Entrainment of the circadian rhythm in the rat pineal N-acetyltransferase activity by melatonin is photoperiod dependent

Entraining effect of melatonin on the circadian rhythm in rat pineal N-acetyltransferase (NAT) activity was studied under various photoperiods. Melatonin administration prior to dark onset for 5 successive days phase-advanced the evening NAT rise under the light:dark (LD) cycle of either LD 10:14 or LD 8:16, but not under LD 12:12. It is assumed that under the latter regime, the end of a light period exhibited a phase-delaying effect on the NAT rise. The light exposure appeared to be a stronger Zeitgeber than melatonin itself. Data show that melatonin applied in the late light period advances the evening NAT rise under a short photoperiod only; under a longer photoperiod, the phase-advancing effect of melatonin may conflict with a phase-delaying effect of the end of a light period, and the effect of light exposure overrides that of melatonin.     

Adenosine 5''-monophosphate enhances the dark and isoproterenol-induced rise in rat pineal N-acetyltransferase activity

Exposure of rats to light during darkness or blockade of the pineal beta-adrenoceptor in stimulated pineal glands results in a rapid fall in pineal N-acetyltransferase (NAT) activity. Maintenance of a high level of NAT activity requires continuous stimulation of the pineal beta-adrenoceptors. It is not known what factors in the pinealocyte are responsible for this rapid inactivation of NAT activity. In the present study we have attempted to investigate a possible regulatory role of 5'-AMP on pineal NAT activity. The results show that 5'-AMP further enhances dark-induced as well as isoproterenol-induced NAT activity by approximately 3-fold but does not alter unstimulated daytime NAT activity. Theophylline, an inhibitor of 5'-AMP synthesis, when administered early in the dark phase, caused a rise in pineal cAMP with a concomitant fall in pineal NAT activity. These findings indicate that 5'-AMP could play a role in the activation of pineal NAT. The possibility that the rapid inactivation of NAT is due to a rapid removal of 5'-AMP by, for example, phosphorylation, remains to be investigated.     

Swimming depresses nighttime melatonin content without changing N-acetyltransferase activity in the rat pineal gland

Recently, it was shown that a 1.5-ml subcutaneous saline injection depressed N-acetyltransferase (NAT) activity and melatonin content in the rat pineal gland at night. The present studies were undertaken to determine if another perturbation, swimming, could duplicate this response. Rats swam at 23.10 h (lights out at 20.00 h) for 10 min and were killed 15 and 30 min after the unset of swimming. Pineal NAT activity was found to be unaffected while melatonin content was depressed dramatically. Hydroxyindole-O-methyltransferase (HIOMT) activity as well as the content of serotonin (5HT), 5-hydroxytryptophan (5HTP) and 5-hydroxyindoleacetic acid (5HIAA) were not changed by this treatment. In a second study, pineal melatonin again was depressed without a concomitant drop in NAT activity. Mean serum melatonin at 15 min after onset of swimming was increased although the rise was not statistically significant. In the final study, it was found that NAT activity was slightly increased in intact rats and unchanged in adrenalectomized rats at 7 min after swimming onset. At 15 min both intact and adrenalectomized animals had NAT activity values similar to those of controls. Pineal melatonin content in intact and adrenalectomized rats plummeted to 50% of control values at 7 min and fell further to 25% at 15 min. While the rate of melatonin synthesis was not directly measured, lack of change in the activities of the enzymes involved in melatonin synthesis and the contents of two melatonin precursors suggests that swimming depresses pineal melatonin content by enhancing melatonin efflux from the gland.     

Melatonin resynchronizes dysregulated circadian rhythm circuitry in human prostate cancer cells

