Melatonin Supports CYP2D-Mediated Serotonin Synthesis in the Brain
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1521-009X/44/3/445–452$25.00 http://dx.doi.org/10.1124/dmd.115.067413
DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:445–452, March 2016
Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics
Melatonin Supports CYP2D-Mediated Serotonin Synthesis in
the Brain
Anna Haduch, Ewa Bromek, Jacek Wójcikowski, Krystyna Gołembiowska,
and Władysława A. Daniel
Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland
Received September 25, 2015; accepted January 7, 2016
ABSTRACT
Melatonin is used in the therapy of sleep and mood disorders and as (a monoaminoxidase inhibitor), the effect of melatonin was not
a neuroprotective agent. The aim of our study was to demonstrate visible in the majority of the brain structures studied but could be
that melatonin supported (via its deacetylation to 5-methoxytrypt- seen in all of them in 5,7-dihydroxytryptamine–lesioned animals
Downloaded from dmd.aspetjournals.org at ASPET Journals on February 13, 2021
amine) CYP2D-mediated synthesis of serotonin from 5-methoxy- when serotonin storage and synthesis via a classic tryptophan
tryptamine. We measured serotonin tissue content in some brain pathway was diminished. Melatonin alone did not significantly
regions (the cortex, hippocampus, nucleus accumbens, striatum, increase extracellular serotonin concentration in the striatum of
thalamus, hypothalamus, brain stem, medulla oblongata, and cere- naïve rats but raised its content in pargyline-pretreated animals
bellum) (model A), as well as its extracellular concentration in the (model B). The CYP2D inhibitor propafenone given intrastructurally
striatum using an in vivo microdialysis (model B) after melatonin prevented the melatonin-induced increase in striatal serotonin in
injection (100 mg/kg i.p.) to male Wistar rats. Melatonin increased those animals. The obtained results indicate that melatonin supports
the tissue concentration of serotonin in the brain structures stud- CYP2D-catalyzed serotonin synthesis from 5-methoxytryptamine in
ied of naïve, sham-operated, or serotonergic neurotoxin the brain in vivo, which closes the serotonin-melatonin-serotonin
(5,7-dihydroxytryptamine)–lesioned rats (model A). Intracerebroven- biochemical cycle. The metabolism of exogenous melatonin to the
tricular quinine (a CYP2D inhibitor) prevented the melatonin-induced neurotransmitter serotonin may be regarded as a newly recognized
increase in serotonin concentration. In the presence of pargyline additional component of its pharmacological action.
Introduction melatonin synthesis in them. Extrapineal melatonin seems not to be
Melatonin is used in the treatment of some sleep disorders, including engaged in the regulation of the photoperiod (Acuña-Castroviejo et al.,
concurrent sleep disturbances in the course of different psychiatric 2014); however, together with pineal melatonin, extrapineal melatonin
diseases such as schizophrenia and major depressive and seasonal protects cells against damage from oxidative stress due to its antioxidant
affective disorders (Dolberg et al., 1998; Dalton et al., 2000; Shamir and anti-inflammatory activity. Moreover, melatonin exerts an anti-
et al., 2000; Singh and Jadhav, 2014). Moreover, high doses of excitotoxic effect in the brain by reducing glutamate activity and
melatonin are recommended for neuroprotection (Venegas et al., increasing that of GABA and it stimulates neurogenesis (Pandi-Perumal
2012; Acuña-Castroviejo et al., 2014). et al., 2008; Acuña-Castroviejo et al., 2014; Singh and Jadhav, 2014).
The synthesis of endogenous melatonin in the pineal gland is Being an amphiphilic substance, peripheral melatonin easily crosses the
governed by the light/dark cycle, which controls circadian and blood–brain barrier (Green et al., 1975). Circulating melatonin is
circannual rhythms. However, melatonin can also be produced in a metabolized mainly in the liver by cytochrome P450 isoforms of the
substantial amount in extrapineal organs, including the gastrointestinal CYP1A subfamily (CYP1A1/A2/B1) to form 6-hydroxymelatonin and
tract (in enterochromaffin cells), where it is not controlled by the then 6-sulfatoxymelatonin (Ma et al., 2005; Hardeland, 2010), but it can
photoperiod (Pandi-Perumal et al., 2008; Venegas et al., 2012; Acuña- also be deacetylated to 5-methoxytryptamine (5-MT) (Rogawski et al.,
Castroviejo et al., 2014). The synthesis of melatonin from tryptophan 1979; Beck and Jonsson, 1981).
via serotonin is catalyzed by N-acetyltransferase and hydroxyindole-O- In the brain, melatonin deacetylation to 5-MT seems to be of minor
methyltransferase, these two enzymes being present in a variety of importance; both melatonin and 5-MT are formed mainly in the pineal
organs/tissues such as the heart, liver, leukocytes, or the brain (e.g., the gland from which they are released to the circulation or to the third
cerebral cortex and striatum), which also suggests the occurrence of ventricle. However, 5-MT formed from gut-derived melatonin in the
liver crosses the blood–brain barrier (Beck and Jonsson, 1981; Acuña-
Castroviejo et al., 2014) and, together with the brain-derived 5-MT,
This research was supported by the European Union and the Polish Ministry of provides a direct substrate for CYP2D to form serotonin. Thus, the gut-
Science and Higher Education [Grant DeMeTer POIG.01.01.02-12-004/09] and by derived or the exogenously supplied melatonin that provides most of
the Polish Academy of Sciences Institute of Pharmacology [Statutory Funds]. the 5-MT for the organism may support serotonin formation by a
dx.doi.org/10.1124/dmd.115.067413. CYP2D-mediated alternative pathway in vivo.
