Brhin Reseat'ch, 528 (1990) 349-352

349

Elsevier

BRES 24303

The chick retinal melatonin receptor revisited: localization and modulation of agonist binding with guanine nucleotides Jarmo T. Laitinen and Juan M. Saavedra Section on Pharmacology, Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD 20892 (U.S.A.)

(Accepted 19 June 1990) Key words: Agonist binding; G-Protein; Melatonin; Receptor; Retina

The chick retinal melatonin receptor was localized to the inner plexiform layer using 2-125I-melatonin and in vitro autoradiographic techniques. This receptor was of high affinity (Kd 19.8 pM) and specificityand showed guanine nucleotide modulation of agonist binding. It thus behaves similarly to other recently characterized high affinity melatonin receptors. Melatonin (MT) is produced in the pineal and retina of several vertebrates in a rhythmic fashion with an increase in synhesis during the night. While the pineal MT is secreted to the body fluids, the retinal MT is thought to act locally within the eye 2'4'13. Picomolar concentrations of MT were shown to selectively inhibit CaE÷-dependent release of dopamine from rabbit and chicken retina in vitro through the activation of a site possessing functional and pharmacological characteristics of a receptor for the indole 7'9. Moreover, specific binding sites for the MT agonist 9"21'24, 2-125I-MT (125IMT) have been extensively characterized in chick retinal membranes dissected free of pigment epithelium (PE) 8'9'1l. The specificity of these sites strongly suggests that they are putative MT receptors. However, the localization of this binding has not been described. Recent work using 125IMT has led to the characterization of high-affinity MT receptors (K d around 50 pM when determined in Tris-buffer at 22-37 °C ) in lizard ~9 and chick2° brain, in sheep 17'18 and rodent pars tuberalis 22-25, as well as in rat postrema ~6 and suprachiasmatic nuclei 14'15. All of these sites show similar affinity and high specificity for MT. Where tested, guanine nucleotides modulated agonist binding to these receptors, suggesting that they are all coupled to G-protein(s). Direct evidence for this coupling has been provided for the MT receptor in the hamster pars tuberalis 5. In contrast, laSIMT binding in chick retinal membranes was not affected by GTP when incubations were carried out at 0 °C (ref. 11). Moreover, the chick retinal MT receptor has considerably lower affinity than the above mentioned MT receptor sites. Whether these discrepancies reflect

differences in methodology (incubation temperature) or in receptor characteristics has not been addressed. We have now localized the chick retinal MT receptor to the inner plexiform layer (IPL) and demonstrate that it shows comparable affinity to the other high-affinity MT receptor sites, as well as guanine nucleotide modulation of agonist binding. Chicks were obtained from Truslow Farms, Inc. (Chestertown, MD). They were kept on a 9.15 h light/dark cycle (lights on at 08.00 h). Whole eyeballs from 2-day-old-chicks, decapitated between 15 and 16 h on the day of arrival, were dissected out and frozen in isopentane at -70 °C. Sagittal sections (16 or 20 p m thick) were cut in Leitz cryostat at -20 °C, thaw-mounted on gelatin-coated glass slides and dried in a desiccator at 4°C for 12h. Sections were preincubated for 15 min at 22 °C in 50 mM Tris-HCl, pH 7.40, containing 4 mM CaCl 2 and incubated in the same buffer at 22 °C for 1 h with 125IMT (Amersham, Arlington Heights, IL; specific activity 1800-2100 Ci/mmol) as described 14-16. Nonspecific binding was assessed in the presence of 1/~M MT (Sigma, St Louis, MO). After incubation, sections were washed twice for 5 min each time in Tris-HCl at 0 °C and for 30 s in distilled H20. We have shown that this protocol is quantitative (intact free and bound ligand, equilibrium binding conditions, no loss of high affinity binding during washing) for the high affinity MT receptors in the rat brain 14'16. Although we did not validate the protocol for the chick retina, the kinetic data from the work of Dubocovich and Takahashi 1° using chick retinal membranes suggests that these binding conditions are likely to

Correspondence: J.T. Laitinen, National Institutes of Health, Building 10, Room 2D-45, 9000 Rockville Pike, Bethesda, MD 20892, U.S.A.

