Gonadotropin-releasing hormone increases melatonin release in the pineal gland of the female rat in vitro.

To evaluate the effect of gonadotropin-releasing hormone (GnRH) on melatonin ( N-acetyl-5-methoxytryptamine) release and its synthesizing enzyme activities in pineal glands, pineals from adult female rats during diestrus were organ-cultured in a medium containing 10 -12, 10 -10, or 10 -8 M GnRH for 6 h. Melatonin release increased significantly in pineals cultured with 10 -10 and 10 -8 M GnRH compared to controls. However, in pineal glands that were organ-cultured in a medium containing 10 -12 to 10 -8 M GnRH, the activity of arylalkylamine N-acetyltransferase, which is the key regulatory enzyme in melatonin biosynthesis, showed no significant difference from controls. Likewise, GnRH at these concentrations had no significant effect on the activity of pineal hydroxyindole- O-methyltransferase, which catalyzes the final step of melatonin biosynthesis. These results show that GnRH stimulates pineal melatonin release, but suggest that GnRH does not affect its melatonin synthesis.     

Evidence that interferon-gamma alters pineal metabolism both indirectly via sympathetic nerves and directly on the pinealocytes.

1. Interferon-gamma (IFN-gamma) has been shown to suppress N-acetyltransferase (NAT) activity in cultured rat pineal glands when stimulated with isoproterenol (ISO). 2. Conversely, IFN-gamma has also been shown to increase the melatonin content of the rat pineal gland in organ culture. 3. Circumstantial evidence leads to a hypothesis that the NAT suppressive effect may be due to the action of IFN-gamma on the sympathetic nerve terminals. 4. To test this hypothesis, pineal glands from intact (INT) and superior cervical ganglionectomized (SCGX) rats, which had been operated 5 days earlier, were cultured with either ISO or ISO + IFN-gamma. 5. The concentration of ISO was 10(-8) M and that of IFN-gamma was 300 antiviral units/ml. 6. The pineals were incubated for a total period of 5.5 hr, after which the activities of NAT and hydroxyindole-O-methyltransferase (HIOMT) and the levels of melatonin and cAMP were estimated. 7. Suppression of NAT by IFN-gamma was observed in the pineals from INT rats, but not in those from SCGX animals. 8. IFN-gamma significantly enhanced melatonin levels over those in ISO-stimulated pineals and culture media from the SCGX animals, but not from the INT animals. 9. IFN-gamma treatment had no effect on either the HIOMT activity or cAMP levels. 10. The results indicate that the IFN-gamma-induced NAT suppression requires the integrity of the sympathetic nerve terminals and the IFN-gamma-induced enhancement of melatonin production is accomplished through its direct action on pinealocytes.     

12-Hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-HPETE stimulate melatonin synthesis in rat pineals

The role of arachidonic acid metabolites in norepinephrine (NE)-induced N-acetyltransferase (NAT) activity and melatonin release was examined from 6 h-incubations of rat pineal glands. A cyclooxygenase inhibitor, indomethacin (5 x 10(-8) - 5 x 10(-6) M) was ineffective on melatonin release, in the presence of absence of NE (5 x 10(-6) M) while a lipoxygenase inhibitor, nordihydroguaiaretic acid (5 x 10(-7) -5 x 10(-5) M) had an inhibitory effect. Among the lipoxygenase metabolites, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-HPETE stimulated both NAT activity and melatonin release in a dose-dependent manner, with a maximal effect occurring at 10(-6) M, while 5-HPETE or hydroxy derivatives of these compounds (12-HETE, 15-HETE and 5-HETE) were ineffective. These results indicate that 12-HPETE and 15-HPETE can be involved in NE-induced melatonin release.     