ABBREVIATIONS: 5,7-DHT, 5,7-dihydroxytryptamine; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; 5-MT, 5-methoxytryptamine; aCSF,
artificial cerebrospinal fluid; DA, dopamine; DMSO, dimethylsulfoxide; DRN, dorsal raphe nuclei; HPLC, high-performance liquid chromatography;
MAO, monoaminoxidase; MRN, median raphe nuclei; NA, noradrenaline.
445446 Haduch et al.
Recent studies have shown that, apart from the classic pathway of Materials and Methods
serotonin formation from L-5-hydroxytryptophan, serotonin may also Chemicals
be synthesized via CYP2D-catalyzed O-demethylation of 5-MT.
Melatonin (hydrochloride), pargyline (hydrochloride), quinine (hydrochlo-
Such a way of serotonin synthesis has been demonstrated for human ride), propafenone (hydrochloride), serotonin (5-hydroxytryptamine or 5-HT;
and rat cDNA-expressed CYP2D isoforms, as well as for brain hydrochloride) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA), dopa-
microsomes (Yu et al., 2003; Haduch et al., 2013). Moreover, using a mine (DA), noradrenaline (NA), 5,7-DHT (a creatinine sulfate salt), and ascorbic
microdialysis model, the formation of serotonin via this alternative acid were purchased from Sigma-Aldrich (St. Louis, MO). Ketamine hydro-
pathway has recently been shown to function in the brain in vivo chloride (Ketamine) and xylazine hydrochloride (Sedazin) came from Biowet
(Haduch et al., 2015). (Puławy, Poland). All organic reagents were of high-performance liquid
The aim of this study was to demonstrate that exogenous melatonin chromatography (HPLC) grade and were supplied by Merck (Darmstadt,
supported (via deacetylation to 5-MT) CYP2D-mediated serotonin Germany).
synthesis from 5-MT in the rat brain in vivo (Fig. 1) by measuring
tissue or extracellular serotonin concentration after intraperitoneal Animals
melatonin injection. Serotonin tissue content was measured in brain All experimental procedures were carried out in accordance with the National
regions containing CYP2D and serotonergic innervation (model A). Institutes of Health Guide for the Care and Use of Laboratory Animals and were
The experiment was carried out with naïve and 5,7-dihydroxytryptamine approved by the Bioethics Commission at the Polish Academy of Sciences
(5,7-DHT)–pretreated animals in the absence/presence of the mono- Institute of Pharmacology, as compliant with the Polish law. The study was
amine oxidase (MAO) inhibitor pargyline. Intraperitoneal pargyline conducted on male Wistar-Han rats (Charles River Laboratories, Sulzfeld,
Downloaded from dmd.aspetjournals.org at ASPET Journals on February 13, 2021
Germany) weighing 300–325 g. The animals were housed in rooms with a
was applied to prevent 5-MT (successively formed from melatonin
controlled temperature and humidity on a 12-hour light/dark cycle and had free
in the liver) and serotonin (formed from 5-MT in the brain) from
access to tap water and standard laboratory food during the study.
the oxidation by MAO and thus to potentiate the effect of melatonin
on the serotonin level in the brain (Prozialek and Vogel, 1978;
The Tissue Levels of Serotonin in the Brain after Intraperitoneal
Suzuki et al., 1981; Galzin and Langer, 1986; Raynaud and Pévet,
Melatonin Administration (Ex Vivo Measurement, Model A)
1991). Intracerebral 5,7-DHT (a neurotoxin specific to serotonergic
neurons) was used to decrease the production of serotonin via a To study the supportive effect of exogenous melatonin on the alternative
classic pathway from tryptophan and serotonin storage in neuronal pathway of serotonin synthesis from 5-MT in a brain tissue, melatonin was
injected intraperitoneally (100 mg/kg i.p.) to naïve and 5,7-DHT–lesioned rats.
terminals of the brain (Rogawski et al., 1979; Beck and Jonsson,
The rats were anesthetized with ketamine HCl (75 mg/kg i.p.) and xylazine
1981) and, consequently, to enhance serotonin derived from the HCl (10 mg/kg i.p.) and were then placed in stereotaxic apparatus (David Kopf
peripherally formed 5-MT (from melatonin), as well as to demon- Instruments, Tujunga, CA). All solutions were freshly prepared on the day of
strate the role of cytochrome P450 present in neurons/terminals in experimentation. 5,7-DHT was dissolved in 0.9% NaCl with 0.05% ascorbic
serotonin formation from 5-MT via an alternative pathway. The acid and was injected into the dorsal raphe nuclei (DRN) and median raphe
extracellular, functional concentration of serotonin in the striatum nuclei (MRN) at a concentration of 10 mg/ml (1 ml, infused at a rate of 1 ml/min)
(involved in motor functions) was measured in the absence/ into both raphe nuclei. The following coordinates were used (Paxinos and
presence of pargyline using an in vivo microdialysis (model B). Watson, 2007): AP (anterior-posterior), –7.9; L (lateral), 0.0 from the bregma;
The animals also received a CYP2D inhibitor to ascertain whether and V (ventral), –7.9 (MRN), –5.9 (DRN) from the surface of the dura. The
CYP2D was engaged in the synthesis of serotonin from melatonin needle stayed in place for 5 minutes after injection before it was slowly
withdrawn. Sham-operated animals were subjected to the same procedure as 5,7-
via 5-MT O-demethylation.
DHT–treated animals, but they received a vehicle (a 0.9% NaCl plus 0.05%
ascorbic acid) instead of 5,7-DHT. Ten days after injection of 5,7-DHT (or
vehicle), the rats received melatonin (100 mg/kg i.p.), an indirect exogenous
substrate for serotonin synthesis and/or pargyline (75 mg/kg i.p., 30 minutes
before melatonin) to prevent the melatonin-produced 5-MT and serotonin
(formed from 5-MT) from MAO oxidation. Because of its insolubility in water,
melatonin was dissolved in a 30% dimethylsulfoxide (DMSO).
