Melatonin binding sites in the goldfish retina were characterized by radioreceptor assay using 2-[125|]iodomelatonin as the radioligand. The specific binding to goldfish retinal membranes is rapid, stable, saturable and reversible. Saturation studies demonstrated that 2-[125|]iodomelatonin binds to a single class of sites with an affinity constant (Kd) of 61.9 ± 5.7 pM, a total binding capacity (Bmax) of 6.52 ± 0.79 fmol/mg protein and Hill coefficients (nH) of 1.07 ± 0.03 (mean ± SEM, n = 6). Competition experiments with various indoles and neurotransmitters revealed the following order of affinities: 2-iodomelatonin > melatonin > 6-hydroxymelatonin > 5-methoxytryptamine = N-acetylserotonin > 5-methoxytryptophol. The other indoles and neurotransmitters tested were much less effective. The order resembles with those reported for the goldfish brain and the ML-1 subtype melatonin receptors in vertebrates. Co-incubation of retinal membranes with a non-hydrolyzable GTP analog, guanosine 5′-O-(3-thiotriphosphate), significantly reduced the specific binding. These results suggest that in the goldfish, ocular melatonin plays neuromodulatory roles in the retina via G protein-coupled melatonin receptors with picomolar affinity.
Melatonin is synthesized in the pineal organ and the retina of vertebrates in a rhythmic fashion with peak production during the dark phase of light-dark (LD) cycles. Melatonin produced in the pineal organ is secreted into the blood and/or the cerebro-spinal fluid and acts as a time keeping hormone that regulates circadian and seasonal rhythms (for review, see Underwood and Goldman, 1987). Melatonin produced in the retina, on the other hand, is considered to act within the retina itself. Melatonin is known to affect several aspects of retinal physiological processes such as dopamine release, retinomotor movements, disc shedding of photoreceptor outer segments, aggregation of the retinal pigment epithelium and sensitivity of horizontal cells (for review, see Pang and Allen, 1986; Cahill et al., 1991).
The actions of melatonin are thought to be mediated through its specific receptors. Although several attempts have been made to characterize them using [3H]melatonin as a radioligand, only the binding sites with low (nanomolar) affinity were labeled (Cohen et al., 1978; Cardinali et al., 1979; Niles et al., 1979; Gern et al., 1980; Wiechmann et al., 1986). The physiological functions of these sites still remain unclear. However, introduction of a melatonin agonist, 2-[125l]-iodomelatonin with high specific radioactivity (Vakkuri et al., 1984), enables us to characterize melatonin binding sites with picomolar affinity by radioreceptor assay and in vitro autoradiography. These sites are considered as functional melatonin receptors in various tissues including the central nervous system and peripheral tissues (for review, see Dubocovich, 1988, 1995; Pang et al., 1993; Morgan et al., 1994). In the vertebrate retina, melatonin binding sites with picomolar affinity were identified in mammals, birds, reptiles and amphibians (Dubocovich and Takahashi, 1987; Vaněček, 1988; Laitinen and Saavedra, 1990; Blazynski and Dubocovich, 1991; Chong and Sugden, 1991; Lu et al., 1991; Wiechmann and Wirsig-Wiechmann, 1991, 1994; Skene et al., 1993; Failace et al., 1995; James et al., 1995).
In teleosts, melatonin is produced in the retina (Quay, 1965; Gern and Ralph, 1979; Wiechmann and Hollyfield, 1988; Nowak et al., 1989; Falcon and Collin, 1991; Grace et al., 1991; Cahill, 1996; ligo et al., 1997a) and known to affect retinal rhythmic processes (Chéze and Ali, 1976; Mangel and Wong, 1996) as well as in other vertebrate classes. Thus, it is probable that melatonin receptors exist in the teleostean retina. However, to our knowledge, melatonin binding sites with picomolar affinity have not yet identified in the retina of fishes.