Abstract: Prostate cancer (PCa) is a major age‐related malignancy as increasing age correlates with increased risk for developing this neoplasm. Similarly, alterations in circadian rhythms have also been associated with the aging population and cancer risk. The pineal hormone melatonin is known to regulate circadian rhythms, which is under the control of a core set of genes: Period 1, 2, 3 (Per 1–3); Cryptochrome 1, 2 (Cry 1, 2); Clock, and Bmal 1, 2. Melatonin levels have been shown to decrease in patients with cancer and exogenous melatonin exhibits antiproliferative effects against certain cancers. In this study, we challenged the hypothesis that melatonin imparts antiproliferative effects in prostate cancer via resynchronization of deregulated core clock circuitry. We found that Clock and Per2 protein levels were downregulated whereas Bmal1 protein levels were upregulated in PCa cells, compared to normal prostate cells. Additionally, employing automated quantitative analysis of a microarray containing human tissues, we found that compared to benign tissues, Clock and Per2 levels were downregulated, whereas Bmal1 levels were upregulated in PCa and other proliferative prostatic conditions. Overexpression of Per2 was found to result in a significant loss of PCa cell growth and viability. Interestingly, melatonin treatment resulted in an increase in Per2 and Clock and a reduction in Bmal1 in PCa cells. Further, melatonin treatment resulted in a resynchronization of oscillatory circadian rhythm genes (Dbp and Per2). Our data support our hypothesis and suggest that melatonin should be thoroughly investigated as an agent for the management of PCa and other age‐related malignancies.     

Melatonin accelerates the reentrainment of the circadian adrenocortical rhythm in inverted illumination cycle

The effect of melatonin, para-chlorophenyl alanine (PCPA), gamma-aminobutyric acid (GABA) and dibutyryl-cAMP (d-cAMP) on the photoentrainment of the circadian adrenocortical rhythm was studied by the intrahypothalamic (near the SCN) implantation of a paraffin pellet mixed with each agent, in the rat. 2 weeks after implantation, the rats were moved to a room with a reversed lighting cycle (from LD to DL). Blood samples were taken from individual rats at 4-hour intervals for 24 h, and 2, 4, 6 and 10 days after movement, by the tail tip incision method. The effects of implantation of PCPA, GABA and d-cAMP on the reentrainment to the illumination cycle reversed were indistinguishable from those due to implantation of a pellet of paraffin alone. In these groups, more than half of the rats did not reentrain to the light cycle reversed in 4 (less than 18.1%) and 6 days (less than 36.3%), but did reentrain in 10 days. However, melatonin caused reentrainment of the circadian adrenocortical rhythm to reversed illumination between 4 (45.4%) and 6 days (72.7%). Pinealectomized rats receiving a melatonin pellet also exhibited an accelerated reentrainment to the light cycle reversed, when compared with the rat which received a pellet of paraffin alone. However, pinealectomized rats which received a pellet of paraffin alone did not require more time than intact rats to reentrain to the light cycle reversed. These results indicate that melatonin accelerates the reentrainment of the circadian adrenocortical rhythm to the light cycle reversed.     

Light-induced changes in pineal gland N-acetyltransferase activity: developmental aspects

In adult rats, light acting via a retino-pineal gland neural pathway influences pineal gland biochemistry in two ways: (1) it entrains endogenous circadian rhythms in melatonin biosynthesis to the environmental photoperiod and (2) exposure to even very brief periods of light during the nighttime rapidly suppresses the high levels of nocturnal melatonin production. The present studies were undertaken to determine precisely when photic stimulation first influences the enzymic activity of N-acetyltransferase (NAT), the pineal gland enzyme which rate-limits the overall biosynthesis of the hormone melatonin, and to examine some of the cellular mechanisms which might mediate light-induced effects in neonatal animals. Rats of different ages were either killed during the light phase or were exposed to darkness or light for 1 min during the dark phase of the lighting cycle, returned to their litters in darkness for 30 min and then killed. Pineal gland NAT activity in animals nocturnally exposed to 1 min of light was suppressed in animals 6 days of age or older. Nocturnal light exposure did not suppress enzyme activity in 3- to 5-day-old rats, even though these animals displayed clear light:dark differences in pineal gland NAT activity. Nocturnal light exposure also did not suppress nighttime levels of NAT activity in 7-day-old animals who had been bilaterally enucleated, suggesting that this effect is retinally mediated. Pretreatment of 7-day-old animals with the beta-noradrenoceptor agonist drug, isoproterenol, prevented the nocturnal light-induced suppression of NAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

HPLC validation of a circadian melatonin rhythm in the pineal gland of inbred mice

Abstract: Production of melatonin in the pineal gland of inbred mice such as C57B1/6J, B ALB/c and AKR strains is still a matter of debate. In previous studies, we and other authors showed that these strains of inbred mice have a clear-cut circadian rhythm of serum melatonin and urinary 6-hydroxy-melatonin-sulphate. In contrast, other groups claimed these mice are unable to synthesize melatonin. These studies were based on RIA measurements and/or estimates of N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) activities. In the present study we validate the presence of melatonin in the pineal gland of C57B1/6, BALB/c, and AKR mice by HPLC determinations. We found a short-term melatonin peak in the middle of the dark period with a pattern which mirrors that found previously in the serum. The possibility remains, although it seems unlikely, that the pineal melatonin rhythm measured here represents melatonin produced elsewhere which then was subsequently taken up by the pineal gland.     

