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GTP modulates [125I]iodomelatonin binding to a picomolar-affinity site in the Syrian hamster hypothalamus
European Journal of Pharntacology - Molecular Pharmacology.Section. 189 (19~0) 95-9~
95
Elsevier EJPMOL 80027 Short communicalion
GTP modulates [m lliodomelatonin binding to a picomolar-affini~, site in the Syrian hamster hypothalamus L e n n a r d P. Nfles D~partment of Biomedical Sciences, Division of Neuroscience, McMaster Unicers:tv, 1200 Atom Street We~l. Hamilton. Ontarto LSN 3Z5, Canada
Received 19 March 1990.accepted 28 March 1990
Saturation binding experiments conducted with [~251]iodomelalonin at 0-4°C in the Syrian hamster hypothalamus, revealed a single nanomolar-affinity site which was not affected by GTP. In comrast, incubation at 30°C revealed two distinct binding sites with picomolar and nanomolar affinities, respectively. GTP caused a significant decrease in the affinity of only the picomolar site but did not alter its density: control: Ka = 43 + 6 pM, Bm~,= 1.7 + 0.3 fmol/mg protein; GTP (1 mM): K a ~ 250 +_52, Bm~x = 3.9 _+2.6 fmol/mg protein. The foregoing indicates that the affinity of the putative melatonin recepter in the hamster hypothalamus is modulated by a regulatory G protein. GTP; G protein; [~251]lodorlelatonin binding; Hypothalamus; (Syrian hamster): (Picomolar-affinity sites)
1. Introduction
Recent studies, using [tzsI]iodomelatonin, have identified putative receptor sites for the pineal hormone, melatonin, in the hamster brain. These sites are particularly enriched in the hamster hypothalamus (Duncan et al., 1989; Picketing and Niles, 1990) in accordance with the view that this is the primary target area for melatonin's neuroendocfine effects. While the functional role of these binding sites awaits clarification, previous studies have indicated that melatonin decreases cyclic A M P (cAMP) levels in the rat hypothalamus (Vacas et at., 1981). ~,~loreover, in earlier single-point studies, we observed that G T P decreases [~251]iodomelatonin binding while, in the presence of this nuclcofide, melatonin inhibits forskolin-stimulated adenylate cyclase activity in the Syrian hamster Correspondence to: Dr. L.P. Nile,s, Department of Biomedical Sciences,Di,,~sionof Neuroscience,McMaster University,1200 Main Street West, Hamilton, Ontario LSN 3Z5, Canada~
hypothalamus (Niles et al., 1988). It was therefore of interest to further examine the effects of G T P on [~25I]iodomelatonin binding in the hypothalamus, since this guanine nucteotide modulates the binding affinity of receptors coupled to adenylate cyclase (Rodbel], 1980).
2. Materials and methods 2.1. Drugs
The radioligand, [l~'-~I]iodomelatonin, specific activity 1900-20f~ C i / m m o L was synthesized as previously reported (Niles et al., 1987). G T P was obtained from Sigma and 6-chloromelatonin was a gift frgm Dr. M. Flaugh (Lilly Research Laborateties). 2.2. Tissue preparation
Mate (2-3 m o n t h old) Syrian hamsters (Charles River) were individua||y housed under a i4 h light/10 h dark cycle with lights on at 0 6 : 0 0 h,
0922-4106/90/$03.50 © 1990 ElsevierScience Publishers B.V. (Biomedical D~Ssic,n)
96 and fed and watered ad fibitum. Animals were decapitated at 5-6 h after lights on and hypothalarrdc tissues were immediately prepared for assays. Tissues were homogenized in 50 mM TrisHCl buffer (pH 7.4 at 25°C) containing 0.1~ ascorbic acid, 1 mM EDTA and 2 mM MgCi 2, and centrifuged at 30 000 x g for 10 min. Homogenates were washed twice by resuspension in the above buffer followed by centrifugation and finally re.suspended in the same buffer at a concentration of abom 0.6-1 mg protdn/ml for binding assays.
