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Neurotransmitters and Receptors: Melatonin Receptors
Neurotransmitters and Receptors: Melatonin Receptors A F Wiechmann, University of Oklahoma College of Medicine, Oklahoma City, OK, USA ã 2010 Elsevier Ltd. All rights reserved.
Glossary Circadian rhythm – The term from the Latin circa which means around and diem which denotes day that is an approximate daily (24-h) periodicity in physiological processes of many organisms. Diurnal – Activities that are repeated every 24 h, but are not necessarily under the control of a biological clock. Orthologs – Homologous sequences which are similar to each other because they originated from a common ancestor. Paracrine – The term from the Latin para, which means near, is a form of cell signaling in which the target cell is located within the same tissues as the cell that releases the chemical signal. Pinealocytes – The main cells of the pineal gland that produce and secrete melatonin into the circulation. Xenopus – The term from the Latin strange foot, which is a genus of frog native to Africa, and commonly used in research as a model organism.
Introduction Melatonin (N-acetyl-5-methoxytryptamine) is an indolamine hormone synthesized by pinealocytes and retinal photoreceptors. The rate of melatonin synthesis, in most species studied, is highest at nighttime, and is considered to be a chemical signal of darkness that entrains circadian rhythms. This hormone synthesized in the pineal gland is secreted immediately into the circulation and acts as an endocrine hormone on distant target sites throughout the body. Melatonin produced in the retina, however, is thought to have a local, or paracrine role. It is thought that melatonin is synthesized and released by the photoreceptors at night, and diffuses throughout the retina to bind to melatonin receptors located on a variety of retinal cells. Since melatonin is a very lipophilic molecule, it diffuses freely through plasma membranes. The three major subtypes of melatonin receptors are members of the superfamily of guanine nucleotide binding (G-protein)-coupled receptors. Most studies have shown that melatonin receptor activation is coupled to an inhibition of adenylate cyclase activity, although many reports
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demonstrate that other signaling mechanisms are conveyed by the melatonin signal. Melatonin receptors have been identified in many different retinal cells, including amacrine cells, horizontal cells, ganglion cells, photoreceptors, and the adjacent retinal pigment epithelium (RPE).
Sites of Retinal Melatonin Synthesis Melatonin Synthesis by Photoreceptors The photoreceptors appear to be the sites of melatonin synthesis in the retina. They express all of the enzymes involved in melatonin synthesis. Melatonin is synthesized from tryptophan in a series of four enzymatic steps: (1) tryptophan is converted into 5-hydroxytryptophan by tryptophan hydroxylase (TPH); (2) the 5-hydroxytryptophan is then converted into 5-hydroxytryptamine (serotonin) by aromatic amino acid decarboxylase; (3) serotonin is then converted into N-acetylserotonin by arylalkylamine N-acetyltransferase (AANAT); and (4) N-acetylserotonin is converted into melatonin (N-acetyl-5-methoxytryptamine) by hydroxyindole-O-methyltransferase (HIOMT). The enzyme activity and messenger RNA (mRNA) encoding TPH and AANAT exhibit circadian rhythms of expression, with highest levels occurring at night. There is strong evidence that identifies the photoreceptors as the sites of retinal melatonin synthesis. Melatonin immunoreactivity is localized in the outer nuclear layer (ONL) of the retina which contains the cell soma of the photoreceptors. HIOMT and AANAT protein and mRNA are localized to photoreceptor cytoplasm, and a cyclic rhythm of AANAT activity persists following chemical lesion of the inner retina. The mRNA encoding TPH is localized to photoreceptors, and the photoreceptor layer of the amphibian retina continues to produce melatonin rhythmically in darkness after isolation from the inner retina. In addition to the photoreceptors, some neurons of the inner retina may have the capacity to produce a small amount of melatonin. Melatonin immunoreactivity is observed in the inner retina, and a low level of AANAT mRNA has been detected in the inner nuclear layer (INL) and ganglion cell layer (GCL). The INL contains the cell soma of amacrine, horizontal, bipolar, and Mu¨ller cells. Since some cells in these layers also have melatonin receptors, the synthesis of melatonin by inner retinal neurons may be involved in the circadian activity of cells of the inner retina.
Neurotransmitters and Receptors: Melatonin Receptors
Phylogenetic Relationships between Photoreceptors and Pinealocytes The ability of photoreceptors and pinealocytes to synthesize melatonin appears to be the consequence of an ancestral relationship between the retina and the pineal gland. Some primitive animals possessed three eyes which may have produced melatonin and were also capable of phototransduction. Pinealocytes of some lower vertebrates are morphologically very similar to retinal photoreceptors, and they synthesize melatonin as well as many proteins that are characteristic of retinal photoreceptors. Furthermore, the photoreceptors of the nonmammalian pineal gland are directly photosensitive, and during the embryologic development of the mammalian pineal gland, the pinealocytes undergo a transient photoreceptor-like differentiation. It is suggested that the middle, or third eye, eventually evolved into an endocrine organ specialized for the secretion of melatonin into the circulation, in which the melatonin-producing pineal photoreceptors eventually lost their phototransduction capabilities, and the melatonin-producing cells of the lateral eyes evolved into photoreceptors specialized for phototransduction, but maintained their ability to synthesize melatonin. Genes expressing melatonin receptors in peripheral tissues may have also become expressed in ocular tissues, which enabled local paracrine signaling by melatonin in the retina.
