Proc. Natl. Acad. Sci. USAVol. 84, pp. 3916-3920, June 1987Neurobiology

Use of 2-[1251]iodomelatonin to characterize melatonin binding sitesin chicken retina

(receptor/radioligand/N-acetyltryptamine/indole ethylamine)

MARGARITA L. DUBOCOVICH*t AND JOSEPH S. TAKAHASHIt*Department of Pharmacology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611; and tDepartment of Neurobiology andPhysiology, Northwestern University, Hogan Hall, Evanston, IL 60201

Communicated by Colin S. Pittendrigh, February 12, 1987

ABSTRACT 2-['25I]Iodomelatonin binds with high affinityto a site possessing the pharmacological characteristics of amelatonin receptor in chicken retinal membranes. The specificbinding of 2-[125I]iodomelatonin is stable, saturable, and re-versible. Saturation experiments indicated that 2-[125I]iodo-melatonin labeled a single class of sites with an affinity constant(Kd) of 434 ± 56 pM and a total number of binding sites (B.,)of 74.0 ± 13.6 fmol/mg of protein. The affinity constantobtained from kinetic analysis was in close agreement with thatobtained in saturation experiments. Competition experimentsshowed a monophasic reduction of 2-[1251]iodomelatonin bind-ing with a pharmacological order of indole amine affinitiescharacteristic of a melatonin receptor: 2-iodomelatonin >6-chloromelatonin : melatonin , 6,7-dichloro-2-methylmela-tonin > 6-hydroxymelatonin > 6-methoxymelatonin > N-acetyltryptamine > N-acetyl-5-hydroxytryptamine > 5-meth-oxytryptamine >>> 5-hydroxytryptamine (inactive). The af-fmities of these melatonin analogs in competing for 2-[1251]iodo-melatonin binding sites were correlated closely with theirpotencies for inhibition of the calcium-dependent release of[3H]dopamine from chicken and rabbit retinas, indicatingassociation of the binding site with a functional responseregulated by melatonin. The results indicate that 2-[1251]iodo-melatonin is a selective, high-affinity radioligand for theidentification and characterization of melatonin receptor sites.

A body of literature suggests that the hormone melatonin(N-acetyl-5-methoxytryptamine) regulates a number ofphys-iological processes in vertebrates. These include the regula-tion of reproduction in photoperiodic mammals (1, 2), thecontrol of circadian rhythms in birds and reptiles (3-5), andthe modulation of retinal physiology. Melatonin has beenfound in the retina of several species (6-10). Retinalmelatonin has been implicated in photoreceptor outer seg-ment disc shedding and phagocytosis (11, 12), melanosomeaggregation in pigment epithelium (13), and cone photorecep-tor retinomotor movement (14). Because so many importantbiological processes are regulated by melatonin, the mecha-nism by which this hormone acts is of great interest. Under-standing of this mechanism has been hampered by severaltechnical difficulties, perhaps the most important of which isthe lack of a soundly based pharmacology. Dubocovich(15-17) has shown that picomolar concentrations ofmelatonin selectively inhibit the calcium-dependent releaseof dopamine from rabbit and chicken retina through activa-tion of a site possessing the pharmacological and functionalcharacteristics of a receptor. Although binding sites for[3H]melatonin have been reported in cytosolic and membranefractions of several central nervous system tissues includingthe retina of lower vertebrates, these sites have not beenassociated with biological effects of melatonin and related

indoles (10, 18-21). Further, the relatively low specificactivity of [3H]melatonin, compared to that of radioiodinatedligands, may have hindered detection of high-affinity bindingsites in tissues with a low density of receptors (10). Toenhance the ability to detect melatonin receptor sites invertebrate retina, we have utilized the radioiodinated ligand2-['25I]iodomelatonin (22). Here we report the characteristicsof binding of 2-[125I]iodomelatonin to chicken retinal mem-branes and demonstrate a pharmacological correlation be-tween the binding site labeled by 2-['25I]iodomelatonin and afunctional response regulated by melatonin in the chickenand rabbit retina.

