Changes in vasopressin-converting aminopeptidase activity in the rat pineal gland during summer: Relationship to vasopressin contents

Vasopressin (VP)-converting aminopeptidase (VP-AP) activity and VP contents were measured in single rat pineal glands during the summer of two successive years. The peptidase activity decreased significantly in August. The lowest activity (±SEM) of 0.18±0.02 pmol·hour⁻¹ was recorded on August 14, compared to the basal activity of 0.25±0.01 pmol·hour⁻¹ in July and September of 1986. The change with similar percentage occurred in the same period of 1987. The specific activity of the enzyme in the crude homogenate, 15,000 g pellet and supernatant fraction of rat pineal glands, exhibited the same pattern of variations. The decrease in peptidase activity coincided with the previously reported dramatic rise in pineal VP content in early August which was confirmed in this series of experiments. Another peptidase, the so-called γ-endorphin generating endopeptidase (γ-EGE) activity, and β-endorphin-related peptides in the pineal gland did not change in this period. The results show that the variations of pineal VP contents and VP-AP activity during summer are not general for other peptides and peptidases. The coincidence of opposite changes in VP content and VP-AP activity of the pineal gland may indicate a role of the peptidase activity to regulate the VP content.     

A day/night rhythm of vasopressin and oxytocin in rat retina, pineal and harderian gland

Arginine vasopressin (AVP), oxytocin (OT) and neurophysins (Np) have been found in the pineal gland and the retina of the rat. Because the retina, pineal gland and Harderian gland (HG) serve analogous functions, we undertook a study to determine the presence of these peptides in these three organs of rats. They were detected by two specific methods: HPLC and specific radioimmunoassays. For Np, total neurophysins (NpT) were measured. To determine a 24 hr rhythm, the animals were maintained under a light/dark cycle of 12 hr/12 hr for 3 weeks. The pineal glands, retinae and HG were collected. Day/night rhythms of AVP, OT and NpT were demonstrated in the retina and HG; but the pineal gland had only AVP rhythm. A significant decrease in the rhythms at 4 a.m. was demonstrated in the retina and HG. The 24 hr variation of AVP in the retina seemed parallel to that of the HG.     

Injections of alpha-melanocyte stimulating hormone affect pineal serotonin, melatonin and N-acetyltransferase activity

To determine if exogenously administered alpha-melanocyte stimulating hormone (alpha-MSH) affects nighttime pineal N-acetyltransferase activity, pineal levels of 5-hydroxytryptophan, serotonin and melatonin, and plasma prolactin levels, adult male hamsters were injected at 1900 hr (lights out 2000-0600 hr) with two doses of the peptide and killed at 0300 hr. The low dose of alpha-MSH (200 ng) produced a significant fall in pineal serotonin, pineal NAT activity and plasma prolactin values. The high dose of the peptide (20 micrograms) increased circulating prolactin titers and pineal serotonin levels and caused a concomitant decrease in pineal melatonin levels.     

Avian Melatonin Synthesis: Photic and Circadian Regulation of Serotonin N-Acetyltransferase mRNA in the Chicken Pineal Gland and Retina

Abstract: The circadian rhythms in melatonin production in the chicken pineal gland and retina reflect changes in the activity of serotonin N -acetyltransferase (arylalkylamine N -acetyltransferase; AA-NAT; EC 2.3.1.87). Here we determined that the chicken AA-NAT mRNA is detectable in follicular pineal cells and retinal photoreceptors and that it exhibits a circadian rhythm, with peak levels at night. AA-NAT mRNA was not detected in other tissues. The AA-NAT mRNA rhythm in the pineal gland and retina persists in constant darkness (DD) and constant lighting (LL). The amplitude of the pineal mRNA rhythm is not decreased in LL. Light appears to influence the phase of the clock driving the rhythm in pineal AA-NAT mRNA in two ways: The peak is delayed by ∼6 h in LL, and it is advanced by >4 h by a 6-h light pulse late in subjective night in DD. Nocturnal AA-NAT mRNA levels do not change during a 20-min exposure to light, whereas this treatment dramatically decreases AA-NAT activity. These observations suggest that the rhythmic changes in chicken pineal AA-NAT activity reflect, at least in part, clock-generated changes in mRNA levels. In contrast, changes in mRNA content are not involved in the rapid light-induced decrease in AA-NAT activity.     

