short- versus long-term pinealectomy in the rat suprachiasmatic nuclei and pars tuberalis. Gauer F, Masson-Ptvet M, Pivet P. Differential regulation of melatonin.
Copyright 0 Munksguurd, 1994
Journal of Pineal Research
J Pineal Res 1994:16:73-76
ISSN 0742-3098
Printed in the United States of Americu--all rights reserved
Differential regulation of melatonin receptors by short- versus long-term pinealectomy in the rat suprachiasmatic nuclei and pars tuberalis Gauer F, Masson-Ptvet M, Pivet P. Differential regulation of melatonin receptors by short- versus long-term pinealectomy in the rat suprachiasmatic nuclei and pars tuberalis. J. Pineal Res. 1994: 16:73-76. Abstract: We have investigated the effects of short- and long-term pinealectomy on the density of melatonin receptors in both the suprachiasmatic nuclei (SCN) and pars tuberalis (PT) of the rat. Short-term pinealectomy (conducted 3-5 days before sacrifice) induced a significant increase in the density of melatonin receptors in both structures. This increase can be interpreted as an up-regulation of melatonin receptors following the absence of plasma melatonin. In contrast, 7 days after pinelectorny, the receptor density began to decrease, reaching a low point 1.5 months after pinealectomy. At this time, the decrease represents a 4045% reduction in binding sites as compared with values measured 3-5 days after pinealectomy. This slow decrease could be related to a slowdown of melatonin receptor synthesis. These results suggest that melatonin has a stimulatory effect on its own receptor synthesis. However, at 1.5 months after pinealectomy a novel equilibrium between synthesis and hydrolysis seems to be reached. Melatonin does not appear to be indispensable for the synthesis of its receptors.
Introduction
The hormone melatonin, secreted from the pineal gland only during the dark period of the 1ight:dark cycle [Rollag and Niswender, 1976; Wilkinson et al., 1977; Reiter, 19911 is known to be implicated in the regulation of many biological rhythms [Goldman and Darrow, 1983; Karsch et al., 1984; Tamarkin et al., 1985; PCvet, 19881. Using 2-['251]melatonin as a ligand [Vakkuri et al., 19841, researchers have shown that melatonin interacts with the neuroendocrine system via high affinity receptors located particularly in the pars tuberalis (PT) [Vanecek et al., 1987; Williams and Morgan, 1988; Morgan and Williams, 1989; Stankov et al., 19911 and the suprachiasmatic nulei (SCN) [Vanecek et al., 1987; Laitinen and Saavedra, 1989; Morgan and Williams, 1989; Stankov et al., 19911. The SCN are known to contain the circadian biological clock in mammals [Ralph et al., 19901 and to be implicated in the regulation of many circadian rhythms [Rusak and Zucker, 1979; Cassone, 1990; Meijer and Rietveld, 19891. The PT is supposed to
Fraqois Gauer,
Mireille Masson-Pevet, and Paul Pevet 'Neurobiologie des Fonctions Rythmiques et Saisonnieres," URA-CNRS 1332, Universite Louis Pasteur, Strasbourg, France
Key words: melatonin receptors-pineal gland-pinealectomy-pars tuberalissuprachiasmatic nuclei Address reprint requests to M. Masson-Pevet. 'Neurobiologie des Fonctions Rythmiques et Saisonnibres," URA-CNRS 1332, Universite Louis Pasteur, 12 Rue de I'Universit6, 67000 Strasbourg, France Received July 2, 1992; accepted September 28, 1993.
transduce the seasonally varying melatonin signal into seasonal functions [De Reviers et al., 1989; Vanecek and Jansky, 1989; Weaver and Reppert, 1990; Stanton et al., 1991; Nakazawa et al., 1991; Skene et al., 1993; Gauer et al., 1993bl. We have previously reported that melatonin directly regulates the density of its own receptors in both the SCN and PT by desensitization mechanisms [Gauer et al., 1993al. Absence of plasma melatonin, as a consequence of either constant light exposure for 3 days or pinealectomy 3 days prior to sacrifice, induced an increase in melatonin receptor density in both structures [Gauer et al., 1992a,b]. This effect could be related to hypersensitization of melatonin receptors due to the absence of its ligand melatonin. However, it has also been reported that 10-17 days after pinealectomy, there is no modification in the density of melatonin receptors in SCN [Laitinen et al., 19921. These results raise the possibility of a differential regulation of melatonin receptors by a short- versus a long-lasting absence of plasma melatonin.
