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Melatonin receptors in brain areas and ocular tissues of the teleostTinca tinca: Characterization and effect of temperature
M.A. Lopez Patino, A.L. Alonso-Gomez, A. Guijarro, E. Isorna, M.J. Delgado *
Departamento de Fisiologıa (Fisiologıa Animal II), Facultad de Biologıa, Universidad Complutense, 28040 Madrid, Spain
Received 25 July 2007; revised 5 November 2007; accepted 7 November 2007
Available online 22 November 2007
Abstract
The aim of the present study was to characterize the central melatonin receptors in brain areas and ocular tissues of the teleost Tincatinca. We investigated the temperature-dependence of 2-iodo-melatonin ([125I]Mel) binding in the optic tectum-tegmentum area and theneural retina. The binding of [125I]Mel showed a widespread distribution in brain and ocular tissues, with the highest density in the optictectum-thalamus and the lowest in hindbrain. The [125I]Mel affinity was similar in all the studied tissues, and it was on the order of thelow pM range. Saturation, kinetic and pharmacological studies showed the presence of a unique MT1-like melatonin binding site. Inaddition, the non-hydrolysable GTP analog, the GTPcS, and sodium cations induced a specific binding decrease in the optic tectumand neural retina, suggesting that such melatonin binding sites in the tench are coupled to G protein. Thus, these melatonin binding sitesin optic tectum and neural retina fulfil the requirements of a real hormone receptor, the specific binding is rapid, saturable, and reversible,and is inhibited by GTP analogs. Additionally, a clear effect of temperature on such central melatonin receptors was found. Temperaturedid not modify the Bmax and Kd, but the kinetics of [
125I]Mel binding resulted in a highly thermosensitive process in both tissues. Bothassociation and dissociation rates (K+1 and K�1) significantly increased with assay temperature (15–30 �C), but the Kd constant (esti-mated as K�1/K+1) remained unaltered. In conclusion, this high thermal dependence of the melatonin binding to its receptors in the tenchcentral nervous system supports the conclusion that temperature plays a key role in melatonin signal transduction in fish.� 2007 Elsevier Inc. All rights reserved.
Keywords: Melatonin; Receptors; Brain; Retina; Temperature; Fish
1. Introduction
Melatonin (N-acetyl 5-methoxytryptamine) is synthe-sized in the vertebrate pineal complex and the retina on adaily rhythmic pattern, with high levels during the darkphase and low levels during the light phase (Falcon,1999). This daily profile exhibits seasonal changes thatcan synchronize a large number of rhythmic physiologicaland behavioural processes (Pevet, 2003). In fish, melatoninhas been involved in the control of seasonal reproduction,ocular functions, pigmentation pattern, motor activity,feeding regulation and osmoregulation (Zachmann et al.,1992; Ekstrom and Meissl, 1997; Pinillos et al., 2001; Kulc-zykowska, 2002). In the last decade, concerted efforts have
been made to characterize the receptors involved in thetransduction of such melatonin effects.
Radioligand studies using the high specific activity radi-oiodinated melatonin agonist, 2-[125I]-iodo-melatonin([125I]Mel), have allowed the characterization of melatoninbinding sites (putative melatonin receptors) in central andperipheral tissues of several fish species. In addition, somecloning studies have supported the existence of three sub-types of high affinity melatonin receptors in fish, MT1,MT2, and Mel 1c, belonging to the G-protein coupledreceptors (Mazurais et al., 1999; Gaildrat et al., 2002; Parket al., 2007). Another binding site, MT3, with low affinitybinding for [125I]Mel, has been identified in mammals asthe enzyme NRH: quinone oxidoreductase (Nosjeanet al., 2000).
Melatonin binding sites in fish brain have been charac-terized in several species, such as goldfish, Carassius aura-
0016-6480/$ - see front matter � 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygcen.2007.11.011
* Corresponding author. Fax: +34 913944935.E-mail address: [email protected] (M.J. Delgado).
www.elsevier.com/locate/ygcen
Available online at www.sciencedirect.com
General and Comparative Endocrinology 155 (2008) 847–856
tus (Martinoli et al., 1991; Iigo et al., 1994), sea bream,Sparus auratus (Falcon et al., 1996), catfish, Silurus asotus(Iigo et al., 1997a), pike, Esox lucius (Gaildrat et al., 1998,2002), European sea bass, Dicentrarchus labrax (Bayarriet al., 2004) and in some salmonids (Pang et al., 1994;Davies et al., 1994; Amano et al., 2003). Nevertheless,almost all of these studies have been conducted using thewhole brain, and not in brain areas. On the other hand,in spite of the well known intraocular actions of melatonin(regulation of dopamine release, retinomotor movements,disc shedding of photoreceptors, melanosome aggregationin the pigment epithelium), only two studies have beenpublished on melatonin binding sites in the fish retina(goldfish, Iigo et al., 1997b, sea bass, Bayarri et al., 2004).
