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JOURNALOF PHARMACOLOGY ANDEXPERIMENTALTHERAPEUTIC8 VoL246, No.3
CopyrightC 1988byThe AmericanSocietyfor Pharmacologyand ExperimentalTherapeutics Printed in U.SA.

Luzindole(N-0774):A NovelMelatoninReceptorAntagonist'

MARGARITA L. DUBOCOVICH
Departmentof Pharmacology,Northwestern University Medical School, Chicago,Illinois
Acceptedfor publicationMay23,1966

ABSTRACT
The pharmaCOlOgiCal potencies of 2-substituted N-acetyltryptam lation-evoked release of rH]do@amlne when added alone. How
ines were determined on the presynaptic melatonin receptor site ever,luzindole(0.1—1 0 @M)shiftedtheconcentrationeffectcurve
of rabbit retina labeled in vitro with [@H]dopamine.Calcium for melatonin to the nght in a parallel fashion. The pA@extrapo
dependent ralease of [3HJdoparT@ne was eliCited by alectrlcal latedfrom the Schildplot (slope,0.91)was 7.7, with a KB=20
stimulation at 3 Hz for 2 mm (20 mA, 2 msec). Melatonin (5- nM. Thedissociationconstantsfor luzindole(K8),determinedin
OCHrN-acetyltryptamine) and 6-chioromelatonin were equipo thepresenceof 6,7-dlchlcro-2-methylmalatonin (10 pM—inM)or
tent In inhibiting the calcium-dependent release of @H]dopamine 6-chloromelatonin(10 pM—100 nM) were 16 and 40 nM, respec
(IC@1,
= 40 pM). 2-Substituted N-acetyltryptamines with a methyl tively.Thesedatasuggestthatluzindoleandthevariousmela
(i.e., 6,7-dlchloro-2-methylmelatonin,
lC@= I 0 pM)or iodine(l.e., toninagonistsarecompetingfor thesamepresynapticmelatonin
2-lodomelatonin, lC@ = 5 pM) group were more potent than receptor site in the rabbit retina. In summary, 2-substituted 5-
melatonin in inhibiting [3H]dopamine release. I report here the methoxy-N-acetyltryptamines (i.e., 6.7-dlchloro-2-methytmela
pharmacological properties of the novel N-acetyltryptamine, 2- tonin, 2-lodomelatonin)are agonists, N-acetyftryptamineIs a
benzyl-N-acetyltryptamine(N-0774, luzindole)on the presynaptic partialagonistand2-benzyl-N-acetyltryptamine(/.e.,
luzindole)is
melatonin receptor of rabbit retina. Luzindole (0.1—10 @M)did a competitivereceptorantagonistat the presynapticmelatonin
not affect the SpOntaneOusoutflow of radIOaCtivityor the stimu receptor of rabbit retina.

Melatonin (5-OCH3 N-acetyltryptamine) is synthesized and
secreted primarily by the pineal gland with highest levels oc
curring during the dark period of a circadian light-dark cycle
(Axeirod, 1974; Klein, 1979). This hormone is involved in the
transduction ofphotoperiodic information, and appears to mod
ulate a variety of neural and endocrine functions in vertebrates,
including the regulation of reproduction, body weight and me
tabolism in photoperiodic mammals (Darrow and Goldman,
1985; Tamarkin et aL, 1975), and the control of circadian
rhythms in birds and reptiles (Underwood and Harless, 1985).
Exogenous melatonin has been found to synchronize circadian
rhythms in rats (Redman et aL, 1983; Cassone et aL, 1986). In
humans, sleep disturbances due to jet lag, considered to be
caused by desynchronization of circadian rhythms, can be
treated by administration of melatonin at scheduled times
(Arendt et a!., 1986).
Classically, the physiological effects ofendogenous melatonin
have been antagonized by exposure of animals to light, which
inhibits the synthesis of this hormone, or by removing the
Receivedfor publicationJanuary 27, 1988.
‘¿This
work was supported by grants from Nelson Research and U.S. Public
Health Service Grants RR 05470 and MH 42922. Portions ofthis work have been
presented in abstract form (International PharmacologyAssociation10th Inter
national Congress of Pharmacology, Abstract 0438, 1987; Society for Neurosci
ence, Neuroscience Abstract 13, p. 1039, 1987).
pines! gland (Darrow and Goldman, 1985; Tamarkin et ci,
1985). However, the understanding of the role of melatonin in
physiological processes as well as clinical pathological condi
tions has been hampered by the lack of potent and selective
melatonin receptor antagonists.
In the past, melatonin responses were studied on amphibian
dermal melanocytes using in uiuo (Quay and Bagnara, 1964;
Quay, 1968; Reed and Finnin, 1970) or in vitro methods (He
ward and Hadley, 1975). In mammals, potential melatonin
agonists have been screened by measuring endocrine response
in vivo (Clemens and Flaugh, 1985). The recent discovery that
melatonin is a potent inhibitor of the calcium-dependent re
lease of dopamine from retinal tissues has led to the develop
ment of a sensitive in vitro functional receptor assay which
permitted for the first time quantitative determination of po
tencies of structurally related melatonin analogs (Dubocovich,
1983, 1984, 1985).
The initial studies on structure-activity on the retinal mela
toninreceptor
confirmed
suggestions
fromthedataofearlier
test systems (Heward and Hadley, 1975), that the most active
agonists are those possessing a 5-methoxy group on carbon 5
of the indole nucleus and an N-acetyl group on the same
position as in melatonin (Dubocovich, 1985). The structure
activity data available also suggest that N-acetyltryptamines
lacking the 5-methoxy group are potential melatonin receptor

ABBREVIATiON 5-HT serotonin.