MATERIALS AND METHODSMaterials. 2-['25I]Iodomelatonin was synthesized by a

modification (J.S.T., S. S. Nikaido, and M.L.D., unpub-lished) of the method of Vakkuri et al. (22). The specificactivity of the radioligand was 1800-2175 Ci/mmol (1 Ci = 37GBq) and was stable for 60 days. Purity ofthe radioligand waschecked by TLC and was >95%. 2-Iodomelatonin wassynthesized by the method of Vakkuri et al. (23) and purifiedby silica gel chromatography. The purity of this compoundwas shown to be greater than 98% by HPLC. 6-Chloromel-atonin, 6,7-dichloro-2-methylmelatonin, and N-acetyltrypt-amine were donated by J. Clemens (Eli Lilly); N-acetyl-5-methoxykynurenamine by D. E. Clark (College of Pharma-cy, University ofHouston, TX); and 6-methoxymelatonin byD. C. Klein (Laboratory of Developmental Endocrinology,National Institutes of Health, Bethesda, MD). Other drugswere obtained from commercial sources or the pharmaceu-tical company of origin.Membrane Preparation. Chickens (4-6 weeks old) main-

tained in a controlled lighting regime (14 hr light/10 hr dark)were decapitated during the light phase. Retinas were dis-sected free of pigment epithelium and homogenized in ice-cold 50 mM Tris HCl buffer, pH 7.5 (250C)/0.1% ascorbicacid with a Brinkmann Polytron PT-S at setting 5 for 10 sec.The homogenate was centrifuged at 50,000 x g for 10 min at40C. The pellet was washed once by resuspension andcentrifugation in Tris-HCl buffer. The retinal membranepellet was resuspended by homogenization at a concentrationof 500 ,ug of protein per ml.Where indicated, retinal homogenates were fractionated

by differential centrifugation at 40C to yield a crude nuclearpellet (P1, 1200 x g for 10 min), a crude mitochondrial pellet(P2, 27,000 x g for 20 min), and a crude microsomal pellet (P3,100,000 x g for 60 min). The pellets were resuspended byhomogenization in ice-cold 50 mM Tris-HCl buffer, pH7.5/0.1% ascorbic acid and used in binding assays.

Binding Assays. For binding assays, 2-[1251]iodomelatoninwas diluted in Tris HCl buffer with 0.01% bovine serumalbumin, and drugs were dissolved in 1 mM HCl with 0.1%

tTo whom reprint requests should be addressed.

3916

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Proc. Natl. Acad. Sci. USA 84 (1987) 3917

bovine serum albumin. Binding was initiated by addition of220-Al aliquots of membranes resuspended in the Tris HClbuffer to tubes containing 20 A.l of appropriate 2-[l251]iodo-melatonin concentrations and 20 1.l of drugs or vehicle.Unless otherwise indicated the binding of 2-['251]iodomela-tonin was routinely measured in duplicate after incubation at0C for 5 hr in the dark. Reactions were terminated byaddition of 5 ml of ice-cold Tris HCl buffer, and the contentswere immediately filtered through glass-fiber filters(Schleicher & Schuell no. 30) soaked in 0.5% (vol/vol)polyethylenimine solution. Each filter was washed twice with5 ml of the cold buffer. Radioactivity was determined in agamma counter. Nonspecific binding, unless otherwise indi-cated, was defined as binding in the presence of 3 AM6-chloromelatonin. Specific binding of 2-[125I]iodomelatoninwas calculated by subtracting nonspecific binding from totalbinding and expressed as fmol/mg of protein. In a typicalexperiment, total 2-[125I]iodomelatonin (67 pM) binding was2542 ± 361 cpm (n = 3), and the nonspecific binding definedwith 3 AM 6-chloromelatonin was 350 ± 35 cpm (n = 3). Thetotal radioactivity bound to filters represented 166 ± 10 cpm(n = 3).

