J. Pineal Res. 2005; 38:176–181
Copyright Blackwell Munksgaard, 2004
Journal of Pineal Research
Doi:10.1111/j.1600-079X.2004.00190.x
Effects of temperature on 2-[125I]-iodomelatonin binding to
melatonin receptors in the neural retina of the frog Rana perezi
Abstract:The present study analyzes the effect of temperature-dependent
modifications on the binding of the analog 2-[125I]-melatonin to melatonin
receptors in isolated neural retina membranes from the greenfrog Rana
perezi. Association and dissociation rate constants (K+1, K)1) were
exponentially increased by the assay temperature. At 15C, association and
dissociation required several hours; meanwhile, at 35C, rate constants were
100- and 34-fold faster, respectively. However, the Kd constant calculated as
K)1/K+1 was unmodified by the assay temperature. When frogs were
acclimated at either 5 or 22C for 1 month, K+1, and K)1 constants
determined at 15 and 25C were identical in both cold- and warm-acclimated
groups. Thus, the binding kinetics of melatonin receptors in frog retinas did
not shown any thermal compensation. Results from saturation curves and
pharmacological profiles of melatonin binding sites support a lack of effect of
assay temperature on the affinity of melatonin receptors in the frog retina.
The inhibition of [125I]Mel binding by GTPcS showed clearly that the
coupling of melatonin receptors to G proteins is temperature-dependent.
Higher concentrations of the GTP analog were needed to inhibit specific
binding when temperature decreased. The temperature effect on binding
kinetics and on the G protein coupling to melatonin receptors suggests that
the melatonin signal could be transduced distinctly depending on the
temperature. Thus, temperature plays a major role, not only on melatonin
synthesis, but also in the transduction of melatonin signal in ectotherms.
Introduction
The daily melatonin rhythm in ectotherm vertebrates shows
pronounced seasonal variations synchronized by environmental photoperiod and temperature [1–3]. In the study of
environmental regulation of melatonin synthesis in poikilotherms, temperature has been investigated as a key factor.
Some early studies demonstrate that temperature is responsible of the amplitude of the nocturnal melatonin peak in
the anuran Rana perezi [2]. High nocturnal temperature
enhances ocular melatonin production, both in vivo [2, 3]
and in vitro [4]. Moreover, melatonin production during the
photophase is low and independent of temperature.
Melatonin receptors have been characterized in several
tissues of vertebrates [5] and three subtypes have been
cloned (MT1, MT2 and Mel-1c), all belonging to the
superfamily of guanine nucleotide-binding protein (G
protein) coupled receptors [6, 7]. Melatonin binding sites
have been characterized recently in brain and neural retina
of the amphibian R. perezi. Saturation and pharmacological studies revealed a single, high affinity, low capacity site,
coupled to a G protein. These characteristics indicate the
existence of a typical Mel 1 receptor [8].
In spite of the large amount of information on the thermal
regulation of melatonin synthesis, little is known about the
effect of temperature on melatonin receptors. Marked
176
Esther Isorna, Ana Guijarro,
Marcos A. López-Patiño, Marı́a
Jesús Delgado, Mercedes
Alonso-Bedate and Angel L.
Alonso-Gómez
Departamento de Fisiologı́a (Fisiologı́a Animal
II), Facultad de Biologı́a, Universidad
Complutense, Madrid, España
Key words: frog, melatonin receptors, neural
retina, Rana perezi, temperature
Address reprint requests to Angel L. Alonso
Gómez, Departamento de Fisiologı́a (Fisiologı́a Animal II), Facultad de Biologı́a, UCM,
Ciudad Universitaria, Madrid 28040, Spain.
E-mail: [email protected]
Received June 9, 2004;
accepted September 20, 2004.
seasonal variations in melatonin functions have been
described in vertebrates, mainly in ectothermic species
[1, 9, 10]. However, no studies have investigated the effects
of temperature on melatonin binding to its receptors. Thus,
the aim of the present work was the thermal characterization
of melatonin receptors in the neural retina of R. perezi. We
have determined association and dissociation kinetic constants (K+1, K)1), affinity (Kd) and density (Bmax) of 2-[125I]melatonin ([125I]Mel) binding on membrane preparations of
frog retinas at different temperatures. We have also studied
possible temperature-dependent changes in pharmacological profiles of [125I]Mel binding. In order to investigate
whether melatonin binding to frog retina exhibits thermal
adaptation, the kinetics of melatonin binding was tested
after a thermal acclimation period. To complete the study,
the thermal dependence of the interaction between melatonin receptors and G protein has been determined.