Commonalities in vasoactive intestinal peptide and peptide N-terminal histidine C-terminal isoleucine stimulation of N-acetyltransferase activity in the rat pineal

Abstract: Exposure of adult rat pineal glands in organ culture to the polypeptides vasoactive intestinal polypeptide (VIP), and peptide N-terminal histidine C-terminal isoleucine (PHI) increases pineal serotonin N-acetyltransferase (NAT) activity and melatonin synthesis. The following research results are taken to indicate that VIP and PHI share common components of the NAT induction system: (1) The effects of the two peptides are additive at concentrations of 10 nM VIP and 100 nM PHI but not at higher peptide concentrations. (2) Pineals from newborns also respond to PHI with a dose dependent increase in NAT activity. NAT responses are additive at the same concentrations as seen with the adult pineals. (3) Light exposure affects the sensitivity of pineals to VIP and PHI stimulation in a similar manner; pineals taken after 3 hr of light are much less sensitive to PHI or VIP than those taken after 13 hr of light. (4) Pineals exposed for 48 hr to either PHI or VIP have a reduced NAT response to either agonist, which is reversible by culture in agonist-free media. (5) Neither VIP nor PHI stimulation of NAT activity is affected by concentrations of the VIP antagonists (N-Ac-Tyr¹, D-Phe²)-GRF(l-29)-NH2 (NAcTDGRF), L-8-K, VIP-Neurotensin Hybrid (VIPNET), or (4Cl-D-Phe⁶, Leu¹⁷)-VIP (4C1PLVIP), which affect VIP binding or function in other tissues.     

Pubertal manifestation of sex difference in circadian rhythm of corticotrophin-releasing activity in the rat hypothalamus.

Post-natal development of the circadian rhythm of hypothalamic content of corticotrophin-releasing factor (CRF) was examined in male and female rats, separately. CRF activity was estimated by the intrapituitary injection technique. The circadian rhythm of the CRF content observed at the third week was without any noticeable sex difference: both male and female rats began their circadian rhythm with higher values in the afternoon than in the morning. Male rats maintained this pattern up to maturity. In contrast, female rats showed a marked change at ages of fifth to sixth week: the CRF rhythm in female rats changed to a female pattern, with higher values in the morning than in the afternoon. During this period, the vaginal opening occurred concurrently with a marked afternoon rise in the plasma corticosterone, characteristic of mature female rats. On the other hand, no essential difference could be observed between male and female rats in the developmental change in the circadian rhythm of locomotor activity. These results indicate that a sex difference in the CRF rhythm is not essentially related to the process of sex differentiation in the central nervous system, but is rather related to changes in ovarian activity following the onset of puberty.     

Melatonin: a major regulator of the circadian rhythm of core temperature in humans.

The circadian rhythm of core body temperature (BTc), with maxima during the day and minima at night, is normally coupled with the sleep-wake cycle. Pineal melatonin secretion occurs contemporaneously during the nighttime hours and is mediated by the activation of beta-adrenergic receptors during darkness. The hypothesis that nocturnal melatonin secretion may be involved in the regulation of the human circadian BTc rhythm was examined. The temporal relationship between melatonin and the circadian BTc rhythm was characterized in 12 young women, normally entrained to the light-dark cycle. Melatonin levels were manipulated through the administration of exogenous melatonin (2.5 mg, orally) during the daytime (n = 6) or suppression of endogenous nocturnal melatonin secretion by the beta-adrenergic antagonist atenolol (100 mg; n = 6) in double blind placebo-controlled experiments conducted during 2 consecutive days. Serum melatonin levels and BTc were monitored at 20- and 10-min intervals, respectively. In a nightshift worker the temporal relationship between the circadian rhythm of melatonin and BTc was investigated before and after entrainment to a reversed wake-sleep cycle. Our data show that in normally entrained subjects, the time course and amplitude of nocturnal melatonin secretion were temporally coupled with the decline of BTc (r = 0.97; P less than 0.00001). The same occurred in the nightshift worker, both during the dissociation and after entrainment to the reversed sleep-wake cycle. Compared with placebo, administration of melatonin significantly reduced daytime BTc (P less than 0.01), and the suppression of melatonin (by atenolol) attenuated the nocturnal decline of BTc (P less than 0.01). Cosinor analysis showed that the amplitude of the circadian BTc rhythm was reduced by about 40% in response to both daytime melatonin administration (P less than 0.05) and nocturnal melatonin suppression (P less than 0.02). In conclusion, circadian rhythms of melatonin and BTc are inversely coupled. The demonstrated hypothermic properties of melatonin are accountable for the generation of at least 40% of the amplitude of the circadian BTc rhythm. Manipulation of melatonin levels might be clinically useful to resynchronize the BTc rhythm under conditions of BTc rhythm desynchronization.     