2.3. Binding assay Single-point assays were carried out by incubating membranes with [125I]iodomelatonin at 0M°C for 60 mill in the absence or presence of GTP (1 raM) and/or NaCI (120 raM). Saturation binding assays utilized [12Sl]iodomelatonin in concentrations of 0.05-8 nM and 5-80 pM in the whole hypothalamas ( - 30 mg wet weight) and ventral hypothalamus (-10-15 mg wet weight), respectively. Assay tubes containing - 100-200 Pg protein ~n'e incubated at 0-4°C for 60 rain or at 30°C for 30 min with or without GTI~,~ (1 raM) as indicated. Specific binding was defined as that displaceable by 1 pM 6-chloromelatonin and represented 65-755g and 40-55~ of total bincing at 0-4°C and 30 ° C, respectively. Bound radioactivity was separated by rapid vacuum-filtration on Whatman G F / B filters and incubation tubes and filters were washed with 4 x 3.5 ml aliquots of ice-cold 50 mM Tris-HC1 (pH 7.4 at 4~C). Radioactivity was measured on a Searle gamma counter ( - 7 1 ~ efficiency). Protein concentrations were measured as previously described (Niles et aL, 1987).
3. Results An examination of the effects of 10 and 100 FM concentrations of various nucleotides on [125I]iodomelatonin ( - 100 pM) binding revealed that GTP and its more stable analog, GTPyS, significantly decreased binding (P < 0.01 vs. control n = 4 ; Student's t-test). In contrast, GMP
and ATP had no effects on binding indicating that the effect of GTP is specific. At an incubation temperature and time of 30 ° C x 30 min, the percent inhibition o! [t2Sl]iodomelatonin binding by GTP and GTPyS was, respectively, GTP (10 #M, 100 #M): 20 + 4, 28 + 6 and GTPyS (10/~M, 100 /tM): 35 + 7, 48 + 12 ( , = 4). In the present study; GTP was used at a concentration of 1 mM, which inhibited binding by about 42 + 8(P < 0.01 vs. control, n = 4), in order to compensa*~e for i~ rapid degradation pariieuI~ly at lfigher incubation temperatures (Oft and Costa, 1989). In single-point experiments carried out at 04°C, GTP (1 mM) decreased binding at all radioligand concentrations examined while NaCI (120 raM) was similarly effective with 60 pM of [~25I]iodomelatonin but not at higher concentrations. In keeping with the well known ability of Na + ions to augment the effects of GTP, the combined effects of these agents were additive but only at a concentration of 60 pM [t25I]iodomelatonin. Binding values at 60, 212 and 890 pM [125I]iodomelatonin were respectively as follows. Control: 1.4 + 0.I, 5.4 + 0.2 and 16.0 + 1.4 fmol/mg protein; NaCl: 0.54 + 0.06 *, 5.02 + 0.73 and 15.91 + 0.11 fmol/mg protein; GTP: 0.67 + 0.06 *, 2.77 + 0.09 * and 6.94 + 0.52 * fmol/mg protein; GTP + NaCI: 0.46 + 0.05 * *, 2.58 + 0.19 and 7.05 + 0.03 fmol/mg protein (* P < 0.01 vs. respective control; ** P < 0.05 vs. GTP, n = 3; Scheffe's test). In order to determine how GTP alters [~25I]iodomelatonin binding, its effects were examined in saturation experiments with incubations conducted at 0-4° C for 60 min. Under these conditions a single class of binding sites of nanomolar affinity was detected and GTP did not aiter the affinity or density of these sites; control: K d = 3.58 + 0.85 nM, Bma~ = 194.76 :[: 73; GTP: K d = 3.54 + 0.33, Bmax = 156.42 + 76 (n -- 3). On the assumption that the relatively high density of lower-affinity (nanomolar) sites might have masked the effects of GTP on the high-affinity (picomolar) sites for melatonin, further studies were conducted at 30°C, since higher incubation temperatures have permitted detection of sites with picomolar affinity in the hamster hypothalamus
97 TABLE 1 Effects of GTP on [z:~I]iodomelatoninbmmng in the hypothalamus. Fresh hypothalamiemembranes were prepared a~ described in Materials and methods and incubations were carried out at 30°C for 30 rain. Means±S,E,M. of three ,'~parate espcrimcnts conducted in duplicate are presented. * * P < 0.017 vs. contro' (unpaired Student's t-test). Treatment
Kdl(pM)
B~I (fmol/mg protein)
Control G T P ( I raM)
43.05:6.3 250 5:52 *~
1.75:0.3 3.9+2.6
(Vanecek and Jansky, 1989; Duncan et al., 1989). Saturation binding experiments conductea at 3 0 0 C for 30 rain (at which time equilibrium is achieved), produced curvilinear Scatchard plots. Computer analysis of saturation data indicated the presence of two binding sites with picomolar and nanomolar affinities, respectively. G T P significantly decreased the affinity of the high-affinity site but did not affect its density. G T P did not alter the affinity or density of the low-affinity site (table I).