Materials and methods
Chemicals
Radioligand 2-[125I]iodomelatonin (2000 Ci/mmol) was
purchased from Amersham International (Buckinghamshire, UK). 2-I-Mel, melatonin, N-acetylserotonin, guanosine 5¢-O-[c-thiotriphosphate] came from Sigma Chemical
Temperature and melatonin receptors in frog retina
Co. (St Louis, MO, USA). 2-Phenylmelatonin, and Nacetyl-2-benzyl-tryptamine (luzindole) were purchased
from Tocris Cookson Ltd (Bristol, UK). The remaining
reagents were of at least analytical grade.
Animals
Adult frogs (R. perezi) from Orense (Spain) were maintained in aquaria with dechlorinated water and were fed
with Calliphora sp. larvae twice a week. Unless otherwise
noted animals were kept under 12L:12D photoperiod and
22 ± 1C temperature conditions for at least 2 weeks
before the experiments.
For the thermal acclimation experiment, frogs were
maintained during 1 month at two temperatures, 5C
(n ¼ 6) or 22C (n ¼ 6), under the same 12L:12D photoperiod. At the end of acclimation, the animals were killed
during the early light phase of the photocycle (ZT ¼ 3).
Membrane preparation
Animals were killed during the light phase of the photocycle
and neural retinas were dissected, frozen on dry ice and
stored at )80C until used. The procedure for membrane
preparation was described previously [8]. Briefly, retinas
were sonicated (six pulses of 3 s at 30 w potency) in 100 lL
of assay buffer (50 mm Tris:HCl, 5 mm MgCl2, pH ¼ 7.4)
and centrifuged for 5 min at 800 g to eliminate melanin
granules. The supernatant was centrifuged for 10 min at
16,000 g to precipitate the cellular membranes. The pellet
was resuspended in 500 lL of assay buffer and centrifuged
again. Finally, the membranes were resuspended in 100 lL
of assay buffer and stored at )80C until the binding assays
were performed. All membrane manipulations were done at
4C. Protein concentration in the membranes was determined according to the Lowry’s method [11]. Neural retina
membranes were pooled for each experiment, except for
Experiment 3, where two pools of membranes were used
(cold- and warm-acclimated frogs, respectively).
[12] assuming a pseudofirst-order exponential rise to a
maximum for association, and a first-order exponential
decay for dissociation. The Kd from kinetic studies was
calculated as Kd ¼ K)1/K+1. The Q10 coefficient was
calculated as the ratio of the parameter measured at a
temperature difference of 10C (Q10 ¼ RT+10/RT) [13]. The
equilibrium dissociation constants (Kd) and binding densities (Bmax) from saturation studies, and IC50 values of
ligands from drug competition and GTPcS assays were
calculated by a nonlinear regression of a 4-parameters
logistic model using the ALLFIT program [14]. Inhibition
constant (Ki) was calculated from IC50 values by the
equation of Cheng and Prusoff [15]. Standard error (S.E.)
of constants was estimated from the residual sum of squares
in the least-squares fit. Statistical differences in Kd, Bmax
and Ki values were evaluated by the extra sum of squares
principle [16].
Results
Effect of temperature on [125I]Mel binding kinetics
Neural retina membranes were incubated at 15, 25 or 35C.