0006-8993/90/$03.50 t~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)

350 be optimal also for the chick retinal binding site. After washes, sections were dried and apposed to Hyperfilm-3H (Amersham) for 5 days together with 16-/~m sections cut from Amersham Autoradiographic [125I] Micro-Scale standards. Developing and quantitation of films were as described earlier 14-16. For emulsion autoradiography, some sections were dipped in autoradiography emulsion (Kodak NTB-2, Rochester, NY), exposed for 10 days at 4 °C and developed in a Kodak D-19 developer. To indentify anatomical structures, sections were stained with Cresyl violet. ATP, GTP and GTPyS were from Sigma and ATPyS from Boehringer Mannheim Biochemicals (Indianapolis, IN). In vitro autoradiography revealed two bands of radioactivity in sagittal sections of the chick eyeball, one restricted to the retina (Fig. 1A, inner band) and the other to the PE (Fig. 1A, outer band). The retinal binding was displaceable with excess of unlabeled MT while the binding to the PE was not (see below). This pattern of radioactivity was observed across the entire cross-section of the eyeball (data not shown). Emulsion

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autoradiography revealed that the specific binding was restricted to the IPL of the retina (Fig. 1B,C). The apparent binding in layers close to the PE was due to 'fogging' from the ligand bound to the PE and was not detected with lower ligand concentrations (15-61 pM) which readily labeled the IPL (data not shown). The binding of 125IMT in the chick IPL was to a single class of high affinity sites (K d 19.8, pM, Bmax 96.7 fmol/mg protein (Fig. 2, upper part). This binding capacity closely agrees with that previously reported for 125IMT binding in PE-free chick retinal membranes incubated at 0 °Cll. However, the affinity obtained here is considerably higher and similar to the affinity of the MT receptor sites mentioned above. The specificity of the chick retinal MT receptor has been extensively characterized 7-93°. We confirmed the order of potency in the IPL being MT > 6-hydroxy-MT > Noacetylserotonin (ICs0 25 pM, 4.5 nM and 97 nM, respectively). Agonist binding to putative MT receptors in several areas is modulated with guanine nucleotides. As shown in

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Fig. 1. Autoradiographic localization of 125IMTbinding sites in the chick eyeballs. A: sagittal sections (16/~m thick) were incubated with 149 pM I25IMTin the absence (total binding) or presence of 1/~M MT (nonspecificbinding) and processed for autoradiography for 5 days. Images are light field photomicrographs from autoradiography film. B: dark field emulsion autoradiography of 12SIMTbinding into chick retinal IPL and PE in an area corresponding to the square shown in A. Sagittal sections (16/~m thick) were incubated as described above, dipped into photoemulsion and developed 10 days later. The displaceable binding was restricted to the IPL. C: sections shown in B after staining with Cresyl violet. Abbreviations: GCL, ganglioniccell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; PE, pigment epithelium.

351 CHICK RETINA 100 I

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Fig. 3. Effect of guanine (GTPyS, GTP) and adenine (ATPyS, ATP) nucleotides on MT-agonist binding at 22 °C in the IPL of chick retina. Sagittal sections (20/~m thick) of the eyeball were incubated with 63 pM 1251MTin the absence (total binding) and presence of increasing concentrations of the nucleotides. Half-maximal inhibition (IC50)was achieved with 3.2/~M GTPyS, 15.8/~M GTP and 2.0 mM ATPTS. Values are means + S.E. of 4 eyeballs.

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Fig. 2. Upper part: saturation isotherm and Scatchard plot of 125IMT binding in the IPL of chick retina. Sagittal sections (16/tm thick) were incubated with increasing concentrations of a25IMT in the absence (total binding) or presence of 1 pM MT (nonspecific binding, A). Specific binding (0) was obtained after substracting nonspecific binding from total binding. Inset: Scatchard plot of the data. The K a was 19.8 pM and the Bma, was 96.7 fmol/mg protein. Lower part: lZSlMTbinding isotherms to the PE of chick eye in the absence (total binding, O) and presence (nonspecific binding, A) of 1 pM MT. In both figures, values are means of 4 eyeballs.