In order to obtain more insights on the physiological functions of melatonin in the fish retina, in the present study, melatonin binding sites in the retina of the goldfish (Carassius auratus) were characterized by radioreceptor assay using 2-[125I]iodomelatonin as the radioligand. This fish was selected as our experimental model because we have studied the regulation of melatonin production in the pineal organ and eye, and characteristics and regulation of melatonin binding sites in the brain in this species (Kezuka et al., 1992; ligo et al., 1994a, b, 1995, 1997a, b).
MATERIALS AND METHODS
2-[125l]lodomelatonin (specific radioactivity, 2,000-2,200 Ci/mmol) was obtained from Amersham (Buckinghamshire, England) or New England Nuclear (Boston, MA). Unlabeled 2-iodomelatonin was obtained from Research Biochemicals Inc. (Natick, MA). The other indole derivatives, norepinephrine, polyethlenimine, and guanosine 5′-O-(3-thiotriphosphate) (GTPγS; tetralithium salt) were obtained from Sigma (St. Louis, MO). Acetylcholine was obtained from Nacalai Tesque, Inc. (Kyoto, Japan). Ordinary chemicals were obtained from commercial sources.
The goldfish (61-164 g in body weight) purchased from a local dealer were maintained in experimental aquaria under LD cycles of 12 hr:12 hr (lights on 06.00-18.00) with controlled temperature at 24-25°C for at least two weeks. Illumination (300-500 Ix at the water surface) was supplied by a white fluorescent bulb (10 W) for the light phase. Fish were fed commercial trout pellets.
The goldfish were anesthetized with 2-phenoxyethanol (0.6 ml/I) and decapitated during the light phase (11.00-16.00) when ocular melatonin contents are low (ligo et al., 1994a, 1997a). The retina (neural retina with the pigment epithelium) was dissected out, immediately frozen on dry ice, and stored at -80°C until used.
Membrane preparation and binding assay
Membrane preparation and binding assay were performed according to the procedure as previously described (ligo et al., 1994b) unless otherwise stated. Briefly, the retina was homogenized in 50 mM Tris-HCI buffer (pH 7.5), centrifuged (40,000 × g for 20 min at 4°C). The pellet was resuspended in the buffer and centrifuged for the second time. The pellet was used for binding assay. Incubation was performed in a total volume of 200 μl and the binding of 2-[125l]iodomelatonin was routinely measured in duplicate after incubation at 25°C for 1.5 hr with the exception of kinetic studies where incubation duration and temperature varied. Protein contents in the incubation mixture were determined by the method of Bradford (1976) with bovine γ-globulin as a standard. Nonspecific binding was defined as the binding in the presence of 10 μM melatonin. Specific binding was calculated by subtracting nonspecific binding from total binding and expressed as fmol/mg protein.
Identification of bound radioligand
Stability of 2-[125l]iodomelatonin during the incubation period was examined since melatonin is known to be metabolized in the eye of nonmammalian vertebrates including the goldfish (Grace et al., 1991). After filtration of a binding assay, filters were removed and extracted with methanol (2 ml). The methanol extract was evaporated under reduced pressure to a volume of 100 μl. An aliquot of this extract (50 μl) was applied to a silica gel plate for thin layer chromatography (TLC) (Whatman LK6F, Whatman International Ltd., Maidstone, England). Melatonin (10 μg) and 2-iodomelatonin (10 μg) were also applied as tracers. Chromatograms were developed with ethyl acetate for 10 cm. The position of melatonin and 2-iodomelatonin on the TLC plate was determined by fluorescence during exposure to UV lights and the Rf values were calculated. Radioactivity of sections of silica gel (1 cm) were counted in a γ-counter. The peak radioactivity in the extracts of the bound radioligand was compared with the position of 2-iodomelatonin.
