melatonin content per 100 gm body weight or the calculated blood melatonin levels (plasma melatonin in the confluens sinuum/body:head ratio) correlated well with the fluctuation of serum melatonin in the systemic circulation. Thus, the developmental changes in the concentrations of melatonin in the general circulation are the result of 1) changes in the rate of pineal melatonin secretion and 2) increase in the dilution factor because of increase in body size. In old rats, levels of plasma melatonin in the confluens sinuum and pineal melatonin content decreased indicating a decline in the rate of pineal melatonin secretion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Secretory Patterns of Pineal Melatonin in the Rat

The release patterns of pineal melatonin were studied by continuously monitoring melatonin levels in the confluens sinuum plasma in sighted and bilaterally enucleated rats in the light and dark periods. Plasma melatonin was determined by radioimmunoassay, and the data were analyzed by a computerized algorithm developed in our laboratory. Pulsations of melatonin levels were found in the confluens sinuum plasma in all the animals studied, suggesting episodic secretion of pineal melatonin in rats. Because the minimum melatonin levels in the confluens sinuum were over three times the melatonin levels in the general circulation, it is postulated that 1) there is an episodic release pattern of pineal melatonin superimposed on a basal release pattern and 2) there are two pools of melatonin in the pineal gland, a readily releasible pool responsible for the basal release and a bound pool responsible for the pulsatile release. In the sighted rats, there was no diurnal difference in mean melatonin concentration, mean pulse amplitude, mean pulse rate, mean minimum melatonin level, and mean maximum melatonin level in the confluens sinuum. In the bilaterally enucleated rats, with the exception of the mean pulse amplitude, diurnal rhythms were demonstrated in all the other parameters studied with, higher values in the dark period. This experimental model should be employed in future investigations on the regulation of secretory patterns of pineal melatonin. Results of these studies may provide important insight into the regulation of pulsatile release of neuroendocrine secretions in general.     

Immunohistochemical Assessment of Melatonin Binding in the Pineal Gland

Melatonin binding in the pineal gland of albino rats is estimated using an immunohistochemical procedure. Binding is saturable, has relatively high affinity (Apparent KD = 2.7 nM), and competition studies indicate binding of indoleamines possessing an N-acetyl group on the terminus of the side chain (N-acetylserotonin and melatonin). These data are consistent with the interpretation that immunohistochemically determined melatonin in unfixed pineal tissue is assessing binding of N-acetylated indolealkylamines to pineal cell components. In albino rats maintained on 12-hour light: 12-hour dark cycles, melatonin binding exhibits a diurnal rhythm with low levels of saturation (30%) early in the light and saturation by endogenous melatonin near the onset of darkness. An annual rhythm of melatonin binding was observed in albino rats with low levels during the summer and high levels during the winter. Other rats were maintained on 12-hour light:dark cycles and fed for 2 hours either early in the light period or early in the dark period. For both morning- and evening-fed animals, melatonin binding was high prior to feeding and dropped immediately after feeding. Changes in melatonin binding that occur in response to alterations of feeding and time of year suggest the possibility that this binding reflects a functional site for melatonin.     

Melatonin Content of the Pineal Gland in Different Mouse Strains

Pineal melatonin content at several times during the day and night was measured in 36 inbred strains of mice (Mus musculus) kept under LD 12:12 cycles. The results have indicated that only five inbred strains have pineal melatonin content, with higher levels during the night and lower levels during the day; the other 31 strains do not contain detectable melatonin in their pineal gland at any of times examined. The former group includes two commonly used strains (C3H/He and CBA/Ms) and three wild-derived strains (Mol-A, Mol-Nis, MOM). C3H and CBA mice showed a similar pattern of pineal melatonin rhythm with a peak at 2 hours before lights on. The peak levels were about 150 pg/gland in both strains. The rhythmic patterns of melatonin content in Mol-A, Mol-Nis, and MOM were slightly different from those in CBA and C3H. In the wild-derived strains, the peak of melatonin content did not occur at 2 hours before lights on but tended to occur at midnight. The peak levels were 67-91 pg/gland at the highest point in these strains.     