Effects of short-term cold exposure on pineal biosynthetic function in rats

In light of recent studies demonstrating stress-induced changes in pineal indoleamine metabolism, we tested the effect of acute cold stress on pineal biosynthetic function. Adult male rats were subjected to 30, 60, or 120 min of cold exposure (Ta = 2 degrees C) during either the light or dark phase of the daily photoperiodic cycle. Controls were kept at room temperature (22 +/- 2 degrees C). Animals were killed by decapitation and pineals were analyzed by radioimmunoassay for melatonin content and by radioenzymeassay for the activity of N-acetyltransferase (NAT). Cold exposure during the day elicited no significant changes in pineal indoleamine metabolism. Exposure to cold for 1 hr during the second hour after lights off slightly increased pineal melatonin content, without a concomitant change in NAT activity. Rats exposed to 2 hr of cold beginning 2 hr after lights off, however, displayed a 50% reduction in NAT activity, whereas pineal melatonin content remained unchanged. The paradoxical response of pineal NAT activity and melatonin content are not uncommon when rats are exposed to adverse stimuli.     

Enzyme cytochemistry of rat organs after uremia with special reference to proteases

Wistar rat organs and tissues were investigated after acute and chronic uremia using enzyme cytochemical means whereby special attention was paid to plasma membrane and lysosomal proteases. Heart muscle, pancreas, spleen, stomach, duodenum, jejunum, colon and skeletal muscle did not show any clear-cut indications of alterations. After acute uremia activities of dipeptidyl peptidase IV, glutamyl aminopeptidase and microsomal alanyl aminopeptidase were decreased in the extraorbital gland and that of dipeptidyl peptidase IV in the submandibular gland. The thymus showed an increased staining for glutamyl aminopeptidase and lysosomal proteases. An activity increase of dipeptidyl peptidase IV, acid phosphatase and beta-N-acetyl-D-glucosaminidase occurred in bronchial lavage cells among which the alveolar macrophages predominated. In addition, their number was comparatively higher. Non-specific esterase activity was lowered in these cells. Alkaline phosphatase activity was drastically enhanced at the biliary pole of hepatocytes. Following chronic uremia all effects were less pronounced except for the lavage cells which were positive for glutamyl aminopeptidase, microsomal alanyl aminopeptidase and gamma-glutamyl transpeptidase and showed increased staining for lysosomal proteases, glycosidases and nonspecific phosphatases.     

Measurement and distribution of vasopressin-converting aminopeptidase activity in rat brain

The aminopeptidase activity in the brain which converts vasopressin into centrally active metabolites, was quantitated on basis of the release of 3H-Phe from the substate [3H-Phe3]vasopressin and separation by hydrophobic interaction chromatography on mini-columns. After subcellular fractionation of whole rat brain homogenates the highest specific activity of the peptidase was recovered in membrane fractions, in particular microsomes and the P3 fraction, and the cytosol. The peptidase activity was present in all brain areas. Highest activity was measured in membranes of the bulbus olfactorius, preoptical area and cerebellum. Lowest activity was found in the medulla oblongata and striatum. The peptidase activity is not restricted to the vasopressin system per se, but may have a more general role in neuropeptide metabolism.     

Pineal glands of immature rats: rise and fall in N-acetyltransferase activity in vitro

N-acetyltransferase activity in the pineal glands of immature rats (12--14 days old) shows a spontaneous rise and fall when the glands are placed in organ culture. The peak of the 28-fold change occurs 5--16 hr after the cultures are initiated. This observation can be interpreted in two ways: (1) the pup pineal gland is responding to norepinephrine released when nerve endings degenerate in culture, or (2) the pup pineal gland has an innate ability which is responsible. Whatever the mechanism, the phenomenon is associated with the development of pineal gland function since pineal glands of adult rats do not show a spontaneous rise and fall in identical experiments.     

