l-;uropean Journal of Pharmacology. 213 (1992) 405-408

~, 1992 Elsevier Science Publishers B.V. All rights reserved (X)14-2999/92/$05.(10

EJP 52376

Effect of putative melatonin receptor antagonists on melatonin-induced pigment aggregation in isolated Xenopus laevis melanophores David Sugden Physiolo~' Group. Biomedical Sciences Di~'ision, King~"College London, Campden Hill Road. London W8 7,4tt. U.K.

Rcceivcd 24 October 1991. revised MS rcccived 27 December 1991, accepted 14 January 1992

The ability of putative melatonin receptor antagonists to antagonise melatonin-induced aggregation of pigment granuk cultured neural crest Xenopus laet'is melanophorcs was examined. Neither ML 23 (N-(2,4-dinitrophenyl)-5-methoxytryptan7 nor 6-methoxy-2-benzoxazolinone showed agonist or antagonist activity. N-Aeetyltryptamine and N-butanoyltryptamine partial agonists; both compounds aggregated pigment granules in some cells, but also reversed melatonin-induced pigr agregation in a fraction of the cells tested. In contrast, 2-benzyl N-acetyltryptamine Cluzindole) did not show agonist activity (~ 10 p,M) but did reverse the aggrcgating action of melatonin at 1 and 10 /sM. Pretreatmcnt of melanophores with luzin shifted the melatonin concentration-response curve to the right. Melatonin; Melatonin receptor antagonists; Xenopus melanophores; Pigment aggregation; Luzindolc

1. Introduction Melatonin (5-methoxy N-acetyltryptamine) regulates seasonal changes in reproduction and metabolism in photopcriodic species, synchronizes circadian rhythms and acts as a local hormone in the eye to regulate various aspects of retinal physiology (Tamarkin et al., 1985: Underwood, 1989; Besharse et ai., 1988). In addition, melatonin participates in the regulation of skin colour in amphibians such as Xenopus laecis (Bagnara, 1960) by causing a condensation of melanincontaining pigment granules in dermal melanophores (for review see Rollag, 1988). This response to melatonin occurs at very low concentrations of the hormone, is very specific and can readily be seen in vitro in isolated melanophores prepared from Xenopus embryos (Jackson et al., 1974; Messenger and Warner, 1977). The model has been used to examine structure-activity relationships at the melatonin receptor (Sugden, 1991) and can be used to test the potency of novel melatonin agonists and antagonists. Several compounds have been reported to be antagonists of melatonin either in vivo or in vitro. These

Correspondence tO: D. Sugden, Physiology Group, Biomedical S c i ences Division, King's College La]ndon, Campden Hill Road. Kensington, London W8 7AH, U.K. Tel. 44.71.836 5454 ext. 4372, fax 44.71.937 7783.

include 6-methoxy-2-benzoxazolinone (Sanders et 1981), N - ( 2 , 4 - d i n i t r o p h e n y l ) - 5 - m c t h o x y t r y p t a r r (ML-23) (Zisapel and Laudon, 1987), N-acetyltry mine (Heward and Hadley, 1975; Dubocovich, 1' and most recently, luzindole (2-benzyl N-acetyltry mine) (Dubocovich, 1988). In the present study action of these compounds on melatonin-induced m e n t granule aggregation in isolated Xen~ melanophores was tested.

2. Materials and Methods

2.1 Tissue cultt~re Xenopus embryos were produced from adults duced to lay by injection of human chorionic nadotrophin (Chorulon, Intervet Laboratories Ltd., 1.U./male, 600 1.U./female). Neural crest from s 20 embryos was dissected out and dispersed as scribed previously (Messenger and Warner, I c, Melanophores which appeared after 2 - 3 days of ture were readily visible among many other ne crest-derived cells. Melanophores were grown in bovitz L-15 medium (Gibco) diluted (I to 1) deionized water containing 10% fetal calf serum, I U / m l penicillin, 2 0 0 / ~ g / m l streptomycin, 2.5 /,~g

amphotericin B. Melanophore experiments were c between 7 and 12 days of culture.