902
1988 MelatoninRceptor Antagonist 903
antagonists (Heward and Hadley, 1975; Dubocovich, 1985). In The stimulatedoverflowof transmitterwas the percentageof total
fact,N-acetyltryptamine
itselfwasreported
tobea melatonintissue radioactivity released above the spontaneous levels during and
receptor antagonist in the frog skin and chicken retina, but it after the period of field stimulation (Dubocovich, 1985) Results are
andHadley, expressed
wasa partialagonistin rabbitretina(Heward as the ratio (S@/S1) of the overflow measured during the
second (S2)and first (S1)periodsof field stimulationwithin the same
1975; Dubocovich, 1984, 1985). This paper describes the phar
experiment. In the followingtext, the phrase “¿overflow
of transmitter―
macological potencies of various N-acetyltryptamine analogs is also referred to as: “¿stimulation-evoked
release of [3Hjdopamine―or
substituted in position 2 ofthe indole nucleus which were found “¿calcium-dependent
releaseof [3H)dopamine.―
to either mimic (i.e., 2-iodomelatonin, 6.7-dichloro-2 methyl The dissociation constant (K5) of luzindole in rabbit retina was
melatonin) or to competitively antagonize (2-benzyl-N-acetyl determined by the method of Arunlakshana and Schild (1959) using
tryptamine, referred to as luzindole) the melatonin-induced the equation:
inhibition of [3H]dopamine release from mammalian retina.
(Rent
In addition, several other compounds that have been reported pA2= log -@;- —¿
1) —¿
log [Ant]
previously to antagonize measurements of melatonin activity
in in vitro and in vivo models were tested. 6-Methoxy-2-ben where ECo is the concentrationof agonist causing 50%inhibition of
zoxazoline (Sanders et ci, 1981; Yuwiler and Winters, 1985), the biologicalresponse;Eant is the concentrationof agonist causing
N-(3,5-dinitrophenyl)-5-methoxytryptamine (Zisapel and Lau 50% inhibition of the biological response in the presence of the antag
don, 1987) and 5-methoxy-indole-N-methyl-3-propionamide, onist; and Ant is the concentration of the antagonist.
the reversed amide of melatonin (Askam et ci, 1985), as well Results are expressed as mean ±S.E.M. Unpaired Student's t test
(two-tailed) was used for statistical comparisons.
asothercompounds
lacking
theacetamidoethyl
group
allfailed Drugs were Obtainedfrom the pharmaceutical company of origin or
to antagonize the melatonin-induced inhibition of [3H]dopa commercial sources. 6-Chloromelatonin and 6,7-di-chloro-2-methyl
mine release from rabbit retina. melatoninwere donatedby Eli Lilly Laboratories(Indianapolis,IN);
2-iodomelatonin by Dupont New England Nuclear (Boston, MA): 5-
Materialsand Methods methoxy-3-(2-morpholinoethyl) indole by Dr. E. W. Taylor (Depart
ment of Pharmacologyand Toxicology,Universityof ArizonaCollege
Albino rabbits (2.5—3.5
kg) maintained on a 14/10 hr light-dark cycle of Pharmacy,Thcson, AZ);5-methoxy-indole-N-methyl-3-pmpionam
weresacrificedby decapitationduringthe light cycle. All experimental ide by Dr. V. Askam (Department of Pharmacology, the Welsh School
procedures were carried out in the light. Rabbit eyes were enucleated of Pharmacy,Cardiff,Great Britain); N-(3,5-dinitrophenyl)-5-meth
and the cornea, lens and vitreous humor were removed carefully. The oxytryptamine by Dr. N. Zisapel (Department of Biochemistry, Tel
remaining eye cup was everted and placed in a vial containing 5 ml of Aviv, Israel); apomorphine by Merck Sharp and Dohme Laboratories
Krebs' solution gassed with 95% 02-5% CO2. The rabbit retina was (Rahway, NJ); N-0437 and N-0774 (luzindole) by Nelson Research,
detached by gentle shaking during which the retina fragmented into (Irvine, CA); and clomdine by Boehringer Ingeiheim Ltd., (Ridgefield,
several pieces. CT). All other drugs were obtained from Sigma Chemical Co. (St.
Retinal pieces were incubated for 20 mm at 37°Cin the presence of Louis, MO).
0.1 @M [3Hjdopamine(specific activity, 55 Ci/mmol; AmershamCor 6-Chioromelatonin, 6,7-dichloro-2-methylmelatonin,2-iodomela
poration, Arlington Heights, IL). Thereafter, the pieces of retina were tonizi and luzindole were dissolved a minimum amount of ethanol
washed in 5 ml of Krebs' solution at 37C. The tissue from each retina (95%)and dilutedin waterto give a stock solutionof 1 mM;additional
was divided into four approximately equal portions and placed in four dilutions were done in water. All solutions were prepared fresh the day
individual cylindrical plastic tubes with a thin nylon mesh on the ofthe experiment.The maximalconcentrationofethanol in the super
bottom. The plastic tubes containing the pieces of retina were trans fusion medium for the highest concentration (10 ,@M)of luzindole (N-
ferred to individual glass superfusion chambers, which each contained 0774) was below 11 mM. This concentration of ethanol when added
apairofplatinum
electrodes
30mmapart.
Theretinal
pieces
were alone before the second period of stimulation did not significantly
superfused by means of a roller pump with 1 mi/rain ofKrebs' solution modifythe calcium-dependent release of [3H]dopaminefrom the rabbit
prewarmed to 37'C, until the spontaneous outflow of radioactivity had retina (see table 1).
leveled off. Samples of the superfusate were collected by means of a
fraction collector at 4-mis intervals. The composition of the Krebs'
solution was (milhimolar):NaC1,108;KC1,4.7; glucose, 11.1;NaHCO3,
Results
25.0;MgCl2,1.2;NaH2PO4,
1.0@,
CaCl2,1.3;ascorbicacid,0.11;and Spontaneous efflux of tritium and calcium-dependent
disodium EDTA, 0.004. release of [3HJdopamine from the rabbit retina labeled
Tritium release was elicited by field stimulation of retinal tissue
in vitro with [3flldopamine. The rabbit retina wasdissected
using 3 Hz, 20 mA, 2 msec duration pulses that were delivered from
the electrode by a Grass Stimulator, model 844. During stimulation,
and labeled in vitro as described under “¿Materials and Meth
pulses were monitored on an oscilloscope. In each experiment two ods.―Superfusate samples were collected in 4-min fractions
periods of field stimulation were applied at 60 (S1) and 100 (S) mm when the spontaneous outflow of radioactivity had leveled off
after the end of the incubation with [2Hjdopamine. Samples of the (56 mAn superfusion) (fig. 1). The percentage of the total
superfusate were collected before, during and after the period of stim radioactivity released in the 4-mm fraction preceding each
ulation. The drugs were added to the perfusion medium either 40 or 20 period of stimulation (Sp) was 1.10 ±0.08% for Sp1 and 0.96 ±
mm before S1 or S@,respectively, and were present throughout the 0.06%, n = 14, for Sp@(table 1). Figure 1A shows the efflux of
remainder ofthe experiment. At the end ofthe experiment, the retinal radioactivity expressed as percentage of total tissue radioactiv
tissue contained in each chamber was solubilized in 0.5 ml of TS-1
ity released in each sample before, during and after each period
(Research Products International, Elk Grove, IL) and the tritium
(S@and S2) of electrical field stimulation. Table 1 shows the
content was determined by liquid scintillation counting.
The outflowof radioactivityin each sample was expressed as the percentage oftotal tissue radioactivity released above the spon
percentage ofthe total tissue radiOaCtivity determined to be present at taneous levels of release during the first (S1) and the second
the beginningofeach samplecollection:total tritiumreleasepersample (S2) periods of stimulation in the controls. In the absence of
divided by total tritiu.m present in the tissue and multiplied by 100 drugs the control ratio SJS1 was not different from one
(Dubocovich, 1985). (fig. 2).
 904 Duboco@th Vol. 246