The study of environmental regulation of melatoninreceptors in vertebrates has been restricted to the roleplayed by photoperiod (for review, see Witt-Enderbyet al., 2003). Daily changes in melatonin binding sites havebeen reported in some fish species (goldfish, Iigo et al.,2003; sea bream, Falcon et al., 1996; pike, Gaildrat et al.,1998, rabbitfish, Park et al., 2007) but not in others (seabass, Bayarri et al., 2004, masu salmon, Amano et al.,2006). Environmental temperature, a key factor in the reg-ulation of daily and seasonal melatonin synthesis in ecto-therm vertebrates (Delgado and Vivien-Roels, 1989;Valenciano et al., 1997; Falcon, 1999), has received very lit-tle attention in relation to its possible role in melatoninreceptors regulation. Thus, the only report of temperatureplaying a major role in the transduction of melatonin signalis for frogs (Isorna et al., 2004b, 2005), and no studies havebeen carried out to investigate the effects of this environ-mental factor on melatonin receptors in fish.
The tench, Tinca tinca, is an economically importantcyprinid widely distributed in Europe. It is a strictly noctur-nal species (Herrero et al., 2005) which exhibits a pro-nounced daily melatonin rhythm in plasma (Vera et al.,2005). In the present study, we have investigated the tenchcentral melatonin receptors to further knowledge of melato-nin’s role in the signalling of environmental information inthis species. First, we have studied the distribution of bind-ing sites in different ocular tissues and brain areas using[125I]Mel as radioligand. Second, we have characterizedsuch melatonin binding sites in optic tectum and neural ret-ina by saturation, kinetics and pharmacological assays, andstudied the coupling of such melatonin receptors to G-pro-teins. Third, we have investigated the effect of temperatureon the affinity, capacity and kinetics of [125I]Mel bindingto its receptors in the optic tectum and neural retina.
2. Methods
2.1. Chemicals
The 2-[125I]-iodo-melatonin ([125I] Mel, specific activity: 2000 Ci/mmol)
obtained from Amersham (Buckinghamshire, UK) was employed as radi-
oligand. 2-iodo-melatonin (2-I-Mel), 2-phenyl-melatonin (2-Ph-Mel), 6-
chloro-melatonin (6-Cl-Mel), melatonin, N-acetyl-2-benzyl-tryptamine
(luzindole), and guanosine 5 0-O-[c-thiotriphosphate] (GTPcS) were from
Sigma (St. Louis, MO). 4-phenyl-2-propionamidotetralin (4-P-PDOT),
5-methoxycarbanylamino-N-acetyl tryptamine (5-MCA-NAT), and 5-
methoxy-N-cyclopropanoil tryptamine (5-MCPT) were from Tocris (Bris-
tol, UK).
2.2. Animals and tissue sampling
Adult tench (T. tinca L.) of both sexes (80–150 g body weight) were
obtained from the ‘‘Centro de Acuicultura Vegas del Guadiana’’ (Badajoz,
Spain) and ‘‘Ipescon’’ (Salamanca, Spain) hatcheries. Fish were kept in
5 m3 circular tanks with continuously supplied water under natural condi-
tions of photoperiod and temperature for 3 weeks prior to the experi-
ments. Fish were fed with commercial extruded pellets (Dibaq,
Diprotec) at a daily rate of 1% body weight.
Fish were sacrificed by decapitation at midday and the whole brain and
eyecups were immediately removed. The eyecups were dissected into the
pigmented epithelium, iris, and neural retina, and the brains into the fol-
lowing four areas: telencephalon, diencephalon, mesencephalic optic tec-
tum-tegmentum, and hindbrain (cerebellum-vestibulolateral lobe). All
the tissues were frozen in solid CO2 and stored at �80 �C until use. The
experiments were performed in accordance with the ‘‘Principles of Labo-
ratory Animal Care’’ (NIH published 86–23, revised 1985) and were
approved by the Animal Experimentation and Ethics Committee of the
Complutense University of Madrid (Spain).
2.3. Membrane preparation and binding assays
Membranes were prepared as previously described for frog brain (Isor-
na et al., 2004a), with the following modifications. Samples were sonicated
in 400 ll of Tris–HCl buffer (50 mM, 5 mM MgCl2, pH 7.4), and centri-
fuged for 10 min at 800g to eliminate melanin granules. The supernatant
was centrifuged for 10 min at 16,000g, and the pellet was resuspended in
400 ll of Tris–HCl buffer and centrifuged again for 10 min. The resultant
pellet was finally resuspended in 100 ll of buffer for the brain areas and
200 ll for the ocular tissues. The membrane extracts obtained were stored
at �80 �C until the binding assays were (Lowry et al., 1951), performed.
Proteins were determined by the Lowry method using bovine serum albu-
min as standard.