The association rates were determined by incubating with
the radioligand until the specific binding of [125I]Mel
reached steady state. To determine the dissociation rates,
1 lm melatonin was added to membranes previously
equilibrated with [125I]Mel, and the remaining specific
binding was quantified throughout incubation. The
[125I]Mel binding kinetics to neural retina of R. perezi is
extremely dependent on assay temperature (Fig. 1). Association of [125I]Mel reached the steady state within 15–20 min
at 35C, while more than 8 hr are needed when retinal
membranes were incubated at 15C. Dissociation is also
highly thermosensitive, [125I]Mel specific binding is displaced by the unlabelled melatonin in 1, 4 and more than
20 hr at 35, 25 and 15C, respectively. Association and
dissociation (K+1 and K)1) rate constants increased substantially as assay temperature increased, rendering very
high Q10 values (e.g. Q10 > 5, Table 1).
Binding assays
Assays were performed in a total volume of 50 lL. We used
[125I]Mel as radioligand, and unlabeled melatonin (1 lm) to
quantify the nonspecific binding. Radioligand concentrations, incubation time and temperature employed, are
specified in each experimental design and the respective
figure legend. The reaction was stopped by the addition of
ice-cold assay buffer (750 lL). Immediate vacuum filtration
through 25-mm glass fiber filters (Millipore, APFC) was
carried out using a Millipore 1225 cell harvester, and filters
were washed with ice-cold assay buffer (4 mL). Then, filter
disks were placed into vials and radioactivity was quantified
in a c counter (LKB, 1275 minigamma) with 75% efficiency.
Specific binding of [125I]Mel of each sample was calculated
by subtracting the nonspecific from the total binding.
Data analysis
The association and dissociation rate constants (K+1, K)1)
from kinetic studies were obtained by nonlinear regression
Influence of the temperature on affinity and density of
[125I]Mel binding
Neural retina membranes were incubated with increasing
[125I]Mel concentrations (3–140 pm) at the three different
temperatures (15, 25 or 35C). The incubation time was
8 hr, 90 min or 45 min, respectively, in order to reach
equilibrium at each temperature. Saturation curves carried
out at 15, 25 and 35C were almost identical (Fig. 2). In
fact, the respective values of Kd and Bmax were not
significantly different among the three temperatures
(Table 1). Thus, the conservation of receptor Kd implies
that the high thermosensitivity of [125I]Mel binding kinetics
must be subjected to a compensatory mechanism. This fact
can be clearly observed in Fig. 3. The correlation of K+1
and K)1 with assay temperature renders parallel lines with a
constant slope, indicating the exponential increase of both
rate constants. By contrast, the Kd values, calculated as
K)1/K+1, showed no correlation with temperature and the
corresponding Q10 values were near 1 (Table 1).
177
Isorna et al.
were estimated at 15 and 25C. The kinetic profiles of the
[125I]Mel binding were very similar in the neural retinas
from both cold- and warm-acclimated frogs. Thermal
acclimation did not change association and dissociation
constants (Table 2), which supports the idea that frog
retinas do not show any thermal compensation in response
to chronic thermal acclimation.
Thermal dependence of the pharmacological pattern
of [125I]Mel binding
Although assay temperature did not modify the receptor
Kd for [125I]Mel, the pharmacological profile could vary
depending on temperature. Competitive binding assays
with different melatonin agonists (2-Ph-Mel, 2-I-Mel,
Mel) and antagonists (Luzindole, NAS) were performed
at 15 or 25C. The displacement of specific [125I]Mel
binding by melatonin analogs is concentration-dependent
in the neural retina at both assay temperatures, and the
potency order of the drugs was the same: 2-Ph-Mel ¼ 2I-Mel > Mel luzindole > NAS. Corresponding Ki
values are shown in Table 3. The difference between
inhibition constants at 15 or 25C was less than a factor
of 2 and were not statistically significant for any of the
drugs tested.
Effect of temperature on G protein activation by
melatonin receptors
Fig. 1. Effect of temperature on the time course of association and
dissociation of [125I]Mel binding to neural retina membranes of
Rana perezi. Radioligand concentrations were in the range
60–75 pm. The arrow indicates the addition of unlabeled melatonin
(1 lm) to initiate dissociation. Each point represents radioligand
binding as the mean ± S.E.M. of triplicate determinations.
(—d—) association, and (- -.- -) dissociation curves.