Development and Regulation of Rhodopsin Kinase in Rat Pineal and Retina

Rhodopsin kinase, once thought to be a retinal enzyme, was recently found at high levels in the pineal gland. In the present study the developmental pattern and the regulation by environmental lighting of this enzyme in both tissues was studied in the rat. Enzyme activity was present in the neonatal pineal gland several days earlier than in the retina, and increased gradually up to 20 days of age and remained at that level thereafter; the retinal enzyme appeared to increase until day 60. Pineal and retinal rhodopsin kinase activities showed a 25% increase in in the middle of the dark and the beginning of the light period, respectively. Exposure to constant light caused a 50% decrease in rhodopsin kinase levels in both tissues. However, only pineal rhodopsin kinase activity declined followed bilateral superior cervical ganglionectomy. This indicates pineal rhodopsin kinase activity is similar to other pineal enzymes in that it is controlled by light acting through the sympathetic nervous system. In contrast, the light-induced decrease in retinal rhodopsin kinase may be due to the direct destructive effect of light on the retina. The finding of neural control of pineal rhodopsin kinase in the pineal gland of adult rats is consistent with a function of the enzyme in the neural regulation of pineal function.     

Light-induced changes in frog pineal gland N-acetyltransferase activity

N-Acetyltransferase (NAT) activity was determined in the pineal gland of frogs (Rana tigrina) of different ages using 2-aminofluorene and p-aminobenzoic acid as substrates, and assayed by high-pressure liquid chromatography. Frogs of different ages were either killed during the light phase or exposed to darkness or light for 1 min during the dark phase of the lighting cycle, then returned to their cages in darkness for 30 min before being killed. The pineal gland NAT activity of 1-month-old frogs was inhibited when the animal was nocturnally exposed to 1 min of light. Nocturnal light exposure did not inhibit NAT activity in 1-month-old frogs, even though these animal displayed clear light-dark differences in pineal gland NAT activity. Nocturnal light exposure did not inhibit night-time levels of NAT activity in 1-month-old animals which had been bilaterally enucleated, thus suggesting that this effect is retinally mediated. Pretreatment of 1-month-old and 6-month-old animals with isoproterenol (a beta-adrenoceptor agonist drug) prevented the nocturnal light-induced inhibition of NAT activity. From the different sensitivity of 1-month-old and 6-month-old animals to different intensities or durations of nocturnal light exposure it was found that the duration or intensity of light exposure was not able to inhibit nocturnal NAT activity. The NAT activity was at least 4–5-fold greater in 1-month-old frogs than in 6-month-old frogs. This is the first demonstration of the retino-pineal gland pathway that appears to produce light-induced changes in pineal glands of frogs 1-month-old or older, but this pathway only functions in 1-month-old frogs, and does not appear to function in 6-month-old frogs.     

大鼠松果体Clock基因和芳烷脘N-乙酰基转移酶基因的昼夜节律性表达及光照影响

探讨12h光照、12h黑暗交替(12h-light：12h-dark cycle,LD)及持续黑暗(constant darkness,DD)光制下松果体Clock基因和芳烷脘N-乙酰基转移酶基因(arylalkylamine N-acetyltransferase gene,NAT)是否存在昼夜节律性表达及其光反应变化。Sprague-Dawley大鼠在LD和DD光制下分别被饲养4周(n=36)和8周(n=36)后,在一昼夜内每隔4h采集一组松果体组织(n=6),提取总RNA,用竞争性定量RT-PCR测定不同昼夜时点样品中Clock及NAT基因的mRNA相对表达量,通过余弦法和ClockLab软件获取节律参数,并经振幅检验是否存在昼夜节律。结果如下：(1)在DD或LD光制下,松果体Clock和NAT基因mRNA的表达均呈现夜高昼低的节律性振荡(P＜0．05)。(2)与DD光制下比较,LD光制下松果体Clock和NAT基因的表达振幅及峰值相的mRNA水平均降低(P＜0．05)。(3)在DD或LD光制下,Clock和NAT基因之间显示相似的节律性表达(P＞0．05)。结果表明,Clock和NAT基因在松果体中存在同步的内源性昼夜节律表达,光照作用可使其表达下调。     

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.     

