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

Abstract

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: [email protected])

Keywords:

• AANAT
• Adrenergic
• Carp
• Cholinergic
• Dopaminergic
• Melatonin
• Pineal

ACKNOWLEDGMENTS

The authors are thankful to the Department of Science and Technology, (SR/SO/AS/72/2006) and the Department of Biotechnology, Government of India (BT/PR11423/AAQ/03/421/2008) for financial assistance and to Dr. Asamanja Chattoraj for his technical support. The authors are also grateful to three anonymous reviewers who have provided a thorough critique, with clear suggestions for adequately improving the earlier version of the manuscript.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.