In vitro effect of cholinergic agonists and antagonists, activators and blockers of voltage-sensitive Ca++-channels and Na+-channels on arylalkylamine N-acetyltransferase 2 mRNA levels in pineal organ of the catfish, Clarias gariepinus

Abstract

The fish pineal organ is innervated by cholinergic nerve fibres. But there is no information on cholinergic regulation of aanat2 gene in the fish pineal organ. Therefore, we investigated in vitro effect of specific agonists and antagonists of cholinergic receptors on aanat2 mRNA levels in the pineal organ of catfish Clarias gariepinus. Acclimatized mature male fish was decapitated and the pineal organ was rapidly removed and placed in culture medium and pre-incubated for 1h. After pre-incubation, the culture medium was replaced with the culture medium containing desired concentrations of specific ago-nists and antagonists of cholinergic receptors, blockers and activators of volt-age-sensitive Ca++ channels and Na+ channels for 1h, after which the pineal organs were collected and processed for measuring relative expression of aanat2 gene by measuring AANAT2 mRNA with the help of Real Time RT-PCR. Acetylcholine and nicotine produced dose-dependent inhibitory and stimulatory effect on aanat2 transcript levels, while carbachol increased aa-nat2 transcript levels in a dose-dependent manner. In vitro treatment of the pineal organ with atropine, hexamethonium, the blockers of voltage-sensitive Ca++ channels and Na+ channels significantly blocked the stimulato-ry effect of carbachol and nicotine on aanat2 transcript levels, respectively. Based on these findings, we suggest that cholinergic mechanism plays an important and direct role in regulation of aanat2 gene expression in the fish pineal organ, and stimulates the gene transcription via both muscarinic and nicotinic receptors. Further, muscarinic and nicotinic receptors seem to in-crease aanat2 transcript levels by affecting voltage-sensitive Ca++ channels and Na+ channels, respectively.