All drugs were given according to the following schedule: 5,7-DHT or vehicle
(raphe nuclei) → after 10 days, pargyline i.p. → after 30 minutes, melatonin i.p.
or 30% DMSO → after 1 hour, decapitation → tissue serotonin (DA, NA).
The rats were divided into 12 experimental groups (Table 1). Groups of
nonoperated rats were as follows: control, 30% DMSO (2 ml/kg i.p.); melatonin
(100 mg/kg i.p.); pargyline (75 mg/kg i.p.) plus 30% DMSO (2 ml/kg i.p.); and
pargyline (75 mg/kg i.p.) plus melatonin (100 mg/kg i.p.). Groups of sham-
operated rats were as follows: vehicle (1 ml, into the raphe nuclei) plus 30%
DMSO (2 ml/kg i.p.); vehicle (1 ml, into the raphe nuclei) plus melatonin (100
mg/kg i.p.); vehicle (1 ml, into the raphe nuclei) plus pargyline (75 mg/kg i.p.)
plus 30% DMSO (2 ml/kg i.p.); and vehicle (1 ml, into the raphe nuclei) plus
pargyline (75 mg/kg i.p.) plus melatonin (100 mg/kg i.p.). Groups of operated
(5,7-DHT–lesioned) rats were as follows: 5,7-DHT (1 ml, into the raphe nuclei)
plus 30% DMSO (2 ml/kg i.p.); 5,7-DHT (1 ml, into the raphe nuclei) plus
melatonin (100 mg/kg i.p.); 5,7-DHT (1 ml, into the raphe nuclei) plus pargyline
(75 mg/kg i.p.) plus 30% DMSO (2 ml/kg i.p.); and 5,7-DHT (1 ml, into the raphe
nuclei) plus pargyline (75 mg/kg i.p.) plus melatonin (100 mg/kg i.p.).
Moreover, an additional group of naïve rats received bilateral intracerebro-
Fig. 1. Metabolism of exogenous melatonin via deacetylation to 5-MT in the ventricular injections of the CYP2D inhibitor quinine (150 mg/5 ml i.c.v.),
liver and subsequent O-demethylation to serotonin in the brain (the investigated melatonin (100 mg/kg i.p.), or quinine (30 minutes before melatonin) plus
pathway). melatonin. The CYP2D competitive inhibitor quinine (Kobayashi et al., 1989)Melatonin Supports Serotonin Formation by Brain CYP2D 447
Nonoperated rats: *P , 0.05 (versus control); **P , 0.01 (versus control); ***P , 0.001 (versus control); ##P , 0.01 (versus PARG); ###P , 0.001 (versus PARG). Operated rats: +P , 0.05 (versus sham-operated rats); ++P , 0.01 (versus sham-operated
rats); +++P , 0.001 versus sham-operated rats (versus sham-operated rats); ^P , 0.05 (versus sham plus PARG); $P , 0.05 (versus lesioned animals); $$P , 0.01 (versus lesioned animals); $$$P , 0.001 (versus lesioned animals); !P , 0.05 (versus lesion plus
was used in an appropriate intracerebral dose to produce an enzyme-specific
24.1$$$ 574.36 27.9$$$ 896.9 6 113.2!!!
54.2$$$ 1063.06 69.1$$$ 1525.0 6 133.0!!
715.0 6 45.0$$$ 946.9 6 56.6!!!
403.7 6 38.7!!!
micromolar concentration of the inhibitor in the brain (Bromek et al., 2010;
176.4 6 25.2!!
854.1 6 61.0!!
540.8 6 35.3!
11.7$$ 406.0 6 15.8$$$ 474.0 6 20.9!
PARG + MEL
19.6$$$ 643.4 6 31.5$$$ 722.6 6 32.4
43.7 6 3.3!
Haduch et al., 2015). Such an application of quinine allowed us to ascertain
whether CYP2D was engaged in the indirect synthesis of serotonin from
melatonin (via O-demethylation of the melatonin-formed 5-MT). The rats
were anesthetized with ketamine HCl (75 mg/kg i.p.) and xylazine HCl
(10 mg/kg i.p.) and were then placed in the Kopf stereotaxic apparatus. The
280.9 6 12.5$$
374.7 6 53.3$
77.4 6 20.5
103.8$ 505.8 6 21.4
30.0 6 2.8$
coordinates based on the Paxinos and Watson (2007) atlas were as follows:
PARG
5,7-DHT–Lesioned Rats
AP, –0.8; L, 61.5 from the bregma; and V, –3.5 from the dura. The freshly
prepared quinine at a concentration of 30 mg/ml was administered (5 ml,
infused at a rate of 1 ml/min) into both lateral ventricles (150 mg per
The effect of melatonin (100 mg/kg i.p.) on serotonin tissue level in the brain structures of naïve, sham-operated, and 5,7-DHT–lesioned rats (model A)
ventricle). The needle was left in place for 5 minutes after injection before its
5.1$$$
45.0$
21.7
26.5
52.8
slow withdrawal. Control rats (sham-operated animals) were subjected to the
MEL
6
6
6
6
6
6
6
6
6
6
same procedure as the quinine-treated group, except that they received a
542.0
242.6
58.4
612.0
550.4
678.3
320.5
952.7
234.4
52.5
vehicle (0.9% NaCl) instead of quinine. The placement of the needle was
histologically verified in 10 rats in a preliminary experiment and was then
The data are expressed as the mean 6 S.E.M. (n = 5–8). Statistical significance was assessed by a two-way (nonoperated rats) or three-way (operated rats) analysis of variance and Fisher’s test.