Vasopressin is a physiological substrate for the insulin-regulated aminopeptidase IRAP

Insulin-regulated aminopeptidase (IRAP) is a membrane aminopeptidase and is homologous to the placental leucine aminopeptidase, P-LAP. IRAP has a wide distribution but has been best characterized in adipocytes and myocytes. In these cells, IRAP colocalizes with the glucose transporter GLUT4 to intracellular vesicles and, like GLUT4, translocates from these vesicles to the cell surface in response to insulin. Earlier studies demonstrated that purified IRAP cleaves several peptide hormones and that, concomitant with the appearance of IRAP at the surface of insulin-stimulated adipocytes, aminopeptidase activity toward extracellular substrates increases. In the present study, to identify in vivo substrates for IRAP, we tested potential substrates for cleavage by IRAP-deficient (IRAP(-/-)) and control mice. We found that vasopressin and oxytocin were not processed from the NH(2) terminus by isolated IRAP(-/-) adipocytes and skeletal muscles. Vasopressin was not cleaved from the NH(2) terminus after injection into IRAP(-/-) mice and exhibited a threefold increased half-life in the circulation of IRAP(-/-) mice. Consistent with this finding, endogenous plasma vasopressin levels were elevated twofold in IRAP(-/-) mice, and vasopressin levels in IRAP(-/-) brains, where plasma vasopressin originates, showed a compensatory decrease. We further established that insulin increased the clearance of vasopressin from control but not from IRAP(-/-) mice. In conclusion, we have identified vasopressin as the first physiological substrate for IRAP. Changes in plasma and brain vasopressin levels in IRAP(-/-) mice suggest a significant role for IRAP in regulating vasopressin. We have also uncovered a novel IRAP-dependent insulin effect: to acutely modify vasopressin.     

Atypical 24-hour rhythms of serotonin N-acetyltransferase activity in the rat pineal gland

Previous long-term studies have shown that in the pineal gland of rats melatonin synthesis is subject to infradian rhythms with periods between 4 and 7 days. Since in these studies melatonin-related parameters were measured at one timepoint of a 24-hr cycle only, the aim of the present investigation was to extend these experiments by more frequent sampling, to characterize the infradian rhythmicity in more detail. Male Sprague-Dawley rats kept under a light schedule of LD 12:12 (lights on at 0700) were killed at 6-hr intervals on 8 consecutive days. After decapitation the pineal gland was rapidly dissected out, followed by measurements of one of the melatonin-forming enzymes, serotonin N-acetyltransferase (NAT) activity. It was found that pineal NAT activity exhibited the well known day/night rhythm, i.e. low activity during daytime and strikingly enhanced activity at night, during the first 4 days of the experiment. On the fifth night (from Saturday to Sunday) an unusually high NAT peak occurred at 2400 hr, followed by two atypical 24-hr cycles. In the first cycle the midnight and 0600 hr values were equal and in the second cycle the 0600 hr value was significantly higher than the midnight value. To investigate whether the unusually high NAT peak was a single event or not, four additional short-term experiments were carried out at 2400 hr on 4 consecutive weekends, from Friday to Monday. In each of the four 4-day experiments a distinctly higher peak of NAT activity was found on Saturday, but with time the peaks became less prominent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum melatonin and pineal indoleamine metabolism in a species with a small day/night N-acetyltransferase rhythm

Ovine serum and pineal melatonin levels are low during the day, increase five to ten-fold at night, decrease during a light pulse at night, and rapidly increase to night levels following the light-dark transition. N-Acetyltransferase activity increases three-fold at night, falls significantly in response to the light pulse, but does not increase following the light pulse. No significant change in N-acetylserotonin occurs under these conditions. These results suggest that the biochemical mechanisms controlling pineal melatonin synthesis in the sheep pineal gland may be different from those in the rat.     