/ .03 ~
L ~
Conl~ (0) GTP (O)
Kd 56 pM 115 pM
nmax 1,4 frnol/n1~Woteirl 2.0 tarot/ragpcOlc/n
.02
......
Kd~ (aM)
B.m2 (fmot/mg protein)
11.84- 3.9 12.2_+3.6
3114-68 247 4-5g
In order to confirm the effects of G T P on picomolar-affinity binding, a thi:d series of experiments was performed using ventral h)~aothalamic membranes, in which the median eminence is an enriched source of the picomolar bin& ing sites for melatonin (Vanecek and Jansky, 1989), Picomolar-affinity binding was saturable between 80-100 p M of the radioligand but substantial binding to lower-affinity sites occurred at radioligand concentrations exceeding 70-80 pM. Therefore, a range of - 2 - 8 0 pM [t~l]iodomelatonin was used in order to examine only high-affinity sites. Computer analysis of binding data indicated a single class of binding sites with picomolar affinity. G T P caused a significant decrease in the affinity of these sites but did not change their density (fig. 1). Binding parameters were for control: K d --- 40 - 7 pM, Bm~ = 1.0 ± 0.2 f m o l / m g prorein and for GTP: K~ = 131 +_ 3 * pM, B=~, = 1.7 + 0.3 f m o l / m g prote:m (* P < 0.001 v~. control, n = 3; unpaired Student's t-te~t). 4. D i s c ~ s i o n
IL
[ ..........
0
0.5
1.0
1.5
2.0
BOUNO 0tool!rag protein)
Fig. 1. Scatchard plots of [z~lJiodomelatonin binding ir~ the ventral hypothalamus with and without GTP (1 raM). Means of duplicate determinations are presented for one of three separate experiments.
The major f i n d i n ~ of this study are that both picomolar and nanomol~; affinity binding sites for [~25I]iodomelatonin are present in the S3~an hamster hypothalamus mM that the modulatory effects of G T P are restricted tc the picomolar site. The effect of G T P is due to a decrease in b i n ~ a g affinity as shown by the more than five-fold iacrease in me apparent dissociation constant (K~) for [i25I]iodomelatonin binding in the presence of this nucleotide while the binding site density ( B ~ ) remained unchanged (table I). The ability of guanine nucleotides to regulate melatonin receptors on the ov~me pars tuberalis (Morgan et ai.,
98 1989) and in lizard brain (Rivkees et al., 1989) has been reported recently. In these studies, a significant GTP S-induced decrease in the density of higla-affinity receptors for melatonin was observed with no change in binding affinity, In contrast, the present study indicates that GTP alters only the binding affinity of the putative melatonin receptor, as has been reported for other GTP-regulated receptors (Peroutka et al., 1979). Although single-point experiments conducted at 0-4°C (with picomolar concentrations of [1251]iodomelatonin) indicated that GTP decreased binding, tiffs effect was not seen in saturation binding studies carried out under similar incubation conditions (0-4°C for 60 min). q-kis appears to be due to the significantly greater density of nanomolar-affinity binding sites which are not affected by GTP and which mask the GTP-sensitive picomolar-affinity sites under these incubation conditions. In contrast, the GTP-sensitive picomolar-affinity binding sites in the hypothalamus were observed when saturation experiments were carded out at an incubation temperature of 30 ° C which favoured detection of high-affinity binding. In earlier studies, 2-iodomelatonin and other drugs produced shallow inhibition curves and low Hill coefficients, suggesting the presence of multiple binding sites in the hamster hypothalamus (Picketing and Niles, 1990). The ability of GTP to modify the affinity of [nsI]iodomelatonin binding sites suggests that our earlier detection of multiple sites in the hamster hypothalamus may be due to the presence of high- and low-affinity states of the putative melatonin receptor in this brain region. However, the existence of other receptor populations cannot be discounted at present. Since GTP plays a pivotal role in mediating the effects of various hormones and neurotransmitters on adenylate cyclase activity, its ability to modulate binding often indicates that the receptors involved are coupled to adenylate eyclase (Rodbell, 1980). Earlier studies have indicated that melatonin decreases cAMP levels in the rat hypothalamus (Vacas et al., 1981). Furthermore, we hav~ observed that p~comolar concentrations of melatoain inhibit forskolinozt~ul.~ted adenylate cyclase activity in the Syrian hamster hypothala-
mus (Niles et at., 1988). Tbfis effect was observed in the presence of GTP, in accordance with the importance of this nucleotide as discussed earlier. Taken together, these findings suggest that the high-affinity (picomolar) binding sites labelie~/by [1251]iodomelatonin in the Syrian hamster hypathalamus are functionally important mdatonin receptor sites negatively coupled to adenylate cyclase via a regulatory G protein.