The inhibition of [125I]Mel binding by various concentrations of GTPcS was used to quantify the coupling efficiency
of retinal melatonin receptors to G proteins. Inhibition
curves were carried out at 15, 25 and 35C, and the IC50 of
GTPcS was determined. GTP analog (GTPcS) reduced the
[125I]Mel binding in a concentration-dependent manner at
the three temperatures assayed (Fig. 4). Moreover, a clear
effect of temperature can be observed. The increase of assay
temperature produced a left-shift of the inhibition curves,
indicating a potentiation of inhibition. The IC50 values
changed from 3.5 lm at 15C to 0.5 lm at 25C and 0.1 lm
at 35C (Table 4). Moreover, the inhibition of the binding
was only partial but was augmented by temperature,
reaching 85% at 35C.
Effect of thermal acclimation on kinetics of binding
To test whether the R. perezi retina deployed mechanisms
of thermal adaptation to compensate the slow kinetics of
[125I]Mel binding at low temperature, frogs were acclimated
to 5C or 22C for 1 month, and the kinetic rate constants
Discussion
Knowledge concerning melatonin receptors in ectothermic
vertebrates has increased in recent years. There are several
studies, mainly in fish, that describe central [125I]Mel
Table 1. Parameters of [125I]Mel binding from saturation and kinetic assays in neural retina of Rana perezi estimated at three different
temperatures
15C
K+1 (1/p m · min) · 10)3
K)1 (1/min) · 10)3
Kinetic Kd (pm)
Kd (pm)
Bmax (fmol/mg prot)
0.050
1.55
31.1
15.29
9.61
± 0.003
± 0.19
± 1.93
± 0.49
Q10
20.4
5.3
0.3
25C
1.02
8.20
8.04
19.74
9.31
± 0.12
± 0.64
± 3.61
± 0.70
Q10
5.3
6.4
1.3
35C
5.14
52.6
10.23
19.44
9.18
± 0.24
± 3.8
± 2.91
± 0.59
Results are shown as the estimation of the parameter ±S.E. Q10 coefficient was calculated for 15–25 and 25–35C temperature ranges.
178
Temperature and melatonin receptors in frog retina
Fig. 2. Effect of temperature on equilibrium saturation curve of
[125I]Mel binding to the membrane preparations from neural retina
of Rana perezi. Each point represents radioligand binding as the
mean ± S.E.M. of triplicate determinations.
Fig. 3. Correlation of kinetic constants of [125I]Mel binding with
assay temperature from the neural retina of Rana perezi. Parameters (K+1, K)1 and Kd) obtained in kinetic assays are represented in
function of the incubation temperature. Lines represent the linear
regression.
binding sites [17–20]. Also, melatonin receptors in some
ectotherms have been cloned [18, 19, 21].
To our knowledge, this is the first study showing the
thermal dependence of [125I]Mel binding to melatonin
receptors in a poikilothermic vertebrate. The importance
of studying temperature as a key factor of the ligandreceptor interaction lies in the fact that the thermodynamics
of the binding in vivo is, in contrast to homeotherms,
determined by environmental temperature.
Mel 1-like receptors have been identified in the brain and
retina of the frog R. perezi and characterized in detail at the
Table 2. Association and dissociation
rate constants of [125I]Mel binding from
neural retina membranes measured of
cold- and warm-acclimated Rana perezi
frogs at 15C and 25C
standard assay temperature of 25C [8]. In the present
work, we extend the receptor characterization to a wide
temperature range (15–35C). Thermal dependence of
[125I]Mel binding association rate has been described in
very few studies. Temperature accelerates the binding
process from several hours at low temperature (0–4C) to
a few minutes at high temperatures (37C) [22, 23]. The frog
retinas exhibited a similar pattern, but compared with rams
or chicken, frog receptors appear to be much more
thermosensitive, an increase of only 10C accelerated the
association rate more than 5 times (Table 1).
Ligand dissociation is also a thermosensitive process
(Table 1, Fig. 3). The Q10 values obtained in the present
study were higher than expected for most physiological
processes in poikilotherms [13]. Hence, it can be assumed
that signal transduction should be strongly affected because
of the very slow kinetics of melatonin receptors at temperatures lower than 15C. Consequently, the reading of the
melatonin daily rhythm may be impaired at such low
temperatures.