Identity of Bound Ligand. Immediately following a filtra-tion binding assay, filters were removed and placed in 1 ml ofmethanol. The methanol extract was evaporated under ni-trogen to a volume of 100 ,Al. Melatonin (10 ,4g) was added asa carrier and 50 jul was applied to a silica gel TLC plate(Kodak 13179). Chromatograms were developed with ethylacetate. The TLC plate was cut into 0.5-cm segments andradioactivity was quantified. The peak of radioactivity wascompared with the position of authentic 2-[125I]iodomelato-nin.

Calculations. Kinetic data were analyzed by the method ofBennett and Yamamura (24), using pseudo-first-order con-ditions to estimate the association rate constant (kj) andaddition of a 1000-fold molar excess of unlabeled ligand toestimate the dissociation rate constant (kL). Data for satu-ration and competition experiments were analyzed by use ofthe EBDA/LIGAND program (25). Ki values were calculatedfrom IC50 values by the method of Cheng and Prusoff (26).

[3H]Dopamine-Release Experiments. Retinas from chickensmaintained on 14 hr light/10 hr dark cycle were dissected freeof pigment epithelium and prepared for superfusion experi-ments as described (17, 27). In brief, pieces of chick retinawere labeled in vitro with [3H]dopamine (specific activity28.4 Ci/mmol) in Krebs' solution with 1.3 mM CaCl2 and thenwere superfused at a rate of 1 ml/min until the spontaneousoutflow of radioactivity leveled off. Tritium release waselicited twice in each experiment by electrical stimulation at3 Hz for 2 min (20 mA, 2-msec duration). The first (Sl) andthe second (S2) periods of stimulation were applied 60 minand 100 min after the end of the incubation with [3H]dopa-mine. Results were calculated as the percentage of the totaltissue radioactivity released in each sample (17).

RESULTSCharacterization of 2-[125I]Iodomelatonin Binding. Binding

of 2-[125I]iodomelatonin was determined in washed chickenretinal membranes in 50 mM Tris HCl buffer (pH 7.5)containing 0.1% ascorbic acid, following 5 hr of incubation at0°C. The highest concentration of 2-[125I]iodomelatonin bind-ing sites was found in membranes from nuclear (43%) andmitochondrial (47%) subcellular fractions. In subcellularfractions, specific binding defined with 3 ,uM 6-chloromela-tonin was 85-90% of total 2-[125I]iodomelatonin binding.Because we could not detect any differences in the binding of2-[1251]iodomelatonin to the crude mitochondrial pellet (P2)and the total particulate fraction, we used the latter forroutine binding studies. The binding of 2-[125I]iodomelatonin

(30 pM) to chicken retinal membranes was linear between 25and 150 pug of protein. Incubation of retinal membranes for 5min in a boiling water bath prior to the binding assaycompletely abolished specific binding; nonspecific bindingincreased from 350 ± 35 cpm to 553 ± 32 cpm (n = 3).Extraction of the bound radioligand after a binding assayshowed that the bound radioligand comigrated on TLC withauthentic 2-[125I]iodomelatonin; no other radiolabeled bandswere observed. Thus, 2-[125I]iodomelatonin is stable duringthe course of a standard binding assay at 00C. Specific2-[1251]iodomelatonin binding to washed chicken retinal mem-branes was not affected by MgCl2 (0.1-10 mM), KCl (5-200mM), or CaCl2 (0.1-10 mM). However, NaCl (100-200 mM)inhibited binding in a concentration-dependent manner [63 +4.3% inhibition (n = 3) for 200 mM NaCl].The binding of 2-[1251]iodomelatonin to chicken retinal