21.7+++
10.0+++
38.9+++
25.3+++
checked in two animals from each experimental group.
7.9+++
38.2+
25.5+
28.7
15.6
1.1
All drugs were given according to the following schedule: quinine i.c.v. or
Lesion
vehicle → after 30 minutes, melatonin i.p. or 30% DMSO → after 1 hour,
6
6
6
6
6
6
6
6
6
6
381.9
185.9
261.0
161.7
305.4
236.3
411.2
129.6
33.5
14.0
decapitation → tissue serotonin.
Downloaded from dmd.aspetjournals.org at ASPET Journals on February 13, 2021
Operated Rats
The rats were divided into four experimental groups (Fig. 2) as follows:
2026.0 6 163.2^
control, vehicle (5 ml i.c.v.) plus 30% DMSO (2 ml/kg i.p.); quinine (150 mg/5
834.5 6 30.4^
908.4 6 27.5^
915.3 6 47.3+++ 1035.0 6 25.9
526.1 6 15.3
428.1 6 22.0
1111.8 6 46.4
897.0 6 41.1
624.7 6 53.2
PARG + MEL
58.8 6 8.2
ml i.c.v.) plus 30% DMSO (2 ml/kg i.p.); vehicle (5 ml i.c.v.) plus melatonin
(100 mg/kg i.p.); and quinine (150 mg/5 ml i.c.v.) plus melatonin (100 mg/kg i.p.).
One hour after melatonin injection, the rats were decapitated and their
brains were removed. The brains were cut into the cerebellum, hypothalamus,
+++
thalamus, nucleus accumbens, striatum, hippocampus, frontal cortex, the rest
523.3 6 17.2+++
413.8 6 19.8+++
623.0 6 64.7+++
773.3 6 23.6+++
+++
+++
+++
58.9 6 2.8+++
1266.4 6 168.1
962.0 6 55.9
1640.0 6 78.9
546.6 6 17.1
of cortex, brain stem, and medulla oblongata. Brain tissues were frozen on dry
PARG
ice and stored at –80C until they were further analyzed.
Serotonin Tissue Level
Sham-Operated Rats
pg/mg of tissue
Melatonin and the other pharmacological substances applied were
administered at the same time of a day to rats coming from all of the
experimental groups. Consequently, all experimental procedures involving
+++
20.7 467.4 6 42.6+++
23.9 821.5 6 67.1+++
19.9 739.7 6 31.2+++
+++
+++
control groups and groups treated with melatonin were carried out at the same
22.7 400.6 6 20.6++
72.1 6 6.7+++
43.3 1045.8 6 134.8
TABLE 1
20.1 685.1 6 31.4+
+
56.8 918.0 6 46.1
69.5 1306.1 6 51.1
26.7 397.7 6 53.3
time of a day (between 10:00 AM and 4:00 PM).
MEL
Extracellular Concentrations of Serotonin in the Striatum after
Intraperitoneal Administration of Melatonin: An In Vivo Microdialysis
(Model B)
2.3
Microdialysis guides were implanted 7 days before melatonin (100 mg/kg
Sham
6
6
6
6
6
6
6
6
6
6
PARG group); !!P , 0.01 (versus lesion plus PARG group); !!!P , 0.001 (versus lesion plus PARG group).
i.p.) administration. The rats were anesthetized with ketamine (75 mg/kg i.m.)
506.6
305.3
252.6
368.8
303.5
417.8
564.2
990.7
256.5
21.8
and xylazine (10 mg/kg i.m.) and were then placed in stereotaxic apparatus
(David Kopf Instruments). Vertical microdialysis guides (Bioanalytical
15.0 753.7 6 54.6*** 794.0 6 35.5*** 1136.0 6 50.1###
683.7 6 20.7###
759.3 6 48.6##
1901.0 6 100.3*** 2091.0 6 151.5
PARG + MEL
1127.0 6 82.7*** 1257.0 6 44.8
570.5 6 18.0
536.2 6 40.6
922.6 6 66.16*** 938.9 6 61.4
646.2 6 47.8
Systems Inc., West Lafayette, IN) were implanted in the striatum using the
81.7 6 4.6
following coordinates (Paxinos and Watson, 2007): AP, +1.2; L, +3.0 from
the bregma; and V, –3.2 from the surface of the dura.
On day 7 after the surgery, brain microdialysis probes (membrane
polyacrylonitrile, 320 mm OD, 4 mm long; Bioanalytical Systems Inc.) were
523.9 6 21.5***
555.7 6 32.4***
581.7 6 55.93**
673.8 6 55.7***
438.8 6 27.2***
80.6 6 4.6***
placed inside the guides implanted in the striatum and were connected to the
Univentor 802 syringe pump (Agn Tho’s AB, Lidingö, Sweden), which
PARG
delivered an artificial cerebrospinal fluid (aCSF) consisting of 145 mM NaCl,
Nonoperated Rats
2.7 mM KCl, 1.0 mM MgCl2, and 1.2 mM CaCl2, pH 7.4, at a flow rate of
2.0 ml/min.
Four baseline samples were collected from freely moving rats at 20-minute
295.9 6 18.2**
20.2 378.8 6 25.8**
25.0 338.1 6 24.8*
13.7 516.4 6 28.5*
33.9 545.6 6 64.8*
intervals after a 165-minute washout period. Then, the appropriate drugs,
34.4 637.2 6 30.7
19.6 417.1 6 21.0
42.6 1124.0 6 28.5
50.1 6 6.6
propafenone (50 mM given using microdialysis probes) and/or melatonin
MEL
(100 mg/kg i.p.), were administered to naïve or pargyline-treated rats
(pargyline 75 mg/kg i.p., 60 minutes before melatonin). The applied effective
doses of melatonin and the other pharmacological substances were chosen on
MEL, melatonin; PARG, pargyline.
