In vitro effects of 5-hydroxytryptophan, indoleamines and leptin on arylalkylamine N-acetyltransferase (AA-NAT) activity in pineal organ of the fish, Clarias gariepinus (Burchell, 1822) during different phases of the breeding cycle

Arylalkylamine N-acetyltransferase (AA-NAT) is the rate-limiting enzyme of melatonin biosynthetic pathway. In vitro effects of 5-hydroxytryptophan (5-HTP) and indoleamines (serotonin, N-acetylserotonin and melatonin) were studied on AA-NAT activity in the pineal organ of the fish, C. gariepinus during different phases of its annual breeding cycle. Further, in vitro effects of leptin on AA-NAT activity in the pineal organ were studied in fed and fasted fishes during summer and winter seasons. Treatments with 5-HTP and indoleamines invariably stimulated pineal AA-NAT activity in a dose-dependent manner during all the phases. However, leptin increased AA-NAT activity in a dose-dependent manner only in the pineal organ of the fed fishes, but not of the fasted fishes irrespective of the seasons.     

Rhythm and soul in the avian pineal

The avian pineal gland, like that of mammals, displays a striking circadian rhythm in the synthesis and release of the hormone melatonin. However, the pineal gland plays a more prominent role in avian circadian organization and differs from that in mammals in several ways. One important difference is that the pineal gland in birds is relatively autonomous. In addition to making melatonin, the avian pineal contains photoreceptors and a circadian clock (thus, an entire circadian system) within itself. Furthermore, avian pineals retain their circadian properties in organ or dispersed cell culture, making biochemical components of regulatory pathways accessible. Avian pinealocytes are directly photosensitive, and novel candidates for the unidentified photopigments involved in the regulation of clock function and melatonin production, including melanopsin, pinopsin, iodopsin, and the cryptochromes, are being evaluated. Transduction pathways and second messengers that may be involved in acute and entraining effects, including cyclic nucleotides, calcium fluxes, and protein kinases, have been, and continue to be, examined. Moreover, several clock genes similar to those found in Drosophila and mouse are expressed, and their dynamics and interactions are being studied. Finally, the bases for acute and clock regulation of the key enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AA-NAT), are described. The ability to study entrainment, the oscillator itself, and a physiological output in the same tissue at the same time makes the avian pineal gland an excellent model to study the bases and regulation of circadian rhythms.     

Chick pineal melatonin synthesis: light and cyclic AMP control abundance of serotonin N-acetyltransferase protein

Melatonin production in the pineal gland is high at night and low during the day. This rhythm reflects circadian changes in the activity of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87], the penultimate enzyme in melatonin synthesis. The rhythm is generated by an endogenous circadian clock. In the chick, a clock is located in the pinealocyte, which also contains two phototransduction systems. One controls melatonin production by adjusting the clock and the other acts distal to the clock, via cyclic AMP mechanisms, to switch melatonin synthesis on and off. Unlike the clock in these cells, cyclic AMP does not appear to regulate activity by altering AA-NAT mRNA levels. The major changes in AA-NAT mRNA levels induced by the clock seemed likely (but not certain) to generate comparable changes in AA-NAT protein levels and AA-NAT activity. Cyclic AMP might also regulate AA-NAT activity via changes in protein levels, or it might act via other mechanisms, including posttranslational changes affecting activity. We measured AA-NAT protein levels and enzyme activity in cultured chick pineal cells and found that they correlated well under all conditions. They rose and fell spontaneously with a circadian rhythm. They also rose in response to agents that increase cyclic AMP. They were raised by agents that increase cyclic AMP, such as forskolin, and lowered by agents that decrease cyclic AMP, such as light and norepinephrine. Thus, both the clock and cyclic AMP can control AA-NAT activity by altering the total amount of AA-NAT protein. Effects of proteosomal proteolysis inhibitors suggest that changes in AA-NAT protein levels, in turn, reflect changes in the rate at which the protein is destroyed by proteosomal proteolysis. It is likely that cyclic AMP-induced changes in AA-NAT protein levels mediate rapid changes in chick pineal AA-NAT activity. Our results indicate that light can rapidly regulate the abundance of a specific protein (AA-NAT) within a photoreceptive cell.     

Turkey retina and pineal gland differentially respond to constant environment

Dynamics of rhythmic oscillations in the activity of arylalkylamine N-acetyltransferase (AA-NAT, the penultimate and key regulatory enzyme in melatonin biosynthesis) were examined in the retina and pineal gland of turkeys maintained for 7 days in the environment without daily light-dark (LD) changes, namely constant darkness (DD) or continuous light (LL). The two tissues differentially responded to constant environment. In the retina, a circadian AA-NAT activity rhythm disappeared after 5 days of DD, while in the pineal gland it persisted for the whole experiment. No circadian rhythm was observed in the retinas of turkeys exposed to LL, although rhythmic oscillations in both AA-NAT and melatonin content were found in the pineal glands. Both tissues required one or two cycles of the re-installed LD for the full recovery of the high-amplitude AA-NAT rhythm suppressed under constant conditions. It is suggested that the retina of turkey is less able to maintain rhythmicity in constant environment and is more sensitive to changes in the environmental lighting conditions than the pineal gland. Our results indicate that, in contrast to mammals, pineal glands of light-exposed galliformes maintain the limited capacity to rhythmically produce melatonin.     