Acknowledgements This study was supported by the Ontario Mental Health Foundation and the Medical Research Council of Canada. The author thanks Ms. Nola Miksza for typing the manuscript.
Referenees Duncan, M.J., J.S. Takahashi and M.L. Dubocovich, 1989, Characteristics and autoradiographic localization of 2l1251]iodomelatonin binding sites in Djungaxian hamster blain, Endocrinology 125, 1011. Morgan, P.J., W. Lawson, G. Davids,on and H.E. Howell. 1~ "3,Guanine nucleotidesregulate the affirdtyof melatonin receptors on the ovine pars tuberalis, Neurocndocrinology 50, 359. Niles, L.P., F.S. Hasheml and B.J. Mason, 1988, GTP raodulates melatoninbindingand its effectson adenylatecyclase in hamsterbrain, Soc. Neuro~i. Abstr. 14, 105. Niles. L.P., D.S. Picketing and B.G. Sa)~er,1987, HPLC-purifled 2-[12511iodomalatonin labels multiple binding sites in hamster brain, Biochem. Biophys. Res. Commun. 147, 949. Ott, S. and T. Costa, 1989, Enzymaticdegradation of GTP and its 'stable" analogues produce apparent isomerization of opioid receptors,J. Recept. Res. 9, 43. Peroutka, SJ., R.M. Lebovitzand S.H. Snyder, 1979, Sero~onin receptor binding sites affected differentially by guanine nucleotides, Mol. Pharmacol. 16, 700. Picketing, D.S. and L.P- Niles, 1990, Pharmacological characterization of melatonin binding sites in Syrian hamster hypothalamus, Euro~an J. Pharmacol. 175, 71. Rivkees. S.A., L.L. Carlson and S.M. Reppert, 1989, Guanine nucteotide-binding protein regulation of melatonin receptoil in lizard brain, Proc. Natl. Acad. Sci. U.S.A. 86, 3882. Rod!~ell, M. 1980, Role of hormone receptors and GTP-regu|atory proteins in membrane transduction, Nature 284, 17. Vacas, M.L, M.I. Keller-Sarmientoand D.P. Cardinali. 1981, MelatonL,l increams cGMP ~nd decreases cAMP levels in rat medial basal hypothalamus in vitro, Brain Res. 225, 207. Vanecek, J. and L. 3ansky. 1989, Short days induce changesin specific melatonin binding in hamster median eminence and anterior pituitary, Brain Res. 477, 387.