7.3
1.8
the basis of the results of our preliminary experiments (data not shown). In
Control
6
6
6
6
6
6
6
6
6
6
that experimental set, intracerebral propafenone (added to the aCSF) was
563.8
394.2
259.0
191.8
370.8
353.6
339.7
950.9
280.9
39.4
used instead of quinine as a specific and competitive CYP2D inhibitor (Xu
et al., 1995; Zhou et al., 2013) to show the efficacy of another enzyme
Nucleus accumbens
inhibitor in preventing melatonin effect. Moreover, in our preliminary
Medulla oblongata
experiment, propafenone was effective at a concentration 10 times lower
Hypothalamus
Rest of cortex
Frontal cortex
Hippocampus
Brain Structure
than quinine (50 mM versus 500 mM), being thus also easier to dissolve in the
Cerebellum
Brain stem
Thalamus
Striatum
aCSF. Dialysate fractions after melatonin injection were collected throughout
180 minutes. All drugs were given according to the following schedule:
microdialysis guide (in the striatum) → after 7 days, pargyline i.p. → after 60448 Haduch et al.
Downloaded from dmd.aspetjournals.org at ASPET Journals on February 13, 2021
Fig. 2. Model A. The effect of intracerebral quinine
administration on the melatonin-induced (100 mg/kg
i.p.) increase in 5-HT tissue content in the following
brain structures: frontal cortex (A), rest of the cortex
(B), hippocampus (C), nucleus accumbens (D),
striatum (E), thalamus (F), hypothalamus (G), brain
stem (H), medulla oblongata (I), and cerebellum (J). The
data are expressed as the mean 6 S.E.M. (n = 5–8).
Statistical significance was assessed by a two-way
analysis of variance and Fisher’s test. *P , 0.05 (versus
control); **P , 0.01 (versus control); ***P , 0.001
(versus control); # P , 0.05 (versus melatonin);
##
P , 0.01(versus melatonin); ###P , 0.001 (versus
melatonin). The control values (in picograms per
milligram of tissue) were as follows: 465.50 6 20.39
(frontal cortex), 352.08 6 17.42 (rest of the cortex),
270.26 6 7.94 (hippocampus), 424.01 6 24.26
(nucleus accumbens), 247.19 6 23.29 (striatum),
577.54 6 30.39 (thalamus), 459.33 6 34.76 (hypo-
thalamus), 704.81 6 25.53 (brain stem), 560.21 6
18.68 (medulla oblongata), and 45.83 6 3.59 (cerebel-
lum). MEL, melatonin; QUIN, quinine.
minutes, propafenone into the striatum and/or melatonin i.p. → in vivo plus melatonin (100 mg/kg i.p.); and pargyline (75 mg/kg i.p.) plus propafenone
extracellular serotonin (dopamine). (50 mM) plus melatonin (100 mg/kg i.p.)
The rats were divided into six experimental groups (Figs. 3 and 4): control, 30% At the end of the experiment, the rats were euthanized and their brains were
DMSO (2 ml/kg i.p.); pargyline (75 mg/kg i.p.) plus 30% DMSO (2 ml/kg i.p.); histologically examined to validate probe placement.
saline (2 mg/kg i.p.) plus melatonin (100 mg/kg i.p.); pargyline (75 mg/kg i.p.) plus Brain microdialysis procedures were carried out in rats from all of the
propafenone (50 mM) plus 30% DMSO (2 ml/kg i.p.); pargyline (75 mg/kg i.p.) experimental groups at the same time of a day (between 10:00 AM and 6:00 PM).Melatonin Supports Serotonin Formation by Brain CYP2D 449
column (3 mm, 100 3 mm; Thermo Scientific, Waltham, MA). The mobile
phase contained 0.1 M KH2PO4, 0.5 mM Na2EDTA, 80 mg/l sodium
1-octanesulfonate, and 4% methanol and was adjusted to pH 3.7 with 85%
H3PO4. The flow rate of the eluent was 0.6 ml/min. The potential of a 3-mm
glassy carbon electrode was set at 0.7 V and a sensitivity of 5 nA/V. The column
temperature was maintained at 30C. The Chromax 2007 program (Pol-
Laboratory, Warsaw, Poland) was applied for the collection and analysis of
data. Neurotransmitter concentrations in the dialysate were measured using a
composition of the mobile phase: 0.1 M KH2PO4, 0.5 mM Na2EDTA, 16 mg/l
sodium 1-octanesulfonate, and 2% methanol, adjusted to pH 3.6 with 85%
H3PO4. The flow rate of the eluent was 0.7 ml/min.
Data Analysis
An average neurotransmitter concentration of four stable samples of the
dialysate fraction prior to drug administration was regarded as a basal value
(100%). The data were statistically analyzed using two-way (nonoperated rats) or
three-way (operated rats) analysis of variance (model A), followed by Fisher’s
least significant differences post hoc test, or by a repeated-measures analysis of
Fig. 3. Model B. The melatonin-produced increase in extracellular serotonin variance (model B), followed by Tukey’s post hoc test (Program Origin 7.5 or
Downloaded from dmd.aspetjournals.org at ASPET Journals on February 13, 2021
concentration in the striatum is prevented by the CYP2D inhibitor propafenone in
pargyline-pretreated rats. Drugs were given as indicated with an arrow. The obtained
Statistica 9, respectively; StatSoft Inc., Tulsa, OK). The results were considered
values are the mean 6 S.E.M. (n = 5–8). A repeated-measures analysis of variance to be statistically significant when P , 0.05.