Classification of Melatonin Receptors Melatonin receptor expression has been identified in the retinas of several species. The major types of melatonin receptors that have been cloned are members of the superfamily of G-protein-coupled receptors. Melatonin receptors have been classified as Mel1a, Mel1b, and Mel1c subtypes. These three subtypes are expressed in tissues, including the retinas, of lower vertebrates such as amphibians, fish, and birds. Mammalian melatonin receptors are classified according to their homology to the nonmammalian receptors, and according to their pharmacological properties. In mammals, the Mel1a and Mel1b receptor subtypes are designated as the MT1 and MT2 receptor subtypes, respectively. The mammalian ortholog of the Mel1c subtype has been designated as GPR50, and does not bind melatonin. The G-alpha proteins coupled to melatonin receptors are inhibitory (Gi) to the activation of adenylate cyclase and cyclic adenosine monophosphate (cAMP) production in most tissues studied. However, receptor coupling to other G-alpha proteins (Gia2, Gia2, Giaq, Gias, Giaz, and Gia16), and hence other signaling pathways, have been reported. Nuclear melatonin receptors, which are members of the RAR-related RZR/ROR
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orphan nuclear receptor superfamily, have been reported to exist in some tissues, and a melatonin-binding site on the enzyme quinine reductase 2 has been identified and is referred to as the mammalian MT3 melatonin receptor. Most G-protein-coupled receptors interact with each other to form homodimers or heterodimers. The mammalian MT1 and MT2 melatonin receptors can exist as homodimers and as heterodimers. Dimerization of G-protein-coupled receptors has important functional consequences in regard to receptor affinity, trafficking, and signaling. The relative expression levels of melatonin receptor subtypes in the retina may have a significant impact on the function of melatonin in the target cells.
Sites of Melatonin Receptors in the Retina Melatonin receptors have been identified not only in several retinal neurons of the inner retina, but also in photoreceptors cells and RPE cells. The identification of melatonin receptors in photoreceptor cells was unanticipated, given that the photoreceptors are the site of retinal melatonin synthesis. The presence of melatonin receptors in photoreceptor cells suggests the possibility of what can be characterized as an intracrine (autocrine) signaling mechanism in response to melatonin, in which a cell synthesizes and releases a signaling molecule, and also has receptors to which the molecule binds and triggers an intracellular response. Another potential role of melatonin receptors in photoreceptor cells is that they may be involved in a negative-feedback mechanism which would enable melatonin to regulate the expression of the receptors to which it binds. Melatonin Receptors in Photoreceptor Cells Mel1a, Mel1b, and Mel1c melatonin receptor subtype protein and mRNA have been identified in the photoreceptors of nonmammalian vertebrates, and MT1 receptors in photoreceptors of the mammalian retina. The receptor immunoreactivity has been identified primarily in the photoreceptor membranes of the inner segments, although some cytoplasmic immunoreactivity has been reported for some subtypes, and may represent newly synthesized receptors that have not yet been transported to the plasma membrane, or receptors that have been internalized after activation. Mel1b and Mel1c melatonin receptor RNA and/or protein are expressed in Xenopus photoreceptors, and the MT1 (Mel1a) receptor is localized to photoreceptors of the human retina. In the chicken retina, Mel1a immunoreactivity and melatonin receptor mRNA expression (Mel1a, Mel1b, and Mel1c) are localized to the photoreceptor layer. In the Xenopus retina, Mel1c immunoreactivity is observed in the plasma membrane of photoreceptor
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inner segments, whereas Mel1b receptor immunoreactivity appears in a punctate pattern in the proximal portion of photoreceptor inner segments. The differential pattern of Mel1b and Mel1c may reflect differential regulation of expression or trafficking of melatonin receptors in photoreceptor cells. Melatonin Receptors in RPE The RPE is a monolayer of cuboidal cells located between the vascular choroid layer and the neural retina. It is very closely associated with the retinal photoreceptor cells. This close association reflects the vital function of the RPE to provide physical and metabolic support to the photoreceptors. Circadian signals may play a role in influencing the coordinated interactions between the RPE and its adjacent tissues. The RPE, photoreceptors, retinal neurons, and choroidal cells interact in a coordinated manner for optimal function. Melatonin may play a role in the timing of the circadian phagocytosis of shed photoreceptor outer segments. The distal tips of rod photoreceptor outer segments are shed on a circadian rhythm as part of a renewal process, with peak shedding occurring early in the light period. The shed outer segment tips are phagocytized by the RPE, and melatonin is thought to be involved in this process. Melatonin secreted from photoreceptors at night may activate melatonin receptors on the RPE to regulate some circadian activities of the RPE that are important for optimal photoreceptor activity. Melatonin inhibits forskolin-stimulated cAMP synthesis in RPE cell cultures, and melatonin affects the RPE membrane potentials and resistances at the apical or basal membrane. The mRNA encoding all three melatonin receptor subtypes is expressed in Xenopus laevis RPE but the Mel1b receptor protein is localized only to the apical surface of the Xenopus RPE, and is not present on the basal surface. The Mel1c receptor protein has also been localized to the Xenopus RPE. The presence of melatonin receptors on the apical microvilli, which directly contact the photoreceptors, but not on the basal membrane of the RPE, suggests that the photoreceptors are more likely to be the source of melatonin that activate melatonin receptors on the RPE rather than melatonin that is produced by the pineal gland and secreted into the general circulation. Melatonin Receptors in Inner Retinal Neurons Using autoradiography with 125I-melatonin, it has been demonstrated that melatonin binding occurs in the inner plexiform layer (IPL) of many species. The IPL contains the synaptic terminals between bipolar cells, amacrine cells, horizontal cells, and ganglion cells. Since melatonin inhibits dopamine release from the retina, and highaffinity melatonin binding occurs in the IPL of the retina, the dopaminergic amacrine cell, which forms synaptic contacts in the IPL, has long been considered to be a
