Norepinephrine activates NF-κB transcription factor in cultured rat pineal gland.
OBJECTIVE
The circadian rhythm in mammalian pineal melatonin secretion is modulated by norepinephrine (NE) released at night. NE interaction with β1-adrenoceptors activates PKA that phosphorylates the transcription factor CREB, leading to the transcription and translation of the arylalkylamine-N-acetyltransferase (AANAT) enzyme. Several studies have reported the interplay between CREB and the nuclear factor-κB (NF-κB) and a circadian rhythm for this transcription factor was recently described in the rat pineal gland. In this work we studied a direct effect of NE on NF-κB activation and the role played by this factor on melatonin synthesis and Aanat transcription and activity.


METHODS
Cultured rat pineal glands were incubated in the presence of two different NF-κB inhibitors, pyrrolidine-dithiocarbamate or sodium salicylate, and stimulated with NE. Melatonin content was quantified by HPLC with electrochemical detection. AANAT activity was measured by a radiometric assay and the expression of Aanat mRNA was analyzed by real-time PCR. Gel shift assay was performed to study the NF-κB activation in cultured rat pineal glands stimulated by NE.


RESULTS
Our results showed that the p50/p50 homodimer of NF-κB is activated by NE and that it has a role in melatonin synthesis, acting on Aanat transcription and activity.


CONCLUSIONS
Here we present evidence that NF-κB is an important transcription factor that acts, directly or indirectly, on Aanat transcription and activity leading to a modulation of melatonin synthesis. NE plays a role in the translocation of NF-κB p50/p50 homodimer to the nucleus of pinealocytes, thus probably influencing the nocturnal pineal melatonin synthesis.
Laboratory of Pharmacology, Butantan Institute, São Paulo, SP, Brazil; Department of Physiology and Biophysics, Institute of Biomedical Science, University of São Paulo, São Paulo, SP, Brazil.
Darine
D
Villela
Molecular Neuropharmacology Laboratory, Department of Pharmacology, Institute of Biomedical Science, São Paulo, SP, Brazil.
Larissa
L
de Sá Lima
Department of Physiology and Biophysics, Institute of Biomedical Science, University of São Paulo, São Paulo, SP, Brazil.
Rafael
R
Peres
Department of Physiology and Biophysics, Institute of Biomedical Science, University of São Paulo, São Paulo, SP, Brazil.
Rodrigo Antonio
RA
Peliciari-Garcia
Department of Physiology and Biophysics, Institute of Biomedical Science, University of São Paulo, São Paulo, SP, Brazil.
Fernanda Gaspar
FG
do Amaral
Department of Physiology and Biophysics, Institute of Biomedical Science, University of São Paulo, São Paulo, SP, Brazil.
José
J
Cipolla-Neto
Molecular Neuropharmacology Laboratory, Department of Pharmacology, Institute of Biomedical Science, São Paulo, SP, Brazil.
Cristóforo
C
Scavone
Laboratory of Pharmacology, Butantan Institute, São Paulo, SP, Brazil. Electronic address: solange.afeche@butantan.gov.br.
Solange Castro
SC
Afeche
D000818:Animals; D047068:Arylalkylamine N-Acetyltransferase / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology; D017362:Cyclic AMP Response Element-Binding Protein / Q000096:biosynthesis / Q000502:physiology; D024202:Electrophoretic Mobility Shift Assay; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D008297:Male; D008550:Melatonin / Q000096:biosynthesis / Q000502:physiology; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000502:physiology; D009638:Norepinephrine / Q000494:pharmacology*; D009924:Organ Culture Techniques; D010870:Pineal Gland / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D011759:Pyrrolidines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction; D012980:Sodium Salicylate / Q000494:pharmacology; D013859:Thiocarbamates / Q000494:pharmacology
D017362:Cyclic AMP Response Element-Binding Protein; D016328:NF-kappa B; D011759:Pyrrolidines; D013859:Thiocarbamates; C020972:pyrrolidine dithiocarbamic acid; D047068:Arylalkylamine N-Acetyltransferase; D008550:Melatonin; D012980:Sodium Salicylate; D009638:Norepinephrine
AANAT activity; Aanat transcription; Melatonin; NF-κB; Norepinephrine; Pineal gland