showed effect of treatment (F = 5.25, P = 0), effect of time (F = 11.27, P = 0), and
interaction between both factors (F = 55.27, P = 0). *P , 0.001 (P = 0.000135;
versus PARG); ^P , 0.001 (P = 0.00014; versus pargyline plus melatonin); !P , 0.1 Results
(P = 0.065; versus pargyline plus propafenone) (repeated-measures analysis of
variance and Tukey’s post hoc test). MEL, melatonin; PARG, pargyline; PFN, The Tissue Levels of Serotonin in the Brain after Intraperitoneal
propafenone. Administration of Melatonin (Ex Vivo Measurement, Model A)
Intact Rats. Melatonin and the applied pharmacological substances
Determination of Brain Neurotransmitters (the MAO inhibitor pargyline and the serotonergic neurotoxin 5,7-
The tissue concentrations of 5-HT, DA, NA, and 5-HIAA were measured DHT) affected the tissue concentration of serotonin in the brain; this
using HPLC with electrochemical detection, according to the method of Haduch effect was structure dependent (Table 1). Melatonin (100 mg/kg i.p.)
et al. (2015). Briefly, brain structures were homogenized in 20 volumes (v/w) of significantly increased serotonin content in the rest of the cortex
ice-cold 0.1 M HClO2 and were centrifuged at 15,000 g for 15 minutes at 4C. (131%), hippocampus (154%), nucleus accumbens (191%), thalamus
The obtained supernatant (5 ml) was injected into the HPLC system. An external (146%), hypothalamus (161%), and medulla oblongata (135%) com-
standard containing NA, DA, 5-HT, and 5-HIAA at concentrations of 50 ng/ml pared with the control (Table 1). A similar tendency was observed in
(or 2.5 ng/ml for the dialysate) was used. The chromatography system comprised other structures. The MAO inhibitor pargyline (75 mg/kg i.p.)
an LC-4C amperometric detector with a cross-flow detector cell (Bioanalytical
potently increased serotonin concentration in all of the investigated
Systems Inc.) as well as a 626 Alltech pump and a Hypersil Gold analytical
brain structures, on average up to 207% of the control (frontal cortex,
200%; rest of the cortex, 202%; hippocampus, 290%; nucleus
accumbens, 201%; striatum, 157%; thalamus, 261%; hypothalamus,
198%; brain stem, 200%; medulla oblongata, 156%; and cerebellum,
205%). However, melatonin administered to pargyline-pretreated rats
did not elevate the serotonin concentration in most of the brain
structures studied, except for the nucleus accumbens, striatum, and
medulla oblongata (an increase up to 143%, 131%, and 156% of the
control, respectively) (Table 1).
Sham-Operated Rats. Melatonin (100 mg/kg i.p.) significantly
elevated serotonin content in all of the brain structures studied in sham-
operated rats: the frontal cortex (135%), the rest of the cortex (131%),
hippocampus (185%), nucleus accumbens (284%), striatum (271%),
thalamus (177%), hypothalamus (163%), brain stem (132%), medulla
oblongata (155%), and cerebellum (331%) (Table 1). Pargyline
potently increased tissue serotonin content in the brain structures of
sham-operated rats, on average up to 209% (frontal cortex, 181%; rest
of the cortex, 171%; hippocampus, 164%; nucleus accumbens, 344%;
striatum, 205%; thalamus, 185%; hypothalamus, 171%; brain stem,
Fig. 4. Model B. The melatonin-produced increase in extracellular dopamine 166%; medulla oblongata, 213%; and cerebellum, 270%) (Table 1).
concentration in the striatum is prevented by the CYP2D inhibitor propafenone in Melatonin administered to pargyline-pretreated sham-operated rats
pargyline-pretreated rats. Drugs were given as indicated with an arrow. The obtained
values are the mean 6 S.E.M. (n = 5–8). A repeated-measures analysis of variance
significantly elevated serotonin concentration up to approximately
showed effect of treatment (F = 5.25, P = 0), effect of time (F = 11.27, P = 0), and 134% in the striatum, 117% in the thalamus, and 124% in the brain stem
interaction between both factors (F = 55.27, P = 0). #P , 0.001 (P = 0.00014; versus compared with pargyline-pretreated animals (Table 1).
control); *P , 0.001 (P = 0, 00014; versus pargyline); ^P , 0.001 (P = 0.00014; 5,7-DHT–Lesioned Rats. The neurotoxin 5,7-DHT (10 mg/1 ml),
versus pargyline plus melatonin); !P , 0.1 (P = 0.053; versus pargyline plus
propafenone) (repeated-measures analysis of variance and Tukey’s post hoc test). injected into the DRN and MRN, decreased tissue serotonin concen-
MEL, melatonin; PARG, pargyline; PFN, propafenone. tration in all of the brain structures studied, on average down to 54% of450 Haduch et al.
the sham-operated animals (frontal cortex, 75%; rest of the cortex, 61%; concentration in pargyline-pretreated rats. Pargyline alone raised the
hippocampus, 13%; nucleus accumbens, 71%; striatum, 53%; thala- extracellular dopamine concentration up to approximately 450% of the
mus, 73%; hypothalamus, 42%; brain stem, 42%; medulla oblongata, basal level. Propafenone did not significantly affect the extracellular
51%; and cerebellum, 64%) (Table 1). The lesion was specific, since the concentration of dopamine in pargyline-pretreated animals (Fig. 4).
concentrations of the catecholaminergic neurotransmitters NA and DA The basal extracellular level of serotonin in the striatum of naïve rats
were not affected (data not shown). Melatonin (100 mg/kg i.p.) was 0.938 6 0.069 pg/10 ml, whereas that of dopamine equalled 7.5 6
significantly enhanced the serotonin level in the brain structures of 0.7 pg/10 ml and did not differ between the experimental groups.