onists and antagonists. Picomolar concentrations of mela

tonin inhibited the calcium-dependent release of [3H]dopamine
(Dubocovich, 1983, 1985, fig. 1). The concentration of mela
tonin inhibiting [3Hjdopamine release by 50% (IC@) was 40
pM. The effect of 0.1 nM melatonin on the spontaneous and
stimulated release of [3HJdopamine from rabbit retina is shown
in figure lB. The inhibition of the calcium-dependent release
of [3Hjdopamine
by0.1nMmelatonin, expressed
astheratio
S2/s1was:0.48±0.04(n = 26)(table1).Stocksolutions
of
melatonin (1 mM) were made in water, however, several of the
compounds tested for their ability to antagonize melatonin
LUZOCOLE
I @I
weredissolvedin ethanoLTable1 showsthat the highest
Fig. 1. Effluxof radioactivityfrom rabbitretinalabeledIn vitro with [@H] concentration
of ethanol(11mM)presentin themediumas
dopamine.Rabbit retinaswere labeledIn vitro with [@H)dopamlne as vehicle during the first (S1) and the second (S2) period of
describedtmder“¿Mate@elS and Methods. Superfus@esam@eswere stimulation did not antagonize the inhibitory effect of mela
collectedIn 4-mIt@ fractions beginning56 mlii after the end of the
k@cubatIon withrH)doparnine.Orclnate:percentageof totaltissuetrftlum tonin.
releasedk@ each4-mmfraction.The calcium-dependent releaseof [@H] Potential melatonin antagonists that did not modify the
dopamine(R, U, @,@: [@H]dopamine overflow,S) was evokedby field spontaneous and calcium-dependent release of tritium (fig. 2)
stimulation(3 Hz,2 mEn,20 mA,2 msec)abovethe spontaneouslevels weretestedagainstmelatonin
usingthesameparadigm
asthat
of tritlumreleasep spontaneousoverflow,S,) at 60 mm(Si) and 100
ruin(Se)OfStq@ffUSl0fl. Melatonin(0.1 nM) E@ B and C was added20 shown in figure 1C for luzindole. Compounds were added 40
mm beforeS@and remainedthroughoutthe experiment.Luzlndole(1 min before the first period (S1) of stimulation and remained
iiM) In C was added40 mmbeforeS1and remelnedpresentdurk@g S present throughout the experiment. Those compounds found
untiltheendof the experiment.Shownaremeanvalues±S.E.M. to antagonize the inhibitory effect of 0.1 nM melatonin were
considered potential melatonin antagonists. Figure 1C shows
Table 1 shows the effect of ethanol on the spontaneous and that 1 @iMluzindole antagonized completely the inhibitory
stimulation-evoked release of [3Hjdopamine as the stock solu effect of melatonin. The pharmacological profile ofluzindole is
tions of some of the compounds studied were made in ethanoL described below.
The compounds were generally dissolved with the minimum Luzindole (N-0774) antagonized the inhibition of the
amount of ethanol given a final concentration in the superfu calcium-dependent release of [8H]dopamine elicited by
sion medium below 11 mM. We have chosen to test the direct melatonin agonists. Luzindole (0.01-10 giM), when added
effect of 11 mM ethanol present in the superfusion medium alone 20 mm before the second period (S2) of electrical stimu
containing the highest concentration of luzindole (10 aiM). lation did not modify the spontaneous outflow of radioactivity
Table 1 shows that a concentration of ethanol of 11 mM added of the calcium-dependent
releaseof [3H]dopamine
(table2;
alone 20 before the second stimulation (S@)or from 40 mm fig. 2).
before the first stimulation (Se) did not modify either the Luzindole was tested on the melatonin-induced inhibition of
spontaneous or the calcium-dependent release of [3H)dopamine the calcium-dependent release of [3H]dopamine as it fulfills the
from rabbit retina. structural requirements of a melatonin receptor antagonist, i.e.,
Experimental model to screen melatonin receptor ag anN-acetyltryptamine
withoutanysubstitution
inthecarbon
TABLE1
Effect of ethanolon ths mslatonlnInducsd-Inhlbltlonof [‘lflclop.mlne
releasefrom rabbItretina

outflow2
DrugsuiS,•n%Tot@flseueRadioectMty@—
Ra@o
Sp2/Spi.SIS,Ra505Control140.88
0.05Melatonin, ±0.022.31 ±0.182.16 ±0.180.98 ±
0.1 nM260.80
0.04*(82)Ethanol, ±0.012.04 ±0.131.09 ±0.09k0.48 ±

