Papel da temporização noradrenérgica na regulação da síntese de melatonina pela glândula pineal em cultura: características funcionais e mecanismos de ação.

Abstract

Silva JA. Role of norepinephrine synchronization on melatonin synthesis regulation of pineal gland culture: function and action mechanisms. [Masters thesis (Human Physiology)]. Sao Paulo: Instituto de Ciencias Biomedicas, Universidade de Sao Paulo; 2013. Melatonin, a hormone synthesized by the pineal gland on the dark phase, shows a circadian and seasonal rhythm of secretion. Since the mammal’s pineal gland is not an autonomous oscillator, the melatonin production requires the release of norepinephrine (NE) on the dark phase. The circadian gene expression in pineal gland is sustained by clock genes. In the standard pineal culture, the gland is extracted and maintained on culture for 48 hours prior to treatment with NE. Under this condition, the glands do not express any functional rhythmic variation. To mimic the physiological pattern of NE release in the pineal gland culture, we develop a synchronized culture with NE. The aim of this study was to investigate the maintenance of circadian clock genes expression within rat pineal gland under acute and NEsynchronized culture. Also we have investigated by which noradrenergic pathway this maintenance occurs. For the in vivo experiments, the animals were sacrificed of a circadian way. For the in vitro, the rats were sacrificed on the transition of light to dark phase, and the glands were submitted to the culture for 72 h under conditions: control (without NE), acute (NE 10 -6 for 12h after 48h), synchronized (since the begin of the culture: 12h presence/12h absence of NE), synchronized with Prasozin (a α1 blocker), synchronized with Propranolol (a β blocker) and synchronized with both drugs. In the last 24h of culture, the glands and medium were collected every 3h. The mRNA expressions of Aanat, Bmal1, Cry1, Cry2, Dbp, Per1, Per2 and Rev-erbα were investigated by qPCR, as well as the AANAT activity and melatonin content by UHPLC method. All genes analyzed showed circadian rhythm expression in vivo. However, under in vitro, control and acute condition, every rhythm observed before were abolished. The presence of circadian rhythmicity, despite the differences of acrofase, was found in all genes in the synchronized group. Further, the synchronization was also able to improve the peak duration of the AANAT activity, as well as increase the melatonin content. The prasozin addition in the synchronized culture reduced the gene expression, and in some cases the rhythm was abolished. On the other hand, the propranolol addition, combined or not with prasozin, abolished every rhythmic expression variation in analyzed genes. Thus, the synchronization was able to maintain the circadian clock expression in the pineal gland, improving the melatonin synthesis. Also, the synchronized norepinephrine acts by β receptor, and the α1 receptor pathway potentiates this. In conclusion, the synchronized culture method showed itself as a useful approach to avoid the disruption of rhythmic variations that are present in the standard culture.