Neural regulation of dark-induced abundance of arylalkylamine N-acetyltransferase (AANAT) and melatonin in the carp (Catla catla) pineal: an in vitro study

In all the vertebrates, synthesis of melatonin and its rhythm-generating enzyme arylalkylamine N-acetyltransferase (AANAT) reaches its peak in the pineal during the night in a daily light-dark cycle, but the role of different neuronal signals in their regulation were unknown for any fish. Hence, the authors used specific agonist and antagonists of receptors for different neuronal signals and regulators of intracellular calcium (Ca(2+)) and adenosine 3',5'-cyclic monophosphate (cAMP) in vitro to study their effects on the abundance of AANAT and titer of melatonin in the carp (Catla catla) pineal. Western blot analysis followed by quantitative analysis of respective immunoblot data for AANAT protein, radioimmunoassay of melatonin, and spectrophotometric analysis of Ca(2+) in the pineal revealed stimulatory effects of both adrenergic (α(1) and β(1)) and dopaminergic (D(1)) agonists and cholinergic (both nicotinic and muscarinic) antagonists, inhibition by both adrenergic and dopaminergic antagonists and cholinergic agonists, but independent of the influence of any agonists or antagonists of α(2)-adrenergic receptors. Band intensity of AANAT and concentration of melatonin in the pineal were also enhanced by the intracellular calcium-releasing agent, activators of both calcium channel and adenylate cyclase, and phophodiesterase inhibitor, but suppressed by inhibitor of calcium channel and adenylate cyclase as well as activator of phophodiesterase. Moreover, an inhibitory effect of light on the pineal AANAT and melatonin was blocked by both cAMP and proteasomal proteolysis inhibitor MG132. Collectively, these data suggest that dark-induced abundance of AANAT and melatonin synthesis in the carp pineal are a multineuronal function, in which both adrenergic (α(1) and β(1), but not α(2)) and dopaminergic signals are stimulatory, whereas cholinergic signals are inhibitory. This study also provides indications, though arguably not conclusive evidence, that in either case the neuronal mechanisms follow a signal-transduction pathway in which Ca(2+) and cAMP may act as the intracellular messengers. It also appears that proteasomal proteolysis is a conserved event in the regulation of AANAT activity in vertebrates.     

Ability of tryptophan derivatives to mimic melatonin's action upon the Syrian hamster reproductive system

The ability of 18 different tryptophan derivatives to induce gonadal regression in Syrian hamsters when injected daily at either midday or late evening has been examined. The compounds chosen have either been identified within mammalian pineal glands or are thought to be possible metabolic derivatives of melatonin. Of the compounds tested, only melatonin and 5-methoxytryptamine were found to possess antigonadotropic activity. 5-Methoxytryptamine's potency, however, was 1/10th that of melatonin and, like melatonin, 5-methoxytryptamine was effective when injected in the evening but not when injected during midday. In addition to the general survey of tryptophan derivatives for antigonadal capability when injected, 5-methoxytryptamine, melatonin, and 5-methoxytryptophol were compared relative to their abilities to prevent photo-induced gonadal regression when administered within beeswax implants. Again 5-methoxytryptamine and melatonin, but not 5-methoxytryptophol, were effective with 5-methoxytryptamine's potency being less than that of melatonin. These results indirectly support the contention that melatonin is the pineal product which mediates photoperiodic effects upon the Syrian hamster reproductive system.     

Nocturnal increase in the sensitivity of the Syrian hamster pineal gland to isoproterenol is darkness dependent

The sensitivity of the Syrian hamster pineal gland to stimulation by isoproterenol is greatly increased in the latter half of the daily dark phase. This increased sensitivity requires a period of dark exposure for up to 6.5 hr. Also, if dark-maintained hamsters are exposed to light in the latter half of the night pineal melatonin levels drop precipitously but can be restimulated by isoproterenol administration. As the interval of light exposure continues, however, the pineal sensitivity to isoproterenol decreases.     

Neural Regulation of Dark-Induced Abundance of Arylalkylamine N-Acetyltransferase (AANAT) and Melatonin in the Carp (Catla catla) Pineal: An In Vitro Study

