Psychopharmacology (2001) 154:403–407 DOI 10.1007/s002130000631
O R I G I N A L I N V E S T I G AT I O N
Yasmina Djeridane · Yvan Touitou
Chronic diazepam administration differentially affects melatonin synthesis in rat pineal and Harderian glands
Received: 4 August 2000 / Accepted: 17 October 2000 / Published online: 23 February 2001 © Springer-Verlag 2001
Abstract Rationale: Pineal and Harderian gland melatonin production as well as plasma melatonin levels were investigated in male Wistar rats (12 weeks old) after administration of diazepam, a benzodiazepine widely used as anxiolytic. Objective: The present study investigates the effects of a chronic administration of diazepam on pineal and Harderian gland melatonin contents. Methods: Diazepam was administered subcutaneously, for 21 days, at a dosage of 3 mg/kg body weight per day, 1 h before the onset of darkness. Results: Diazepam clearly affected pineal melatonin biosynthesis and plasma melatonin levels. Diazepam reduced the pineal melatonin content (by a factor of 2) and the activity of N-acetyltransferase (NAT) (by a factor of 3.5), as well as plasma melatonin levels (by a factor of 1.5), but had no effects on pineal hydroxyindole-O-methyltransferase activity. By contrast to the pineal gland, diazepam failed to affect the Harderian gland melatonin content. Conclusions: Our results suggest that the inhibition of melatonin production induced by diazepam in vivo may be due to a direct action of this benzodiazepine on the pineal gland, through its action on NAT, the key enzyme of melatonin synthesis, and that the control of melatonin production in the Harderian glands may be different from that observed in the pineal gland. Keywords Diazepam · Melatonin · Pineal gland · Harderian gland · N-Acetyltransferase · Hydroxyindole-O-methyltransferase
Y. Djeridane is a postdoctoral fellow at the Faculté de Médecine de la Pitié-Salpêtrière, Paris, France Y. Djeridane (✉) · Y. Touitou Service de Biochimie Médicale, Faculté de Médecine Pitié-Salpêtrière, 91 Boulevard de l’Hôpital, 75634 Paris Cedex 13, France e-mail:
[email protected] Tel.: +33-1-40779667, Fax: +33-1-40779665
Introduction Many psychiatric disorders are thought to be related to changes in pineal gland function. Benzodiazepines are psychotropic drugs widely used in the treatment of anxiety, insomnia and other physiologic disorders (Rickels et al. 2000). Diazepam potentiates the action of the neurotransmitter γ-aminobutyric acid (GABA) through benzodiazepine receptors (Wakabayashi et al. 1991b). Melatonin is the major secretory product of the pineal gland (Reiter 1991) and displays benzodiazepine-like pharmacological properties (Philo and Berkowitz 1984; Lewis et al. 1990). Several investigations have been undertaken to elucidate the relationship between melatonin and diazepam (Zatz and Brownstein 1979; Matthew et al. 1984; Lowenstein et al. 1985; Morton 1986; Oxenkrug and Harris 1988; Monteleone et al. 1989; Wakabayashi et al. 1989, 1991a, 1991b; Oxenkrug and Requintina 1990; Winters et al. 1991; Atsmon et al. 1996). Benzodiazepine receptors are divided into high affinity (nM) “central” and lower (µM) affinity “peripheral” receptors (Iversen 1978; Surany-Cadotte et al. 1987). The rat pineal gland is considered to have peripheral-type receptors (Mattew et al. 1984; Lowenstein et al. 1985), while bovine and human pineal glands are thought to have both peripheral and central types (Lowenstein et al. 1984; Surany-Cadotte et al. 1987). Several in vivo studies in humans and rodents suggest that administration of diazepam alters pineal function, and consequently melatonin biosynthesis (Zatz and Brownstein 1979; Morton 1986; Oxenkrug and Harris 1988; Monteleone et al. 1989; Wakabayashi et al. 1989, 1991a, 1991b; Oxenkrug and Requintina 1990; Winters et al. 1991). All of these reports, however, studied the acute effects of diazepam and we could not find in the literature studies on the chronic effects of diazepam. Since diazepam is administered on a long-term basis in therapeutics, we thought it worthwhile to investigate the chronic effects of the drug on plasma and pineal melatonin and associated enzyme activities, N-acetyltransferase (NAT) and hydroxyindoleO-methyltransferase (HIOMT). The effect of diazepam
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on the melatonin content in the Harderian glands, orbital structures (Djeridane 1992, 1994, 1996) considered to be part of the retinal-pineal axis and known as melatonin producing organs (Djeridane et al. 1998, 1999a, 1999b), has also been examined.
Materials and methods Animals and experimental design Male albino Wistar rats (Iffa Credo, L’Arbresle, France), 6 weeks old (200–250 g) on arrival at the laboratory, were used in the experiments. Animals were housed in a chronobiologic animal facility (Enceinte Autonome d’Animalerie, Ref. A 110-SP-6, ESI Flufrance, Arcueil, France) with food and water available ad libitum. The chronobiologic facility was equipped with equally spaced, sound-proof, temperature-controlled (21±1.0°C) compartments provided with independent light-dark cycles. The rats were synchronized with a light:dark cycle regimen of 12:12 under reverse lighting schedule (lights on from 11:00 p.m. to 11:00 a.m.) which allowed dark span sampling during the day. The rats were synchronized to this lighting regimen for 3 weeks prior to the experiments. Diazepam (Roche) was administered subcutaneously to rats (n=5), daily, 1 h before lights off, at a dosage of 3 mg/kg body weight per day, for 3 weeks. This dosage was chosen since it has been shown that acute administration of the same dose of diazepam to rats, 1 h before lights off, significantly suppressed nocturnal elevations of NAT and melatonin content in the pineal gland (Wakabayashi et al. 1991a). We therefore used the same dosage of diazepam (3 mg/kg body weight per day) for 3 weeks to compare the acute (data found in the literature) and the chronic (our present study) effects of diazepam on melatonin synthesis. Animals which received no drug but only aqueous vehicle (Roche) equal in volume to the diazepam solution served as controls (n=5). Rats were killed by decapitation at the age of 12 weeks, 7 h after the last injection (in the middle of the dark phase, i.e. at 5:00 a.m.), under red lighting. The pineal and Harderian glands were removed, frozen on dry ice and subsequently stored at –80°C until processed for the assay of melatonin and pineal enzyme activities of NAT and HIOMT. Trunk blood was collected in heparin and plasma was collected following centrifugation of the blood and stored at –20°C until assayed for the hormone. Animal experimentation was performed in agreement with the principles of Laboratory Animal Care (NIH) and French National Laws. Biochemical measurements
Fig. 1 Effect of a chronic diazepam administration (3 mg/kg) on rat pineal melatonin content, and enzyme activities of NAT and HIOMT documented in the middle of the dark phase. Diazepam was injected subcutaneously 1 h before the onset of darkness for 21 days. Each value is the mean±SEM of five rats. *P