95
Elsevier EJPMOL 80027 Short communicalion
GTP modulates [m lliodomelatonin binding to a picomolar-affini~, site in the Syrian hamster hypothalamus L e n n a r d P. Nfles D~partment of Biomedical Sciences, Division of Neuroscience, McMaster Unicers:tv, 1200 Atom Street We~l. Hamilton. Ontarto LSN 3Z5, Canada
Received 19 March 1990.accepted 28 March 1990
Saturation binding experiments conducted with [~251]iodomelalonin at 0-4°C in the Syrian hamster hypothalamus, revealed a single nanomolar-affinity site which was not affected by GTP. In comrast, incubation at 30°C revealed two distinct binding sites with picomolar and nanomolar affinities, respectively. GTP caused a significant decrease in the affinity of only the picomolar site but did not alter its density: control: Ka = 43 + 6 pM, Bm~,= 1.7 + 0.3 fmol/mg protein; GTP (1 mM): K a ~ 250 +_52, Bm~x = 3.9 _+2.6 fmol/mg protein. The foregoing indicates that the affinity of the putative melatonin recepter in the hamster hypothalamus is modulated by a regulatory G protein. GTP; G protein; [~251]lodorlelatonin binding; Hypothalamus; (Syrian hamster): (Picomolar-affinity sites)
1. Introduction
Recent studies, using [tzsI]iodomelatonin, have identified putative receptor sites for the pineal hormone, melatonin, in the hamster brain. These sites are particularly enriched in the hamster hypothalamus (Duncan et al., 1989; Picketing and Niles, 1990) in accordance with the view that this is the primary target area for melatonin's neuroendocfine effects. While the functional role of these binding sites awaits clarification, previous studies have indicated that melatonin decreases cyclic A M P (cAMP) levels in the rat hypothalamus (Vacas et at., 1981). ~,~loreover, in earlier single-point studies, we observed that G T P decreases [~251]iodomelatonin binding while, in the presence of this nuclcofide, melatonin inhibits forskolin-stimulated adenylate cyclase activity in the Syrian hamster Correspondence to: Dr. L.P. Nile,s, Department of Biomedical Sciences,Di,,~sionof Neuroscience,McMaster University,1200 Main Street West, Hamilton, Ontario LSN 3Z5, Canada~
hypothalamus (Niles et al., 1988). It was therefore of interest to further examine the effects of G T P on [~25I]iodomelatonin binding in the hypothalamus, since this guanine nucteotide modulates the binding affinity of receptors coupled to adenylate cyclase (Rodbel], 1980).
2. Materials and methods 2.1. Drugs
The radioligand, [l~'-~I]iodomelatonin, specific activity 1900-20f~ C i / m m o L was synthesized as previously reported (Niles et al., 1987). G T P was obtained from Sigma and 6-chloromelatonin was a gift frgm Dr. M. Flaugh (Lilly Research Laborateties). 2.2. Tissue preparation
Mate (2-3 m o n t h old) Syrian hamsters (Charles River) were individua||y housed under a i4 h light/10 h dark cycle with lights on at 0 6 : 0 0 h,
0922-4106/90/$03.50 © 1990 ElsevierScience Publishers B.V. (Biomedical D~Ssic,n)
96 and fed and watered ad fibitum. Animals were decapitated at 5-6 h after lights on and hypothalarrdc tissues were immediately prepared for assays. Tissues were homogenized in 50 mM TrisHCl buffer (pH 7.4 at 25°C) containing 0.1~ ascorbic acid, 1 mM EDTA and 2 mM MgCi 2, and centrifuged at 30 000 x g for 10 min. Homogenates were washed twice by resuspension in the above buffer followed by centrifugation and finally re.suspended in the same buffer at a concentration of abom 0.6-1 mg protdn/ml for binding assays.
2.3. Binding assay Single-point assays were carried out by incubating membranes with [125I]iodomelatonin at 0M°C for 60 mill in the absence or presence of GTP (1 raM) and/or NaCI (120 raM). Saturation binding assays utilized [12Sl]iodomelatonin in concentrations of 0.05-8 nM and 5-80 pM in the whole hypothalamas ( - 30 mg wet weight) and ventral hypothalamus (-10-15 mg wet weight), respectively. Assay tubes containing - 100-200 Pg protein ~n'e incubated at 0-4°C for 60 rain or at 30°C for 30 min with or without GTI~,~ (1 raM) as indicated. Specific binding was defined as that displaceable by 1 pM 6-chloromelatonin and represented 65-755g and 40-55~ of total bincing at 0-4°C and 30 ° C, respectively. Bound radioactivity was separated by rapid vacuum-filtration on Whatman G F / B filters and incubation tubes and filters were washed with 4 x 3.5 ml aliquots of ice-cold 50 mM Tris-HC1 (pH 7.4 at 4~C). Radioactivity was measured on a Searle gamma counter ( - 7 1 ~ efficiency). Protein concentrations were measured as previously described (Niles et aL, 1987).