On the contrary, association and dissociation rates were
accurately compensated. In fact, the kinetic Kd, indicative
of binding affinity, was almost constant in the 15–35C
range (Q10 values are near 1). This thermal conservation of
receptor Kd is supported by the saturation curves obtained
at different temperatures (Fig. 2, Table 1). This fact has
thermodynamic consequences on binding. The changes in
binding enthalpy and entropy induced by temperature must
be compensated exactly in order to keep the standard Gibbs
free energy, and consequently the Kd, constant.
The results obtained in R. perezi retinas differ substantially from data from homeotherms [24]. In chicken brain
and retina and rabbit retina, the affinity of [125I]Mel binding
sites increases steadily with the temperature. There is a 10fold decrease of Kd in the range 0–37C, that is, the two rate
constants are not compensated, with the association rate
more thermosensitive than the dissociation rate in these
homeotherms.
Nevertheless, the temperature effect appears to be more
complex. Although the maximal capacity of [125I]Mel
binding sites (Bmax) in chicken brain membranes was not
affected by temperature, Kd showed a minimum at 21C
with higher values at both higher and lower temperatures
giving curvilinear van’t Hoff plots [23]. This result revealed
a distinct [125I]Mel binding thermal sensitivity in chicken
and frogs. In the case of poikilotherms, their body
temperature changes with the environmental temperature,
and adaptive strategies were developed to maintain the
receptor functionality, at least in the physiological range of
Assay temperature
15C
25C
Acclimation temperature
22C
5C
22C
5C
K+1 (1/p m · min) · 10)3
K)1 (1/min) · 10)3
Kinetic Kd
0.11 ± 0.01
2.06 ± 0.17
18.78
0.11 ± 0.01
1.64 ± 0.21
14.84
0.31 ± 0.04
10.49 ± 0.76
34.42
0.36 ± 0.04
7.72 ± 1.10
21.57
Results are shown as the estimation of the parameter ±S.E.
179
Isorna et al.
Table 3. Comparison of inhibition constants of melatoninergic
ligands in neural retina from Rana perezi determined at 15 and
25C
Ligands
Ki (pm)
2-Phenyl-melatonin
2-Iodo-melatonin
Melatonin
Ki (nm)
Luzindole
N-acetyl-serotonin
15C
25C
1.25 ± 0.28
8.55 ± 1.67
137.40 ± 23.53
1.93 ± 0.38
7.98 ± 1.39
208.25 ± 40.61
241.20 ± 39.37
556.03 ± 85.75
317.76 ± 47.33
487.17 ± 77.51
Results are shown as the estimation of parameter ±S.E.
Fig. 4. Inhibition by GTPcS of [125I]Mel binding from retinal
membranes of Rana perezi at three different temperatures. Data are
expressed as a percentage of specific binding in the absence of
GTPcS at each assay temperature. Each point represents the
mean ± S.E.M. of triplicate determinations.
Table 4. IC50 values and maximal inhibition of specific [125I]Mel
binding by GTPcS from neural membranes of Rana perezi at
different temperatures
Assay temperature
Maximal
inhibition (%)
IC50 (lm)
15C
25C
35C
69.5 ± 7.4
71.5 ± 4.9
84.5 ± 2.2
3.53 ± 0.83
0.48 ± 0.16
0.095 ± 0.014
Results are shown as the estimation of parameter ±S.E.
temperatures (15–35C). On the contrary, the receptors of
homeotherms do not require this thermal compensation.
The conservation of the Kd was demonstrated for the
ligand [125I]Mel. Nevertheless, other melatoninergic ligands, like melatonin itself, may show differential affinity
changes dependent on temperature. This attribute has been
described in other G-protein coupled receptors, such as
dopamine receptors [25], where binding of agonists is
enthalpy-driven (dependent on temperature). Meanwhile
binding of antagonists is less temperature-sensitive and
entropy-driven. The ligands tested in neural retina of R.
perezi have very different chemical structure and functional
properties. 2-Ph-Mel, 2-I-Mel and Mel are full agonists,
NAS is a weak partial agonist and luzindole is an
antagonist [24]. However, the potency order of such drugs
on the inhibition of [125I]Mel binding was identical at 15
and 25C, in agreement with the classic pharmacology of
180
Mel 1-like receptors [8]. There were no significant differences in Ki values at the two assay temperatures for any of
the drugs tested. These results reinforce the hypothesis that
the affinity of melatonin receptors does not change with
temperature, regardless of the ligand structure. Hence, the
hydrophobic and nonhydrophobic interactions of the
ligand-receptor must be well compensated in frogs, in
contrast to other species [23].