membranes was reversible and temperature-dependent. At0C the binding of 2-[125I]iodomelatonin (40 pM) reached asteady state by 3 hr and was stable for 10 hr (Fig. 1). Theassociation rate constant (kj) determined from the pseudo-first-order equation was 5.5 x 107 M-1 min'. Specific2-[125I]iodomelatonin binding was reversed (t1/2 = 36.6 min)by the addition of excess competing ligand (3 /iM 6-chloromelatonin). The rate constant for dissociation (kL1)was 0.0188 min'. The kinetic dissociation constant (Kd) for2-[1251]iodomelatonin calculated from the ratio kL1/k, was 342pM. The total binding of 2-[1251]iodomelatonin at equilibriumwas similar at 0°, 25°, and 370C; however, a steady state wasreached at 30 min and at 9 min in binding assays at 25TC and370C, respectively.

Concentration-dependent binding of 2-[125I]iodomelatonin(0.05-1.5 nM) to chicken retinal membranes was saturableand resulted in linear Scatchard plots suggesting binding to asingle class of sites (Fig. 2). The apparent Kd for 2-[1251]iodomelatonin from the Scatchard analysis was 434 ± 56pM (n = 5) and the total number of binding sites was 74.0 +13.6 fmol/mg of protein (n = 5). The Kd values determinedin kinetic (342 pM) and saturation (434 pM) experiments werein close agreement.The pharmacological characterization of 2-[125I]iodomela-

tonin binding to chicken retinal membranes was carried outwith tracer concentrations of radioligand (30-60 pM). N-Acetyltryptamines, which are potent inhibitors of calcium-dependent release of [3H]dopamine from rabbit retina (17),were most effective in competing for 2-[125I]iodomelatoninbinding sites (Table 1 and Fig. 3). All four N-acetyltrypt-amines shown in Fig. 3 inhibited the binding of 2-[125I]iodo-melatonin to the same extent. N-Acetyl-5-methoxykynuren-amine, an endogenously occurring metabolite of melatoninpossessing a non-indolic structure, was as potent as 6-methoxymelatonin in competing for 2-[1251]iodomelatoninbinding sites in chicken retinal membranes. N-Acetyltrypt-amine, a putative melatonin receptor antagonist in chickenretina (16, 17), and N-acetyl-5-hydroxytryptamine were less

.so Q

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00

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IL

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5

I T

F~ ~ i

2 4 6 8 10Time, hr

FIG. 1. Reversible binding of 2-[1251]iodomelatonin to chickenretinal membranes at 0C. Specific binding of 2-[125I]iodomelatonin(40 pM) was reversible upon addition of 3 puM 6-chloromelatonin(arrow). Values are means + SEM of three determinations.

Neurobiology: Dubocovich and Takahashi

3918 Neurobiology: Dubocovich and Takahashi

'E c 60-ct

v

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v - m.E'-40-- 0E0

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-6

2-[125I]Iodomelatonin,nM

10

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010 20 40

2-[ 1251] lodomelatoninspecifically bound,fmol/mg of protein

Table 1. Pharmacological profile of the melatonin binding site ofchicken retina

Ki (nM) for IC50 (nM) for2-['251]iodomelatonin [3H]dopamine

Inhibitor binding* releaser

60

FIG. 2. Scatchard analysis of 2-[1251]iodomelatonin binding tochicken retinal membranes. Membranes were incubated with variousconcentrations of 2-[1251]iodomelatonin (0.05-1.2 nM) for 5 hr at 00C.Nonspecific binding (v) was measured in the presence of 3 AiM6-chloromelatonin. Specific binding (s) is defined as total binding (a)minus nonspecific binding. Values shown are means from a repre-sentative experiment performed in triplicate. (Left) Saturation curve.(Right) Transformation of the saturation curve by the method ofScatchard; this analysis gave a dissociation constant (Kd) of 0.26 nMand a total number of binding sites (Bmax) of 55.6 fmol/mg of protein.

potent than the corresponding 5-methoxyindoles (Table 1).Replacement of the acetamido group of melatonin with aprimary amino group, as in 5-methoxytryptamine, with ahydroxyl group, as in 5-methoxytryptophol, or with a tertiaryamino group, as in 5-methoxy-N,N-dimethyltryptamine, sig-nificantly reduced the inhibition of 2-[125I]iodomelatoninbinding (Table 1 and ref. 17). Antagonists of serotonin anddopamine receptors and of a- and P-adrenergic receptorswere fairly poor inhibitors of 2-[125I]iodomelatonin binding(Table 1). 2-[1251]Iodomelatonin binding was not affected byinhibitors of the neuronal uptake of dopamine or norepineph-rine (Table 1).