11 mM30.85 ±0.352.31
0.03(82)Ethanol,llmM30.85±0.012.50±0.112.38±0.080.96±0.07(S1,S@)Ethanol,
±0.032.14 ±0.381.08 ±

11 mM30.75
O.Or,@(S1,S@)Melatonin, ±0.022.37 ±0.350.91 ±0.16k0.38 ±

nM(82) 0.1

a Ethanol (1 1 mM) was added to the superfusion medium eWw 20 mm before S@ or 40 before 8, and remained present throughout the experiment.
@ C outflow of rathoactMty was calculated as the percentage of tot@ tissue radioactivity released during the 4 mm preceding the first (Sp,) and the
@ second (Sp2) of stimulation.and expressed as the ratio Sp,JSp@.Inthe controls,the percentage of total tissue radioactivityrelease duringSp@was 1.0 ±0.08%
@ and duringSp2was 0.96 ±0.06% (n 3).The radioactivityretainedby the controltissue after 120 mmsuperfuslonwas 66.8 ±6.3 n@iper chanber(n 14).
C Tt@m (@HJdopamine overflow was calculated as the percentage of the total tissue radioactivfty released @ove the spontaneous levels elicited by field stimulation at 3

Hz (20 mA, 2 msec)duringa 2-mmperiod.

‘¿
Ratio of the percentage of the total tissue radioactMty released during the second (Se) to that released during the first stimulation period (Si).
. P < .001 when compared with the corresponding controlt P .< @c@1
when compared with melator@n alone (Student's t test).
 1988 Melatonin Receptor Antagonist 905

shifted the concentration-effect curves for 6-chioromelatonin

and 6,7-dichloro-2-methylmelatonin to the right without
changing their maximal inhibitory effects. The inhibition of
‘¿-I the calcium-dependent release of [3H]dopamine from rabbit
retina elicited by 100 pM 2-iodomelatonin was also antagonized
by 1 @iMluzindole. The equilibrium dissociation constant cal
@ Is culated from the Schild equation (see “¿Materials
and Methods―)
using 6-chioromelatonin was 40 nM and using 6,7-dichloro-2-
methylmelatonin was 16 nM (table 3).
Effect of luzindole on apomorphine- and clonidine
induced inhibition of [3H]dopamine release. Studies on
the rabbit retina have shown that [3H]dopamine release can be
.1 1 C 0.01 0.1 1 10
inhibited by activation of at least four presynaptic receptors:
LUZI*@OLE($4) LUZDCOL.E(.iøO
D-2 dopamine autoreceptors, a@pIw-2adrenergic, opiate or me
latonin receptors (Dubocovich and Weiner, 1981, 1983; Dubo
Fig. 2. Effect of luzindole on the spontaneous and calcium-dependent
covich, 1983, 1984, 1985). Luzindole, in concentrations up to
releaseof tritiumfrom rabbitretina.Ordinates:A, spontaneousoutflow
of tritiumrepresentsthe percentageof totaltissuerad@activfty released 10 @M,did not modify the calcium-dependent release of [3H]
during the 4 mm preceding the first (Sp1)and the second (Sp2)period of dopamine from rabbit retina (table 2; fig. 2B), suggesting that
stimulation,expressed as the ratio Sp@/Sp1.B, [3Hjdopamineoverflowis it did not activate inhibitory presynaptic receptors modulating
the percentageof the totaltissue radioactivityreleasedabove the spon the release of [3H]dopamine from the retina. Luzindole (1 MM)
taneousbasallevel,elicitedby fieldstimulationat 3 Hz (20mA,2 msec)
during a 2-mm period. LUzindOlein the concentration indicated was had no effect on the inhibition ofthe calcium-dependent release
added 20 mm before the second period of stimulation (82). Shown are of [3H]dopamine elicited by apomorphine (0.01-1 MM) or N-
meanvalues±S.E.M. 0437 (0.01 MM) (fig. 5), nor did it antagonize the inhibitory
effect of clonidine (0.1—1 MM) (fig. 6) (Dubocovich, 1984; Du
5 of the indole nucleus (Dubocovich, 1985). Concentration
bocovich and Weiner, 1985; van der Weide et ci, 1988). These
effect curves of the agonist melatonin were performed in the
results suggest that luzindole specifically antagonized the me
absence and presence of several concentrations (0.1—10@M) of
latonin-induced inhibition of [3H]dopamine release, and that
luzindole (fig. 3A). Luzindole at 0.1, 1 and 10 sM shifted the
its mechanism of action does not involve interaction with D-2
concentration-effect curve for melatonin (IC@,o 40 pM) to the
right (fig. 3A). The Schild plot gave a straight line with a slope dopamine or a@pha-2adrenergic receptors.
of 0.91 suggesting a competitive interaction between melatonin Effect of various compounds on the melatonin-induced
and luzindole at the melatonin receptor site (Arunlakshana and inhibition of [3Hjdopamine release from rabbit retina.
Schild, 1959; Kenakin, 1987). The pA2 extrapolated from the Table 4 lists compounds of various chemical classes that did
Schild plot was 7.7, with a corresponding dissociation constant not alter the inhibitory effect of melatonin (0.1 nM) on [3HJ
(KB) of2O nM. dopamine release from rabbit retina (Dubocovich, 1983, 1985).
The dissociation constant (KB) for luzindole was also deter This list includes various indoles, tetrahydro-$-carbolines, neu
mined using the melatonin agonists 6-chloromelatonin (10 pM ronal uptake inhibitors, benzodiazepines and antagonists of
100 nM) and 6,7-dichloro-2-methylmelatonin (10 pM-i nM) serotonin, dopamine, c4pha adrenergic and opiate receptors.
which did not modify the spontaneous outflow of radioactivity A series of compounds have been shown recently to antago
from rabbit retina. 6-Chioromelatonin (IC@= 40 pM) and 6,7- nize various functional measurements of melatonin activity.
dichloro-2-methylmelatonin (IC@ = 10 pM) inhibited in a One such compound, 6-methoxy-2-benzoxazoline was reported
concentration-dependent manner the calcium dependent re to antagonize the physiological effects of melatonin in vivo
lease of [3H]dopamine when added before the second period of (Sanders et at., 1981; Yuwiler and Winters, 1985). In the rabbit
stimulation (S2), with maximal inhibitory (75-80%) effects retina, 6-methoxy-2-benzoxazoline did not modify the sponta
obtained at 1 nM (fig. 4, A and B; table 3). Luzindole (1 tiM) neous or calcium-dependent release oftritium (fig. 7A), nor did