candidate for the site of action of melatonin in the inner retina. Another candidate cell for melatonin receptor expression is the GABAergic amacrine cell (GABA, gamma aminobutyric acid) of the INL since GABAA receptor antagonists block melatonin-induced suppression of dopamine release. This suggests that the effect of melatonin on dopamine release may not be mediated only by direct action on dopaminergic cells, but that indirect action on GABAergic amacrine cells may also contribute to the inhibition of dopamine release via melatonin. The autoradiographic localization of melatonin-binding sites in cells of the inner retina has been confirmed by both in situ hybridization and immunocytochemistry. In Xenopus, Mel1b and Mel1c RNA expression is localized to the INL, GCL, and photoreceptor inner segments. In the chicken retina, the mRNA encoding the Mel1a, Mel1b, and Mel1c receptor subtypes is present in the INL, GCL, and photoreceptor inner segments. The INL contains the cell soma of bipolar, amacrine, horizontal, and Mu¨ller cells. In the human retina, Mel1b receptor mRNA is much more highly expressed than is Mel1a receptor RNA, suggesting that the Mel1b receptor has a more significant role in human retinal physiology. Using antibodies against specific melatonin receptor subtypes for immunocytochemistry, all three melatonin receptor subtypes (Mel1a, Mel1b, and Mel1c) have been observed in the outer plexiform layer (OPL; the layer that contains the synaptic contacts between photoreceptors, bipolar cells, and horizontal cells) and the IPL. The MT1 (Mel1a) receptor has been localized to horizontal cells in several mammalian species, including human. All three melatonin receptor subtypes appear to be present in horizontal cells of fish and Xenopus retina. The MT1 (Mel1a) receptor has also been localized to AII amacrine and GABAergic amacrine cells of the mammalian retina. Some Mel1a and Mel1c receptor immunoreactivity co-localizes with GABAergic and dopaminergic amacrine cells in the Xenopus retina. The presence of Mel1a and Mel1c receptors on dopaminergic and GABAergic amacrine cells is consistent with the observation that melatonin modulates the cyclic release of GABA and dopamine from retinal amacrine cells. In contrast, Mel1b receptor immunoreactivity does not appear to co-localize with markers for dopaminergic and GABAergic neurons in the Xenopus retina, suggesting that melatonin does not act directly on GABAergic and dopaminergic amacrine cells through the Mel1b receptor in this species. In the Xenopus retina, the Mel1a, Mel1b, and Mel1c receptor proteins are differentially distributed throughout the retina. In the OPL, for example, presumptive horizontal cell processes are immunoreactive for Mel1a and Mel1b receptor subtypes, but the immunoreactive labels appear to be in different cell processes. Cell somas in the INL are immunoreactive either for Mel1b or Mel1a, or for Mel1a or Mel1c, but not for both. All three melatonin receptor
Neurotransmitters and Receptors: Melatonin Receptors
subtypes appear to be expressed in different populations of ganglion cells in the Xenopus retina, and the MT1 subtype is present in ganglion cells of the human and macaque retina. Melatonin receptor mRNA and protein are rhythmically expressed in Xenopus and chicks, with peak levels of Mel1c expression occurring in the day. In chicks, the rhythms of Mel1a and Mel1b receptor protein generally appear to be the opposite to that of Mel1c, with lowest levels occurring in the early morning and higher levels in the evening. The patterns of cyclic rhythms appear to be distinctive for each receptor subtype in the retina. Circadian rhythms in melatonin receptor expression may perhaps be superimposed on the rhythm in retinal melatonin levels to provide an additional level of regulation of the responsiveness of retinal target to melatonin.
Effects of Melatonin on Retinal Function Modulation of Neurotransmitter Release Melatonin released from photoreceptors at night diffuses into the extracellular milieu and binds to melatonin receptors on dopaminergic amacrine cells. The activation of the melatonin receptors results in a decrease of dopamine release at nighttime. Thus, retinal dopamine levels are higher during the day and lowest during the night due to the circadian release of melatonin. Resultant lower dopamine levels at night cause a reduction in D2 dopamine receptor activity on photoreceptors, causing an increase in photoreceptor intracellular cAMP levels that in turn causes an increase in coupling of gap junctions between rod and cone photoreceptors so that rod input dominates the cone horizontal cells. The increased sensitivity of horizontal cells to light at nighttime is therefore mediated at least in part by activation of D2-like receptors by dopamine released from amacrine cells. A reduction in endogenous retinal dopamine levels causes hyperpolarization of horizontal cells and enhanced dark adaptation. D1 dopamine receptors, which are positively coupled to cAMP synthesis, are located on horizontal cells. Melatonin may therefore postsynaptically regulate horizontal cell activity by inhibiting the stimulation of cAMP synthesis in response to D1 receptor activation. It may bind to receptors on GABAergic amacrine cells, stimulating them to inhibit dopamine release from nearby dopaminergic amacrine cells. In addition, since horizontal cells express melatonin receptors, and melatonin increases horizontal cell sensitivity to light, melatonin may act directly on horizontal cells to increase gap-junctional coupling of horizontal cells. Increased horizontal cell coupling would cause an increase in receptive field size, which would potentially increase the sensitivity of the retina to light during the dark period, since more second-order neurons would respond to a light stimulus.
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Melatonin may therefore modulate dopaminergic transmission by a combination of directly reducing dopamine release from amacrine cells, and indirectly by stimulating GABAergic amacrine cells to inhibit dopamine release from dopaminergic amacrine cells, both of which would increase horizontal cell coupling. The resulting increased visual sensitivity at nighttime could be due to increased rod–cone coupling through dopamine binding to D2 receptors on photoreceptors, or to horizontal cell coupling stimulated by the binding of melatonin to melatonin receptors on horizontal cells. A summary diagram of the known locations of melatonin receptors and the possible interactions with the various target cells is presented in Figure 1. Melatonin increases horizontal cell sensitivity to light in salamander retina, and also potentiates glutamateinduced currents from isolated cone-driven horizontal cells in carp retina by increasing the efficacy and affinity of the glutamate receptor. These observations suggest that melatonin acts directly on melatonin receptors of horizontal cells. Melatonin modulates cyclic guanosine monophosphate (cGMP)-dependent glutaminergic transmission from cones to cone-driven horizontal cells by activation of the Mel1a receptor, causes a depolarization of the H1 horizontal cell membrane potential, and reduces its light responses. These observations suggest that melatonin enhances the circadian sensitivity of rod photoreceptor signaling. Melatonin has been shown in fish retina to potentiate responses of rod ON bipolar cells to simulated light flashes. This action of melatonin is mediated by the Mel1b receptor, and increases cGMP levels by inhibiting phosphodiesterase activity. Melatonin may bind directly to Mel1b receptors on rod ON bipolar cells to improve the signal/noise ratio for rod signals by enhancing signal transfer from rod photoreceptors to rod bipolar cells. The presence of melatonin receptors by immunocytochemistry has not yet been definitively established in bipolar cells. Melatonin treatment of isolated rat retinal ganglion cells potentiates glycine-induced currents by increasing the efficacy and channel conductance of a glycine receptor. The inhibitory modulation of glycinergic inputs to ganglion cells may thus be strengthened by stimulation of melatonin receptor activation. This suggests that melatonin may regulate circadian changes in receptive field organization and light sensitivity by binding to melatonin receptors on ganglion cells.