Binding assays were performed in a final volume of 50 ll containing
the membrane sample (10–25 lg of proteins), the [125I]Mel as radioligand,
and unlabeled melatonin (1 lM) to determine the non-specific binding
(NSB). The radioligand concentrations ranged from 5 to 300 pM for sat-
uration studies, while for kinetic and pharmacological assays the radioli-
gand concentration used was 55 pM. The incubation time for kinetic
studies varied from 6 to 480 min for association and from 10 to
1620 min for dissociation. Kinetic studies were performed at three incuba-
tion temperatures, 15, 25, and 30 �C. The reaction was stopped with 750 ll
of ice-cold Tris–HCl buffer, and membranes were collected by immediate
vacuum filtration through glass fibre filters (Millipore, USA). The filters
were washed with 4 ml of ice-cold buffer, placed into vials, and radioactiv-
ity was measured using a gamma counter (Perkin Elmer, USA) with 82%
efficiency. Specific binding was calculated by subtracting the non-specific
from the total binding and expressed as fmol/mg protein.
2.4. Data analysis
In the equilibrium saturation analysis, data were fitted to the equation
of a rectangular hyperbola, and the calculation of the binding densities
(Bmax) and equilibrium dissociation constants (Kd) were performed by a
non-linear regression of a four-parameters logistic model using the ALL-
FIT program (De Lean et al., 1978). Scatchard plots determined if a single
or multiple binding sites were present in the same sample, considering sig-
nificant P values of regression lower than 0.05. Association and dissocia-
tion rate constants (K+1, K�1) were obtained by non-linear regression
(Duggleby, 1981) with the assumption of a pseudo first-order exponential
rise for association, and a first-order exponential decay for dissociation.
The Kd from kinetic studies was calculated as the ratio K�1/K+1. The
848 M.A. Lopez Patino et al. / General and Comparative Endocrinology 155 (2008) 847–856
IC50 values in the competitive binding assays were calculated as above
described for equilibrium dissociation constants, and the inhibition con-
stants (Ki) from IC50 values were determined by the Cheng and Prusoff,
equation (1973). Standard error (SE) of constants was estimated from
the residual sum of squares in the least-squares fit. The Q10 coefficients
for 15–25 �C and 25–30 �C temperature ranges were calculated by using
the Van’t Hoff equation (Prosser, 1986).
Statistical differences in Kd, Bmax, and IC50 values were tested by the
extra sum of squares principle (Draper and Smith, 1998).
3. Results
3.1. Distribution of central melatonin binding sites
The relative densities of [125I]Mel binding sites in differ-ent brain areas and ocular tissues of the tench are shown inFig. 1. The highest densities were found in the optic tectumand the neural retina (30–50 fmol/mg prot), whereas thelowest densities were measured in the hindbrain, dienceph-alon, and telencephalon (<5 fmol/mg prot). The pigmentedepithelium and the iris exhibited intermediate densities (14–19 fmol/mg prot). Based on these higher densities andlower non-specific binding, we selected the optic tectumand neural retina as targets of brain and ocular tissues,respectively, in order to characterize in depth central Melbinding sites in the tench.
3.2. Saturation, kinetics, and pharmacological
characterization of melatonin binding sites
Saturation studies, using [125I]Mel concentrations rang-ing from 5 to 600 pM, demonstrated that the specific bindingincreases with radioligand concentration and is saturableabove at 100 pM in both optic tectum and neural retina(Fig. 2a). Similar results were obtained in the other oculartissues: pigmented epithelium and iris (data not shown). Alinear Scatchard plot (Fig. 2a, inset) revealed that [125I]Mel
binds to a single class site in the optic tectum and neural ret-ina. The affinity of binding sites for the radioligand was inthe picomolar range (Kd values, 22.31 pM for the optic tec-tum; and 17.47 pM for the neural retina, Table 1).
Time course of the association of [125I]Mel binding wasdetermined by incubation membranes from optic tectumand neural retinas with the radioligand at 25 �C. Associa-tion of [125I]Mel to tench membranes was rapid andreached a steady state in 60 min for both tissues(Fig. 2b). Binding was stable at least for 2 h of incubation.Then, dissociation was initiated by the addition of melato-nin (1 lM) and the remaining specific binding was quanti-fied at different time intervals. Dissociation was ended after9 and 14 h of unlabeled melatonin addition in optic tectumand neural retina membranes, respectively (Fig. 2b).Kinetic constants (K+1 and K�1) and the dissociation con-stant (Kdc) derived from kinetic assays were in the lowpicomolar range (Table 1).