Daily oscillation in melatonin synthesis in the Turkey pineal gland and retina: diurnal and circadian rhythms

The aim of the present study was to examine arylalkylamine N-acetyltransferase (AANAT) activity and melatonin content in the pineal gland and retina as well as the melatonin concentration in plasma of the turkey (Meleagris gallopavo), an avian species in which several physiological processes, including reproduction, are controlled by day length. In order to investigate whether the analyzed parameters display diurnal or circadian rhythmicity, we measured these variables in tissues isolated at regular time intervals from birds kept either under a regular light-dark (LD) cycle or under constant darkness (DD). The pineal gland and retina of the turkey rhythmically produced melatonin. In birds kept under a daily LD cycle, melatonin levels in the pineal gland and retina were high during the dark phase and low during the light phase. Rhythmic oscillations in melatonin, with high night-time concentrations, were also found in the plasma. The pineal and retinal melatonin rhythms mirrored oscillations in the activity of AANAT, the penultimate enzyme in the melatonin biosynthetic pathway. Rhythmic oscillations in AANAT activity in the turkey pineal gland and retina were circadian in nature, as they persisted under conditions of constant darkness (DD). Transferring birds from LD into DD, however, resulted in a potent decline in the amplitude of the AANAT rhythm from the first day of DD. On the sixth day of DD, pineal AANAT activity was still markedly higher during the subjective dark than during the subjective light phase; whereas, AANAT activity in the retina did not exhibit significant oscillations. The results indicate that melatonin rhythmicity in the turkey pineal gland and retina is regulated both by light and the endogenous circadian clock. The findings suggest that environmental light may be of primary importance in the maintenance of the high-amplitude melatonin rhythms in the turkey.     

Maternal stress induces adult reduced REM sleep and melatonin level

We have previously reported that neonatal maternal deprivation (MD) resulted in a decrease of total sleep and an increase of orexin A in adult rats. Now, we characterized features of sleep, activity, and melatonin levels in rats neonatally treated with MD and control (MC) procedures. Adult male Sprague-Dawley rats were treated with either MD or MC procedures for 10 days starting at postnatal day 4. At 3 months of age, sleep was recorded for 48 h in one set of MD and MC rats, while another set of MD and MC rats was measured for locomotor activity (under LD = 12:12). Melatonin levels in the blood, pineal gland, and hypothalamus were measured as well as clock protein level in the hypothalamus. Compared to the MC rats, REM sleep in the MD rats was significantly reduced in the light periods but not in the dark periods. Both quiet wake and total wake in the MD rats were significantly increased during the light period compared to the MC rats. The weight of the pineal gland of the MD rats was significantly smaller than in MC rats. Melatonin levels of the MD group were significantly reduced in the pineal gland and hypothalamus compared to the MC group. No significant difference was identified between groups in the expression of the clock protein in the hypothalamus. Neonatal MD resulted in reduced REM sleep and melatonin levels, without changes of circadian cycle of locomotor activity and levels of clock protein.     

Effect of long-term exposure to constant dim light on the circadian system of rats

The newly discovered multi-oscillatory nature of the mammalian circadian clock system and the cloning of the genes involved in the molecular mechanism that generates circadian rhythmicity have opened new approaches for understanding how mammals are temporally organized and how the mammalian circadian system reacts to the lack of normal synchronization cues. In the present study we investigated the effects of long-term exposure to constant red dim light on the pattern of the expression of Period 1 in the suprachiasmatic nuclei of the hypothalamus and of Arylalkylamine N-acetyltransferase(Aa-nat) in the retina and pineal gland. Our data demonstrate that Period 1 mRNA expression in the suprachiasmatic nuclei of the hypothalamus was not affected by exposure to constant red dim light for 60 days, whereas Aa-nat mRNA expression in the retina and in the pineal gland was significantly affected, since in some animals (20-30%) Aa-nat mRNA levels were found to be higher during the subjective day. A circadian rhythm of serum melatonin and locomotor activity was present in all the animals tested. In 4 animals serum melatonin levels were high during the subjective day. Our data suggest that long-term exposure to constant red dim light may induce desynchronization between the circadian rhythm of locomotor activity and serum melatonin levels.