Circadian rhythms of indoleamines and serotonin N-acetyltransferase activity in the pineal gland

Conclusion The circadian rhythm of melatonin synthesis in the pineal glands of various species has been summarized. The night-time elevation of melatonin content is in most if not all cases regulated by the change of N-acetyltransferase activity. In mammals, the N-acetyltransferase rhythm is controlled by the central nervous system, presumably by suprachiasmatic nuclei in hypothalamus through the superior cervical ganglion. In birds, the circadian oscillator that regulates the N-acetyltransferase rhythm is located in the pineal glands. The avian pineal gland may play a biological clock function to control the circadian rhythms in physiological, endocrinological and biochemical processes via pineal hormone melatonin.     

Changes in the electrophoretic mobility of cells from chick blastoderms during early development

A change in cell surface charge density during early avian development is shown with a free-flow electrophoresis apparatus. The blastoderms of freshly laid eggs consist of two electrophoretically distinct cell populations. After the onset of gastrulation a third cell population with an intermediate electrophoretic mobility appears. With increasing time of incubation there is a shift in the proportion of these populations and an increase in the mobility of the fastest fraction.     

Effect of lithium on the melatonin production in the pineal gland of viscacha

Melatonin is synthesized primarily in the pineal gland. Lithium affects the circadian rhythms that may explain its therapeutic effectiveness in the treatment of bipolar disorder. The objective of this study was to investigate the effect of lithium on the biochemical parameters involved in melatonin synthesis in the pineal gland of viscacha. Viscachas were daily intraperitoneally injected with lithium chloride or saline solution for one month. Pineal mRNAs encoding β₁-adrenoceptor and arylalkylamine-N-acetyltransferase enzyme (AA-NAT) were studied by in situ hybridization. Pineal melatonin concentrations were determined by radioimmunoassay, and AA-NAT and hydroxyindol-O-methyltransferase (HIOMT) activities were investigated by radiometric assays. The only parameters that decreased significantly were the expression of AA-NAT mRNA and pineal melatonin levels. Our data suggest that lithium treatment may decrease melatonin synthesis in the viscacha pineal gland by a complex mechanism that involves currently unknown events that are beyond a decrease in the expression of AA-NAT enzyme.     

In vitro effects of steroid hormones on arylalkylamine N-acetyltransferase (AA-NAT) activity in the pineal of fish, Clarias gariepinus (Burchell, 1822) during different phases of breeding cycle

In vitro effects of gonadal hormones (testosterone, 17beta-estradiol estriol and estrone) and corticosteroid hormones (corticosterone and cortisol) were studied on arylalklyamine N-acetyltransferase (AA-NAT) activity in the pineal organ of the fish, C. gariepinus during quiescent, progressive, breeding and regressive phases of its annual breeding cycle. The pineals were collected under dim red light, maintained in organ culture for 7 hr and incubated with three concentrations (10(-6), 10(-5) and 10(-4) M) of hormones for 6 hr. The treatments with gonadal hormones and corticosteroid hormones inhibited pineal AA-NAT activity in a dose-dependent manner during all the phases of the breeding cycle. AA-NAT activity was comparatively more sensitive to the inhibitory effects of the gonadal hormones during the regressive phase and less sensitive during the quiescent phase. Further, the enzyme activity was more sensitive to the inhibitory effects of corticosteroid hormones (corticosterone and cortisol) during the breeding phase and less sensitive during the quiescent phase. These findings seem to suggest that gonadal hormones and corticosteroid hormones have direct inhibitory influence on AA-NAT activity and, hence melatonin synthesis in the photoreceptive pineal organ of C. gariepinus.     

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.     

Mitogen-activated protein kinase phosphatase-1 (MKP-1): >100-fold nocturnal and norepinephrine-induced changes in the rat pineal gland

The norepinephrine-driven increase in mitogen-activated protein kinase (MAPK) activity is part of the mechanism that regulates arylalkylamine N-acetyltransferase (AA-NAT) activity in the rat pineal gland. We now report a marked nocturnal increase in the expression of a MAPK phosphatase, MAP kinase phosphatase-1 (MKP-1), that was blocked by maintaining animals in constant light or treatment with propranolol. MKP-1 expression was regulated by norepinephrine acting through both alpha- and beta-adrenergic receptors. These results establish a nocturnal increase in pineal MKP-1 expression that is under the control of a photoneural system. Because substrates of MKP-1 can influence AA-NAT activity, our findings suggest the involvement of MKP-1 in the regulation of the nocturnal AA-NAT signal.