Modulation of Photoreceptor Function Several reports support the concept of a direct action of melatonin on retinal photoreceptor function. Melatonin induces membrane conductance changes in isolated frog rod photoreceptors, binds with low affinity to structures in the OPL in frog retina, enhances the rate of photoreceptor
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Neurotransmitters and Receptors: Melatonin Receptors
Melatonin receptor D1 D2
D1 dopamine receptor RPE
D2 dopamine receptor
GABA receptor GABA Dark D2
PH
(–) H
(–)
MEL
H D1
DA
(–) MEL A
DA
B
MEL (+) A
(+)
GABA
(–) G
GABA
Figure 1 Summary diagram of locations of retinal melatonin receptors and potential interactions among target cells. Melatonin (MEL) is produced by photoreceptors (PH) at nighttime, and diffuses to target cells within the retina. Arrows represent the movement of melatonin to the target cells. The melatonin receptors are represented by a black crescent symbol. Dopamine and GABA receptors are represented by crescent symbols with D1, D2, or GABA as identifiers. Melatonin may bind to amacrine cells (A) that release GABA or dopamine (DA) as their neurotransmitter. Melatonin is thought to stimulate (þ) GABA release and/or inhibit () dopamine release from these cells. GABA inhibits dopamine release from dopaminergic amacrine cells. A lower rate of dopamine release from amacrine cells at nighttime results in lower stimulation of D1 receptors on horizontal cells (H), which leads to increased coupling of horizontal cells through gap junctions (¼), which could cause an increase in receptive field size and increased retinal sensitivity to light. The decreased binding of dopamine at nighttime to D2 receptors on photoreceptor cells results in an increase in melatonin synthesis, since dopamine inhibits melatonin synthesis in photoreceptors. Melatonin may potentially bind to horizontal cells to directly inhibit the cellular response to D1 receptor binding. Melatonin may also bind to receptors located on the photoreceptor membrane, which could directly increase rod sensitivity to light, increase rod–cone coupling, and/or regulate synthesis of melatonin. Melatonin may bind to receptors on the apical membrane of retinal pigment epithelial (RPE) cells, to coordinate circadian interactions with photoreceptor outer segments. The possible expression of melatonin receptors on bipolar (B) cells, and the confirmed expression of melatonin receptors on ganglion cells (G) are not indicated by arrows.
outer segment disk shedding, and increases the degree of light-induced photoreceptor cell death. It causes a stimulation of the amplitude of the a-wave (rod photoreceptors) and the b-wave (inner retinal cells responding to the photoreceptor input) of electroretinogram (ERG) recordings of transgenic frogs that overexpress the Mel1c receptor in rod photoreceptors. This suggests that melatonin acts directly on rod photoreceptors to increase retinal sensitivity to light as part of a dark-adaptation mechanism. The role of melatonin in dark adaptation suggests a potential mechanism by which melatonin increases the degree of light-induced photoreceptor cell death. Since melatonin appears to increase the sensitivity of the retina to light as part of a dark-adaptation mechanism, an undesirable consequence of this may be an increased sensitivity to the deleterious effects of light. Although signals from the inner retina obviously play a significant role in the circadian activities of retinal photoreceptors, direct action of melatonin on receptors located on photoreceptors may contribute substantially to the functions of melatonin in circadian-regulated activities of the retina.
See also: Chick Metabolism in the Chick Retina; Circadian Photoreception; The Circadian Clock in the Retina Regulates Rod and Cone Pathways; Circadian Regulation of Ion Channels in Photoreceptors; Neurotransmitters and Receptors: Dopamine.
Further Reading Besharse, J. C. and Dunis, D. A. (1983). Methoxyindoles and photoreceptor metabolism: Activation of rod shedding. Science 219: 1341–1342. Boatright, J. H., Rubim, N. M., and Iuvone, P. M. (1994). Regulation of endogenous dopamine release in amphibian retina by melatonin: The role of GABA. Visual Neuroscience 11: 1013–1018. Dubocovich, M. L. (1983). Melatonin is a potent modulator of dopamine release in the retina. Nature 306: 782–784. Dubocovich, M. L., Cardinali, D. P., Guardiola-Lemaitre, B., et al. (1998). Melatonin receptors. In: Girdlestone, D. (ed.) The IUPHAR Compendium of Receptor Characterisation and Classification, vol. I, pp. 188–193. London: UCPHAR Media. Fujieda, H., Scher, J., Hamadanizadeh, S. A., et al. (2000). Dopaminergic and GABAergic amacrine cells are direct targets of melatonin: Immunocytochemical study of mt1 melatonin receptor in guinea pig retina. Visual Neuroscience 17: 63–70.
Neurotransmitters and Receptors: Melatonin Receptors Huang, H., Lee, S. C., and Yang, X. L. (2005). Modulation by melatonin of glutamatergic synaptic transmission in the carp retina. Journal of Physiology 569: 857–871. Iuvone, P. M., Tosini, G., Pozdeyev, N., et al. (2005). Circadian clocks, clock networks, arylalkylamine N-acetyltransferase, and melatonin in the retina. Progress in Retinal and Eye Research 24: 433–456. Lundmark, P. O., Pandi-Perumal, S. R., Srinivasan, V., and Cardinali, D. P. (2006). Role of melatonin in the eye and ocular dysfunctions. Visual Neuroscience 23: 853–862. Reppert, S. M., Godson, C., Mahle, C. D., et al. (1995). Molecular characterization of a second melatonin receptor expressed in human retina and brain: The Mel1b melatonin receptor. Proceedings of the National Academy of Sciences of the United States of America 92: 8734–8738. Scher, J., Wankiewicz, E., Brown, G. M., and Fujieda, H. (2002). MT1 melatonin receptor in the human retina: Expression and localization. Investigative Ophthalmology and Visual Science 43: 889–897.
