Tryptophan hydroxylase is modulated by L-type calcium channels in the rat pineal gland Roseli Barbosa a , Julieta Helena Scialfa b , Ilza Mingarini Terra b , José Cipolla-Neto b , Valérie Simonneaux c , Solange Castro Afeche a, ⁎ a Laboratory of Pharmacology, Butantan Institute, São Paulo, SP, Brazil b Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil c Neurobiologie des Rythmes, Institut de Neurosciences Cellulaires et Intégratives, CNRS/Université Louis Pasteur, Strasbourg, France Received 25 October 2007; accepted 12 December 2007 Abstract Calcium is an important second messenger in the rat pineal gland, as well as cAMP. They both contribute to melatonin synthesis mediated by the three main enzymes of the melatonin synthesis pathway: tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O- methyltransferase. The cytosolic calcium is elevated in pinealocytes following α 1 -adrenergic stimulation, through IP 3 -and membrane calcium channels activation. Nifedipine, an L-type calcium channel blocker, reduces melatonin synthesis in rat pineal glands in vitro. With the purpose of investigating the mechanisms involved in melatonin synthesis regulation by the L-type calcium channel, we studied the effects of nifedipine on noradrenergic stimulated cultured rat pineal glands. Tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O- methyltransferase activities were quantified by radiometric assays and 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin contents were quantified by HPLC with electrochemical detection. The data showed that calcium influx blockaded by nifedipine caused a decrease in tryptophan hydroxylase activity, but did not change either arylalkylamine N-acetyltransferase or hydroxyindole-O-methyltransferase activities. Moreover, there was a reduction of 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin intracellular content, as well as a reduction of serotonin and melatonin secretion. Thus, it seems that the calcium influx through L-type high voltage-activated calcium channels is essential for the full activation of tryptophan hydroxylase leading to melatonin synthesis in the pineal gland. © 2007 Elsevier Inc. All rights reserved. Keywords: L-type calcium channel; Melatonin; Arylalkylamine N-acetyltransferase; Tryptophan hydroxylase; Hydroxyindole-O-methyltransferase; Pineal gland Introduction Melatonin is the main hormone synthesized and released from the pineal gland following sympathetic stimulation during the night. Norepinephrine interacts with α-and β-adrenoceptors in the pinealocyte membranes and, consequently, elevates intracel- lular cAMP and calcium levels (Sugden et al., 1986; Sugden, 1989). These two second messengers participate in some way in the synthesis and activation of the three most important enzymes in the melatonin biosynthetic pathway (Sugden et al., 1986; Ehret et al., 1989, 1991; Klein et al., 1970; Ribelayga et al., 1997, 1999; Yu et al., 1993). Tryptophan hydroxylase (TPH, EC 1.14.16.4) is the first enzyme, catalyzing the transformation of tryptophan into 5-hydroxytryptophan, and is the rate-limiting step in the serotonin synthesis. Serotonin accumulates in the pineal gland during the day and, only at night, due to the activation of the enzyme arylalkylamine N-acetyltransferase (AA-NAT; EC 2.3.1.87), it is converted to N-acetylserotonin, that is then transformed into melatonin by the catalysis of hydroxyindole-O-methyltransferase enzyme (HIOMT, EC 2.1.1.4) (Sugden, 1989; Simonneaux and Ribelayga, 2003). Available online at www.sciencedirect.com Life Sciences 82 (2008) 529 – 535 www.elsevier.com/locate/lifescie ⁎ Corresponding author. Laboratory of Pharmacology, Butantan Institute, Av. Vital Brazil, 1500, São Paulo, SP, 05503-900,Brazil. Tel.: +55 11 3726 7222; fax: +55 11 3091 7629. E-mail address: soafeche@butantan.gov.br (S. Castro Afeche). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.12.011