DAILY VARIATION OF CONSTITUTIVELY ACTIVATED NUCLEAR FACTOR KAPPA B (NFKB) IN RAT PINEAL GLAND

Abstract

In mammals, the production of melatonin by the pineal gland is mainly controlled by the suprachiasmatic nuclei (SCN), the master clock of the circadian system. We have previously shown that agents involved in inflammatory responses, such as cytokines and corticosterone, modulate pineal melatonin synthesis. The nuclear transcription factor NFKB, detected by our group in the rat pineal gland, modulates this effect. Here, we evaluated a putative constitutive role for the pineal gland NFKB pathway. Male rats were kept under 12 h:12 h light-dark (LD) cycle or under constant darkness (DD) condition. Nuclear NFKB was quantified by electrophoretic mobility shift assay on pineal glands obtained from animals killed throughout the day at different times. Nuclear content of NFKB presented a daily rhythm only in LD-entrained animals. During the light phase, the amount of NFKB increased continuously, and a sharp drop occurred when lights were turned off. Animals maintained in a constant light environment until ZT 18 showed diurnal levels of nuclear NFKB at ZT15 and ZT18. Propranolol (20 mg/kg, i.p., ZT 11) treatment, which inhibits nocturnal sympathetic input, impaired nocturnal decrease of NFKB only at ZT18. A similar effect was observed in free-running animals, which secreted less nocturnal melatonin. Because melatonin reduces constitutive NFKB activation in cultured pineal glands, we propose that this indolamine regulates this transcription factor pathway in the rat pineal gland, but not at the LD transition. The controversial results regarding the inhibition of pineal function by constant light or blocking sympathetic neurotransmission are discussed according to the hypothesis that the prompt effect of lights-off is not mediated by noradrenaline, which otherwise contributes to maintaining low levels of nuclear NFKB at night. In summary, we report here a novel transcription factor in the pineal gland, which exhibits a constitutive rhythm dependent on environmental photic information. (Author correspondence: [email protected])

Keywords:

• Circadian rhythm
• Melatonin
• Propranolol
• Nuclear factor kappa B
• Pineal gland

ACKNOWLEDGMENTS

We are indebted to Dr. Maria Christina Werneck de Avellar, Section of Experimental Pharmacology, Department of Pharmacology, Federal University of São Paulo for all of the assistance in setting up EMSA. We also acknowledge Dr. Maria Ines Nogueira from the Department of Anatomy, Institute of Biomedical Science of the University of São Paulo for kindly allowing the use of the rest/activity rhythm detection apparatus. The invaluable help of the graduate student Vanderlei Amadeu da Rocha, and the constant technical assistance of Debora Aparecida Moura were essential for developing this work. RPM is a senior fellow of CNPq, LP and PF are post-doc fellows, and EC is graduate fellow of FAPESP.