TABLE2
Effectof luzindoleonthecalcium-dependent
releaseof tritlumfromrabbitretina
% Tots Tissue RacflcactMty

Drugsii @,• n ‘¿H-SpoManeousoutflow― [@H@DOPanrnO

overflow'
Sp@ Sp, S@ 5,
Control 3 0.95 ±0.04 0.81 ±0.05 2.14 ±0.35 2.31 ±0.38
Luzindole,
0.01MM 3 1.06±0.05 0.84±0.01 2.34 ±0.58 2.10 ±0.34
Luzindole, 0.1 MM 3 0.88 ±0.04 0.79 ±0.04 1.66 ±0.15 1.59 ±0.2
Luzindole,1 MM 6 0.76 ±0.07 0.63 ±0.06 1.73 ±0.08 2.05 ±0.32
Luzindole,10 MM 5 0.93 ±0.08 0.84 ±0.07 1.53 ±0.20 1.75 ±0.33
a Luzindole was dissolved in the minimum amount of ethanol (95%). The concentration of ethanol in the superfusion medium containing 0.01 , 0.1 , 1 and 10 @M
luzindole was 0.02, 0.22, 2.2 and 11 mM, respectively. Ethanol (11 mM) in the control or IuzWdole in the concentrations indicated was added to the supertusion medium
20 mmbeforeS,.
bft.,@ spontaneous outflow of radioactivity was calculated as the percentage of total tissue radioactivity released during the 4 mm preceding the first (Sp,) and the

second(Spa)periodofStImUlatiOn.
Theradioactivity
retainedbythecontroltissueafter120mmsuperfuslon
was75.7±0.6nCiperchamber
(n= 3).
C The [@H)dopamine overflow was calculated as the percentage of the total tissue radioactivity released above the spontaneous levels @ited by field stimulation at 3

Hz (20 mA, 2 msec) during a 2-mm period.

@ -H@

906 Dubocovlsh Vol. 246

it have any effect on melatonin-induced inhibition on [3HJ

‘¿
T LLJZIMJOLE dopamine overflow (fig. 7B).
1.0
V@:%%@.
A0.1@ * * a. SCHILD PLOT
Another compound, N-(3,5-dinitrophenyl)-5-methoxytryp
@ all tamine, was reported recently to be a melatonin antagonist in
I (jell_La 51fl4 in vitro and in vivo models (Zisapel and Laudon, 1987). In the
2.5
rabbit retina, 1 MM of this compound inhibited the calcium
dependent release of [3H]dopamine by 50% (fig. 7A), but did

p
2.0
0.5
15
not antagonize the inhibitory effects of melatonin (fig. 7B).
Finally, 5-methoxy-3-(2-morpholincethyl) indole, known to
1.0 interactwithserotoninreceptors(Tayloret ci, 1986),was
-I
0.5 screened as potential melatonin receptor antagonist because of
its structural similarity to the melatonin molecule. When this
-:@i-;o-@ -e -8 -7 -6 -5 compound was tested at a concentration of 1 MMin the rabbit
11$ DELATONIPII
00 LOG [LUZINDOLEI(N) retina it was found to have no effect on the spontaneous release
Fig. 3. Luzlndoleantagonizesthe melatonin-Induced
inhibitionof the of radioactivity
or on the calcium-dependent
releaseof [3H1
calcium-dependentrelease of [@H]dopamlnefrom rabbit retina. A, Ordi dopamine when added alone (fig. 7A). Furthermore, the same
nate:[@H)dopamlneoverfIow Isthepercentageoftotal tissueradioactMty concentrationof 5-methoxy-3-(2-morpholinoethyl)-indole did
releasedby field stimulation(3 Hz, 2 mm,20 mA, 2 msec)abovethe
spontaneouslevelsof release.Two periodsof fieldstimulation(S1,82)@ not antagonize the inhibitory effects of melatonin (0.1 nM) on
40 mliiapart,wereappliedperexperiment. Resuftsareexpressedas [3H]dopamine release (fig. 7B).
the ratio obtainedbetweenthe second(82)and first (S1)periodsof
stimulationwithinthesameexperiment.Abscissae:molarconcentrations
of melatonin(closedsymbols)(logarithmicscale).Luzlndole(A, 0.1 MM; Discussion
., 1MM;
•¿
10MM)
ifltheconcentrations
lndlcated
wasaddedtothe
medium40 mEnbeforeS@andremainedpresentthroughoutthe experi In this study the pharmacological potencies of a series of 2-
ment.B, Ordinate:Schildplotobtainedby transforming
thedatafrom substituted N-acetyltryptamines were determined for the me
concentratIon-response curvesto melatoninin the absenceor presence latonin receptor site which mediates the inhibition of [3HJ
of different concentrations of luzindole. Ordinate: log [Dose Ratio —¿1]
dopamine release from the rabbit retina labeled in vitro with
wheredoseratioIs thefactorby whichthe agonistconcentrationhasto
be Increasedin the presenceof the antagonistto obtain a response [3H]dopamine. Luzindole was shown to be a competitive antag
IdenticalInmagnftudeto that recordedInthe absenceof the antagonist. onist of mammalian melatonin receptors; the first discovered to
Abscissae:molar concentrationsof luzindole(logarithmicscale).The date. Both 2-iodomelatonin and 6,7-dichloro-2-methylmela
regressionof log (DoseRatio-1] on log [luzindole]yieldsa stralghtline tonin were found to be more potent than melatonin in inhib
wlthaslope=0.91 andapA@valueof7.7.
iting dopamine release from rabbit and chicken retina (table 1;
Dubocovich et ci, 1986; Dubocovich and Takahashi, 1987).
Thus, agonist activity at the retinal presynaptic receptor ap
pears to be enhanced when N-acetyltryptamines are substituted
in position 2 by an iodine or methyl group. In studies measuring
a. a. inhibition of ovulation in rats, substitutions at position 2 of
1.0
the melatonin nucleus (e.g., 6,7-dichloro-2-methylmelatonin)
also were found to result in an increased potency as compared
to melatonin. One possible reason for the enhancement of