The pharmacological characterization of melatoninbinding sites in the neural retina and the optic tectumwas carried out with different concentrations of melatoninanalogs in competitive binding assays using [125I]Mel (70–80 pM) as radioligand (Fig. 2c). The displacement of spe-cific [125I]Mel binding by drugs is concentration-dependentin both tissues. Regarding the Ki values (Table 2), theassayed ligands can be classified into the following threegroups. High (upper picomolar range: 2-I-Mel and 2-Ph-Mel), intermediate (low nanomolar range: melatonin,6-Cl-Mel, and 5-MCPT), and low affinity ligands (lowmicromolar range: 4-P-PDOT and luzindole). The 5-MCA-NAT was unable to displace specific [125I]Mel bind-ing in the neural retina. The relative order of potencyamong the drugs (based on the Ki values) was: 2-I-Mel = 2-Ph-Mel >> melatonin > 6-Cl-Mel = 5-MCPT >>4-P-PDOT = Luzindole (retina), 2-I-Mel = 2-Ph-Mel >>6-Cl-Mel = melatonin >> Luzindole > 4-P-PDOT (optictectum).
A comparison of the pharmacology reveals the higheraffinity (10-fold approximately) of 6-Cl-Mel for optic tec-tum receptors and of 4-P-PDOT for retinal receptors.
3.3. Effect of sodium and a non-hydrolysable GTP analog
(GTPcS)
The neural retina and optic tectum membranes wereused to determine the [125I]Mel binding in the presence ofGTPcS. We found a concentration-dependent, but incom-plete, inhibition of [125I]Mel binding in both tissues(Fig. 3). At a high GTPcS concentration (100 lM), signif-icant [125I]Mel binding remains in the membranes. Themaximal inhibition of GTPcS was slightly higher in neuralretina (81%) than in optic tectum (62%). Inhibitorypotency, expressed as IC50 values, was in the submicromo-lar range (retina: 0.122 lM and optic tectum: 0.261 lM)and was similar in both tissues.
We also tested the effect of the addition of GTPcS(30 lM) and monovalent cation (Na+, 250 mM), on the
Hb Di Tel OT PE Ir Ret
[125I]
Me
l b
ind
ing
(fm
ol/
mg
pro
t)
0
10
20
30
40
50
60
BRAIN OCULAR TISSUES
Fig. 1. Specific [125I]Mel binding (fmol/mg prot) in brain areas and ocular
tissues from the tench (Tinca tinca). Data are expressed as means ± SEM
(n = 5). Hb, hindbrain (cerebellum-vestibulolateral lobe); Di, diencepha-
lon; Tel, telencephalon; OT, mesencephalic optic tectum-tegmentum; PE,
pigmented epithelium; Ir, iris; Ret, neural retina.
M.A. Lopez Patino et al. / General and Comparative Endocrinology 155 (2008) 847–856 849
c
[125
I]Mel (pM)
0 100 200 300 400 500 600 700[125I]
Me
l b
ind
ing
(fm
ol/m
g p
rot)
0
10
20
30
40
50
60
70
B (fmol/mg prot)
0 10 20 30 40 50 60
B/F
0
1
2
3
Mel 1 M
NEURAL RETINA
Time (min)
0 100 200 300 400 500 600 700
[125I]
Me
l b
ind
ing
(fm
ol/m
g p
rot)
0
10
20
30
40
50
0
Log concentration (M)
-11 -10 -9 -8 -7 -6 -5 -4 -3
% [
125I]
Mel b
ind
ing
0
20
40
60
80
100
120
1 M Mel
Time (min)
0 200 400 600 800 1000[125I]
Me
l b
ind
ing
(fm
ol/m
g p
rot)
0
10
20
30
40
50
[125
I]Mel (pM)
0 100 200 300 400 500 600 700[125I]
Mel b
ind
ing
(fm
ol/m
g p
rot)
0
10
20
30
40
50
B (fmol/mg prot)0 10 20 30 40
B/F
0.0
0.5
1.0
1.5
OPTIC TECTUM
Log concentration (M)
-11 -10 -9 -8 -7 -6 -5 -4 -3
% [
125I]
Mel b
ind
ing
0
20
40
60
80
100
120
0
1 M Mel
b
a
Fig. 2. Characterization of [125I]Mel binding sites in membranes of neural retina and optic tectum of the tench (Tinca tinca). (a) Saturation curve of
[125I]Mel binding. Insert graph depicts the Scatchard plot of data, B (concentration of bound ligand), B/F (ratio of bound/free ligand). Specific
binding; total binding; non-specific binding. (b) Time course of [125I]Mel binding. The arrow indicates the addition of unlabeled
melatonin (1 lM) to initiate dissociation. association; dissociation. Each point represents individual values of [125I]Mel binding.
Assays in (a) and (b) were done in triplicate (c) Displacement of specific [125I]Mel binding by melatonin analogs. (d) melatonin; (s) 2-I-Mel; (.) 2-Ph-
Mel; ($) 6-Cl-Mel; (j) Luzindole; (h) 4-P-PDOT; (�) 5-MCPT; (}) 5-MCA-NAT. Data are expressed as means ± SEM (n = 3) of specific binding with or
without agonists or antagonists.