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Gauer et al.
To test this hypothesis, we have investigated the effects of both short-term pinealectomy (e.g., conducted 3-5 days before sacrifice) and long-term pinealectomy (e.g., conducted several weeks or months before sacrifice) on the density of rat melatonin receptors in both the SCN and PT.
a 401
*
*
+
30
5D
7D
Material and methods
Thirty-five 1-month-old male Wistar rats (IffaCredo, 1' Arbresle, France) were maintained for 3 months prior to sacrifice in a 12-hr light:12-hr dark regime (LD 12:12, lights on at 0700). Food and water were provided at libitum. Rats were pinealectomized 3 months, l .5 months, 15 days, 7 days, 5 days, and 3 days before sacrifice. These animals and an intact group of the same age were sacrificed at the same dates by decapitation between 1530 and 1630. Brains were rapidly removed, frozen in -30°C isopentane and then kept at -30°C. Serial coronal sections (20-pm thick) of the hypothalamic regions containing the PT or SCN were cut on a cryostat, thaw-mounted onto gelatin-coated slides, and kept at -30°C until use. Sections were preincubated in 100 mM Tris buffer containing 4 mM CaCI, (pH 7.4) for 15 min at 4"C, incubated for 1 hr in the same buffer containing 2-[ '2sI]-melatonin (synthesized according to the method of Vakkuri [Vakkuri et al., 19841, and purified by HPLC) at room temperature. Afterward, sections were washed twice with agitation for 30 sec in assay buffer and then for 30 sec in distilled water at 4°C with agitation. Sections were air dried and apposed to hyperfilm (3H, Amersham France, les Vlis, France.) for 10 days together with 20-pm thick ','I microscale standards (Amersham). Quantitative analysis of the autoradiograms was performed as described previously [Gauer et al., 1993~1.The data obtained in fmol/mg polymer were converted in fmolimg protein according to the method of Nazarali et al. [ 19891. The saturation curve was analyzed by the equation Y = AX/B X, where A = B,,,, and B = K, (Graph-Pad). Statistical analysis of the data involved a one way analysis of variance followed by Duncan's multiple range test. Results were expressed as means ? SEM (n = 5).
+
Results
A marked increase in the density of melatonin receptors is observed in both the SCN and PT of animals 3 days and 5 days after pinealectomy (Fig. 1, Pt; Fig. 2, SCN). Seven days after pinealectomy, melatonin receptor density started to decrease. After 15 days, the specific binding measured was not 74
C
150
1.5M
3M
Fig. I . Specific binding of 2-[12'1]-melatonin in the PT of intact rats (C) and of animals pinealectomized 3 days before sacrifice (3D), 5 days (5D), 7 days (7D), 15 days (15D), 1.5 month ( 1 .SM), or 3 months before sacrifice (3M). 2-[ '*'I]Melatonin specific activity and concentration were: 1722 Ci/ mmol and 181 pM, respectively. Each point is the mean ? SEM of five animals. *P < 0.05, when compared with values of the control group; +f < 0.05, when compared with values of the 1.5 M and 3 M groups, + + P < 0.01, when compared with values of the 3D and 5D groups.
C
3D
5D
7D
15D
++
++
1.5M
3M
Fig. 2. Specific binding of 2-[1251]-melatoninin the SCN of intact rats (C) and of animals pinealectomized 3 days before sacrifice (3D), 5 days (5D), 7 days (7D), 15 days (15D), 1.5 month (ISM), or 3 months before sacrifice (3M). 2-['*'1]Melatonin specific activity and concentration were: 1722 Ci/ mmol and 181 pM, respectively. Each point is the mean ? SEM of five animals. * P < 0.05, when compared with values of the control group; + P < 0.05, when compared with values of the 1.5M and 3M groups; ++P < 0.01, when compared with values of the 3D, 5D, and 7D groups.
different from that found in intact animals. This decrease was maximal 1.5 month after pinealectomy. Values represented 58% (for SCN) and 55% (for PT), respectively, of binding measured in animals pinealectomized 3-5 days before sacrifice (Fig. 1,PT; Fig. 2, SCN). All variations of specific binding resulted in changes in B,,, without any change in K, (Tables 1, 2).