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Wiechmann, A. F. and Smith, A. R. (2001). Melatonin receptor RNA is expressed in photoreceptors and displays a cyclic rhythm in Xenopus retina. Molecular Brain Research 91: 104–111. Wiechmann, A. F. and Summers, J. A. (2008). Circadian rhythms in the eye: The physiological significance of melatonin receptors in ocular tissues. Progress in Retinal and Eye Research 27: 137–160. Wiechmann, A. F., Vrieze, M. J., Dighe, R. K., and Hu, Y. (2003). Direct modulation of rod photoreceptor responsiveness through a Mel1c melatonin receptor in transgenic Xenopus laevis retina. Investigative Ophthalmology and Visual Science 44: 4522–4531. Wiechmann, A. F., Udin, S. B., and Summers Rada, J. A. (2004). Localization of Mel1b melatonin receptor protein expression in ocular tissues of Xenopus laevis. Experimental Eye Research 79: 585–594. Young, R. W. and Bok, D. (1969). Participation of the retinal pigment epithelium in the rod outer segment renewal process. Journal of Cell Biology 42: 392–403.
Glossary Circadian rhythm – The term from the Latin circa which means around and diem which denotes day that is an approximate daily (24-h) periodicity in physiological processes of many organisms. Diurnal – Activities that are repeated every 24 h, but are not necessarily under the control of a biological clock. Orthologs – Homologous sequences which are similar to each other because they originated from a common ancestor. Paracrine – The term from the Latin para, which means near, is a form of cell signaling in which the target cell is located within the same tissues as the cell that releases the chemical signal. Pinealocytes – The main cells of the pineal gland that produce and secrete melatonin into the circulation. Xenopus – The term from the Latin strange foot, which is a genus of frog native to Africa, and commonly used in research as a model organism.
Introduction Melatonin (N-acetyl-5-methoxytryptamine) is an indolamine hormone synthesized by pinealocytes and retinal photoreceptors. The rate of melatonin synthesis, in most species studied, is highest at nighttime, and is considered to be a chemical signal of darkness that entrains circadian rhythms. This hormone synthesized in the pineal gland is secreted immediately into the circulation and acts as an endocrine hormone on distant target sites throughout the body. Melatonin produced in the retina, however, is thought to have a local, or paracrine role. It is thought that melatonin is synthesized and released by the photoreceptors at night, and diffuses throughout the retina to bind to melatonin receptors located on a variety of retinal cells. Since melatonin is a very lipophilic molecule, it diffuses freely through plasma membranes. The three major subtypes of melatonin receptors are members of the superfamily of guanine nucleotide binding (G-protein)-coupled receptors. Most studies have shown that melatonin receptor activation is coupled to an inhibition of adenylate cyclase activity, although many reports
136
demonstrate that other signaling mechanisms are conveyed by the melatonin signal. Melatonin receptors have been identified in many different retinal cells, including amacrine cells, horizontal cells, ganglion cells, photoreceptors, and the adjacent retinal pigment epithelium (RPE).
Sites of Retinal Melatonin Synthesis Melatonin Synthesis by Photoreceptors The photoreceptors appear to be the sites of melatonin synthesis in the retina. They express all of the enzymes involved in melatonin synthesis. Melatonin is synthesized from tryptophan in a series of four enzymatic steps: (1) tryptophan is converted into 5-hydroxytryptophan by tryptophan hydroxylase (TPH); (2) the 5-hydroxytryptophan is then converted into 5-hydroxytryptamine (serotonin) by aromatic amino acid decarboxylase; (3) serotonin is then converted into N-acetylserotonin by arylalkylamine N-acetyltransferase (AANAT); and (4) N-acetylserotonin is converted into melatonin (N-acetyl-5-methoxytryptamine) by hydroxyindole-O-methyltransferase (HIOMT). The enzyme activity and messenger RNA (mRNA) encoding TPH and AANAT exhibit circadian rhythms of expression, with highest levels occurring at night. There is strong evidence that identifies the photoreceptors as the sites of retinal melatonin synthesis. Melatonin immunoreactivity is localized in the outer nuclear layer (ONL) of the retina which contains the cell soma of the photoreceptors. HIOMT and AANAT protein and mRNA are localized to photoreceptor cytoplasm, and a cyclic rhythm of AANAT activity persists following chemical lesion of the inner retina. The mRNA encoding TPH is localized to photoreceptors, and the photoreceptor layer of the amphibian retina continues to produce melatonin rhythmically in darkness after isolation from the inner retina. In addition to the photoreceptors, some neurons of the inner retina may have the capacity to produce a small amount of melatonin. Melatonin immunoreactivity is observed in the inner retina, and a low level of AANAT mRNA has been detected in the inner nuclear layer (INL) and ganglion cell layer (GCL). The INL contains the cell soma of amacrine, horizontal, bipolar, and Mu¨ller cells. Since some cells in these layers also have melatonin receptors, the synthesis of melatonin by inner retinal neurons may be involved in the circadian activity of cells of the inner retina.
Neurotransmitters and Receptors: Melatonin Receptors
Phylogenetic Relationships between Photoreceptors and Pinealocytes The ability of photoreceptors and pinealocytes to synthesize melatonin appears to be the consequence of an ancestral relationship between the retina and the pineal gland. Some primitive animals possessed three eyes which may have produced melatonin and were also capable of phototransduction. Pinealocytes of some lower vertebrates are morphologically very similar to retinal photoreceptors, and they synthesize melatonin as well as many proteins that are characteristic of retinal photoreceptors. Furthermore, the photoreceptors of the nonmammalian pineal gland are directly photosensitive, and during the embryologic development of the mammalian pineal gland, the pinealocytes undergo a transient photoreceptor-like differentiation. It is suggested that the middle, or third eye, eventually evolved into an endocrine organ specialized for the secretion of melatonin into the circulation, in which the melatonin-producing pineal photoreceptors eventually lost their phototransduction capabilities, and the melatonin-producing cells of the lateral eyes evolved into photoreceptors specialized for phototransduction, but maintained their ability to synthesize melatonin. Genes expressing melatonin receptors in peripheral tissues may have also become expressed in ocular tissues, which enabled local paracrine signaling by melatonin in the retina.