Correlation Between Ability to Inhibit 2-[251]IodomelatoninBinding and Ability to Inhibit the Calcium-Dependent Releaseof [3H]Dopamine from Retina. Melatonin and related indolesinhibited the calcium-dependent release of [3H]dopaminefrom chicken retina with a similar order of potency to thatfound in rabbit retina (Table 1 and ref. 17). The iodinatedligand is biologically active, since 2-iodomelatonin inhibitedthe calcium-dependent release of [3H]dopamine from chickenretina in vitro (Table 2). 2-lodomelatonin was about 10 timesmore potent than melatonin in inhibiting the calcium-depen-dent release of [3H]dopamine and did not affect the sponta-neous release of radioactivity from chicken retina. Theseresults demonstrate that iodination of melatonin in the 2position of the indole ring did not reduce the biologicalactivity of the molecule and suggest that 2-[1251]iodomelato-nin may bind to a melatonin receptor site in the retina (15-17).

In the chicken retina, the melatonin analogs that were themost potent inhibitors of [3H]dopamine release were thosepossessing a methoxy group on carbon 5 ofthe indole nucleusand an acetamidoethyl group on the same position as inmelatonin. The relative potencies (IC50) of 5-methoxyindolesin inhibiting [3H]dopamine release from chicken retina wereas follows: 2-iodomelatonin : 6-chloromelatonin : mela-tonin > 6-hydroxymelatonin > 6-methoxymelatonin > 5-methoxytryptamine (Table 1). This order of potencies forinhibition of [3H]dopamine release is identical to the onereported by Dubocovich (17) for rabbit retina. N-Acetyltrypt-amine, a putative melatonin receptor antagonist, competi-tively antagonized the inhibitory effect of melatonin inchicken retina (16, 17).

In support of an association of the melatonin binding sitelabeled by 2-[1251]iodomelatonin with a functional melatoninreceptor, a highly significant correlation was found betweenthe potency of indoles to inhibit the calcium-dependent

5-Methoxyindoles2-Iodomelatonin6-ChloromelatoninMelatonin6,7-Dichloro-2-methyl-

melatonin6-Hydroxymelatonin6-Methoxymelatonin5-Methoxytryptamine5-Methoxytryptophol5-Methoxy-N,N-di-

methyltryptamine5-Methoxytryptophan5-Methoxyindole-3-acetic

acid5-Methoxyindole

5-HydroxyindolesN-Acetyl-5-hydroxy-

tryptamine5-Hydroxytryptophol5-Hydroxytryptamine5-Hydroxytryptophan5-Hydroxyindole-3-acetic

acidIndolesN-AcetyltryptamineTryptamineL-TryptophanN-AcetyltryptophanIndole-3-acetic acidIndomethacin

Miscellaneous compoundsN-Acetyl-5-methoxy-kynurenamine

6-MethoxyharmalanMethysergideMethiothepineYohimbineBufotenineFluphenazineNomifensineDesipraminePropranololColchicine