Release and effect ofγ-Aminobutyric acid (GABA) on rat pineal melatonin productionin vitro

1. 3H-gamma-Aminobutyric acid (GABA) release elicited by a depolarizing K+ stimulus or by noradrenergic transmitter was examined in rat pineals in vitro. 2. The release of 3H-GABA was detectable at a 20 mM K+ concentration in medium and increased steadily up to 80 mM K+. 3. In a Ca2+-free medium 3H-GABA release elicited by 30 mM K+, but not that elicited by 50 mM K+, became blunted. 4. Norepinephrine (NE; 10(-6)-10(-4) M) stimulated 3H-GABA release from rat pineal explants in a dose-dependent manner. 5. The activity of 10(-5) M NE on pineal GABA release was suppressed by equimolecular amounts of prazosin or phentolamine (alpha 1- and alpha 1/alpha 2-adrenoceptor blockers, respectively) and was unaffected by propranolol (beta-adrenoceptor blocker). 6. The alpha 1-adrenoceptor agonist phenylephrine (10(-7)-10(-5) M) and the beta-adrenoceptor agonist isoproterenol (10(-5) M) mimicked the GABA releasing activity of NE, while 10(-7) M isoproterenol failed to affect it; the alpha 2-adrenoceptor agonist clonidine (10(-7)-10(-5) M) did not modify 3H-GABA release. 7. The addition of 10(-4) M GABA or of the GABA transaminase inhibitor gamma-acetylenic GABA or aminooxyacetic acid inhibited the melatonin content and/or release to the medium in rat pineal organotypic cultures. 8. GABA at concentrations of 10(-5) M or greater partially inhibited the NE-induced increase in melatonin production by pineal explants. 9. The depressant effect of GABA on melatonin production was inhibited by the GABA type A receptor antagonist bicuculline; bicuculline alone increased the pineal melatonin content. Baclofen, a GABA type B receptor agonist, did not affect the pineal melatonin content or release. 10. The decrease in serotonin (5-HT) content of rat pineal explants brought about by NE was not modified by GABA; GABA by itself increased 5-HT levels. 11. These results indicate that (a) GABA is released from rat pineals by a depolarizing stimulus of K+ through a mechanism which is partially Ca2+ dependent; (b) NE releases rat pineal GABA via interaction with alpha 1-adrenoceptors; (c) GABA inhibits melatonin production in vitro via interaction with GABA type A receptor sites; and (d) GABA's effect on NE-induced melatonin release does not correlate with the lack of effect on the NE-induced decrease in pineal 5-HT content.     

Ontogeny of N-acetyltransferase activity rhythm in pineal gland of chick embryo

1. N-acetyltransferase was present in pineal glands of 14-day-old chick embryos though no rhythm either in LL, DD or LD 12:12 was observed in this age. 2. Daily rhythm in pineal NAT activity was found in 18-day-old embryos incubated under LD 12:12 and LD 16:8 but no NAT rhythm was detected in DD or LL. 3. NAT rhythm persists for 2 days in constant darkness and it may be circadian in nature. 4. Presence of melatonin (85 +/- 8 pg/mg tissue) was detected in pineals of 18-day-old chick embryos.     

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.     

Progesterone inhibits, on a circadian basis, the release of melatonin by rat pineal perifusion

The effects of 10(-6) and 10(-9) M of progesterone were documented on isoproterenol-stimulated melatonin release by perifused pineal glands removed from female rats in diestrous at two different times of a 12 : 12 h light/dark cycle, 7 and 19 h after light onset (which corresponds to daytime and nighttime, respectively), to look for the existence of a circadian stage-dependence of the hormone effects. Three weeks before the experiment, the rats were synchronized with a 12 : 12 lighting regimen. Progesterone decreased by approximately 50% the release of melatonin during the light span, but not during the dark span. These results show the direct effects of this ovarian hormone on pineal melatonin release and strongly suggest a time-related effect of progesterone on pineal function.     