Classification of Melatonin Receptors Melatonin receptor expression has been identified in the retinas of several species. The major types of melatonin receptors that have been cloned are members of the superfamily of G-protein-coupled receptors. Melatonin receptors have been classified as Mel1a, Mel1b, and Mel1c subtypes. These three subtypes are expressed in tissues, including the retinas, of lower vertebrates such as amphibians, fish, and birds. Mammalian melatonin receptors are classified according to their homology to the nonmammalian receptors, and according to their pharmacological properties. In mammals, the Mel1a and Mel1b receptor subtypes are designated as the MT1 and MT2 receptor subtypes, respectively. The mammalian ortholog of the Mel1c subtype has been designated as GPR50, and does not bind melatonin. The G-alpha proteins coupled to melatonin receptors are inhibitory (Gi) to the activation of adenylate cyclase and cyclic adenosine monophosphate (cAMP) production in most tissues studied. However, receptor coupling to other G-alpha proteins (Gia2, Gia2, Giaq, Gias, Giaz, and Gia16), and hence other signaling pathways, have been reported. Nuclear melatonin receptors, which are members of the RAR-related RZR/ROR
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orphan nuclear receptor superfamily, have been reported to exist in some tissues, and a melatonin-binding site on the enzyme quinine reductase 2 has been identified and is referred to as the mammalian MT3 melatonin receptor. Most G-protein-coupled receptors interact with each other to form homodimers or heterodimers. The mammalian MT1 and MT2 melatonin receptors can exist as homodimers and as heterodimers. Dimerization of G-protein-coupled receptors has important functional consequences in regard to receptor affinity, trafficking, and signaling. The relative expression levels of melatonin receptor subtypes in the retina may have a significant impact on the function of melatonin in the target cells.
Sites of Melatonin Receptors in the Retina Melatonin receptors have been identified not only in several retinal neurons of the inner retina, but also in photoreceptors cells and RPE cells. The identification of melatonin receptors in photoreceptor cells was unanticipated, given that the photoreceptors are the site of retinal melatonin synthesis. The presence of melatonin receptors in photoreceptor cells suggests the possibility of what can be characterized as an intracrine (autocrine) signaling mechanism in response to melatonin, in which a cell synthesizes and releases a signaling molecule, and also has receptors to which the molecule binds and triggers an intracellular response. Another potential role of melatonin receptors in photoreceptor cells is that they may be involved in a negative-feedback mechanism which would enable melatonin to regulate the expression of the receptors to which it binds. Melatonin Receptors in Photoreceptor Cells Mel1a, Mel1b, and Mel1c melatonin receptor subtype protein and mRNA have been identified in the photoreceptors of nonmammalian vertebrates, and MT1 receptors in photoreceptors of the mammalian retina. The receptor immunoreactivity has been identified primarily in the photoreceptor membranes of the inner segments, although some cytoplasmic immunoreactivity has been reported for some subtypes, and may represent newly synthesized receptors that have not yet been transported to the plasma membrane, or receptors that have been internalized after activation. Mel1b and Mel1c melatonin receptor RNA and/or protein are expressed in Xenopus photoreceptors, and the MT1 (Mel1a) receptor is localized to photoreceptors of the human retina. In the chicken retina, Mel1a immunoreactivity and melatonin receptor mRNA expression (Mel1a, Mel1b, and Mel1c) are localized to the photoreceptor layer. In the Xenopus retina, Mel1c immunoreactivity is observed in the plasma membrane of photoreceptor
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inner segments, whereas Mel1b receptor immunoreactivity appears in a punctate pattern in the proximal portion of photoreceptor inner segments. The differential pattern of Mel1b and Mel1c may reflect differential regulation of expression or trafficking of melatonin receptors in photoreceptor cells. Melatonin Receptors in RPE The RPE is a monolayer of cuboidal cells located between the vascular choroid layer and the neural retina. It is very closely associated with the retinal photoreceptor cells. This close association reflects the vital function of the RPE to provide physical and metabolic support to the photoreceptors. Circadian signals may play a role in influencing the coordinated interactions between the RPE and its adjacent tissues. The RPE, photoreceptors, retinal neurons, and choroidal cells interact in a coordinated manner for optimal function. Melatonin may play a role in the timing of the circadian phagocytosis of shed photoreceptor outer segments. The distal tips of rod photoreceptor outer segments are shed on a circadian rhythm as part of a renewal process, with peak shedding occurring early in the light period. The shed outer segment tips are phagocytized by the RPE, and melatonin is thought to be involved in this process. Melatonin secreted from photoreceptors at night may activate melatonin receptors on the RPE to regulate some circadian activities of the RPE that are important for optimal photoreceptor activity. Melatonin inhibits forskolin-stimulated cAMP synthesis in RPE cell cultures, and melatonin affects the RPE membrane potentials and resistances at the apical or basal membrane. The mRNA encoding all three melatonin receptor subtypes is expressed in Xenopus laevis RPE but the Mel1b receptor protein is localized only to the apical surface of the Xenopus RPE, and is not present on the basal surface. The Mel1c receptor protein has also been localized to the Xenopus RPE. The presence of melatonin receptors on the apical microvilli, which directly contact the photoreceptors, but not on the basal membrane of the RPE, suggests that the photoreceptors are more likely to be the source of melatonin that activate melatonin receptors on the RPE rather than melatonin that is produced by the pineal gland and secreted into the general circulation. Melatonin Receptors in Inner Retinal Neurons Using autoradiography with 125I-melatonin, it has been demonstrated that melatonin binding occurs in the inner plexiform layer (IPL) of many species. The IPL contains the synaptic terminals between bipolar cells, amacrine cells, horizontal cells, and ganglion cells. Since melatonin inhibits dopamine release from the retina, and highaffinity melatonin binding occurs in the IPL of the retina, the dopaminergic amacrine cell, which forms synaptic contacts in the IPL, has long been considered to be a