Characterization and GTPγS modulation of melatonin binding sites in the goldfish retina
Saturation studies were performed using a range of 2-[125l]iodomelatonin concentration from 4.4 to 269.9 pM. Kinetic and competition studies were performed using a 2-[125l]iodomelatonin concentration of 49.9 and 49.6 pM, respectively. To study the effects of GTPγS on melatonin binding sites in the goldfish retina, the specific binding of 2-[125l]iodomelatonin (68.9 pM) was determined in the presence or absence of GTPγS (10-4 M).
Data obtained from kinetic, saturation and competition experiments were analyzed as previously described (ligo et al., 1994b) to calculate association rate constants (K1), dissociation rate constants (K−1), Kd, Bmax, nH and Ki values. Correlation between the specific binding and the protein concentration in the incubation mixture, and between log Ki values of some indoles obtained in the present study and those reported for the goldfish brain (ligo et al., 1994b) was analyzed by linear-regression analysis. The specific binding in the GTPγS-treated group was compared with the vehicle-treated control by paired t-test.
At first, the time course of the association of 2-[125l]-iodomelatonin (50.0 pM) to goldfish retinal membranes at 0, 25 and 37°C was compared (data not shown). The specific binding at equilibrium was similar at 0 and 25°C but the equilibrium was reached faster at 25°C (1 hr) than at 0°C (12 hr). At 37°C, the specific binding did not show a steady state. Therefore, further experiments were performed at 25°C.
In a preliminary study, retinal homogenates were prepared in 50 mM Tris-HCI buffer (pH 7.5) containing 0.32 M sucrose and fractionated by differential centrifugation at 4°C to yield a crude nuclear pellet (P1, 1,000 × g for 10 min), a crude mitochondrial pellet (P2, 40,000 × g for 10 min), and a crude microsomal pellet (P3, 100,000 × g for 60 min) to study the intracellular distribution of melatonin binding sites. The specific binding of 2-[125l]iodomelatonin (50.0 pM) in these membrane fractions was found to be greatest in the P2 fraction (7.35 fmol/mg protein, 42% of the specific binding in the total retinal membranes) as compared to the P1 fraction (2.01 fmol/mg protein, 28%) and the P3 fraction (4.40 fmol/mg protein, 30%). These results indicate that the 40,000 × g pellet used in the present study contained 70% of the specific binding in the total retinal membranes.
Linear regression analysis revealed that there was a significant relationship between the specific binding of 2-[125l]iodomelatonin (50.0 pM) and the protein concentration in the incubation mixture at least up to 0.35 mg protein/tube (r=0.999, P<0.001, n=6; Fig. 1).
Identification of bound radioligand
Radioactivity of the bound radioligand, extracted from filters after a binding assay with methanol, comigrated with synthetic 2-iodomelatonin. The Rf values for synthetic melatonin and 2-iodomelatonin was 0.32 and 0.55, respectively. Peak radioactivity (>96% of total) recovered between Rf values of 0.5 and 0.6.
At 25°C, the specific binding was rapid, stable and reversible (Fig. 2). The K1, determined from the pseudo-first-order plot was 4.69 × 108± 0.74 × 108 M-1 min-1 (n=6). The specific binding was stable from 1-3 hr of incubation. After 1.5 hr of incubation with 2-[125l]iodomelatonin, dissociation was initiated by addition of melatonin (8 μM) to some tubes. The K-1 calculated from the first-order regression analysis was 1.05 × 10-2±0.08x 10-2 min-1 (n=6). The kinetic dissociation constant calculated from the ratio K-1/K1 was 24.2 ±3.0 pM.
Saturation experiments using a range of 2-[125l]-iodomelatonin concentrations from 4.4 to 269.9 pM demonstrated that the specific binding reached a plateau at approximately 130 pM (Fig. 3A). Scatchard analysis of the data (Fig. 3B) revealed that 2-[125l]iodomelatonin binds to a single class of sites with the Kd of 61.9 ±5.7 pM, the Bmax of 6.52 ±0.79 fmol/mg protein, the nH of 1.07 ±0.03 (n=6).