k
potency in uwo is that such substituents block the metabolism
of these indoles by pyrrolase which attacks at position 2 (Cle
mens and Flaugh, 1985; Hirata et ci, 1974). The potency of
0.5 melatonin analogs in inhibiting the calcium-dependent release
of dopamine from the retina does not change with chlorination
at position6 (Dubocovich,
1985;Dubocovich
andTakahashi,
1987). However, 6-halomelatonin derivatives are more potent
antiovulatory agents than the corresponding unsubstituted an
alogs, because 6-halogenation effectively blocks metabolism to
@ —¿11
—¿10
—¿9
-8 —¿7 @.- cn'@-12-11-10 -9 -6 6-OH-melatonin in vivo (Flaugh et at., 1979; Kopin et ci, 1960,
LOG(6-cL-$ELATONDfl00 LOG(2-cH@tL
-wEuTo'a'il 00 1961).
Fig.4. Luzindoleantagonizesthe melatoninagonist-induced
inhibitionof From previous structure-activity studies on the melatonin
the calcium-dependentreleaseof rHjdopaminefromrabbitretina.Ordi receptor, it was suggested that N-acetyltryptamine analogs
nate: [‘H]dopamlne overflow Is the percentage of total tissue radloactMty lacking the 5-methoxy group would be potential melatonin
releasedby field stimulation(3 Hz, 2 mm,20 mA, 2 msec)abovethe receptor antagonists (Heward and Hadley, 1975; Dubocovich,
spontaneouslevelsof release.Two periodsof fieldstimulation(S1,82)@ 1984; Dubocovich and Takahashi, 1987). N-acetyltryptamine
40 mmapart,wereappliedperexperiment. Resultsareexpressedas
the ratio obtained between the second (82) and first (S1) periods of is a competitive melatonin receptor antagonist in chicken ret
stimulationwithin the same experiment. Abscissae:molar concentrations ma, and a partial melatonin agonist in rabbit retina (Dubocov
of melatoninagonists(Iogarithmioscale).Luzindole(0, ) was addedto ich, 1984, 1985). Luzindole, 2-benzyl-N-acetyltryptamine, was
the medium 40 mm before S1 and remained present throughout the found to be a potent and competitive melatonin receptor an
experiment. 6-Chioromelatonin (@, A, ) in A and 6,7-dichloro-2-methyl
@ melatonin •¿, •¿;
2-CH@-CL-melatonln) in B were added to the super tagonist in rabbit retina. Luzindole, in concentrations up to 10
fusion medium 20 mm before 82 in the concentrations indicated. Shown MM, did not modify either the spontaneous outflow or the
are mean values ±S.E.M. calcium-dependent release of [3Hjdopamine but it antagonized
 1988 MelatonlnRscsptorAntagonist 907

TABLE3
5tructure activity relationshipsof 2'.substltutedN-ac.tYItrYPtamInOs
ND,notdetermined.
Overlow@AgonetK.vMs'0pMriMHcol@t,_i@.
chem@ @m
1c50w@p_

CHCHP1H@CN3MELATONIN40ND0I

HCO CHCHNHCCH

3@c*LoRoIELATcI1N40ND024000i.ELATOt4I45ND0
@@1II@:1c;J
22

@ Hco CI@CH2NHCCH3

ci@yLcslsU-D@-CHLOR0.24@ETHYLMELAT0NJN1

0ND@[@@l2@3P@ACE1VLTRYPTAMpE5,600Melatonin

@rA@@()2BENZVLN.ACETVLTRYPTALUNE6-Chioromelatonin40(LUZSNDOLE.
20(@_@.C!@c$NJCH Melatonin9
5.0774)>10,000,0006,7-Dlchloro-2-methylmelatonin16

a N-fr@ypt@nines were tested on I'Hjdopemine release from rabbft retina as described under Materials end Methods.'
@ a @@10
v@ we the concentratIon of compounds necees@y to kihlbft the caiokim-dependent release of [@‘H)dopemine
by 50%. The values were determined
graphicety
fromconcentration-effect
curves(fig.3;DEocovich,1985).
°Thed@sodatlonconstent(K@r N-acetyftrypten@ne
was obtainedfromDubocovioh(1985).The dissodatlon constants for kizlndde were calculatedby the method
of Arunlakehana end Schild (1959).