2.54.06.3

1074

4604,600

46,400

>100,000>100,000

>100,000>100,000

3,00030,000

>100,000>100,000

0.10.51

30100200

>10,000

>10,000

>10,000>10,000

300

>10,000

>100,000

1,600>100,000>100,000>100,000>100,000>100,000

>1,000*

5001,6006,3006,30016,00020,00030,000

>100,000>100,000>100,000>100,000

*K1 values were calculated, from IC50 values obtained from compe-tition curves, by the method of Cheng and Prusoff (26). Inhibitionof specific binding of 2-['251]iodomelatonin (30-60 pM) in chickenretinal membranes was determined for 11 concentrations of com-peting drugs. Results are mean values of 3-8 independent deter-minations.tEach indole compound was tested, at three to seven concentrations,for its effect on the [3H]dopamine release evoked by electricalstimulation (3 Hz, 2 min) of chicken retina. The IC50 value is theconcentration of drug required to inhibit the calcium-dependentrelease of [3H]dopamine by 50%. The IC50 values were determinedgraphically from concentration-effect curves.tKB value for the putative melatonin receptor antagonist N-acetyltryptamine is 33 nM (16, 17).

release of [3H]dopamine from chicken and rabbit retina andthe potency to compete for 2-[1251]iodomelatonin binding inchicken retinal homogenates (Fig. 4). This correlation strong-ly suggests that 2-[1251]iodomelatonin labels a site with thepharmacological and functional characteristics ofa melatoninreceptor. At this time, however, we cannot exclude the

Proc. Natl. Acad. Sci. USA 84 (1987)

Proc. Natl. Acad. Sci. USA 84 (1987) 3919

C

-v-

-C

-CoC

L-

C-

100 - c/ I,-

50 c

d \ \

...\i \e

nI,,, I Ix12 10 8 6 4

-log[inhibitorl (M)

FIG. 3. Competition curves for inhibition of 2-[1251]iodomelatoninbinding by various melatonin agonists in chicken retinal membranes.Washed chicken retinal membranes were incubated with 30-60 pM2-[1251]iodomelatonin and various concentrations of 5-hydroxytrypt-amine (curve a, *), N-acetyl-5-hydroxytryptamine (curve b, v),6-hydroxymelatonin (curve c, *), 6-chloromelatonin (curve d, 0), ormelatonin (curve e, *). Values are means of 3-8 independentdeterminations.

possibility that 2-[1251]iodomelatonin may label melatoninreceptor binding sites regulating other processes in retina,such as photoreceptor outer segment disc shedding andphagocytosis (11, 12) and cone retinomotor movements (14),in addition to those sites modulating [3H]dopamine release.

DISCUSSIONOur results strongly suggest that 2-[1251]iodomelatonin is aselective, high-affinity ligand for the identification and char-acterization of melatonin receptor sites. The specific bindingof 2-[1251]iodomelatonin fulfills all the criteria for binding toa receptor site, being stable, reversible, saturable, and ofhighaffinity. 2-[1251]Iodomelatonin appears to label a single classof sites in membranes isolated from chicken retina. Theapparent affinity constant derived from kinetic analysis wasin close agreement with the Kd obtained from saturationexperiments. Competition curves were monophasic and

Table 2. Effect of melatonin and 2-iodomelatonin on the calcium-dependent release of [3H]dopamine from chicken retina

% total tissue

Drug present radioactivitytduring S2* n S1 S2 S2/Sj ratio:

None (control) 8 1.54 ± 0.28 1.25 ± 0.16 0.87 ± 0.06Melatonin

(10 nM) 8 1.45 ± 0.26 0.56 ± 0.14§ 0.36 ± 0.06§2-lodomelatonin

(2 nM) 11 1.13 ± 0.09 0.51 ± 0.080 0.43 ± 0.05$*Drugs were added to the medium 20 min before the second periodof stimulation (S2). In the controls, the spontaneous outflow ofradioactivity calculated as the percentage of total tissue radioac-tivity released during the 4 min preceding the first period ofstimulation (Sl) was 1.27% + 0.10% (n = 8). (S)-Sulpiride (0.1 /LM)was present in every experiment from 40 min before S1. Melatoninor 2-iodomelatonin, present in the superfusion medium from 20 minbefore S2, did not modify the spontaneous outflow of radioactivityat the concentrations indicated. Radioactivity retained by thecontrol tissue after 120 min of superfusion was 62.4 ± 12.7 nCi perchamber (n = 8).tPercentage of the total tissue radioactivity released above thespontaneous levels elicited by field stimulation at 3 Hz (20 mA, 2msec) during a 2-min period.tRatio of the percentage of total tissue radioactivity released duringS2 to that released during S1.§P < 0.01 and ¶P < 0.001 when compared with corresponding control(Student's t test).