Table 1
Saturation and kinetic constants of [125I]melatonin binding sites from
tench neural retina and optic tectum-tegmentum at 25 �C assay
temperature
Neural retina Optic tectum
Kd (pM) 17.47 ± 1.22 22.31 ± 1.64
Bmax (fmol/mg prot) 48.07 ± 0.89 33.76 ± 0.69
K+1 (pM�1 min�1) 0.74 ± 0.05 (·10�3) 0.36 ± 0.02 (·10�3)
K�1 (min�1) 5.71 ± 0.30 (·10�3) 2.95 ± 0.25 (·10�3)
Kdc 7.72 8.15
Values are shown as constant estimation ± SE. Bmax and Kd were calcu-
lated by a non-linear regression of a four-parameters logistic model. K+1
and K�1 were obtained by non-linear regression assuming a pseudofirst-
order reaction for association, and a first-order reaction for dissociation.
The Kdc was calculated as the ratio K�1/K+1.
Table 2
Pharmacological profile of [125I]Mel binding from neural retina and optic
tectum-tegumentum of tench (Tinca tinca)
Ki Neural retina Optic tectum
2-l-Mel 118.4 pM 52.5 pM
2-Ph-Mel 132.0 pM 87.0 pM
Mel 3.7 nM 1.6 nM
6-CI-Mel 16.7 nM 1.3 nM
5-MCPT 19.3 nM —
4-P-PDOT 0.96 lM 8.31 lM
Luzindole 1.54 lM 2.61 lM
Inhibition constant (Ki) of each ligand has been calculated from IC50 values
by application of Cheng and Prusoff equation: Ki = IC50/(1 + [L]/Kd).
850 M.A. Lopez Patino et al. / General and Comparative Endocrinology 155 (2008) 847–856
displacement curve of melatonin using neural retina mem-branes (Fig. 4). Both Na+ and GTPcS, reduced signifi-cantly the apparent Bmax value (4-fold and 10-fold,respectively). However, the affinity of retinal receptors for[125I]Mel (expressed as IC50) was not modified by any ofthese agents (Fig. 4).
3.4. Effect of temperature on [125I]Mel binding
To test the effect of the assay temperature on the affinityand density of [125I]Mel binding, we used an unique mem-brane pool for the neural retina and for the optic tectum.Membranes were incubated with increasing [125I]Mel con-centrations (5–300 pM) at three different temperatures(15, 25, and 30 �C). The saturation curves obtained in bothtissues are represented in the Fig. 5. There were no signif-icant differences in Kd values at the three temperaturestested. The Bmax values remained unaltered in the optic tec-tum, but were significantly reduced (p < 0.01) at 30 �Cassay temperature in the retina membranes.
The effect of these three assay temperatures (15, 25,and 30 �C) on [125I]Mel binding kinetics in both neuralretina and optic tectum of the tench is shown in Fig. 6.It is clear that the [125I]Mel binding kinetics to both tis-sues is a highly thermosensitive process. The associationof [125I]Mel required 120, 60, and 40 min at 15, 25, and30 �C, respectively, to reach the steady state (Fig. 6a).Dissociation (Fig. 6b) also showed a high dependence
on assay temperature. Thus, the time needed to displace[125I]Mel specific binding by the unlabeled melatonindecreased significantly when assay temperature increasedin both, neural retina and optic tectum. The temperaturecoefficients (Q10) in relation to the association (K+1) anddissociation (K�1) rate constants are summarized in Table3. The Q10 values, near 2 or higher, support such highthermosensitivity of the [125I]Mel binding kinetics in theneural retina of the tench in the 15–30 �C temperaturerange. The [125I]Mel binding in optic tectum membraneswas also highly thermosensitive in the 15–25 �C range.Nevertheless, in the 25–30 �C range the association anddissociation rates were apparently unresponsive to assaytemperature.
4. Discussion
The present study demonstrates for the first time thepresence of melatonin binding sites in brain areas and theeye of the tench. Moreover, the specific binding of[125I]Mel to tench optic tectum and neural retina mem-branes fulfils the criteria for a functional receptor site.The present results on brain location of melatonin bindingsites in the tench are consistent with previous reports inother teleosts, such as Salmo salar (Ekstrom and Vanecek,1992), Oncorhynchus mykiss (Davies et al., 1994; Mazuraiset al., 1999), Carassius auratus (Iigo et al., 1994), Silurusasotus (Iigo et al., 1997a), Raja erinacea (Verdanakiset al., 1998), and Dicentrarchus labrax (Bayarri et al.,2004), but most of these studies on melatonin binding sites
Log [GTP S (M)]
% [
125I]
Me
l b
ind
ing
0
20
40
60
80
100
120
Neural retina
Optic tectum
NEURAL RETINA OPTIC TECTUM
Bmax (fmol/mg prot) 47.1 ± 3.7 17.0 ± 1.1
% Inhibition 81.4 ± 1.6 62.2 ± 3.8
IC50 (mM) 0.12 ± 0.02 0.26 ± 0.10
-8 -7 -6 -5 -4
Fig. 3. Inhibition of specific [125I]Mel binding to neural retina and optic
tectum membranes of tench (Tinca tinca) by GTPcS. Data are expressed as
percentage of Bmax (means ± SEM; n = 3). (d) neural retina; (s) optic
tectum. Insert table shows Bmax values, percentage of maximal inhibition,
and IC50 values (estimation ± SE).