Stimulation of Melatonin Synthesis in Ovine Pineals In Vitro

Static and superfused pineal slices (750 micron) have been used to study the control of melatonin synthesis by ovine pineals. Static incubates show a time-dependent accumulation of melatonin in the medium; this is significantly increased by stimulation with norepinephrine (NE) (10(-5) M), reaching 300% above control levels after 4 h. Perifused pineal slices show a rapid rise in melatonin release within 12-18 min in response to NE stimulation. This reaches a 3.5-4.5-fold increase in melatonin released within 30 min. Withdrawal of NE is associated with a rapid return to prestimulated levels within 12-18 min. These time-course characteristics compare favorably to those changes seen in vivo. The formation of [14C]melatonin from [14C]-tryptophan shows a linear increase with time. In the presence of NE (10(-5) M), the rate of synthesis is increased, albeit after an initial time lag of at least 30 min. The latter may reflect an N-acetyltransferase-independent mechanism of synthesis and release. In static incubations, propranolol (10(-5) M) inhibited NE-induced melatonin production by about 60%, but prazosin (10(-5) M) had no effect. As dibutyryl cyclic AMP (10(-3) M) stimulated melatonin production, it is concluded that beta-receptors are of primary importance to the control of melatonin production, as in the rat. The role of alpha 1-receptors is less clear, but the stimulatory action of phorbol 12-myristate 13-acetate on melatonin release implicates a receptor linked to phosphatidylinositol turnover.     

Regulation of pineal enzymes by photoperiod, gonadal hormones and melatonin in Coturnix quail.

The photoperiodic and hormonal regulation of melatonin-synthesizing enzymes was determined in pineals of Coturnix quail. N-Acetyl transferase (NAT) and hydroxyindole-O-methyl transferase (HIOMT) were twofold higher in pineals of female and male Coturnix quail during exposure to darkness (16L:8D). Castration decreased pineal HIOMT activity in both female and male Coturnix, while selective gonadal steroids restored activity. NAT was not affected by castration or gonadal steroids. Implantation of melatonin into female Coturnix decreased both HIOMT and NAT activities. These results suggest that NAT is regulated primarily by photoperiodicity, while HIOMT activity is a consequence of the external perceptive environment and the internal hormonal milieu, with both enzymic activities modulated by the feedback inhibitory influence of endogenous melatonin.     

12-Hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-HPETE stimulate melatonin synthesis in rat pineals

The role of arachidonic acid metabolites in norepinephrine (NE)-induced N-acetyltransferase (NAT) activity and melatonin release was examined from 6 h-incubations of rat pineal glands. A cyclooxygenase inhibitor, indomethacin (5×10⁻⁸ − 5×10⁻⁶ M) was ineffective on melatonin release, in the presence of absence of NE (5×10⁻⁶ M) while a lipoxygenase inhibitor, nordihydroguaiaretic acid (5×10⁻⁷ −5×10⁻⁵ M) had an inhibitory effect. Among the lipoxygenase metabolites, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-HPETE stimulated both NAT activity and melatonin release in a dose-dependent manner, with a maximal effect occuring at 10⁻⁶ M, while 5-HPETE or hydroxy derivatives of these compounds (12-HETE, 15-HETE and 5-HETE) were ineffective. These results indicate that 12-HPETE and 15-HPETE can be involved in NE-induced melatonin release.     

Neuroendocrine integrative mechanisms in mammalian pineal gland: effects of steroid and adenohypophysial hormones on melatonin synthesis in vitro

The time course for the decrease in norepinephrine concentration of rat pineal explants in culture indicated a significant fall starting at the 4th hour and completed after 16-24 h of incubation. Significant decreases of serotonin and 5-hydroxyindoleacetic acid (HIAA) levels in tissue, an increase of HIAA/serotonin ratio, and an increase of melatonin production rate in vitro were also observed as a function of the incubation time. Estradiol (10(-7)-10(-5) M) increased rat pineal melatonin content, testosterone (10(-5) M) decreased it and progesterone was devoid of activity when incubated with explants for up to 6 h. The in vitro stimulatory effect of estradiol on rat pineal methoxyindole synthesis was blocked by propranolol but not by phentolamine; propranolol also blocked the increase of nuclear estradiol-receptor complex produced by estrogen exposure of pineal explants. TSH (1-100 ng/ml), growth hormone (10-100 ng/ml) and LH (10 ng/ml) augmented rat pineal melatonin content while 100 ng/ml of FSH decreased it significantly. Prolactin exerted a biphasic effect on rat pineal explants, the lowest concentration augmenting melatonin content while the high concentration depressed it. Deep, intermediate and superficial segments of guinea-pig pineal glands showed an increase in melatonin concentration after a 6-h incubation in the presence of 10(-7)-10(-5) M estradiol.     