A

_- '; -c

4-C 4

C_- E 'be 6

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1_CI 1()

I

11 9 7 5

B

k

12 10 8 6

-log WCs(, (M) for inhibition of 1HIdoparmine release

FIG. 4. Correlation between the affinities of compounds for2-['25I]iodomelatonin binding sites in chicken retina and their abilityto inhibit [3H]dopamine release from chicken retina (A) and rabbitretina (B). Ki values for inhibition of 2-[1251]iodomelatonin binding inchicken retinal membranes and IC50 values for inhibition of thecalcium-dependent release of [3H]dopamine from chicken retinawere obtained from Table 1. IC50 values for inhibition of thecalcium-dependent release of [3H]dopamine from rabbit retina wereobtained from ref. 17 and unpublished observations. Linear regres-sion of a logarithmic transformation of the data yielded a slope of0.952 and a correlation coefficient of 0.968 (P < 0.01, n = 7) in A anda slope of 0.767 and a correlation coefficient of 0.969 (P < 0.01, n =

11) in B. Compounds are indicated as follows: a, 2-iodomelatonin; b,6-chloromelatonin; c, melatonin; d, 6-hydroxymelatonin; e, 6-methoxy-melatonin; f, N-acetyl-5-hydroxytryptamine; g, 5-methoxytryptamine;h, 6,7-dichloro-2-methylmelatonin; i, N-acetyl-5-methoxykynuren-amine; j, N-acetyltryptamine; k, 5-methoxytryptophol.

Scatchard plots were linear. The number of 2-[1251]iodomel-atonin binding sites found in chicken retina (Bmax = 74.0 +13.6 fmol/mg of protein) was almost identical with thenumber of sites reported for [3H]melatonin in the frog retina(10). However, the affinity of the melatonin binding site offrog retina, determined by use of [3H]melatonin, was <0.001times (10) the affinity of the melatonin binding site of chickenretina determined by use of 2-[1251]iodomelatonin.

2-[1251I]Iodomelatonin offers distinct advantages for thecharacterization of melatonin binding sites. The binding of2-[1251]iodomelatonin to chicken retinal membranes was 90%specific, even with protein concentrations as low as 30 jig perassay, allowing detection of melatonin binding sites in smalltissue samples. The pharmacological characterization of themelatonin receptor site labeled by 2-[1251]iodomelatonin in-dicated that the radioligand selectively binds to a site with thepharmacological characteristics of a melatonin receptor,since melatonin and related indoles inhibited 2-[1251]iodomel-atonin binding with the same order of potency found forinhibition of release of [3H]dopamine from chicken (Table 1)and rabbit (17) retina. In rabbit retina, 2-[1251]iodomelatoninalso appears to label a melatonin receptor site, since a highlysignificant correlation was obtained between the relativepotencies of melatonin analogs in inhibiting 2-[1251]iodomel-atonin binding and in inhibiting calcium-dependent release of[3H]dopamine from rabbit retina (r = 0.799, P < 0.01, n = 8;ref. 28).