Temperature plays an important role in melatonin
rhythm regulation in poikilotherms. It is known that
melatonin synthesis is reduced at low temperatures in R.
perezi [2–4], and low acclimation temperatures increased
the diurnal and nocturnal in vitro activity of ocular
serotonin N-acetyltransferase (AA-NAT), an essential
enzyme in the pathway of melatonin synthesis. This
response was interpreted as an adaptation to low temperature [3, 26, 27]. However, the conservation of the association and dissociation rates in thermal acclimation
experiment (Table 2) precludes a thermal adaptation of
melatonin receptor to compensate the slow kinetic binding
at low temperature.
In R. perezi, it has been demonstrated that melatonin
binding sites are sensitive to GTP analogs and pertussis
toxin [8]. As an attempt to investigate how temperature
affects the following step of the signal transduction (G
protein activation) after melatonin binding, we measured
the inhibition of specific [125I]Mel binding to retinal
membranes of R. perezi by GTPcS at different temperatures. The IC50 values (Table 4) showed that the coupling
of melatonin receptors to G proteins is temperature
dependent. Higher concentrations of the GTP analog are
necessary to inhibit specific binding when temperature
decreases (Fig. 4). Then, this response suggests that temperature might affect the transduction of melatonin signal,
at least in frogs.
In conclusion, we propose that the effect of the temperature on seasonal melatonin actions in R. perezi [9, 10] may
be due not only to changes in melatonin production but
also to a direct effect of temperature on the melatonin
signaling pathway. Temperature does not affect the steadystate properties of a receptor (Bmax or Kd) in frog retinas,
but the kinetics of binding and the coupling of G protein to
melatonin receptors were strongly affected. Second messengers (G proteins) could have different responses to
melatonin signals depending on the temperature, and in
consequence the same melatonin concentration could have
different effects on the target tissue as a consequence of
environmental temperature. Further studies are needed to
determine how temperature modifies the physiological
response to melatonin in poikilotherms.
Acknowledgments
This study was supported by Project No. BFI2001-1368
from the Spanish MCT. E. Isorna is a recipient of a
doctoral fellowship from the Spanish MECD.
References
1. Falcón J. Cellular circadian clocks in the pineal. Prog Neurobiol 1999; 58:121–162.
Temperature and melatonin receptors in frog retina
2. Delgado MJ, Vivien-Roels B. Effect of environmental
temperature and photoperiod on the melatonin levels in the
pineal, lateral eye, and plasma of the frog, Rana perezi:
importance of ocular melatonin. Gen Comp Endocrinol
1989; 75:46–53.
3. Delgado MJ, Alonso-Gómez AL, Gancedo B et al. Serotonin N-acetyltransferase (NAT) and melatonin levels in the
frog retina are not correlated during the seasonal cycle. Gen
Comp Endocrinol 1993; 92:143–150.
4. Valenciano AI, Alonso-Gómez AL, Alonso-Bedate M
et al. Effect of constant and fluctuating temperature on daily
melatonin production by eyecups from Rana perezi. J Comp
Physiol B 1997; 167:221–228.
5. Vanecek J. Cellular mechanisms of melatonin action. Physiol
Rev 1998; 78:687–721.
6. Ebisawa T, Karne S, Lerner MR et al. Expression cloning of
a high-affinity melatonin receptor from Xenopus dermal melanophores. Proc Natl Acad Sci USA 1994; 91:6133–6137.
7. Reppert SM, Weaver DR, Godson C. Melatonin receptors
step into the light: cloning and classification of subtypes.
Trends Pharmacol Sci 1996; 17:100–102.