5,7-DHT–lesioned rats: frontal cortex (142%), nucleus accumbens
(234%), striatum (340%), thalamus (222%), brain stem (232%),
medulla oblongata (181%), and cerebellum (375%) (Table 1). A similar Discussion
tendency was observed in the hippocampus (174%), hypothalamus Serotonergic projections from the raphe nuclei of the brain stem
(136%), and rest of the cortex (130%). Pargyline markedly increased innervate almost all of the brain structures that control important
serotonin level in the brain structures of 5,7-DHT–lesioned rats, on physiologic functions, including the hypothalamus (food intake,
average up to 227% of the neurotoxin-lesioned animals (frontal circadian rhythm, and thermoregulation), basal ganglia (motor func-
cortex, 187%; rest of the cortex, 151%; hippocampus, 231%; nucleus tions), thalamus (sleep and epilepsy), hippocampus (stress, learning and
accumbens, 194%; striatum, 355%; thalamus, 211%; hypothalamus, memory), and cortex (sleep and mood) (Törk, 1990; Di Giovanni et al.,
159%; brain stem, 259%; medulla oblongata, 313%; and cerebellum, 2008). Recent studies revealed that, apart from the classic pathway of
214%). Melatonin administered to the lesioned rats pretreated with serotonin formation from L-5-hydroxytryptophan, serotonin may also
Downloaded from dmd.aspetjournals.org at ASPET Journals on February 13, 2021
pargyline elevated the serotonin concentration in all of the brain be synthesized in the brain via CYP2D-catalyzed O-demethylation of
structures tested, on average up to 150% of the pargyline-pretreated 5-MT. Serotonin that was formed in that alternative way was shown in
lesioned rats (frontal cortex, 132%; rest of the cortex, 144%; vitro in microsomes derived from different brain structures (Haduch
hippocampus, 228%; nucleus accumbens, 169%; striatum, 156%; et al., 2013) and was found to function in vivo when we measured its
thalamus, 112%; hypothalamus, 144%; brain stem, 143%; medulla tissue and extracellular concentrations in selected structures of the brain
oblongata, 117%; and cerebellum, 146%). after intracerebral administration of 5-MT (Haduch et al., 2015).
Effect of Quinine. In another experimental set, the specific CYP2D Our study provides further evidence for the role of cytochrome P450
inhibitor quinine, given to both lateral ventricles of the brain (150 mg/5 (CYP2D) in serotonin synthesis in the brain in vivo by showing that
ml i.c.v.) 30 minutes before melatonin (100 mg/kg i.p.), prevented the exogenous melatonin administered intraperitoneally supports (via
melatonin-evoked elevation in tissue serotonin content in the following deacetylation to 5-MT) serotonin formation by cytochrome P450. To
brain structures of naïve rats: the rest of the cortex, hippocampus, observe serotonin formation from melatonin in vivo, in our experiment
striatum, thalamus, hypothalamus, brain stem, medulla oblongata, and we used its relatively high dose (100 mg/kg i.p.), which is pharmaco-
cerebellum (Fig. 2). Such an effect of quinine was not observed in the logically acceptable. Such high doses of melatonin (up to 200 mg/kg a
frontal cortex and nucleus accumbens. Quinine alone did not affect the day) were shown to have low toxicity (Barchas et al., 1967; Jahnke
tissue content of serotonin. et al., 1999; Acuña-Castroviejo et al., 2014) and were used for
psychopharmacological behavioral tests in animals (Papp et al., 2003,
2006). Furthermore, high doses of melatonin are recommended because
Extracellular Concentrations of Serotonin and DA in the Striatum of its protective properties as an antioxidant in saturating its in-
after Intraperitoneal Administration of Melatonin: An In Vivo tracellular therapeutic targets (Venegas et al., 2012; Acuña-Castroviejo
Microdialysis (Model B) et al., 2014). On the other hand, the rate of drug metabolism is much
Melatonin (100 mg/kg i.p.) did not significantly affect the extracel- faster in rats (rodents) than in humans, so higher doses are necessary to
lular concentration of serotonin in the striatum of naïve rats (control reach similar plasma or tissue concentration in the two species. The
rats). For that reason, the animals were pretreated with pargyline to dose of melatonin used in our experiment was found to be effective in
inhibit the oxidation of melatonin-produced 5-MT in the liver and of elevating tissue and extracellular concentrations of serotonin in the rat
serotonin in the brain and, consequently, to enhance the effect of brain.
melatonin on the intraneuronal and, possibly, also extraneuronal, Melatonin increases the tissue content of serotonin in the brain
serotonin level. As expected, when administered to animals pretreated structures of naïve, sham-operated, or 5,7-DHT–lesioned rats (model
with pargyline (75 mg/kg i.p.), melatonin increased serotonin concen- A). In the case of the brain stem (containing serotonergic neurons),
tration up to approximately 1000% of the pargyline-treated animals melatonin is considerably more effective in 5,7-DHT–lesioned rats,
(Fig. 3). The CYP2D inhibitor propafenone (50 mM), given locally which suggests the presence of a relatively larger pool of serotonin
through a microdialysis probe, prevented the melatonin-induced in- formed from tryptophan via a classic pathway than from 5-MT (formed
crease in serotonin concentration, having decreased it to approximately from melatonin) via a CYP2D pathway, as well as its considerable
25% of the pargyline- plus melatonin-treated animals. Pargyline alone reduction by the neurotoxins. As shown in naïve rats, the CYP2D
raised the extracellular serotonin concentration up to approximately inhibitor quinine (Boobis et al., 1990; Bromek et al., 2010), given to
325% of the basal level; when single points were compared, the lateral ventricles of the brain, prevents this effect in some brain
pargyline-treated group was significantly different from the control structures (e.g., cortex, hippocampus, striatum, thalamus, hypothala-
group (P , 0.0001, t test). Propafenone did not affect the extracellular mus, brain stem, medulla oblongata, and cerebellum), which indicates
concentration of serotonin in pargyline-pretreated animals. contribution of CYP2D to the melatonin-produced elevation in
Like in the case of serotonin, melatonin did not change the serotonin concentration.