DHEA-sulfate causes a phase-dependent increase in melatonin secretion: a study of perifused rat pineal glands

Steroid hormones affect various metabolic activities, including melatonin synthesis, in mammals and nonmammals. We report here the effects of dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S), two steroids with weak androgen potency, on the levels of isoproterenol-stimulated melatonin released by perifused rat pineal glands removed in the middle of the light and dark spans [7 and 19 Hours After Light Onset (HALO), respectively] in a L/D 12:12 regimen. DHEA-S but not DHEA was found to have a direct action on beta-adrenergic-stimulated melatonin release. DHEA-S increased melatonin secretion (by 50-80%) dose-dependently in pineals obtained during the light span. This effect depended on the circadian stage, because at night (19 HALO), only the highest concentration (10(-3) M) of DHEA-S increased melatonin secretion (by 25%). In contrast, DHEA had no effect on melatonin release in pineals obtained during the light span. This work shows that DHEA-S but not DHEA was able to stimulate melatonin secretion by adrenergic-stimulated pineals removed during the light phase. It also suggests that the effects observed, or their intensity, or both depend on the circadian stage.     

Cannabinoids attenuate norepinephrine-induced melatonin biosynthesis in the rat pineal gland by reducing arylalkylamine N-acetyltransferase activity without involvement of cannabinoid receptors

Cannabinoids modulate neuronal and neuroendocrine circuits by binding to cannabinoid receptors acting upon cAMP/Ca(2+)-mediated intracellular signaling cascades. The rat pineal represents an established model to investigate intracellular signaling processes because a well defined input, the neurotransmitter norepinephrine, is transformed via cAMP/Ca(2+)-dependent mechanisms into an easily detectable output signal, the biosynthesis of melatonin. Here we investigated the impact of cannabinoids on norepinephrine-regulated melatonin biosynthesis in the rat pineal. We demonstrated that treatment of cultured rat pineals with 9-carboxy-11-nor-delta-9-tetrahydrocannabinol (THC), cannabidiol or cannabinol significantly reduced norepinephrine-induced arylalkylamine N-acetyltransferase (AANAT) activity and melatonin biosynthesis. These effects were not mimicked by the cannabinoid receptor agonist WIN55,212-2 and were not blocked by cannabinoid 1 and 2 receptor antagonists. The cannabinoids used did not affect norepinephrine-induced increases in cAMP/Ca(2+) levels. Notably, cannabinoids were found to directly inhibit AANAT activity in lysates of the pineal gland. This effect was specific in so far as cannabinoids did not influence the activity of hydroxyindole-O-methyltransferase (HIOMT), the last enzyme in melatonin biosynthesis. Taken together, our data strongly suggest that cannabinoids inhibit AANAT activity and attenuate melatonin biosynthesis through intracellular actions without involvement of classical cannabinoid receptor-dependent signaling cascades.     

Neonatal Hormone Treatment and Maturation of the Pineal Noradrenergic System: Hydrocortisone and Thyroxine

Abstract The circadian release of norepinephrine from nerve terminals in the pineal gland drives acetyl-CoA:serotonin N -acetyltransferase (NAT; EC 2.3.1.5) activity in the adult pineal from a daytime low to a nighttime high. In the newborn, enzyme activity is intermediate between the adult's daily extremes and has only a small circadian fluctuation. With age, these fluctuations increase in amplitude until the adult pattern is attained at about days 10–12. Treatment of neonates with thyroxine for the first 3 days of life accelerated, whereas administration of hydrocortisone acetate at birth retarded the developmental decline in daytime serotonin-N-acetyltransferase activity. Maximal differences in daytime enzyme activity of controls and thyroxine-treated animals were seen at day 4 and between controls and steroid-treated pups at day 8. Desipramine treatment increased NAT activity in 8-day-old animals; hydrocortisone-treated animals were least affected. Freshly cultured pineals from steroid-treated animals were more responsive to low, and less responsive to high, concentrations of norepinephrine than glands from thyroxine-treated or control animals. They were also less responsive to isoproterenol both in acute and 48-h organ culture. Pineals from hydrocortisone-treated animals in culture accumulated less exogeneous norepinephrine than glands from controls but released a greater fraction of their content on transfer to fresh medium. Normal and steroid-treated animals released the same fraction of their norepinephrine contents into the medium when reuptake was blocked by desipramine (DMI).     