8. Isorna E, Guijarro AI, Delgado MJ et al. Characterization of melatonin binding sites in the brain and retina of
the frog Rana perezi. Gen Comp Endocrinol 2004; 135:259–
267.
9. Alonso-Gómez AL, Tejera M, Alonso-Bedate M et al.
Response to pinealectomy and blinding in vitellogenic female
frogs (Rana perezi) subjected to high temperature in autumn.
Can J Physiol Pharmacol 1990; 68:94–98.
10. Delgado MJ, Alonso-Gómez AL, Alonso-Bedate M. Role
of environmental temperature and photoperiod in regulation
of seasonal testicular activity in the frog Rana perezi. Can J
Physiol Pharmacol 1992; 70:1348–1352.
11. Lowry OH, Rosenbrough NJ, Farr AL et al. Protein
measurement with the folin phenol reagent. J Biol Chem 1951;
193:265–275.
12. Duggleby RG. A nonlinear regression program for small
computers. Anal Biochem 1981; 110:9–18.
13. Rome LC, Stevens ED, John-Alder HB. The influence of
temperature and thermal acclimation on physiological function. In: Environmental Physiology of the Amphibians. Feder
ME, Burggren WW eds. The University of Chicago Press,
Chicago, 1992; pp. 183–205.
14. De Lean A, Munson PJ, Rodbard D. Simultaneous analysis
of families of sigmoidal curves: application to bioassay, radi-
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
oligand assay, and physiological dose–response curves. Am J
Physiol 1978; 235:E97–E102.
Cheng YC, Prusoff WH. Relationship between the inhibition
constant (KI) and the concentration of inhibitor which causes
50 per cent inhibition (I50) of an enzymatic reaction. Biochem
Pharmacol 1973; 22:3099–3108.
Draper NR, Smith H. Applied Regression Analysis. Wiley,
New York, 1966.
Gaildrat P, Ron B, Falcón F. Daily and circadian variations in 2-125I-iodomelatonin binding sites in the pike brain
(Esox lucius). J Neuroendocrinol 1998; 10:511–517.
Mazurais D, Brierley I, Anglade I et al. Central melatonin
receptors in the rainbow trout: comparative distribution of
ligand binding and gene expression. J Comp Neurol 1999;
409:313–324.
Gaildrat P, Falcón F. Melatonin receptors in the pituitary
of a teleost fish: mRNA expression, 2-125I-iodomelatonin
binding and cyclic AMP response. Neuroendocrinology 2000;
72:57–66.
Amano M, Iigo M, Ikuta K et al. Daily variations in melatonin binding sites in the masu salmo brain. Neurosci Lett
2003; 350:9–12.
Wiechmann AF, Campbell LD, Defoe M. Melatonin
receptor RNA expression in Xenopus retina. Mol Brain Res
1999; 63:297–303.
Pelletier J, Castro B, Roblot G et al. Characterization of
melatonin receptors in the ram pars tuberalis: influence of
light. Acta Endocrinol 1990; 123:557–562.
Chong NWS, Sugden D. Thermodynamic analysis of agonist
and antagonist binding to the chicken brain melatonin receptor. Br J Pharmacol 1994; 111:295–301.
Krause DN, Dubocovich ML. Melatonin receptors. Annu
Rev Pharmacol Toxicol 1991; 31:549–568.
Agui T, Amlaiky N, Caron MG et al. Binding of [125I]-it N(p-aminophenethyl) spiroperidol to the D-2 dopamine receptor
in the neurointermediate lobe of the rat pituitary gland: a
thermodynamic study. Mol Pharmacol 1988; 33:163–169.
Alonso-Gómez AL, Alonso-Bedate M, Delgado MJ.
Thermal sensitivity and effect of temperature acclimation on
ocular serotonin N-acetyltransferase activity in Rana perezi.
Neurosci Lett 1992; 142:187–190.
Alonso-Gómez AL, Alonso-Bedate M, Delgado MJ. The
inhibition by indoleamines (tryptamine and serotonin) of
ocular serotonin N-acetyltransferase from Rana perezi is temperature-dependent. Neurosci Lett 1993; 155:33–36.
181