candidate for the site of action of melatonin in the inner retina. Another candidate cell for melatonin receptor expression is the GABAergic amacrine cell (GABA, gamma aminobutyric acid) of the INL since GABAA receptor antagonists block melatonin-induced suppression of dopamine release. This suggests that the effect of melatonin on dopamine release may not be mediated only by direct action on dopaminergic cells, but that indirect action on GABAergic amacrine cells may also contribute to the inhibition of dopamine release via melatonin. The autoradiographic localization of melatonin-binding sites in cells of the inner retina has been confirmed by both in situ hybridization and immunocytochemistry. In Xenopus, Mel1b and Mel1c RNA expression is localized to the INL, GCL, and photoreceptor inner segments. In the chicken retina, the mRNA encoding the Mel1a, Mel1b, and Mel1c receptor subtypes is present in the INL, GCL, and photoreceptor inner segments. The INL contains the cell soma of bipolar, amacrine, horizontal, and Mu¨ller cells. In the human retina, Mel1b receptor mRNA is much more highly expressed than is Mel1a receptor RNA, suggesting that the Mel1b receptor has a more significant role in human retinal physiology. Using antibodies against specific melatonin receptor subtypes for immunocytochemistry, all three melatonin receptor subtypes (Mel1a, Mel1b, and Mel1c) have been observed in the outer plexiform layer (OPL; the layer that contains the synaptic contacts between photoreceptors, bipolar cells, and horizontal cells) and the IPL. The MT1 (Mel1a) receptor has been localized to horizontal cells in several mammalian species, including human. All three melatonin receptor subtypes appear to be present in horizontal cells of fish and Xenopus retina. The MT1 (Mel1a) receptor has also been localized to AII amacrine and GABAergic amacrine cells of the mammalian retina. Some Mel1a and Mel1c receptor immunoreactivity co-localizes with GABAergic and dopaminergic amacrine cells in the Xenopus retina. The presence of Mel1a and Mel1c receptors on dopaminergic and GABAergic amacrine cells is consistent with the observation that melatonin modulates the cyclic release of GABA and dopamine from retinal amacrine cells. In contrast, Mel1b receptor immunoreactivity does not appear to co-localize with markers for dopaminergic and GABAergic neurons in the Xenopus retina, suggesting that melatonin does not act directly on GABAergic and dopaminergic amacrine cells through the Mel1b receptor in this species. In the Xenopus retina, the Mel1a, Mel1b, and Mel1c receptor proteins are differentially distributed throughout the retina. In the OPL, for example, presumptive horizontal cell processes are immunoreactive for Mel1a and Mel1b receptor subtypes, but the immunoreactive labels appear to be in different cell processes. Cell somas in the INL are immunoreactive either for Mel1b or Mel1a, or for Mel1a or Mel1c, but not for both. All three melatonin receptor
Neurotransmitters and Receptors: Melatonin Receptors
subtypes appear to be expressed in different populations of ganglion cells in the Xenopus retina, and the MT1 subtype is present in ganglion cells of the human and macaque retina. Melatonin receptor mRNA and protein are rhythmically expressed in Xenopus and chicks, with peak levels of Mel1c expression occurring in the day. In chicks, the rhythms of Mel1a and Mel1b receptor protein generally appear to be the opposite to that of Mel1c, with lowest levels occurring in the early morning and higher levels in the evening. The patterns of cyclic rhythms appear to be distinctive for each receptor subtype in the retina. Circadian rhythms in melatonin receptor expression may perhaps be superimposed on the rhythm in retinal melatonin levels to provide an additional level of regulation of the responsiveness of retinal target to melatonin.
Effects of Melatonin on Retinal Function Modulation of Neurotransmitter Release Melatonin released from photoreceptors at night diffuses into the extracellular milieu and binds to melatonin receptors on dopaminergic amacrine cells. The activation of the melatonin receptors results in a decrease of dopamine release at nighttime. Thus, retinal dopamine levels are higher during the day and lowest during the night due to the circadian release of melatonin. Resultant lower dopamine levels at night cause a reduction in D2 dopamine receptor activity on photoreceptors, causing an increase in photoreceptor intracellular cAMP levels that in turn causes an increase in coupling of gap junctions between rod and cone photoreceptors so that rod input dominates the cone horizontal cells. The increased sensitivity of horizontal cells to light at nighttime is therefore mediated at least in part by activation of D2-like receptors by dopamine released from amacrine cells. A reduction in endogenous retinal dopamine levels causes hyperpolarization of horizontal cells and enhanced dark adaptation. D1 dopamine receptors, which are positively coupled to cAMP synthesis, are located on horizontal cells. Melatonin may therefore postsynaptically regulate horizontal cell activity by inhibiting the stimulation of cAMP synthesis in response to D1 receptor activation. It may bind to receptors on GABAergic amacrine cells, stimulating them to inhibit dopamine release from nearby dopaminergic amacrine cells. In addition, since horizontal cells express melatonin receptors, and melatonin increases horizontal cell sensitivity to light, melatonin may act directly on horizontal cells to increase gap-junctional coupling of horizontal cells. Increased horizontal cell coupling would cause an increase in receptive field size, which would potentially increase the sensitivity of the retina to light during the dark period, since more second-order neurons would respond to a light stimulus.
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Melatonin may therefore modulate dopaminergic transmission by a combination of directly reducing dopamine release from amacrine cells, and indirectly by stimulating GABAergic amacrine cells to inhibit dopamine release from dopaminergic amacrine cells, both of which would increase horizontal cell coupling. The resulting increased visual sensitivity at nighttime could be due to increased rod–cone coupling through dopamine binding to D2 receptors on photoreceptors, or to horizontal cell coupling stimulated by the binding of melatonin to melatonin receptors on horizontal cells. A summary diagram of the known locations of melatonin receptors and the possible interactions with the various target cells is presented in Figure 1. Melatonin increases horizontal cell sensitivity to light in salamander retina, and also potentiates glutamateinduced currents from isolated cone-driven horizontal cells in carp retina by increasing the efficacy and affinity of the glutamate receptor. These observations suggest that melatonin acts directly on melatonin receptors of horizontal cells. Melatonin modulates cyclic guanosine monophosphate (cGMP)-dependent glutaminergic transmission from cones to cone-driven horizontal cells by activation of the Mel1a receptor, causes a depolarization of the H1 horizontal cell membrane potential, and reduces its light responses. These observations suggest that melatonin enhances the circadian sensitivity of rod photoreceptor signaling. Melatonin has been shown in fish retina to potentiate responses of rod ON bipolar cells to simulated light flashes. This action of melatonin is mediated by the Mel1b receptor, and increases cGMP levels by inhibiting phosphodiesterase activity. Melatonin may bind directly to Mel1b receptors on rod ON bipolar cells to improve the signal/noise ratio for rod signals by enhancing signal transfer from rod photoreceptors to rod bipolar cells. The presence of melatonin receptors by immunocytochemistry has not yet been definitively established in bipolar cells. Melatonin treatment of isolated rat retinal ganglion cells potentiates glycine-induced currents by increasing the efficacy and channel conductance of a glycine receptor. The inhibitory modulation of glycinergic inputs to ganglion cells may thus be strengthened by stimulation of melatonin receptor activation. This suggests that melatonin may regulate circadian changes in receptive field organization and light sensitivity by binding to melatonin receptors on ganglion cells.