Pineal sensitivity to nighttime swimming stress changes during the active season in Richardson's ground squirrels (Spermophilus richardsonii)

Melatonin synthesis in the pineal gland, which is primarily regulated by the environmental lighting regime, can also be influenced by other factors that elicit modifications in sympathetic tone. The objectives of this study were to determine if forced swimming alters the normal pattern of melatonin production in the pineal gland of the Richardson's ground squirrel (Spermophilus richardsonii). In early June, the squirrels were forced to swim for 10 min during the photophase or during the scotophase. In mid-July squirrels swam only during the scotophase. Animals were sacrificed 15, 30, or 60 min after the onset of swimming. Activities of pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) were assessed by radioenzyme assay, and pineal melatonin content was measured by radioimmunoassay. Daytime swimming elicited no major changes in enzyme activity or pineal melatonin. In June, swimming at night prevented the normal rises in NAT activity and pineal melatonin seen in nonswimming controls. In contrast, the pineals of squirrels that were tested 6 weeks later in mid-July did not appear to be as sensitive to nighttime swimming, as there were only minor differences in both NAT activity and melatonin content compared to controls. These results demonstrate that forced nighttime swimming, unlike several other aversive stimuli, can evoke changes in the normal pattern of pineal melatonin production in this species. Furthermore, the pineal's response to such stimuli may not be stable over the course of the active season.     

Circadian Rhythm in Pineal N-Acetyltransferase Activity: Rapid Phase Reversal and Response to Shorter than 24-Hour Cycles (IV)

N-Acetyltransferase (NAT) is an enzyme whose rhythmic activity in the pineal gland and retina is responsible for circadian rhythms in melatonin. The NAT activity rhythm has circadian properties such as persistence in constant conditions and precise control by light and dark. Experiments are reported in which chicks (Gallus domesticus), raised for 3 weeks in 12 h of light alternating with 12 h of dark (LD12:12), were exposed to 1-3 days of light-dark treatments during which NAT activity was measured in their pineal glands. (a) In LD12:12, NAT activity rose from less than 4.5 nmol/pineal gland/h during the light-time to 25-50 nmol/pineal gland/h in the dark-time. Constant light (LL) attenuated the amplitude of the NAT activity rhythm to 26-45% of the NAT activity cycle in LD12:12 during the first 24 h. (b) The timing of the increase in NAT activity was reset by the first full LD12:12 cycle following a 12-h phase shift of the LD12:12 cycle (a procedure that reversed the times of light and dark by imposition of either 24 h of light or dark). This result satisfies one of the criteria for NAT to be considered part of a circadian driving oscillator. (c) In less than 24-h cycles [2 h of light in alternation with 2 h of dark (LD2:2), 4 h of light in alternation with 4 h of dark (LD4:4), and 6 h of light in alternation with 6 h of dark (LD6:6)], NAT activity rose in the dark during the chicks' previously scheduled dark-time but not the previously scheduled light-time of LD12:12. In a cycle where 8 h of light alternated with 8 h of dark (LD8:8), NAT activity rose in both 8-h dark periods, even though the second one fell in the light-time of the prior LD12:12 schedule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of near-ultraviolet (UV-A) light on melatonin biosynthesis in vertebrate pineal gland

The effects of near-ultraviolet (UV-A) irradiation on nocturnal activity of serotonin N-acetyltransferase (NAT; a key regulatory enzyme in melatonin biosynthesis) in the pineal gland of the rat and chick were investigated. Exposure of the animals to UV-A during the 4th or 5th hour of the dark phase of the 12:12 h light-dark (LD) cycle suppressed the night-driven NAT activity in a time-dependent manner, the effects being generally more pronounced in rats than in chicks. The UV-A-evoked suppression of the nocturnal NAT activity was completely restored within 2 h (chicks) or 3 h (rats) in animals which, after irradiation, were returned to darkness. When a short UV-A pulse was applied to the animals after midnight, it induced a decrease in the enzyme activity in both species; yet, the effect was readily reversible only in chicks. The results presented here, as well as other data, demonstrate that UV-A light is a powerful signal affecting the pineal melatonin-generating system both in mammals and avians, and that the involved mechanisms may differ in the tested species.     