3. Results An examination of the effects of 10 and 100 FM concentrations of various nucleotides on [125I]iodomelatonin ( - 100 pM) binding revealed that GTP and its more stable analog, GTPyS, significantly decreased binding (P < 0.01 vs. control n = 4 ; Student's t-test). In contrast, GMP
and ATP had no effects on binding indicating that the effect of GTP is specific. At an incubation temperature and time of 30 ° C x 30 min, the percent inhibition o! [t2Sl]iodomelatonin binding by GTP and GTPyS was, respectively, GTP (10 #M, 100 #M): 20 + 4, 28 + 6 and GTPyS (10/~M, 100 /tM): 35 + 7, 48 + 12 ( , = 4). In the present study; GTP was used at a concentration of 1 mM, which inhibited binding by about 42 + 8(P < 0.01 vs. control, n = 4), in order to compensa*~e for i~ rapid degradation pariieuI~ly at lfigher incubation temperatures (Oft and Costa, 1989). In single-point experiments carried out at 04°C, GTP (1 mM) decreased binding at all radioligand concentrations examined while NaCI (120 raM) was similarly effective with 60 pM of [~25I]iodomelatonin but not at higher concentrations. In keeping with the well known ability of Na + ions to augment the effects of GTP, the combined effects of these agents were additive but only at a concentration of 60 pM [t25I]iodomelatonin. Binding values at 60, 212 and 890 pM [125I]iodomelatonin were respectively as follows. Control: 1.4 + 0.I, 5.4 + 0.2 and 16.0 + 1.4 fmol/mg protein; NaCl: 0.54 + 0.06 *, 5.02 + 0.73 and 15.91 + 0.11 fmol/mg protein; GTP: 0.67 + 0.06 *, 2.77 + 0.09 * and 6.94 + 0.52 * fmol/mg protein; GTP + NaCI: 0.46 + 0.05 * *, 2.58 + 0.19 and 7.05 + 0.03 fmol/mg protein (* P < 0.01 vs. respective control; ** P < 0.05 vs. GTP, n = 3; Scheffe's test). In order to determine how GTP alters [~25I]iodomelatonin binding, its effects were examined in saturation experiments with incubations conducted at 0-4° C for 60 min. Under these conditions a single class of binding sites of nanomolar affinity was detected and GTP did not aiter the affinity or density of these sites; control: K d = 3.58 + 0.85 nM, Bma~ = 194.76 :[: 73; GTP: K d = 3.54 + 0.33, Bmax = 156.42 + 76 (n -- 3). On the assumption that the relatively high density of lower-affinity (nanomolar) sites might have masked the effects of GTP on the high-affinity (picomolar) sites for melatonin, further studies were conducted at 30°C, since higher incubation temperatures have permitted detection of sites with picomolar affinity in the hamster hypothalamus
97 TABLE 1 Effects of GTP on [z:~I]iodomelatoninbmmng in the hypothalamus. Fresh hypothalamiemembranes were prepared a~ described in Materials and methods and incubations were carried out at 30°C for 30 rain. Means±S,E,M. of three ,'~parate espcrimcnts conducted in duplicate are presented. * * P < 0.017 vs. contro' (unpaired Student's t-test). Treatment
Kdl(pM)
B~I (fmol/mg protein)
Control G T P ( I raM)
43.05:6.3 250 5:52 *~
1.75:0.3 3.9+2.6
(Vanecek and Jansky, 1989; Duncan et al., 1989). Saturation binding experiments conductea at 3 0 0 C for 30 rain (at which time equilibrium is achieved), produced curvilinear Scatchard plots. Computer analysis of saturation data indicated the presence of two binding sites with picomolar and nanomolar affinities, respectively. G T P significantly decreased the affinity of the high-affinity site but did not affect its density. G T P did not alter the affinity or density of the low-affinity site (table I).
/ .03 ~
L ~
Conl~ (0) GTP (O)
Kd 56 pM 115 pM
nmax 1,4 frnol/n1~Woteirl 2.0 tarot/ragpcOlc/n
.02
......