Log [Mel] (M)
-11 -10 -9 -8 -7 -6
% [
12
5I]
Me
l b
ind
ing
0
20
40
60
80
100
120
Control
Na+ (250 mM)
GTP S (30 M)
NEURAL RETINA
IC50 (nM) Apparent Bmax
(fmol /mg prot)
Control 8.7 ± 2.6 25.2 ± 1.6
Na+ (250 mM) 9.2 ± 5.6 6.3 ± 1.3
GTP S (30 µM) 6.6 ± 3.9 2.5 ± 1.4
Fig. 4. Effect of Na+ (250 mM) and GTPcS (30 lM) on melatonin
displacement curve of tench (Tinca tinca) neural retina membranes. Data
are expressed as percentage of Bmax (means ± SEM; n = 3). The IC50
values are expressed as estimation ± SE).
M.A. Lopez Patino et al. / General and Comparative Endocrinology 155 (2008) 847–856 851
characterization in fish have been done on whole brain.The high density of melatonin binding sites in the optic tec-tum and the neural retina of the tench support the involve-ment of melatonin in the processing of visual signals in thisteleost, as suggested for other fish species (Mazurais et al.,1999; Bayarri et al., 2004). This melatonin function ishighly conserved, and in fact, the expression of melatoninreceptors in the tectal region of the brain is reported evenin deep sea gadiform fish, where an absence of solar lightexists (Priede et al., 1999). A significant binding capacityis found in other brain areas, such as telencephalon, dien-cephalon and hindbrain (Fig. 1), which supports a rolefor melatonin in the entrainment of different rhythmic(daily or seasonal) functions controlled by these brainareas. This widespread distribution of melatonin bindingsites in the tench brain is coincident with previous studies
in other non-mammalian vertebrates (Rivkees et al.,1989; Wiechmann and Wirsig-Wiechmann, 1994; Isornaet al., 2004a), but differs from studies in mammals, wherea more discrete distribution in the brain has been reported(Vanecek, 1998).
The present results demonstrate that from a pharmaco-logical and biochemical perspective T. tinca has a singleclass of [125I]Mel binding sites with nearly identical charac-teristics in both the neural retina and optic tectum. Thesesites fulfil the criteria required for a functional receptor,and the binding is rapid, saturable (Fig. 2a), stable, andreversible (Fig. 2b), and is inhibited by structurally relatedcompounds (Fig. 2c). The affinity (Kd = 17.47 and 22.31pM in neural retina and optic tectum, respectively) anddensity (Bmax = 48.07 and 33.76 fmol/mg prot. in neuralretina and optic tectum, respectively) for [125I]Mel indicates
NEURAL RETINA OPTIC TECTUM
15ºC
Kd = 22.50 ± 2.25
Bmax = 68.19 ± 2.97
[125
I]Mel (pM)
0 20 40 60 80 100 120 140 160
[125I]
Me
l b
ind
ing
(fm
ol/
mg
pro
t)
0
20
40
60
80
25ºC
Kd = 29.83 ± 3.58
Bmax = 74.09 ± 3.86
[125
I]Mel (pM)
0 20 40 60 80 100 120 140 160
[125I]
Mel
bin
din
g (
fmo
l/m
g p
rot)
0
20
40
60
80
**
30ºC
Kd = 27.37 ± 3.98
Bmax = 47.43 ± 3.05
[125
I]Mel (pM)
0 20 40 60 80 100 120 140 160
[125I]
Me
l b
ind
ing
(fm
ol/
mg
pro
t)
0
20
40
60
80
[125
I]Mel (pM)
0 20 40 60 80 100 120 140 160 180
[125I]
Me
l b
ind
ing
(fm
ol/
mg
pro
t)
0
10
20
30
40
50
60
7015ºC
Kd = 26.66 ± 2.54
Bmax = 59.33 ± 2.49
[125
I]Mel (pM)
0 20 40 60 80 100 120 140 160 180
[125I]
Mel
bin
din
g (
fmo
l/m
g p
rot)
0
10
20
30
40
50
60
70
25ºC
Kd = 42.49 ± 7.15
Bmax = 70.97 ± 5.47
[125
I]Mel (pM)
0 20 40 60 80 100 120 140 160 180
[125I]
Me
l b
ind
ing
(fm
ol/
mg
pro
t)
0
10
20
30
40
50
60
7030ºC
Kd = 33.01 ± 5.29
Bmax = 64.25 ± 4.44
a
b
c
Fig. 5. Effect of assay temperature on [125I]Mel binding. Saturation curves of neural retina and optic tectum of tench determined at (a) 15 �C, (b) 25 �C
and (c) 30 �C. Non-specific binding was determined by addition of 1 lM unlabeled melatonin. Each point represents individual values of [125I]Mel binding.
specific binding; total binding; non-specific binding. **p < 0.01 respect to other temperatures.