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The response to melatonin of individual melanophores was quantitated by measuring the area of each cell occupied by pigment using computer assisted image analysis. All drugs were dissolved in M e O H except for luzindole (dimethylsulphoxide), diluted with deionised water just before use and added from 100 × concentrated stock solutions. At the maximal concerttration used (0.1% v / v ) neither of these solvents produced pigment granule aggregation,

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Mclatonin and 6-mcthoxy-2-benzoxazolinonc were purchased from Sigma Chemical Co., Peele, U.K. Luzindole was provided by Dr. S.M. Reppert, Boston, U.S.A. and ML 23 by Dr. N. Zisapel, Tel Aviv University, Tel Aviv, Israel. N-Acetyl and N-butanoyltryptamine were synthesized from tryptamine as described previously H e et al. (1968) and characterised by thinlayer chromatography a n d N M R (WM250 NMR, Univcrsity of London Intercollegiate Research Service at King's College).

3. Results A previous study (Sugdcn, 1991) has shown that melatonin produces a concentration-related aggregation of pigment granules in Xenopus melanophores (ECs, = 10 pM). When melanophores are fully aggregated pigment occupies = 20% of the initial pigmented cell area. All of the purported melatonin antagonists were first tested for agonist activity. Neither 6-methoxy-2-benzoxazolinone, ML 23 nor luzindole aggregated Xenopus melanophores at concentrations up to 10 /xM (fig. 1). However, N-acetyl and Nbutanoyltryptamine did produce pigment aggregation at high concentrations ( 1 - 1 0 / x M ) but only in some of the cells tested. Of the 12 cells treated with Nacetyltryptamine, two aggregated completely (pigmerited area < 20% of the initial cell area) at a concentration of 10 /zM, yet the remainder showed little response. Similarly with N-butanoyltryptamine (1 /,tM) three of 12 cells clearly responded. Antagonist activity was examined by first aggregating pigment granules with a concentration of melatonin (10 riM) sufficient to give a maximum response (pigmerited a r e a < 20%), then measuring pigmented a r e a after addition of increasing concentrations of the putative antagonists. The pigment granules of cells treated with melatonin (10 nM) remained aggregated for a prolonged period, and did not spontaneously disperse, ML 23 (fig. 2) and 6-methoxy-2-benzoxazolinone (data not shown) showed no antagonist activity at concentra-

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log [AGONIST] (M) Fig. 1. Effect of putative melatonin antagonists on Xenopus l~ melanophores. The area of each cell occupied by pigment measured and determined as a percentage of the initial pigmel area. Responses to N-acetyltryptamine (aT, e), N-butanoyltry mine (bT, o), ML 23 ( • ) and luzindole ( v ) are shown. Each p represents the m e a n + S . E . M , of 12-13 cells. Error bars are omi

when less than 5%. tions up to 10 /xM. Luzindole produced a conccw tion-related reversal of melatonin-induced pigment gregation in all cells tested (fig. 2). At the higl" 12o --" O ~E O o "6 ~ < uJ w < ~_ zLLI ~ tO E

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Fig 2. Antagonism of melatonin-induced pigment aggregatiot putative melatonin receptor antagonists. The pigmented are~ individual Xenopus melanophores was measured before treatr then after addition of melatonin (10 nM, 30 min) which indue maximal aggregation of pigment (pigment area < 20% of ir area); increasing concentrations of putative antagonists were added and pigmented area measured after 20 min. Response luzindole ( A , n = 11), ML 23 ( v , n = 7), N-acctyltryptamine n = 12) and N-butanoyltryptamine ( I t , n = 8) are shown. Each l: represents the mean _+S.E.M. Where the error is < 5% no error