the ability of melatonin to inhibit [3Hjdopamine release in a
competitive fashion, with a K8 of 20 nM. The Schild regression
gave a straight line with a slope of0.91 suggesting
of a competitiveinteractionbetweenmelatoninandluzindole
at the level of the melatoninreceptor(Kenakin,1987).The
calculated dissociation constants (KB) for luzindole determined
using the agonist 6-chloromelatonin or 6,7-dichloro-2-methyl
melatonin were very similar to that seen with melatonin. These
results suggest that these melatonin agonists activate the same
presynaptic melatonin receptor site to mediate inhibition of
calcium-dependent release of [@HJdopamine from rabbit retina.
It is tempting to speculate that the 2-benzyl substitution con
fers properties ofa competitive antagonist
tamine molecule.
The calcium-dependent release of dopamine from the rabbit
retina is modulated through activation of D-2 dopamine auto
receptors, and alpha-2 adrenergic and melatonin heterorecep
tore (Dubocovich and Weiner, 1981, 1985; Dubocovich, 1984).
Luzindole's selectivity for the melatonin receptor site is shown
the existence by the fact that at a concentration (1 @M)very effective in
antagonizing the melatonin-induced inhibition of [3H)dopa
mine release, it did not affect the inhibition of release mediated
by either the dopamine agonists, apomorphine and N-0437, or
the ajpha-2 adrenergic agonist, clonidine (Dubocovich and Wei
ner,1981,1985;Dubocovich,1984).Furtherevidenceforluzin
dole's selectivity for melatonin receptors is provided by binding
studies. Luzindole, in concentrations ranging from 1 nM to 100
MM, did not affect the binding of specific radioligands to a
variety of receptors in brain membranes including alpha-i and
on the N-acetyltryp alpha-2 adrenergic, beta-i and beta-2 adrenergic, D-1 and D-2
dopaminergic, serotonergic 5-HT-1 and 5-HT-2 , muscarinic,
adenosine-1 or benzodiazepine receptors (D. N. Krause and M.
L Dubocovich, in preparation).
A variety of other compounds with possible antagonistic
 908 Dubocovich Vol.246
TABLE4
LUZINDOLE1pM1,(S1.
&.@..
—¿I Ust of compounds• that do not antagonize the molatonin-Induced
@ 1.0 S2) Inhibition of (@H]dopamlnerelease from rabbit retina
w 22 -.-
Indoiss Alpha adrenerglc antagonists
w
TI2C
5-Methoxyindole (0.1 MM) Phentolamine
.< ‘¿I
N-acetyl-5-OH-tryptamine(0.03 Yohimbine
a. C,, MM) Prazosin
0
@ N 5-Methoxy-N,N-di-CHrtryptaminsBenzodiazepines
, U)
5-Methoxytryptophol Clorazepate
5-Methoxymorpholinyltryptamine Diezepan
@-4
6-Methoxy-2-benzoxazolinone Uptakeinhibitors
‘¿-4
N-(3,5-dinitrophenyl)-5-methoxy Nomifensine(30 MM)
tryptamine Imipramine(3 pM)
5-HT Others
0.0 Methysergide Naloxone
C 0.01 0.1 1 0.01 C 0010.1 1 0.01 aN
@ I I I I @_j I I i Methiothepine 6-OCHrtetrahydro-@-car
APON0@4IP€P4-0437 APON0@IItE P4-0437 Spiperone boline
(se) (Se) (Sa) (S2) Dopamineantagonists Tetrahydro-@9-carboline
Fig. 5. Effect of luzindole on the apomorphine- and N-0437-induced S-sulpiride
inhibition of the calcium-dependent release of [3Hjdopamine from rabbit Fluphenazine
retina. Ordinate: [3Hjdopamineoverflow is the percentage of total tissue Spiperone
radioactivityreleased by fieldstimulation(3 Hz, 2 mm,20 mA,2 msec) a The COmpOundS were tested alone and in com@natlon with rnelatorm (0.1 nM)
abovethespontaneouslevelsof release.Twoperiodsof fieldstimulation on the caldum-dependent release of [‘H]dopamineelIcited by field stimulation at 3
(S1, 82). 40 mm apart, were appliedper experiment.Resultsare ex Hz (20 mA, 2 msec) from rabbit retina. MI but two of the COmpOundS, at the
pressedas the ratio obtainedbetweenthe second(8,) and first (S1) concentration tested. failed to inhibit the calcium-dependent release of [@H)dopa
periodsof stimulationwithinthe sameexperiment.Apomorphineor N- minewhenaddedalonebeforeS,, theexceptionsbedgN-acety@5-OH-tryptamlne
0437 (A and B) at the concentrationsindicatedwere added to the (0.03 pM) and N-(3,5.dlnttrophenyl)-5-methoxytryptamlne.However, they did not
antagonize the inhibition of the caldum-dependent release of [@HJdopamlneelicited
superfusion medium 20 mm before the second (82)period of stimulation. by 0.1 nM melatonin. The effects of some of these compounds on melatonin
Luzindole1 pM (B) was added to the medium40 mm before S1 and induced Inhibitionof[3H]dopamine release was reported eader(Dubocovich, 1983,
remainedpresentuntil the end of the experiment.Shown are mean 1985). Aftcompounds were tested at a concentration of 1 @M with the exception
@ values ±S.E.M. P < .05 when compared with the corresponding of 5-OCH,-IndOIe
(0.1pM),N-acetyl-50H-tryptamine
(0.03MM),
nomifensine
(30
control (C). @M)
andimipramine
(3pM).