852 M.A. Lopez Patino et al. / General and Comparative Endocrinology 155 (2008) 847–856
that they belong to the low-capacity high affinity Mel1receptors. Moreover, Scatchard plots, linearity, and mono-phasic inhibition curves indicate that a homogeneous pop-ulation of melatonin binding sites exists in both the neuralretina and optic tectum of the tench. All these results agreewith previous studies carried out in the optic tectum andneural retina of Dicentrarchus labrax (Bayarri et al.,2004). Most studies in fish reported affinity values in thelow picomolar range (Kd < 90 pM), within the physiologi-cal range of circulating melatonin, but the density of recep-tors varies among teleost species, which is consistent withthe different plasma melatonin levels reported in fish (Fal-con, 1999).
Association (K+1) of [125I]Mel to its receptor in theoptic tectum and the neural retina of the tench occurs fas-
ter than dissociation (K�1). Maximum binding wasachieved within 60 min, but the time course of dissocia-tion of the labeled agonist was very low, as also occursin sea bream (Falcon et al., 1996) and pike (Gaildratet al., 1998). The K+1 and K�1 values are similar to thosereported in the same brain regions of the sea bass (Bayar-ri et al., 2004) and in the whole brain in pike (Gaildratet al., 1998) and the masu salmon (Amano et al., 2003).The dissociation constants (Kd), estimated from thekinetic studies for both optic tectum and neural retina,are similar to the Kd values obtained from saturationstudies and on the same order as ocular and circulatingmelatonin in this species (Guijarro, 2004), which supportsthe conclusion that such melatonin binding sites are func-tional receptors.
NEURAL RETINA
Time (min)
0 10 20 30 40 50 60 200
% [
125I]
Me
l b
ind
ing
0
20
40
60
80
100
120ASSOCIATION
Time (min)
0 100 200 300 400 500 1600
% [
125I]
Me
l b
ind
ing
0
20
40
60
80
100
120
DISSOCIATION
OPTIC TECTUM
Time (min)
0 20 40 60 80 250
% [
125I]
Me
l b
ind
ing
0
20
40
60
80
100
120ASSOCIATION
Time (min)
0 200 400 600 800 1600
% [
125I]
Me
l b
ind
ing
0
20
40
60
80
100
120
DISSOCIATION
15ºC
25ºC
30ºC
15ºC
25ºC
30ºC
a
b
Fig. 6. Effect of assay temperature (15, 25, and 30 �C) on the kinetics of [125I]Mel binding. (a) Association curves based on a pseudofirst-order exponential
rise, (b) dissociation curves based on a first-order exponential decay. Each point represents individual values of [125I]Mel binding. Dissociation curves were
started by addition of 1 lM unlabeled melatonin to membranes previously saturated with [125I]Mel.
Table 3
Parameters of [125I]Mel binding from saturation and kinetic assays in neural retina and optic tectum-tegmentum of Tinca tinca estimated at three different
temperatures
15 �C Q10 (15–25 �C) 25 �C Q10 (25–30 �C) 30 �C
K+1 (pM�1 min�1) · 10�3
Neural retina 0.371 ± 0.029 1.93 0.718 ± 0.045 4.05 1.446 ± 0.102
Optic tectum 0.214 ± 0.027 1.85 0.396 ± 0.020 1.27 0.446 ± 0.074
K�1 (min�1) · 10�3
Neural retina 0.860 ± 0.068 6.12 5.261 ± 0.328 2.97 9.075 ± 1.153
Optic tectum 0.446 ± 0.074 7.17 3.20 ± 0.27 0.74 2.751 ± 0.271
Results are shown as the estimation of rate constant (K+1, K�1) ± SE. Q10 coefficient was calculated for 15–25 �C and 25–35 �C temperature ranges by
using the Vant Hoff equation.