Fig. 3, micromolar concentrations of GTPyS and GTP dose-dependently inhibited 125IMT binding in the chick IPL (IC50 3.2 and 15.8 pM, respectively), whereas micromolar concentrations of adenine nucleotides were without effect. We also screened for 125IMT binding sites in the rat retina. In the Wistar IPL, specific binding was < 0.5 fmol/mg protein (limit of sensitivity) and became visible only after prolonged exposure time (data not shown). This agrees with data reporting low density (0.35 fmol/mg protein) of 12SlMT binding using Wistar retinal membranes 22. No saturation of 125IMT binding to the PE was achieved using ligand concentrations that readily saturated the IPL site (Fig. 2, lower part). In the presence of

1/~M MT, a rightward shift, parallel to the total binding isotherm resulted. No Scatchard transformation of the PE binding could be produced. Similarly, 125IMT bound in a nondisplaceable manner to the PE of pigmented rats, mice and hamsters (data not shown). We have localized the chick retinal MT receptor to the IPL. This receptor has comparable affinity to the other MT receptor sites when similar incubation temperature was used. Further, G T P modulation of agonist binding to the MT receptor in the IPL suggests that this receptor may also be coupled to G-protein(s). Additional evidence supporting this arises from studies showing similarities in cation modulation of 125IMT binding to the MT receptors in the chick retina l°J1 and in the rat postrema 16 and suprachiasmatic nuclei TM. The localization of the retinal MT receptor to the IPL provides the anatomical basis for understanding of the actions of MT within the chick eye. Several important synaptic connections, e.g. those between the dopaminergic amacrine cells and those to the ganglion cells are in the IPL 6J2. The MT-elicited inhibition of Ca2+-depen dent dopamine release is likely to occur through the MT receptors in the IPL. We acknowledge a preliminary report localizing specific 125IMT binding in the rabbit retina mainly to the PE and IPL 3. Earlier retinal work using 3HMT in vitro localized specific binding associated with melanosomes of the frog PE-choroid, as well as the OPL of the retina 26. In both

352 sites, this binding had low affinity and high capacity, in contrast to what has been described for the putative M T receptors. O u r data also suggest nonsaturable, low affinity binding of 125IMT to the PE. Melanin, a key c o m p o n e n t of the PE, has been shown to non-specifically bind several small molecules, e.g. the benzazepines 1. We suspect that this might also be the fate of 125IMT in the chick PE. Interestingly, although chick and frog retinae readily synthesize MT, nocturnal content and release of the indole are low in the cultured pigmented eyecups 4. As a possible explanation for this low output from the frog eyecup, retinal M T was recently shown to be metabolized within the eye 4. A n additional explanation might emerge from the present findings suggesting low affinity, high capacity binding to the PE in vitro. In an analogous

manner, the PE might act in vivo as a 'black hole' capturing most of the released MT, thus resulting in low content and release of the indole. Several reports suggest a role for pharmacological levels of M T in the PE, including shedding p h e n o m e n a of rod outer segments and in vitro phagocytosis reaction of P E cells 2'13. While we cannot exclude these proposed roles, it is unlikely that they are mediated via a high-affinity M T receptor. The relatively high density of the M T receptors in the chick retina suggests a potential use of this tissue for studies on MT signal transduction mechanism.

1 Aqui, T., Bryant, G., Kebabian, J.W., Larson, S., Saavedra, J.M., Shigematsu, K., Yamamoto, T. and Yokoyama, K., x2sI-Iodinated benzazepines bind to melanin: implications for the noninvasive localization of pigment melanomas, Nucl. Med. BioL, 14 (1987) 133-141. 2 Besharse, J.C., The daily light-dark cycle and rhythmic metabolism in the photoreceptor-pigment epithelial complex. In N. Osborne and G. Chader (Eds.), Progress in Retinal Research, Vol. 1, Pergamon Press, Oxford, 1982, pp. 81-124. 3 Blazynski, C, Beaty, C.A., Chung, K.C., Jones, Z.J., Woods, C. and Dubocovich, M.L., Localization of 2-[125I]-iodomelatonin binding sites in mammalian retina. In 19th Annual Meeting of the Society for Neuroscience, Phoenix, AZ 1989, p. 1395 (Abstract). 4 Cahill, G.M. and Besharse, J.C., Retinal melatonin is metabolized within the eye of Xenopus leavis, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 1098-1102. 5 Carlson, L.L., Weaver, D.R. and Reppert, S.M., Melatonin signal transduction in hamster brain: inhibition of adenylyi cyclase by a pertussis toxin-sensitive G protein, Endocrinology, 125 (1989) 2670-2676. 6 Daw, N.W., Jensen, R.J. and Brunken, W.J., Rod pathways in mammalian retinae, Trends Neurosci., 13 (1990) 110-115. 7 Dubocovich, M.L., Melatonin is a potent modulator of dopamine release in the retina, Nature (Lond.), 306 (1983) 782-784. 8 Dubocovich, M.L., Characterization of a retinal melatonin receptor, J. Pharmacol. Exp. Ther., 234 (1985) 395-401. 9 Dubocovich, M.L., Pharmacology and function of melatonin receptors, FASEB J., 2 (1988) 2765-2733. 10 Dubocovich, M.L. and Takahashi, J.S., Use of 2-[125I]-iodomelatonin to characterize melatonin binding sites in chicken retina, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 3916--3920. 11 Dubocovich, M.L., Shankar, G. and Mickel, M., 2-[1251]iodomelatonin label sites with identical pharmacological characteristics in chicken brain and chicken retina, Eur. J. Pharmacol., 162 (1989) 289-299. 12 Ehinger, B. and Dowling, J.E., Retinal neurocircuitry and transmission. In A. Bjrrkhind, T. Hrkfelt and L.W. Swanson (Eds.), Handbook of Chemical Neuroanatomy, Vol 5, Elsevier, Amsterdam,1987, pp. 389-446. 13 Iuvone, P.M., Neurotransmitters and neuromodulators in the retina: regulation, interactions, and cellular effects. In R. Adler and D. Farber (Eds.), The Retina: A Model for Cell Biology Studies, Vol. 2, Academic Press, London, 1986 pp. 1-72.