[3H]Melatonin binding sites have been demonstrated inseveral tissues. Saturable binding of [3H]melatonin has beenshown in bovine hypothalamic membranes (18) and in cyto-solic fractions of brain from several species (19, 21).[3H]Melatonin also binds to membranes of bovine pinealgland (29) and trout and frog retinas (10, 20). In these studiesthe binding site labeled by [3H]melatonin was of relativelylow affinity and the abilities of the melatonin-related indolesto compete for this binding site were not correlated with anyfunctional response to melatonin. Moreover, the order ofpotency of melatonin and related indoles on the [3H]melato-nin binding site of bovine hypothalamus (18) differs consid-erably from the pharmacological characteristics of the mel-atonin receptor in rabbit and chicken retina (ref. 17; present

Neurobiology: Dubocovich and Takahashi

VI

3920 Neurobiology: Dubocovich and Takahashi

results). The most distinct difference is that of 6-hydroxy-melatonin, which is very potent in inhibiting [3H]dopaminerelease and competing for the receptor labeled by 2-[125I]-iodomelatonin in rabbit and chicken retina (17, 28) but is apoor inhibitor of [3H]melatonin binding in bovine hypothal-amus (18). In contrast, indoles such as 5-methoxytryptophol,5-methoxyindole-3-acetic acid, 5-hydroxytryptamine (sero-tonin), and 5-hydroxytryptophol, which are inactive or pooragonists on the melatonin receptor of chicken and rabbitretina (refs. 17 and 28; present results), are among the mostpotent competitors of [3H]melatonin binding in bovine hy-pothalamus (18). Similar discrepancies in the order of poten-cy of melatonin and related indoles on the melatonin bindingsite are observed between our results obtained with 2-['25I]iodomelatonin and those reported for retinas of lowervertebrates with [3H]melatonin (10, 20). In contrast, we haverecently found that, in hamster brain membranes, 2-[125I]_iodomelatonin labels a melatonin binding site whose phar-macological characteristics are almost identical to thosefound for the binding site on retinal membranes (30). Thedifferences in the pharmacological characteristics of the siteslabeled by [3H]melatonin and 2-['25I]iodomelatonin in mem-branes prepared from central nervous system tissues appearto reflect different binding sites.Using a functional measurement of melatonin activity,

Dubocovich (17) showed that the efficacy of melatonin andrelated indoles in inhibiting the calcium-dependent release of[3H]dopamine from rabbit retina is determined by the moiety(methoxy) on carbon 5 of the indole nucleus, whereas theaffinity for the receptor is determined primarily by the moiety(acetamidoethyl) on carbon 3. In support of this conclusion,we have found that the most potent inhibitors of 2-['1251]io-domelatonin binding in retina were the N-acetyltryptamines.On the basis ofboth the 2-[1251]iodomelatonin binding and thefunctional responses to indole amines, we suggest that amelatonin receptor is characterized by the following phar-macological order of affinities: 2-iodomelatonin > 6-chloro-melatonin : melatonin : 6,7-dichloro-2-methylmelatonin >6-hydroxymelatonin : 6-methoxymelatonin > N-acetyltryp-tamine > N-acetyl-5-hydroxytryptamine > 5-methoxy-tryptamine >>> 5-hydroxytryptamine (inactive).

In addition to its role in the retina, melatonin has beenimplicated in the regulation of seasonal reproduction inmammals (1, 2) and in the control of circadian rhythms inbirds and reptiles (3-5). Although it is generally assumed thatmelatonin acts centrally upon the hypothalamopituitary axisin mammals (31) the anatomical loci ofmelatonin receptors inthe central nervous system have not been identified. We haverecently characterized melatonin binding sites labeled by2-[1251]iodomelatonin in hamster brain membranes (30). Thisbinding site exhibits pharmacological characteristics similarto those ofthe melatonin receptor ofchicken and rabbit retina(17, 28). In summary, the radioligand 2-[125I]iodomelatoninshould provide a useful probe for the localization andcharacterization ofcentral melatonin receptors, as well as theelucidation of the mechanism of action of melatonin.

We thank Patrick Rita for technical assistance, Selene S. Nikaidofor preparing the 2-iodomelatonin, and Dr. Marilyn J. Duncan forcritical comments on the manuscript. This work was supported byPublic Health Service Grants EY04788, RR05470, and MH39592; by

National Science Foundation Grant DCB-8451642; and by SearleScholars Award 85-H-107.

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