N-Acetylation of Serotonin Is Correlated with α2- but Not with β-Adrenergic Regulation of Cyclic AMP Levels in Cultured Chick Pineal Cells

We investigated the role of cyclic AMP (cAMP) in alpha 2- and possible beta-adrenergic regulation of arylalkylamine-N-acetyltransferase (NAT), the penultimate enzyme in the biosynthesis of melatonin. The study was performed on primary cultures of dispersed chick pineal cells. Electron microscopy indicated that approximately 70% of the dispersed cells were modified photoreceptors. A similar proportion of melatoninergic cells was detected by immunocytochemical labeling of hydroxyindole-O-methyltransferase, the final enzyme in the biosynthesis of melatonin. Adrenergic agonists caused a sustained 50% inhibition of forskolin-augmented cAMP levels and NAT activity, with an alpha 2-adrenergic potency order of UK 14,304 greater than or equal to clonidine greater than norepinephrine greater than phenylephrine. Noradrenergic inhibition of 3-isobutyl-1-methylxanthine-augmented cAMP levels and NAT activity was reversed by yohimbine (an alpha 2-adrenergic antagonist) but not by prazosin (an alpha 1-adrenergic antagonist). The alpha-adrenergic inhibition of cAMP accumulation and NAT activity was prevented by pertussis toxin. Addition of propranolol (a beta-adrenergic antagonist) was necessary to observe an inhibitory effect of norepinephrine on cAMP levels but not on NAT activity. Similarly, the beta-adrenergic agonist isoproterenol transiently increased cAMP levels but did not affect NAT activity. The data indicate that the alpha 2-adrenergic inhibition of NAT activity in chick pineal cells is strongly correlated with an inhibition of cAMP accumulation. The lack of beta-adrenergic effect on NAT suggests that beta-adrenoceptors might be on a subset of cells that do not produce melatonin or that the beta-adrenergic-induced increase in cAMP levels is too transient to affect NAT.     

Serotonin N-acetyltransferase (NAT) induction in mammalian retina: role of cyclic AMP and calcium ions.

In retinas and pineal glands of rat, rabbit and hen, activities of the penultimate (and key regulatory) enzyme in melatonin biosynthesis, serotonin N-acetyltransferase (NAT), display distinct diurnal variations, with high and low values during dark and light phase of a 12-h dark: 12-h light illumination cycle. Two-hour incubation (during daytime hours in light) of isolated pineal glands of the studied vertebrates, or the retinas, with 50 microM forskolin (plus 100 microM 3-isobutyl-1-methylxanthine, IBMX-a phosphodiesterase inhibitor), and 1 mM dibutyryl-cAMP, markedly increased the tissue NAT activity. The same procedures significantly enhanced the enzyme activity of rat retina in light, however, only during nighttime hours. The forskolin (+ IBMX)-induced increase of NAT activity in rat retina was significantly lower in a calcium-free medium, and substantially enhanced when calcium concentration was raised from 1.3 mM to 3.9 mM. Treatment of rats with IBMX or aminophylline, and rabbits with aminophylline, increased NAT activity in their pineal glands irrespective of the time of the day, whereas both phosphodiesterase inhibitors significantly increased the enzyme activity of rat retina only when injected during the subjective dark hours. It is concluded that, by analogy to vertebrate pineal gland, in vertebrate retina an increase of NAT activity (and consequently melatonin formation), stimulated both physiologically (i. e. at night), or pharmacologically, involves a cAMP- and calcium dependent process of the enzyme induction.