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presence of luzindole (I ~ M , +" 10 ,u,M, ,x). Each point represents the m e a n of 8 - 1 6 cells. S.E.M. were < 10~. Curves were drawn t A L L F I T ( D e L e a n et al., 1978). The slope of each curve was constrained to be equal as this did not significantly degrade the quality of the I

concentration of luzindole (10 p,M) aggregation was inhibited by 70%. The responses to N-acetyl and Nbutanoyltryptamine as antagonists of melatonin again varied between cells. Four of the 12 cells tested showed clear reversal of melatonin-induced pigment aggregation at a high concentration of N-acetyltryptamine (10 p,M). Aggregation was reversed in three of eight cells treated with N-butanoyltryptamine (10 p.M). The antagonist action of luzindole was further examined by constructing concentration-response curves to melatonin in the presence and absence of different concentrations of luzindolc (fig. 3). The ECs0 for melatonin in this series of experiments was 49 pM. Luzindole (10 and 1 ,uM) produced a rightward shift in the concentratkm-response curve to melatonin. A lower concentration of luzindolc (0.1 p,M) did not shift the melatonin concentration-response curve,

4. Discussion 6-Methoxy-2-benzoxazolinone, which was reported to antagonize the physiological effects of melatonin in vivo (Sanders et al., 1981), had no activity as either agonist or antagonist at the melanophore melatonin receptor, even when tested at a high concentration (10 p,M). This compound also had no effect on melatonininduced inhibition of [3H]dopamine release from tabbit retina (Dubocovich, 1988), a well-characterized response known to be mediated by physiologically important melatonin receptors, ML 23 (N-(2,4-dinitrophenyl)-5-methoxytryptamine) has also been reported to be a melatonin antagonist in in vivo and in vitro models (Zisapel and Laudon, 1987). On melanophores, ML 23 was neither an agonist nor

an antagonist. ML 23 did not show antagonist acti in the retinal [3H]dopamine release model but have some agonist activity (Dubocovich, 1988). ' lack of effect of these two compounds agrees radioligand binding data using 2-[~2~I]iodomelatonil identify melatonin binding sites as neither compo inhibits high affinity binding to chicken brain m branes at concentrations up to 10 p,M (Sugden, unl: lished data). N-Acetyltryptamine has been reported previousl be an antagonist of melatonin-induced skin-lighte~ in Rana piniens (Heward and Hadley, 1975) and antagonist of [3H]dopamine release in chicken re (Dubocovich, 1984). In the present experiments isolated Xenopus melanophores, N-acetyltryptan induced pigment aggregation in some cells, yet able to antagonise aggregation induced by melatoni others. Thus N-acetyltryptamine acts as a partial nist at this melanophore receptor. The response tained probably reflects differences in the dcnsit' melatonin receptors present on the surface of indi ual melanophores. N-Butanoyltryptamine, which more potent inhibitor of 2-[~2~l]iodomelatonin bim than N-acetyltryptamine (K~ values 68 and 730 respectively; Sugden and Chong, 1991), was als partial agonist at this receptor. As expected from binding data, it appeared to be more potent ( N-acetyltryptamine, although again only a propor of cells responded. Luzindole did not have any agonist activity at melanophore melatonin receptor at concentrat upto 10 p,M. However, it did antagonise the pign aggregating action of mclatonin. This is in agreen with data from the retina (Dubocovich, 1988) w shows that luzindole lacks agonist activity, but

4118

competitive antagonist of melatonin-induced inhibition of [3H]dopamine release. It would a p p e a r that in the retina luzindole is somewhat more potent (Kn = 20 nM) than in melanophores (apparant K B = 90 nM). In contrast, Howell and Morgan (1991) found no evidence that luzindole was a mclatonin antagonist in sheep pars tuberalis cells as it did not antagonize the melatonininduced inhibition of forskolin stimulation of cyclic AMP. Radioligand binding studies on mcmbranes from several tissues, including sheep pars tuberalis and chick retina, indicate that luzindolc has only a weak affinity in competition experiments for the 2-[~2Sl]iodomelatonin binding sitc; in sheep pars tubcralis, K i = 1.35 IzM (Howell and Morgan, 1991), in chick retina, K i ~ 1 l,tM (calculated from fig. 6 in Siuciak et al., 1991), and chick brain, K i = 11.6-0.9 /xM (Siuciak et al., 1991). Our data from competition binding assays are in gcneral agreement with these values (sheep pars tuberalis, chick brain and chick neural retina membranes, K i = 1.0, 1.6 and 1.7 ~ M respectively, Sugden, unpublished), Despite this low affinity, luzindole has been shown to exert an antidepressant-like activity in the mouse behavioural despair test (Dubocovich et al., 1990), an effect which has been proposed to be due to its ability t o antagonise endogenous mclatonin. In contrast, luzindole did not antagonise melatonin-induced testicular regression in adult Siberian hamsters - a photoperiodic species in which pineal melatonin is known to regulate seasonal breeding (Duncan et al., 1990; Carter and Goldman, 1985).