Specificity of melatonin binding sites in the goldfish retina is shown in Fig. 4 and Table 1. Competition experiments carried out using a concentration of 2-[125l]iodomelatonin (49.6 pM) with several indole compounds and neurotransmitters revealed the following order of potency to inhibit the specific binding: 2-iodomelatonin > melatonin > 6-hydroxymelatonin > 5-methoxytryptamine = N-acetylserotonin > 5-methoxy-tryptophol. Even at a concentration of 10-4 M, 5-methoxyin-dole-3-acetic acid and serotonin exhibited less than 50% inhibition, and 5-hydroxytryptophol, 5-hydroxyindoIe-3-acetic acid, norepinephrine and acetylcholine showed no inhibition (data not shown).
Specificity of melatonin binding sites in the goldfish retina
Linear regression analysis demonstrated a significant correlation between the log Ki values obtained in the present study and those reported for the goldfish brain (slope=1.038, r=0.965, P<0.01,n=6; Fig. 5).
Effects of GTPγS
Effects of GTPγS (10-4 M) on the specific binding of 2-[125l]iodomelatonin (68.9 pM) to goldfish retinal membranes are exhibited in Fig. 6. The specific binding of the GTPγS-treated group was significantly reduced as compared with the vehicle-treated control (P<0.01).
Presence of melatonin and its synthesizing enzymes have been demonstrated in the eye of several teleost species (Quay, 1965; Gern and Ralph, 1979; Wiechmann and Hollyfield, 1988; Nowak et al., 1989; Falcón and Collin, 1991; Grace et al., 1991; Cahill, 1996; ligo et al., 1997a, c, d; Sanchez-Vazquez et al., 1997). To investigate the sites of melatonin action in the fish retina, in the present study, existence of melatonin binding sites in the goldfish retina was investigated by radioreceptor assay using 2-[125l]iodomelatonin as the radioligand.
We successfully detected the specific binding of 2-[125l]iodomelatonin to the goldfish retinal membranes as in the goldfish brain membranes (ligo et al., 1994b). The nonspecific binding was relatively high as compared with those in the brain. This might be caused by the binding of the radioligand to the melanosomes in the retinal pigmented epithelium (James et al., 1995).
The specific binding of 2-[125l]iodomelatonin to goldfish retinal membranes fulfills all the criteria for binding to a receptor site: the binding was rapid, stable, saturable, reversible, and of high specificity, low capacity, and high affinity. The radioligand was stable during the incubation period and appeared to label a single class of binding sites as is evident from linear Scatchard plots, monophasic inhibition curves and the nH close to unity. The Kd values obtained from the kinetic and saturation experiments and the Ki values obtained from the competition study were consistent with picomolar affinity as those in the goldfish brain (Martinoli et al., 1991; ligo et al., 1994b, 1995). These values are also well within the physiological range of ocular melatonin contents in the goldfish (0.1-2.5 ng/eye) (ligo et al., 1994a, 1997a), suggesting that the sites labeled with 2-[125l]iodomelatonin are physiological melatonin receptors as in the retina of chicken and rabbit (Dubocovich and Takahashi, 1987; Blazynski and Dubocovich, 1991).
Pharmacological specificity of melatonin binding sites in the goldfish retina was determined using several indoles and neurotransmitters. The results indicate that the binding sites are highly specific for 2-iodomelatonin and melatonin. Precursors and metabolites of melatonin were much less effective. Furthermore, compounds known to act at serotonergic, adrenergic or cholinergic sites were ineffective. The specificity of melatonin binding sites in the retina and brain of the goldfish are highly correlated, and resembles with those of the ML-1 but not with the ML-2 melatonin receptor subtype (Dubocovich, 1988, 1995). These results indicate that melatonin binding sites in the goldfish retina and brain belong to the ML-1 subtype.