Rat pineal N-acetyltransferase activity: stimulation, exhaustion, and recovery

Rat pineal N-acetyltransferase activity (NAT) has a circadian rhythm, with a peak in the dark time. The rhythm of NAT was studied in young rats (26-40 days old). In particular, the possibility of maintenance of prolonged low NAT or prolonged high NAT was studied. NAT (given in nanomoles per pineal gland/h) had peak activity (33.0) in the dark time. Forty-eight hours of constant light suppressed the peak and maintained low NAT (0.5 or less). In vivo, in constant light, NAT was stimulated (to 13.1-29.4) by single injections of isoproterenol. Similarly, NAT was stimulated by isoproterenol or (Bu)2cAMP in vitro. In vivo, multiple injections of isoproterenol stimulated NAT for 14 h (to peak 31.9), but NAT was exhausted and fell to less than 5 over the next 8 h. Similarly, isoproterenol and (Bu)2cAMP exhausted NAT in vitro. High NAT was not maintained. NAT recovered [could be restimulated by dark in vivo, isoproterenol in vivo or in vitro, or (Bu)2cAMP in vitro]. Exhaustion was not permanent. Light can keep NAT at low levels, but dark cannot maintain peak NAT beyond 14 h. The results are interpreted as meaning that the duration of NAT is fixed.     

The effects of near-ultraviolet light on serotonin N-acetyltransferase activity in the chick pineal gland

Effects of near-ultraviolet light (UV-A; 325-390 nm, peak at 365 nm) on the activity of the pineal serotonin N-acetyltransferase (NAT; a key regulatory enzyme in melatonin biosynthesis) were examined in chicks. Acute exposure of dark-adapted animals to UV-A radiation produced a marked decline in NAT activity of the pineal gland. The magnitude of this suppression was dependent upon duration of the light pulse and the age of the animals. The decrease in the nighttime NAT activity evoked by a 5 min pulse of UV-A light applied during the fourth hour of the dark phase of the 12 hr light:12 hr dark cycle (LD) gradually deepened during the first 40 min after the return of animals to constant darkness, then the enzyme activity began to rise, reaching control values by 2 hr. Exposure of chicks to a 5 min pulse of UV-A light during the ninth hour of the dark phase produced a marked decline in pineal NAT activity, which was reversible after 15 min of darkness. Pretreatment of animals with an inhibitor of catecholamine synthesis, alpha-methyl-p-tyrosine (300 mg/kg, i.p.), or with a blocker of alpha2-adrenergic receptors, yohimbine (2 mg/kg, i.p.), antagonized the suppressive effect of UV-A light on nighttime NAT activity of the chick pineal gland. It is concluded that UV-A irradiation, similar to visible light, potently suppresses melatonin biosynthesis in the chick pineal gland, with an alpha2-noradrenergic signal playing the role of an intermediate in this action.     