Acknowledgements 1 am grateful to Dr. Steve Reppert and Dr. Nava Zisapel for the supply of luzindolc and ML 23, and am indebted to Professor Anne Warner and Dr. Sally Rowe, University College, l_xmdon for their help with the Xenopus cultures.

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Carter, D.S. and B.D. Goldman, 1983. Antigonadal effects of ti melatonin infusion in pinealectomised male Djungarian ham:

( P h o d o p usungorus s sungorus): duration is the critical param,

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Physiol. 235, E97. Dubocovich.M.L., 1984. N-Acetyltryptamine antagonizes the n tonin-induccd inhibition of [~tt]dopamine release from re Eur. J. Pharmacol. 1115, 193. Dubocovich. M.L.. 1088, Luzindole (N-07741: A novel molar, rcccptor antagonist, J. Pharmacol. Exp. Ther. 246, 902. Dubocovich.M.L.. E. Mogilnicka and P.M. Areso, 19911,Antider sant-like activity of the melatonin antagonist, luzindolc(N-0" in the mouse bchavioural despair test, Eur. J. Pharmacol. 313.

Duncan, M.J., J.M. Fang and M.L. Dubocovich, 199{I, Effect melatonin agonists and antagonists on reproduction and I weight in the Siberian hamster, J. Pineal Rcs. 9, 231. lleward, C.B. and M.E. Hadley, 1975, Structure-activity relations of mclatonin and related indoleamines, l,ife Sci. 17, 1167. llo, B.T.. W.M. Mclsaac, l,.W. Tansey and P.M. Kralik, 1 Itydroxyindole-O-methyltransferasc 11. Inhibitoo' activitie~

some N-acyltryptamines,J. Pharm. Sci. 57. 1988. tIowell,II.E. and P.J. Morgan, 1991, Luzindole (2-benzyl acetyltryptaminc), 5-methoxytryptamine, N-acetyltryptamine 6-methoxy-2-benzoxazolinone activity in ovinc pars tubcralis ¢ Adv. Pineal Res. 5, 2115. Jackson,P.A.. E.A. Messenger and A.E. Warner, 1974, Differcl tion of amphibian embryonic cells in vitro, J.Physiol. (Lon, 246. 9P. Messenger, E.A. and A.E. Warner, 1977. The action of melatoni single amphibian pigment cells in tissue culture, Br. J. Pharm~ 61, 6117. Rollag, M.D., 1988, Response of amphibian melanophores to n tonin, Pineal Res. Rcv. 6, 67. Sanders, E . H . P . D . Gardner, P.J. Berger and N.C. Negus, 1 6-Methoxybenzoxazolinone: A plant derivative that stimul reproduction in Mictotus montanus, Science 214, 67. Siuciak, J.A.. D.N. Krause and M.L. Dubocovich, 1991, Quantit; pharmacological analysis of 2-1"sI-iodomelatonin binding site discrete areas of the chicken brain. J. Neurosci. 11, 2855. Sugden, D., 1991, Aggregation of pigment granules in single cult~ Xenopus h'acis melanophores by melatonin analogues, BI Pharmacol. 104, 922. Sugden, D. and N.W.S. Chong, 1991, Pharmacological identit 2-[125I]iodomelatonin binding sites in chicken brain and s] pars tuberalis. Brain Res. 539. 151. Tamarkin, L., C.J. Baird and O.F.X. AImcida, 1985. Melatoni coordinating signal fi)r mammalian reproduction'?, Science 714. Underwood, H., 1989, The pineal and melatonin: regulator circadian function in lower vertebrates, Experientia 45, 914. Zisapel, N. and M. Laudon, 1987, A novel melatonin antag~ affects melatonin-mediated processes in vitro and in vivo, Et Pharmacol. 136. 259.