Pineal Dependence of the Syrian Hamster''s Nocturnal Serum Melatonin Surge

The usual nocturnal surge of pineal melatonin content was blocked by bilateral superior cervical ganglionectomy in male Syrian hamsters. Ganglionectomy and pinealectomy each prevented the nocturnal rise of serum melatonin concentration seen in control animals. The normal nocturnal surge of circulating melatonin in this species appears to depend on the pineal gland and its sympathetic innervation.     

Melatonin in Serum and the Pineal of Spontaneously Hypertensive Rats

Involvement of melatonin in the blood pressure regulation as an endogenous central hypotensive factor has been suggested in rats and in man. We studied the relationship between melatonin and the development of hypertension in 5- and 15-week-old spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats, by measuring serum and the pineal concentrations with a sensitive and specific radioimmunoassay coupled with a novel extraction method. Serum melatonin concentration at midnight in young SHR rats was significantly higher than that in age-matched WKY rats (P < 0.01), whereas it was decreased in the adult SHR (P < 0.01). No such differences were observed at noon. Pineal content of melatonin at midnight in 5-week-old SHR rats was lower than in age-matched WKY rats (P < 0.01). These data demonstrate that melatonin in the nocturnal serum of SHR rats is elevated at prehypertensive stage while it is decreased after the development of hypertension. The role of melatonin in the hypertensive process in SHR rats requires further study.     

Melatonin Metabolism: Neural Regulation of Pineal Serotonin: Acetyl Coenzyme A N-Acetyltransferase Activity

There is a diurnal rhythm in the activity of serotonin N-acetyltransferase in the rat pineal gland. In the normal rat, the nocturnal enzyme activities are 15-to 30-fold greater than are daytime activities. This rhythm is abolished by decentralization or removal of the superior cervical ganglia, procedures that interrupt the only source of central neural input to the pineal gland. This effect of superior cervical sympathectomy on the N-acetyltransferase rhythm cannot be attributed to changes occurring in the denervated pineal parenchymal cells. When chronically denervated glands are placed in organ culture with norepinephrine, the neurotransmitter normally located in sympathetic terminals in the gland, N-acetyltransferase activity increases 10- to 20-fold. These data indicate that superior cervical sympathectomy abolishes the N-acetyltransferase rhythm by elimination of the input of central signals to the gland. These signals appear to regulate the N-acetyltransferase rhythm in the normal rat by regulation of the release of norepinephrine from the sympathetic terminals within the pineal gland.     

No Correlation of Pineal "Synaptic" Ribbon Numbers and Melatonin Formation in Individual Rat Pineal Glands

As previous circadian studies of pineal "synaptic" ribbon numbers and melatonin formation suggested that a positive correlation of the two variables exists, in the present investigation this problem was examined in individual pineal glands of rats killed at 1200 h and 2400 h, respectively. For this purpose, one half of the gland was processed for electron microscopy and the ribbons were counted in an area of 20,000 micron2 tissue; in the other half serotonin N-acetyltransferase (NAT) activity and melatonin content were determined. No correlation was found to exist between ribbon numbers and pineal NAT activity, pineal melatonin levels and serum melatonin levels, either at day- or at nighttime. It is concluded that the ribbons may perhaps be more closely related to the innervation of the pineal gland than to melatonin formation.     

Constant Pineal Output and Increasing Body Mass Account for Declining Melatonin Levels During Human Growth and Sexual Maturation