14 Laitinen, J.T. and Saavedra, J.M., Characterization of melatonin receptors in the rat suprachiasmatic nuclei: modulation of affinity with cations and guanine nucleotides, Endocrinology, 126 (1990) 2110-2115. 15 Laitinen, J.T., Castren, E., Vakkuri, O. and Saavedra, J.M., Diurnal rhythm of melatonin binding in the rat suprachiasmatic nucleus, Endocrinology, 124 (1989) 1585-1587. 16 Laitinen, J.T., Fliigge, G. and Saavedra, J.M., Characterization of melatonin receptors in the rat area postrema: modulation of affinity with cations and guanine nucleotides, Neuroendocrinology, 51 (1990) 619-624. 17 Morgan, P.J. Lawson, W., Davidson, G. and Howell, H.E., Guanine nucleotides regulate the affinity of melatonin receptors on the ovine pars tuberalis, Neuroendocrinology, 50 (1989) 359-362. 18 Morgan, P.J., Williams, L.M., Davidson, G., Lawson, W. and Howell, E., Melatonin receptors on ovine pars tuberalis: characterization and autoradiographical localization, J. Neuroendocrinol., 1 (1989) 1-4. 19 Rivkees, S.A., Carlson, L.L. and Reppert, S.M., Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 3882-3886. 20 Rivkees, S.A., Cassone, V.M., Weaver, D.R. and Reppert, S.M., Melatonin receptors in chick brain: characterization and localization, Endocrinology, 125 (1989) 363-368. 21 Vakkuri, O., L/ism~i, E., Rahkamaa, E., Ruotsalainen, H. and Lepp~iluoto, J., Iodinated melatonin: preparation and characterization of molecular structure by mass and 3H-NMR spectrometry, Anal. Biochem., 284-289. 22 Van~ek, J., Melatonin binding sites, J. Neurochem., 51 (1988) 1436--1440. 23 Vanr~ek, J., Pavia, A. and Illnerov~t, H., Hypothalamic melatonin receptor sites revealed by autradiography, Brain Research, 435 (1987) 359-362. 24 Weaver, D.R., Namboodiri, M.A.A. and Reppert, S.M., Iodinated melatonin mimics melatonin action and reveals discrete binding sites in fetal brain, FEBS Lett., 228 (1988) 123-127. 25 Weaver, D.R., Rivkees, S.A. and Reppert, S.M., Localization and characterization of melatonin receptors in rodent brain by vitro autoradiography, J. Neurosci., 9 (1989) 2581-2590. 26 Wiechmann, A.E, Bok, D. and Horwitz, J., Melatonin-binding in the frog retina: autoradiographic and biochemical analysis, Invest. Ophthalmol. Visual Sci., 27 (1986) 153-163.

We thank Dr. Martin Zatz for help in obtaining the chicks and Mr. Quang Tran for typing the manuscript. J.T.L. is a Fogarty Fellow from the Department of Physiology, University of Kuopio, Finland.