-I
U-
LU
D —¿J
LU U-
LU
.@ -I D
o_ C,@ U)

@ N N
I U) .@
In 0.
8
In
@-4
m
@-4

0.0 C BZ NT 4. (Se) NELATONIN

0.1 i@N (S2)
@ C 0.1 I ,$4 C 0.1 ,
D.ONIDINE D..ONIOINE C BZ NT 4. (S,.S2)
(S2) (S3) Fig. 7. Effects of various compounds on the melatonin-induced inhibition
Fig. 6. Effect of luzindole on the clonidine-induced inhibition of the of [‘H]dopamine
release from rabbit retina. Ordinate: [3H]dopammne
calcium-dependentreleaseof [3Hjdopaminefrom rabbit retina. Ordinate: overflow is the percentage of tissue radioactivity released by field stim
[3H]dopamineoverflow is the percentageof total tissue radioactivity ulation (3 Hz, 2 mm 20 mA, 2 msec) above the spontaneous levels of
released by field stimulation (3 Hz, 2 mm, 20 mA, 2 msec) above the release.ResultsareexpressedastheratioObtainedbetweenthesecond
spontaneouslevelsof release.Two periodsof fieldstimulation(S1,82). (82)andfirst (S1)periodsof stimulationwithinthe sameexperiment.0,
40 mm apart, were applied per experiment. Results are expressed as control; @A, 6-OCH@-2-benzoxazolinone (BZ, 1 pM); @,5-methoxy-3-(2-
the ratio obtained between the second (82) and first (S1) periods of morpholmnoeth@1@ndoie
(MT. 1 pM); W 5-OCH, (3,5-dinitrophen@l)
tryp
stimulation within the same experiment. Clonidine (A and B) at the tamine(ML,1 pM).InA, the variouscompoundswereaddedbeforethe
concentrationsindicatedwereaddedto the superfusionmedium20 mm second period of stimulation (82). In B, the effect of 0.1 nM melatonin
beforethe second(82)periodof stimulation.Luzindole1 pM (B) was (U) added before 82 was tested in the presence of the compounds
added to the medium 40 mm before S1 and remained present until the indicated which were added 40 mm before S1 and remained present
@ end of the experiment. Shown are mean values ± P < .05 when throughout 82 until the end of the penmen2 P < .05 when compared
compared with the corresponding control (C). with the correspondingcontrol.

activity at melatonin receptors also were examined in the been implicated in either inhibiting melatonin synthesis or
present study. However, none showed the competitive antago blocking melatonin receptors which control gonadal activity
nism seen with luzindole. Various compounds from different (Yuwiler and Winters, 1985; Sanders et al., 1981). This com
chemical classes, which lack a 3-acetoamidoethyl group, have pound, however, did not activate or block melatonin receptor
been reported to antagonize functional measurements of me sites in the rabbit retina, and therefore its effect in reproduction
latonin activity both in vivo and in vitro. 6-Methoxy-2-benzox may be unrelated to direct blockade of melatonin receptors.
azoline, which induces gonadal stimulation in the vole, has Similarly, N-(3,5-dinitrophenyl)-5-methoxytryptamine, which
1988 Melatonin Receptor Antagonist 909
was reportedpreviouslyto antagonizeweaklythe melatonin effects of endogenous melatonin inasmuch as this hormone is
induced inhibition of [3Hjdopamine release from rat hypothal also secreted by extrapineal tissues (Cardinali, 1981).
amus and also regression ofprostatal development (Zisapel and Recently, several groups of investigators have used bright
Laudon, 1987), did not antagonize the inhibition of [3H]dopa light to treat chronobiologic sleep and mood disorders in hu
mine release induced by melatonin in the rabbit retina in the mans (Lewy and Sack, 1986; Lewy et aL, 1987; Rosenthal et aL,
present study. Interestingly, both N-(3,5-dinitrophenyl)-5- 1984). If the therapeutic effects of light are related to the
methoxytryptamine (Zisapel and Laudon, 1987) and 5-meth suppression of melatonin secretion, the melatonin receptor
oxy-indole-N-methyl-3-propionamide (Askam et ci, 1985), antagonist luzindole may be useful in treating chronobiologic
which were reported to antagonize functional measurements of disorders involving changes in the pattern of melatonin secre
melatonin activity, can by themselves inhibit the calcium tion.In summary,the competitivemelatoninreceptorantago
dependent release of [3Hjdopamine from retina (fig. 7, unpub nist luzindole appears to block the activation of melatonin
lished observations). Whether this inhibition is mediated receptors in vivo providing a new experimental tool to investi
through activation of melatonin or monoamine presynaptic gate further the physiological role ofthis hormone in mammals.
receptors in the retina remains to be seen. Because melatonin Acknowledgments
is structurally similar to its metabolic precursor 5-HT, 5-HT The authorwouldliketo thankDrs.D. N. Krause,J. PeckandA. Heirnyfrom
antagonists such as 5-methoxy-3-(2-morpholinoethyl) indole Nelson Research for helpfUl discussions while conducting this research and for
methiothepine, methysergide or spiperone also were evaluated supplyingluzindole(N-0774)and N-0437.I am also grateful to Dr. J. Clement.
for supplying the 6-chioromelatonin and 6,7-dichlom-2-methylmelatonin to Dr.
but failed to antagonize the effects of melatonin in the rabbit E. W. Taylor for supplying 5-methoxy-3-(2-morpholinoethyl)indole; Dr. V.
retina.This is in keepingwith the fact that 5-HT does not Askam for supplying 5-methoxy-N-methyl-3 propionamide and Dr. N. Zisapel
affect the release of dopamine from the retina (Dubocovich, for supplying N-(3,5-dinitrophenyl-5-methoxytryptamine. I also thank Mr. Pa
trick Rita for excellent technical assistance, Dr. N. Krause for critical comments
1983, 1985). The putative melatonin receptor antagonists dis on the manuscript and Ms. VickyJames-Houffforexcellent secretarial assistance.
cussed above all lack an acetamidoethyl group which appears
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