Twenty-four-h urine samples, divided into two fractions representing night- and daytime melatonin production, were collected from 115 healthy individuals between the ages of 3 and 80, of known height and weight, and assayed for 6-hydroxy melatonin sulphate (SaMT), a major urinary metabolite of melatonin, by gas chromatography mass spectrometry. The population was divided for analytical purposes into children (boys aged 3-10.99, girls aged 3-9.59), adolescents (males aged 11-17.99, females aged 9.60-17.99), and adults (men and women over 18). The results showed approximately the same excretion over 24 h in all 3 groups but that the night/day ratio was considerably greater in children and adolescents compared to adults (P less than 0.001). However, when the results were expressed as a function of body weight (BW), body surface area (BSA), or creatinine excretion (CE), nocturnal SaMT was higher in children than in adults (P less than 0.001 for all 3 parameters) or adolescents (BW, P less than 0.001; BSA, P less than 0.002; CE, P less than 0.001) and was higher in adolescents than in adults (BW and BSA, P less than 0.001). Children also excreted more during the day than adults (BW, P less than 0.01; CE, P less than 0.001) or adolescents (BW alpha CE, P less than 0.02). Our results show that pineal output barely changes during childhood and adolescence. However, there is an age related decrease in SaMT excretion/unit body mass which correlates with an age-related increase in body mass. We therefore conclude that the decrease in circulating levels of melatonin during growth and sexual maturation is brought about by an increase in body mass.     

Pineal Function in Burns: Melatonin Is Not a Marker for General Sympathetic Activity

Burn injury in humans or rats is a model of marked elevation of general sympathetic activity for weeks, manifested in part by increased heart rate, metabolic rate, core temperature, and plasma and urinary catecholamines. Plasma melatonin was sampled at 2-h intervals for 24 h in 9 control subjects and 11 patients with severe burn injury. Daytime melatonin was not different between the groups, but nighttime values were significantly lower in the burn patients. A nocturnal surge was still significant in the patients. Resting heart rate and rectal temperature were elevated in the burn patients. In male Sprague-Dawley rats, pineal melatonin content did not differ between controls and those with an experimental burn at 4 h into the light phase nor during the nocturnal surge. Male Syrian hamsters with burns had lower daytime pineal melatonin content than did controls, but the nocturnal surge in pineal melatonin was not significantly different between groups, nor was daytime morning serum melatonin. Sympathetic activity appears partitioned, with that controlling melatonin (nocturnal surge) regulated independently. In agreement with our previous findings in other models, melatonin is not a marker for general sympathetic activity, even following severe burn injury.     

Effects of Timed Melatonin Infusion on Prolactin Secretion in Pineal Denervated Goat

The effects of timed melatonin infusion on prolactin secretion were examined in the pineal denervated goat. Ovariectomized Shiba goats (n = 5) were subjected to bilateral superior cervical ganglionectomy (SCGX); this procedure resulted in complete abolition of endogenous melatonin release during the dark phase. SCGX goats failed to coordinate their prolactin secretion with the prevailing photoperiod. Melatonin was infused (20 micrograms/h, s.c.) daily into these goats either for 8 h (the long-day-type infusion) or for 16 h (the short-day-type infusion) to mimic the nocturnal profile of plasma melatonin under long days or short days, respectively. The long-day-type melatonin infusion for 9 days, in comparison with control saline infusion, accelerated prolactin secretion, inducing a nocturnal rise in plasma prolactin; this was comparable to that seen in the pineal intact goats under long photoperiods. On the other hand, the short-day-type melatonin infusion suppressed prolactin secretion throughout the day as the short-day treatment did in intact goats. The prevailing photoperiod appeared to have no distinct effect on these prolactin responses to exogenous melatonin, which were indistinguishable under 16L8D and 8L16D conditions. The results indicate that the information about external light-dark cycles is converted by the pineal gland into the endocrine signal as a daily pattern of melatonin secretion, which eventually regulates prolactin secretion from the pituitary gland of the goat.     

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.     