extracellular concentration of dopamine in the striatum of naïve rats. As mentioned above, pargyline was applied to prevent 5-MT (formed
However, when given to animals pretreated with pargyline, melatonin from melatonin in the liver) and serotonin (formed from 5-MT in the
increased dopamine concentration up to approximately 500% of brain) from the MAO oxidation to reinforce the effect of melatonin on
the pargyline-pretreated animals. (Fig. 4). The CYP2D inhibitor the serotonin level in the brain. In contrast with melatonin, both 5-MT
propafenone prevented the melatonin-evoked increase in the dopamine and serotonin are rapidly metabolized MAO substrates, the enzymeMelatonin Supports Serotonin Formation by Brain CYP2D 451
being present in a large amount in the liver and brain (Suzuki et al., Miguez et al., 1995), since its low doses (#1 mg/kg i.p.) were found to
1981; Galzin and Langer, 1986; Raynaud and Pévet, 1991; Hardeland, increase serotonin concentration in some brain structures (Anton-Tay
2010). However, the effect of melatonin on serotonin level is not visible et al., 1968; Miguez et al., 1994).
in the majority of structures of the pargyline-pretreated, nonlesioned The results presented herein are in agreement with some previous
animals (both controls and sham-operated), since the amount of the findings suggesting that the CYP2D-mediated formation of serotonin
tissue serotonin synthesized physiologically from endogenous sub- from 5-MT may take place in vivo. Thus humanized CYP2D6
strates (mainly from tryptophan, but also from endogenous 5-MT) and transgenic mice show a higher concentration of serotonin and its
protected from MAO probably largely dominates the serotonin formed metabolite 5-HIAA in blood plasma (Yu et al., 2003) and the brain
indirectly from exogenous melatonin (via melatonin deacetylation to (Cheng et al., 2013), and intracerebral administration of 5-MT increases
5-MT and subsequent O-demethylation by CYP2D). However, the the extracellular concentration of serotonin in the brain, as has been
melatonin-induced increase in the tissue level of serotonin can be seen shown using a brain microdialysis in living rats (Haduch et al., 2015).
in all of the brain structures studied of pargyline-treated rats with a Moreover, individuals with no or a defective CYP2D6 gene (expressing
partial lesion of the serotonergic system. Under these experimental a poor metabolizer phenotype) are more anxiety prone (Bertilsson et al.,
conditions, the physiologic synthesis and storage of serotonin is 1989; González et al., 2008; Cheng et al., 2013), such a behavior being
diminished by the neurotoxin 5,7-DHT specific to the serotonergic provoked by a low serotonin level in the limbic system of the brain
system, and the amount of serotonin formed by an alternative pathway (Hensler, 2006).
(via 5-MT O-demethylation by CYP2D) is raised due to a supply of an In conclusion, our study indicates that exogenous melatonin supports
exogenous substrate (i.e., 5-MT formed from exogenous melatonin in CYP2D-catalyzed serotonin synthesis from 5-MT in vivo. CYP2D-
Downloaded from dmd.aspetjournals.org at ASPET Journals on February 13, 2021
the liver) (Rogawski et al., 1979; Beck and Jonsson, 1981), which catalyzed serotonin synthesis from the melatonin-derived 5-MT closes
crosses the blood–brain barrier (Acuña-Castroviejo et al., 2014). the serotonin-melatonin-serotonin biochemical cycle. Hence, the
Moreover, both peripheral and brain 5-MT and serotonin formed from therapeutic effect of melatonin may stem not only from its action on
it are protected by intraperitoneal pargyline against MAO oxidation. melatonin receptors and from its radical scavenger properties (Singh
Melatonin also increases the functional, extracellular concentration and Jadhav, 2014) but also from its metabolism to the monoaminergic
of serotonin under specific conditions (model B). In fact, the neurotransmitter serotonin. The latter mechanism may be regarded as a
extracellular level of the neurotransmitter depends not only on its newly recognized additional component of the pharmacological action
amount synthesized in the neuron but also on its release into the of melatonin. These findings are of both physiologic and pharmaco-
synaptic cleft and reuptake into the neuron. The effect of melatonin on logical importance, since melatonin can be formed endogenously and
these presynaptic processes has not yet been well recognized, but the administered as a drug in the therapy of sleep and mood disorders, as
available data suggest such a possibility (Pandi-Perumal et al., 2008). well as a neuroprotective agent.
Thus, exogenous melatonin does not significantly increase extracellular
serotonin in naïve rats but raises it in pargyline-pretreated animals. In
the striatum of pargyline-pretreated rats, melatonin potently elevates Authorship Contributions
serotonin concentration, yet such a strong effect does not appear when Participated in research design: Haduch, Daniel.
the tissue content of serotonin is measured (model A). However, in this Conducted experiments: Haduch, Bromek, Wójcikowski, Gołembiowska.
Performed data analysis: Haduch, Gołembiowska, Daniel.
experimental model and in these conditions (model B), the vesicle-
Wrote or contributed to the writing of the manuscript: Haduch, Daniel.
stored neurotransmitter does not mask the amount of serotonin
synthesized from exogenous melatonin. In addition, as mentioned
above, the 5-MT freshly synthesized from melatonin in the liver and the References
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