M.A. Lopez Patino et al. / General and Comparative Endocrinology 155 (2008) 847–856 853
The very similar kinetics of melatonin binding in tenchand frogs (Isorna et al., 2004a) leads us to suggest the exis-tence of similar receptor subtypes in both groups of verte-brates. Pharmacological characterization (Fig. 2c)supports such a hypothesis. Present results from neural ret-ina and optic tectum show that melatonin binding sites oftench have a high specificity for both 5-metoxy and N-acylsubstitutes (2-Ph-Mel, 2-I-Mel, 6-Cl-Mel, Mel), with thehighest affinity to the 2-substitutes (2-I-Mel and 2-Ph-Mel). Inhibition potency is lowest in other substitutes suchas 5-MCA-NAT and luzindole. The relative potencies ofanalogs in competition studies agree generally with previousresults carried out in the whole brain of other teleosts (Gail-drat et al., 1998; Amano et al., 2003; Bayarri et al., 2004).Moreover, the inhibition constants of 2-I-Mel, 2-Ph-Mel,6-Cl-Mel, and Mel are in agreement with the Ki valuesreported for frogs (Isorna et al., 2004a), birds (Reppertet al., 1995), and mammals (Pevet, 2003). The lack of inhibi-tion of [125I]Mel binding by the 5-MCA-NAT, a melatoninanalog selective for the low affinity melatonin receptor(MT3) in mammals, allows us to discard the possible exis-tence of aMT3 subtype receptor in the neural retina of tench.Moreover, the higherKi values for 4-P-PDOT and luzindolefound in tench with respect to the low-nanomolar range forthe mammalianMT2 subtype (Dubocovich et al., 1997) sup-port the lack of an MT2-like receptor in both neural retinaand optic tectum of tench. All these displacement studiesstrongly support the existence of a single class of melatoninreceptors in the optic tectum and the retina of the tench,which would correspond with the MT1 subtype. Neverthe-less, the pharmacology of melatonin receptors in lower ver-tebrates is unknown to date, and it could be different incomparison to homologous mammalian receptors. In fact,the distinct pharmacology exhibited by a MT2-like receptorcloned from the fish brain in relation to themammalianMT2
subtype (Gaildrat et al., 2002) supports such a hypothesis. Inthis sense, the slight but significant differences on receptorproperties in tench, such as the pharmacology of both 6-Cl-Mel and 4-P-PDOT, and thermokinetics at 25–30 �C,may suggest the existence of several molecular-related mem-bers of the same receptor subfamily in this species. Thisreceptor diversity has also been described in the Europeansea bass (Bayarri et al., 2004).
It is well known that in the G-protein coupled receptorsfamily, binding of an agonist to the receptor is decreased inthe presence of GTP or analogs. Our results on the inhibi-tion of the specific binding of [125I]Mel in tench neural ret-ina and optic tectum by the addition of GTPcS or sodium(Figs. 3 and 4) indicate that melatonin receptors in both tis-sues might correspond to receptors coupled to a hetero-meric G-protein. This result agrees with previous studiesin other fish species (Falcon et al., 1996; Gaildrat et al.,1998; Iigo et al., 1997a; Bayarri et al., 2004). The differentconcentrations of GTPcS inducing inhibition in tench com-pared to other teleost, like pike and sea bream, could be dueto the use of brain regions in tench versus the use of wholebrain preparations in those species. Moreover, the number
of G-protein coupled receptors in basal conditions can bespecies-dependent, as seen by comparing tench with pikeand sea bream, and tissue-dependent, indicated in tenchby comparison of the neural retina and the optic tectum.The binding of many G-protein coupled receptors is highlysensitive to the presence of monovalent cations in the bufferassay. Specifically, Na+ addition can modify receptor con-formation only in Gi/o protein coupled receptors (Reisine,1985). The Na+, independently of GTPcS, decreases Bmax
in the tench neural retina. This specific effect of NaCl addi-tion has not been previously studied in teleosts, only in theamphibian Rana perezi (Isorna et al., 2004a) and in the ratsuprachiasmatic nucleus (Laitinen and Saavedra, 1990),with similar results to those obtained in the tench.
Our results demonstrate, for the first time in fish, that tem-perature is a key factor in the interaction of melatonin withits receptor in both the neural retina and optic tectum (Figs.5 and 6). Thermal dependence of [125I]Mel binding to mela-tonin receptors has been reported in the frog R. perezi (Isor-na et al., 2004b, 2005), the chicken brain (Chong andSugden,1994), the ram pars tuberalis (Pelletier et al., 1990) and rabbitretina (Krause and Dubocovich, 1991), where temperatureaccelerates association process, in agreement with presentresults in fish. Association is more thermosensitive than dis-sociation in homeotherms, and thus, the affinity of [125I]Melbinding sites increases with temperature. However in poi-kilotherms like fish (present results) and frogs (Isornaet al., 2005) bothmelatonin association anddissociation pro-cesses are equally thermosensitive, and consequently, amechanism of thermal compensation is accurately produced(theKd remained constant throughout the temperature inter-val studied). This conservation of receptor Kd implies thatthe high thermosensitivity of ligand binding kinetics are sub-jected to a compensatory mechanism. In fact, the saturationcurves found at different temperatures (Fig. 5) in neural ret-ina and optic tectum support this thermal conservation ofreceptor affinity. Thus, it can be concluded that the thermo-dynamics of the melatonin binding to its receptors in theoptic tectum and retina is strongly dependent on tempera-ture in this teleost. Consequently, this environmental factormust be considered as a key component in the transductionof melatonin, especially as a seasonal signal.
Acknowledgments
This study was supported by the Spanish MEC (projectAGL 2004-08137-C04-01). Special thanks to the CentroNacional de Ciprinicultura ‘‘Vegas del Guadiana’’ for thedonation of the tench.
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