The effects of benzodiazepines on basal and isoproterenol-stimulated N-acetyltransferase activity by the rat pineal gland, in vivo and in vitro

The present study examined the effects of "peripheral," "central," and "mixed" benzodiazepine agonists and antagonists on the nocturnal rise in rat pineal NAT activity in vivo and the isoproterenol-stimulated NAT activity of pineals in organ culture. Administration of the central agonist clonazepam or the mixed agonist-antagonist diazepam, 4 hr after dark, at a dose of 25 mg/kg each, inhibited nocturnally elevated NAT 20 min later, while this same dose of the peripheral agonist RO 54864 elevated NAT activity. In a second study these agents were administered in vivo 1 hr before dark, at a dose of 3, 10, or 25 mg/kg i.p. and tested 4 hr after dark for in vitro rat pineal NAT activity. None of these agents affected NAT activity at the 3- or 10-mg/kg dose, but RO 54864 25 mg/kg did induce elevated activity. In a third study, all of these agents prolonged the time period for NAT induction by isoproterenol in rat pineals cultured for 48 hr before stimulation. The data suggests that benzodiazepine stimulation of NAT activity in vitro is not specific to "central" or "peripheral" benzodiazepine receptors and that inhibition of melatonin production in vivo occurs either at some step before NAT induction or is involved with the inhibition of pineal HIOMT activity.     

Daily rhythm of aspartate aminopeptidase activity in the retina, pineal gland and occipital cortex of the rat.

The levels of specific soluble aspartyl aminopeptidase activity were assayed in retina, occipital cortex, anterior pituitary, posterior pituitary, pineal gland and serum of adult male rats, using Asp-2-naphthylamide as substrate, in a 12:12 h light:dark cycle (7-19 h light). Significant diurnal variations appeared in retina, pineal gland, occipital cortex and serum. In addition, different patterns of diurnal variation of the enzymatic activity were observed in the tissues analyzed. A regular increase of the activity was noticed at the end of the dark period in all the tissues as a common feature, except in serum, in which the enzymatic activity reached a peak in the middle of the light period, decreasing progressively during the dark hours. After the last hours of darkness, the pattern of variation in the activity differed in each tissue. These diurnal variations in aspartyl aminopeptidase activity could reflect the functional status of its putative endogenous substrates, such as angiotensin II, and it may also suggest the existence of differential regulatory mechanisms associated with each location.     

Endogenous circadian rhythm in human motor activity uncoupled from circadian influences on cardiac dynamics

The endogenous circadian pacemaker influences key physiologic functions, such as body temperature and heart rate, and is normally synchronized with the sleep/wake cycle. Epidemiological studies demonstrate a 24-h pattern in adverse cardiovascular events with a peak at ≈10 a.m. It is unknown whether this pattern in cardiac risk is caused by a day/night pattern of behaviors, including activity level and/or influences from the internal circadian pacemaker. We recently found that a scaling index of cardiac vulnerability has an endogenous circadian peak at the circadian phase corresponding to ≈10 a.m., which conceivably could contribute to the morning peak in cardiac risk. Here, we test whether this endogenous circadian influence on cardiac dynamics is caused by circadian-mediated changes in motor activity or whether activity and heart rate dynamics are decoupled across the circadian cycle. We analyze high-frequency recordings of motion from young healthy subjects during two complementary protocols that decouple the sleep/wake cycle from the circadian cycle while controlling scheduled behaviors. We find that static activity properties (mean and standard deviation) exhibit significant circadian rhythms with a peak at the circadian phase corresponding to 5–9 p.m. (≈9 h later than the peak in the scale-invariant index of heartbeat fluctuations). In contrast, dynamic characteristics of the temporal scale-invariant organization of activity fluctuations (long-range correlations) do not exhibit a circadian rhythm. These findings suggest that endogenous circadian-mediated activity variations are not responsible for the endogenous circadian rhythm in the scale-invariant structure of heartbeat fluctuations and likely do not contribute to the increase in cardiac risk at ≈10 a.m.     

Retinal rhythms in chicks: circadian variation in melantonin and serotonin N-acetyltransferase activity.

There is a large-amplitude circadian rhythm of indoleamine metabolism in the retina-pigment epithelium of the chicken. N-Acetyltransferase activity (arylamine acetyltransferase; acetyl-CoA:arylamine N-acetyltransferase, EC 2.3.1.5) and melatonin content are 15-fold higher at night than during the day in a cycle of a 4-fold increase during the subjective night. Light at midnight inactivates N-acetyltransferase and lowers melatonin. N-Acetyltransferase activity is found predominantly in the retina. The circadian rhythm of this enzyme activity persists in pinealectomized chicks. Thus the pineal is not responsible for retinal indoleamine rhythms. Retinal and pineal levels of N-acetyltransferase activity behave similarly under several conditions. In the chicken, the eye is a major site of rhythmic indoleamine metabolic activity.