Kd~ (aM)
B.m2 (fmot/mg protein)
11.84- 3.9 12.2_+3.6
3114-68 247 4-5g
In order to confirm the effects of G T P on picomolar-affinity binding, a thi:d series of experiments was performed using ventral h)~aothalamic membranes, in which the median eminence is an enriched source of the picomolar bin& ing sites for melatonin (Vanecek and Jansky, 1989), Picomolar-affinity binding was saturable between 80-100 p M of the radioligand but substantial binding to lower-affinity sites occurred at radioligand concentrations exceeding 70-80 pM. Therefore, a range of - 2 - 8 0 pM [t~l]iodomelatonin was used in order to examine only high-affinity sites. Computer analysis of binding data indicated a single class of binding sites with picomolar affinity. G T P caused a significant decrease in the affinity of these sites but did not change their density (fig. 1). Binding parameters were for control: K d --- 40 - 7 pM, Bm~ = 1.0 ± 0.2 f m o l / m g prorein and for GTP: K~ = 131 +_ 3 * pM, B=~, = 1.7 + 0.3 f m o l / m g prote:m (* P < 0.001 v~. control, n = 3; unpaired Student's t-te~t). 4. D i s c ~ s i o n
IL
[ ..........
0
0.5
1.0
1.5
2.0
BOUNO 0tool!rag protein)
Fig. 1. Scatchard plots of [z~lJiodomelatonin binding ir~ the ventral hypothalamus with and without GTP (1 raM). Means of duplicate determinations are presented for one of three separate experiments.
The major f i n d i n ~ of this study are that both picomolar and nanomol~; affinity binding sites for [~25I]iodomelatonin are present in the S3~an hamster hypothalamus mM that the modulatory effects of G T P are restricted tc the picomolar site. The effect of G T P is due to a decrease in b i n ~ a g affinity as shown by the more than five-fold iacrease in me apparent dissociation constant (K~) for [i25I]iodomelatonin binding in the presence of this nucleotide while the binding site density ( B ~ ) remained unchanged (table I). The ability of guanine nucleotides to regulate melatonin receptors on the ov~me pars tuberalis (Morgan et ai.,
98 1989) and in lizard brain (Rivkees et al., 1989) has been reported recently. In these studies, a significant GTP S-induced decrease in the density of higla-affinity receptors for melatonin was observed with no change in binding affinity, In contrast, the present study indicates that GTP alters only the binding affinity of the putative melatonin receptor, as has been reported for other GTP-regulated receptors (Peroutka et al., 1979). Although single-point experiments conducted at 0-4°C (with picomolar concentrations of [1251]iodomelatonin) indicated that GTP decreased binding, tiffs effect was not seen in saturation binding studies carried out under similar incubation conditions (0-4°C for 60 min). q-kis appears to be due to the significantly greater density of nanomolar-affinity binding sites which are not affected by GTP and which mask the GTP-sensitive picomolar-affinity sites under these incubation conditions. In contrast, the GTP-sensitive picomolar-affinity binding sites in the hypothalamus were observed when saturation experiments were carded out at an incubation temperature of 30 ° C which favoured detection of high-affinity binding. In earlier studies, 2-iodomelatonin and other drugs produced shallow inhibition curves and low Hill coefficients, suggesting the presence of multiple binding sites in the hamster hypothalamus (Picketing and Niles, 1990). The ability of GTP to modify the affinity of [nsI]iodomelatonin binding sites suggests that our earlier detection of multiple sites in the hamster hypothalamus may be due to the presence of high- and low-affinity states of the putative melatonin receptor in this brain region. However, the existence of other receptor populations cannot be discounted at present. Since GTP plays a pivotal role in mediating the effects of various hormones and neurotransmitters on adenylate cyclase activity, its ability to modulate binding often indicates that the receptors involved are coupled to adenylate eyclase (Rodbell, 1980). Earlier studies have indicated that melatonin decreases cAMP levels in the rat hypothalamus (Vacas et al., 1981). Furthermore, we hav~ observed that p~comolar concentrations of melatoain inhibit forskolinozt~ul.~ted adenylate cyclase activity in the Syrian hamster hypothala-
mus (Niles et at., 1988). Tbfis effect was observed in the presence of GTP, in accordance with the importance of this nucleotide as discussed earlier. Taken together, these findings suggest that the high-affinity (picomolar) binding sites labelie~/by [1251]iodomelatonin in the Syrian hamster hypathalamus are functionally important mdatonin receptor sites negatively coupled to adenylate cyclase via a regulatory G protein.
Acknowledgements This study was supported by the Ontario Mental Health Foundation and the Medical Research Council of Canada. The author thanks Ms. Nola Miksza for typing the manuscript.
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