Kinetic Study of Melatonin Release From Rat Pineal Glands Using a Perifusion Technique

In previous studies, noradrenaline was found to elicit a rise of melatonin secretion through activation of typical beta-adrenergic receptors. In the present study, a perifusion system was developed to characterize the kinetics of melatonin release from rat pineal glands. Isolated pineal glands from adult male rats were continuously perifused for 15 h in a Krebs-Ringer solution, and the concentration of melatonin in the effluent perifusate was monitored using a specific radioimmunoassay. The rate of release of melatonin declined during the first 3-4 h of perifusion and then remained fairly stable for at least 11 h. The spontaneous release of melatonin was around 20 pg per min and per gland. When pineal glands were stimulated with isoproterenol, melatonin release output linearly increased for at least 2 h after the stimulation. The increase in melatonin release depended on the isoproterenol concentration and on the duration of the stimulation. The analysis of the pattern of melatonin secretion by a single rat pineal gland showed that the secretion was irregular but did not present a clear feature of pulsatile or oscillatory release over a 11 h-long study. The perifusion system was found useful in order to follow the characteristics of melatonin release from pineal glands and should allow investigations of neuronal or hormonal control of pineal gland activities.     

Effect of Selective Monoamine Oxidase Inhibitors on Rat Pineal Melatonin Synthesis In Vitro

Freshly cultured pineal glands respond to the monoamine oxidase A (MAO A) inhibitor clorgyline and to high concentrations of the MAO B inhibitor, deprenyl, with an increase in serotonin N-acetyltransferase activity and N-acetylated indoles and a fall in 5-hydroxylated serotonin degradation products. Glands cultured for 48 hours before challenge respond less. Response is absent in glands cultured for 72 hours and in glands from ganglionectomized animals cultured for 48 hours before challenge. These data are consistent with the hypothesis that these MAO inhibitors stimulate melatonin synthesis by protecting norepinephrine from degradation.     

Rhythmic Melatonin Response of the Syrian Hamster Pineal Gland to Norepinephrine In Vitro and In Vivo

Norepinephrine (NE, 10(-6) M) stimulated melatonin accumulation in the incubation medium of rat (but not Syrian hamster) pineals taken at the end of the light phase. However, NE elevated melatonin accumulation in the medium of pineals taken after 20 min of light exposure of animals of either species at 6 h into the 10-h dark phase. A dose response to 10(-7)-10(-5) M NE was observed in both the medium and pineals upon incubation of pineals taken from rats at 4 h into the light phase and from hamsters after 20 min light exposure at 6 h into the dark phase. Approximately 95% of the melatonin present was in the medium. The incubation time was 4 h in all cases. Subcutaneous injection of 1 microgram/g NE (either at the end of the light phase or after 30 min of light at 6 h into the dark phase) did not stimulate in vivo Syrian hamster pineal melatonin content determined 1 or 2 h after injection, whether the hamsters were placed in light or darkness after the injection. However, after 30 min of light beginning at 6 h into dark, injection of 5 micrograms/g desipramine (DMI, a blocker of catecholamine uptake into nerve endings) allowed a dramatic hamster pineal melatonin response to additional injection of 1 microgram/g NE, observed at 1 and 2 h in light after injection. A small effect of DMI alone was seen. DMI also potentiated the effect of NE (each 10(-6) M) on melatonin accumulation in the medium of incubated hamster pineals taken after a short light exposure at night. No significant stimulatory effect of NE and/or DMI was seen in vivo or in vitro near the middle of the light phase. Measurement of melatonin in the incubation medium is a useful method for studying pineal function. The Syrian hamster pineal has rhythm of sensitivity to NE (sensitivity evident at night) and even at night is protected by neuronal uptake from circulating NE-induced stimulation of melatonin production. NE appears to be the neurotransmitter for stimulation of pineal melatonin production in the Syrian hamster. The sensitivity rhythm and uptake protection might provide specificity of control of the nightly melatonin signal by reducing the chance of a melatonin response during the day or a response to circulating catecholamines from general sympathetic stimuli.     

重组人生长激素在成人生长激素缺乏症的应用

近20年来,重组生长激素已可以无限量的制备。近年来的研究已阐明成人生长激素缺乏的影响及生长激素替代治疗的益处。成人生长激素缺乏症对人影响最大的是生活质量、骨骼健康及包括血清脂质谱、身体构成变化的心血管危险因子的改变,而这也正是生长激素替代治疗获益最显著之处。大量的证据显示了重组人生长激素治疗的疗效,尤其在改善骨代谢和改善心血管危险因素等多方面发挥作用,并且是安全的。