Long-term pinealectomy and melatonin receptors TABLE 1. Maximal density (Bmax)and Kd values of melatonin receptors in the SCN of intact (control) rats and rats pinealectomized for 3 days or 3 months before sacrificea Group
,,B,
(fmollmg .protein)
Control
5.4
3 Days 3 Months
7.7 f 0.4* 4.1 t 0.6**
&
0.7
Kd
(pM)
a7 t 14 111 t 9
74 2 32
a2-['251]-Melatonin specific activity and concentrations were: 1414 Ci/rnmol and 25-360 pM, respectively. Each value is the mean f SEM of five animals. * P < 0.05,when compared with control values. * * P < 0.01, when compared with 3 days values. TABLE 2. Maximal density (Bmax) and K, values of melatonin receptors in the PT of intact (control) rats or rats pinealectomized for 3 days or 3 months before sacrificea Group Control
3 Days 3 Months
,,B,
(fmol/mg protein)
56.0 ? 7.0 95.0 2 15.5' 45.6 -t 5.6+
Kd
(pM)
100 4 22 140 t 37 116t 30 .~
a2-['251]-Melat~ninspecific activity and concentrations were: 1414 Ci/mmol and 25-360 pM, respectively. Each value is the mean t SEM of five animals. *P< 0.05,when compared with control values. TP < 0.05,when compared with 3 days values.
of the pineal. This decrease, which is not due to age differences, since all rats were 4 months old, is too slow to be explained by desensitization mechanisms, but suggests a progressive diminution of receptor synthesis, which then, eventually, does not compensate any more receptor hydrolysis. Thus, this induces a decrease in the total number of receptors. After 1.5 months of continuous absence of plasma melatonin, a new equilibrium is apparently reached between receptor synthesis and hydrolysis, which keeps the receptor number constant. The level reached then is about 55% lower than the original level. These results seem to indicate that melatonin may play a role in stimulating the synthesis of its own receptors, but that its presence does not appear to be a requirement for this synthesis. A direct demonstration of a modulatory role of melatonin on the synthesis of its own receptors in the PT as well as in the SCN would, however, require the analysis of possible receptor mRNA fluctuations. Molecular cloning of melatonin receptor(s) is then a crucial step in understanding the regulatory mechanisms of the melatonin message. Acknowledgments
Discussion
Our present results demonstrate that short- and long-term pinealectomies do not have the same effect on the density of melatonin receptors in both the SCN and PT. Short-term pinealectomy (3-5 days before sacrifice), induces an increase in receptor density. This increase cannot be considered a result of a surgical stress, since a similar increase was demonstrated in both the SCN and PT of animals exposed to constant light for 3 days [Gauer et al., 1991a,b, 1993al. Furthermore, in both structures, this increase was reversed by a single melatonin injection [Gauer et al., 1993al. Thus, these results can be interpreted as an up-regulation of receptors caused by the suppression of plasma melatonin shortly after pinealectomy . Such recycling of receptors in absence of their endogenous ligand is well documented for a large number of G-protein-coupled receptors [Sibley et al., 19871. Since the melatonin receptors belong to the family of G-protein-coupled receptors [Carlson et al., 1989; Morgan et al., 1989, Laitinen and Saavedra, 1990; Gauer et al., 1992b; Skene et al., 19921, the initial melatonin up-regulation may be due to the unavailability of endogenous ligand. Approximately 7 days after pinealectomy , melatonin receptor density slowly starts to decrease until a minimal value is reached 1.5 months after removal
The authors wish to thank Drs. J. Stehle and V. Simonneaux for their critical comments and Mr. D. Bonn for taking care of the animals.
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