In all the vertebrates, synthesis of melatonin and its rhythm-generating enzyme arylalkylamine N-acetyltransferase (AANAT) reaches its peak in the pineal during the night in a daily light-dark cycle, but the role of different neuronal signals in their regulation were unknown for any fish. Hence, the authors used specific agonist and antagonists of receptors for different neuronal signals and regulators of intracellular calcium (Ca²⁺) and adenosine 3',5'-cyclic monophosphate (cAMP) in vitro to study their effects on the abundance of AANAT and titer of melatonin in the carp (Catla catla) pineal. Western blot analysis followed by quantitative analysis of respective immunoblot data for AANAT protein, radioimmunoassay of melatonin, and spectrophotometric analysis of Ca²⁺ in the pineal revealed stimulatory effects of both adrenergic (α₁ and β₁) and dopaminergic (D₁) agonists and cholinergic (both nicotinic and muscarinic) antagonists, inhibition by both adrenergic and dopaminergic antagonists and cholinergic agonists, but independent of the influence of any agonists or antagonists of α₂-adrenergic receptors. Band intensity of AANAT and concentration of melatonin in the pineal were also enhanced by the intracellular calcium-releasing agent, activators of both calcium channel and adenylate cyclase, and phophodiesterase inhibitor, but suppressed by inhibitor of calcium channel and adenylate cyclase as well as activator of phophodiesterase. Moreover, an inhibitory effect of light on the pineal AANAT and melatonin was blocked by both cAMP and proteasomal proteolysis inhibitor MG132. Collectively, these data suggest that dark-induced abundance of AANAT and melatonin synthesis in the carp pineal are a multineuronal function, in which both adrenergic (α₁ and β₁, but not α₂) and dopaminergic signals are stimulatory, whereas cholinergic signals are inhibitory. This study also provides indications, though arguably not conclusive evidence, that in either case the neuronal mechanisms follow a signal-transduction pathway in which Ca²⁺ and cAMP may act as the intracellular messengers. It also appears that proteasomal proteolysis is a conserved event in the regulation of AANAT activity in vertebrates. (Author correspondence: dgp_skmaitra@yahoo.co.in)     

Two arylalkylamine N-acetyltransferase genes mediate melatonin synthesis in fish

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT, EC 2.3.1.87) is the first enzyme in the conversion of serotonin to melatonin. Large changes in AANAT activity play an important role in the daily rhythms in melatonin production. Although a single AANAT gene has been found in mammals and the chicken, we have now identified two AANAT genes in fish. These genes are designated AANAT-1 and AANAT-2; all known AANATs belong to the AANAT-1 subfamily. Pike AANAT-1 is nearly exclusively expressed in the retina and AANAT-2 in the pineal gland. The abundance of each mRNA changes on a circadian basis, with retinal AANAT-1 mRNA peaking in late afternoon and pineal AANAT-2 mRNA peaking 6 h later. The pike AANAT-1 and AANAT-2 enzymes (66% identical amino acids) exhibit marked differences in their affinity for serotonin, relative affinity for indoleethylamines versus phenylethylamines and temperature-activity relationships. Two AANAT genes also exist in another fish, the trout. The evolution of two AANATs may represent a strategy to optimally meet tissue-related requirements for synthesis of melatonin: pineal melatonin serves an endocrine role and retinal melatonin plays a paracrine role.     

Signal transduction and regulation of melatonin synthesis in bovine pinealocytes: impact of adrenergic,peptidergic and cholinergic stimuli

Limited studies of the regulation of pineal melatonin biosynthesis in ungulates indicate that it differs considerably from that in rodents. Here we have investigated several signal transduction cascades and their impact on melatonin synthesis in bovine pinealocytes. Norepinephrine increased the intracellular calcium ion concentration ([Ca2+]i) via alpha(1)-adrenergic receptors. Activation of beta-adrenergic receptors enhanced cAMP accumulation and rapidly elevated arylalkylamine N-acetyltransferase (AANAT) activity and melatonin secretion. The beta-adrenergically evoked increases in AANAT activity were potentiated by alpha(1)-adrenergic stimulation, but this was not seen with cAMP or melatonin production. PACAP treatment caused small increases in cAMP, AANAT activity and melatonin biosynthesis, apparently in a subpopulation of cells. VIP and glutamate did not influence any of these parameters. Activation of nicotinic and muscarinic acetylcholine receptors increased [Ca2+]i, but did not alter cAMP levels, AANAT activity or melatonin production. Our study reveals that discrete differences in pineal signal transduction exist between the cow and rodent, and emphasizes the potential importance that the analysis of ungulate pinealocytes may play in understanding regulation of pineal melatonin biosynthesis in primates and man, whose melatonin-generating system appears to be more similar to that in ungulates than to that in rodents.     

Hormonal modulation of pineal melatonin synthesis in rats and Syrian hamsters: effects of streptozotocin-induced diabetes and insulin injections

Pineal levels of tryptophan, 5-hydroxytryptophan, serotonin, N-acetylserotonin, melatonin, 5-hydroxyindoleacetic acid and the enzyme activities of N-acetyltransferase and hydroxyindole-O-methyltransferase were determined in male albino rats and Syrian hamsters that were injected with insulin twice daily for three days, or injected with streptozotocin to induce diabetes. Neither insulin injections nor streptozotocin diabetes had any effect on pineal melatonin production in rats. In hamsters, diabetes reduced the nocturnal peak of pineal melatonin content by approximately one half, while insulin injections had no effect on pineal melatonin levels; however, insulin injections did cause a slight increase in pineal N-acetyltransferase activity. These findings indicate that the pineal gland of the hamster may be more sensitive to alterations in plasma insulin levels than the same organ in rats.