During the course of this study, cDNAs encoding melatonin receptors with picomolar affinity were cloned. The results indicate that there are at least three subtypes of melatonin receptors among the ML-1 subtype, namely Mel1a, Mel1b and Mel1c subtypes (for review, see Reppert and Weaver, 1995). However, no melatonin analogue is available to distinguish differences in the pharmacological properties among these three subtypes (Pickering et al., 1996). Thus, molecular biological studies are necessary to characterize the subtype(s) of melatonin receptors expressed in a given tissue. As far as teleost fish are concerned, 5 different DNA fragments of putative melatonin receptors (2 clones belong to the Mel1a, 2 to the Mel1b and 1 to the Mel1c subtype) were isolated from genomic DNA of the zebrafish (Reppert et al., 1995, 1996). However, it is still controversial whether these are functional melatonin receptors and in which tissues they are expressed. Thus, further molecular biological approach will be required to determine which subtype is expressed in the goldfish retina.
In the present study, the nature of melatonin binding sites-G protein coupling was examined in the goldfish retinal membranes by the use of a non-hydrolyzable GTP analog, GTPγS, because several authors have failed to detect the effects of GTP and its analogues on 2-[125l]iodomelatonin binding to retinal membranes (Dubocovich and Takahashi, 1987; Chong and Sugden, 1991). The treatment of GTP7S significantly reduced the specific binding of 2-[125l]iodomelatonin to the goldfish retinal membranes, indicating that melatonin binding sites in the goldfish retina are coupled to G protein as those in the goldfish brain (ligo et al., 1997b) and in the retina, brain and pars tuberalis of other vertebrate species (Skene et al., 1993; Morgan et al., 1994; James et al., 1995). These results are also consistent with the recent results of cDNA cloning; melatonin receptors are members of the G protein-coupled receptor superfamily that contain seven hydrophobic segments (Reppert and Weaver, 1995). Further studies to identify the type(s) of G protein and the effector enzyme associated with melatonin receptor-G protein complex will help to elucidate the function of melatonin in the goldfish retina.
In the retina of rabbit, chicken, lizard and frog, melatonin binding sites were localized mainly in the inner plexiform layer as revealed by in vitro autoradiography (Laitinen and Saavedra, 1990; Blazynski and Dubocovich, 1991; Wiechmann and Wirsig-Wiechmann, 1991, 1994). Furthermore, localization of functional melatonin receptors are reported in the iris-ciliary processes of rabbit and in the retinal pigmented epithelium of human and rat (Osborne and Chidlow, 1994; Nash and Osborne, 1995). These structures may be possible sites of melatonin action in the goldfish retina. By the use of an in vitro autoradiographic technique, we have localized the specific binding in the neural retina of the goldfish (our unpublished results). However, precise localization was not apparent because of low resolution caused by high nonspecific binding in the retinal pigmented epithelium. Further studies should be required to elucidate this subject.
In the goldfish, melatonin produced in the retina does not contribute to its blood levels and is metabolized in the eye (Quay, 1965; Grace et al., 1991; Kezuka et al., 1992; ligo et al., 1997a), suggesting that melatonin may have local neuromodulatory roles in the retina. Visual sensitivity and inputs from photoreceptor to horizontal cells have been reported to exhibit circadian rhythms in the goldfish (Bassi and Powers, 1987; Wang and Mangel, 1996) and melatonin is considered to be involved in the regulation of these retinal events (ligo et al., 1997a). Recently, melatonin is proved to increase rod input but decrease cone input to the L-type cone horizontal cells in the goldfish (Mangel and Wong, 1996). These processes may be mediated by high-affinity melatonin binding sites characterized in the present study.
This study was supported in part by Grants-in-Aid from the Ministry of Agriculture, Forestry, and Fisheries (BMP 97-ll-2-1) and from the Ministry of Education, Science, Sports and Culture of Japan.