Modulation of Photoreceptor Function Several reports support the concept of a direct action of melatonin on retinal photoreceptor function. Melatonin induces membrane conductance changes in isolated frog rod photoreceptors, binds with low affinity to structures in the OPL in frog retina, enhances the rate of photoreceptor
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Neurotransmitters and Receptors: Melatonin Receptors
Melatonin receptor D1 D2
D1 dopamine receptor RPE
D2 dopamine receptor
GABA receptor GABA Dark D2
PH
(–) H
(–)
MEL
H D1
DA
(–) MEL A
DA
B
MEL (+) A
(+)
GABA
(–) G
GABA
Figure 1 Summary diagram of locations of retinal melatonin receptors and potential interactions among target cells. Melatonin (MEL) is produced by photoreceptors (PH) at nighttime, and diffuses to target cells within the retina. Arrows represent the movement of melatonin to the target cells. The melatonin receptors are represented by a black crescent symbol. Dopamine and GABA receptors are represented by crescent symbols with D1, D2, or GABA as identifiers. Melatonin may bind to amacrine cells (A) that release GABA or dopamine (DA) as their neurotransmitter. Melatonin is thought to stimulate (þ) GABA release and/or inhibit () dopamine release from these cells. GABA inhibits dopamine release from dopaminergic amacrine cells. A lower rate of dopamine release from amacrine cells at nighttime results in lower stimulation of D1 receptors on horizontal cells (H), which leads to increased coupling of horizontal cells through gap junctions (¼), which could cause an increase in receptive field size and increased retinal sensitivity to light. The decreased binding of dopamine at nighttime to D2 receptors on photoreceptor cells results in an increase in melatonin synthesis, since dopamine inhibits melatonin synthesis in photoreceptors. Melatonin may potentially bind to horizontal cells to directly inhibit the cellular response to D1 receptor binding. Melatonin may also bind to receptors located on the photoreceptor membrane, which could directly increase rod sensitivity to light, increase rod–cone coupling, and/or regulate synthesis of melatonin. Melatonin may bind to receptors on the apical membrane of retinal pigment epithelial (RPE) cells, to coordinate circadian interactions with photoreceptor outer segments. The possible expression of melatonin receptors on bipolar (B) cells, and the confirmed expression of melatonin receptors on ganglion cells (G) are not indicated by arrows.
outer segment disk shedding, and increases the degree of light-induced photoreceptor cell death. It causes a stimulation of the amplitude of the a-wave (rod photoreceptors) and the b-wave (inner retinal cells responding to the photoreceptor input) of electroretinogram (ERG) recordings of transgenic frogs that overexpress the Mel1c receptor in rod photoreceptors. This suggests that melatonin acts directly on rod photoreceptors to increase retinal sensitivity to light as part of a dark-adaptation mechanism. The role of melatonin in dark adaptation suggests a potential mechanism by which melatonin increases the degree of light-induced photoreceptor cell death. Since melatonin appears to increase the sensitivity of the retina to light as part of a dark-adaptation mechanism, an undesirable consequence of this may be an increased sensitivity to the deleterious effects of light. Although signals from the inner retina obviously play a significant role in the circadian activities of retinal photoreceptors, direct action of melatonin on receptors located on photoreceptors may contribute substantially to the functions of melatonin in circadian-regulated activities of the retina.
See also: Chick Metabolism in the Chick Retina; Circadian Photoreception; The Circadian Clock in the Retina Regulates Rod and Cone Pathways; Circadian Regulation of Ion Channels in Photoreceptors; Neurotransmitters and Receptors: Dopamine.
Further Reading Besharse, J. C. and Dunis, D. A. (1983). Methoxyindoles and photoreceptor metabolism: Activation of rod shedding. Science 219: 1341–1342. Boatright, J. H., Rubim, N. M., and Iuvone, P. M. (1994). Regulation of endogenous dopamine release in amphibian retina by melatonin: The role of GABA. Visual Neuroscience 11: 1013–1018. Dubocovich, M. L. (1983). Melatonin is a potent modulator of dopamine release in the retina. Nature 306: 782–784. Dubocovich, M. L., Cardinali, D. P., Guardiola-Lemaitre, B., et al. (1998). Melatonin receptors. In: Girdlestone, D. (ed.) The IUPHAR Compendium of Receptor Characterisation and Classification, vol. I, pp. 188–193. London: UCPHAR Media. Fujieda, H., Scher, J., Hamadanizadeh, S. A., et al. (2000). Dopaminergic and GABAergic amacrine cells are direct targets of melatonin: Immunocytochemical study of mt1 melatonin receptor in guinea pig retina. Visual Neuroscience 17: 63–70.
Neurotransmitters and Receptors: Melatonin Receptors Huang, H., Lee, S. C., and Yang, X. L. (2005). Modulation by melatonin of glutamatergic synaptic transmission in the carp retina. Journal of Physiology 569: 857–871. Iuvone, P. M., Tosini, G., Pozdeyev, N., et al. (2005). Circadian clocks, clock networks, arylalkylamine N-acetyltransferase, and melatonin in the retina. Progress in Retinal and Eye Research 24: 433–456. Lundmark, P. O., Pandi-Perumal, S. R., Srinivasan, V., and Cardinali, D. P. (2006). Role of melatonin in the eye and ocular dysfunctions. Visual Neuroscience 23: 853–862. Reppert, S. M., Godson, C., Mahle, C. D., et al. (1995). Molecular characterization of a second melatonin receptor expressed in human retina and brain: The Mel1b melatonin receptor. Proceedings of the National Academy of Sciences of the United States of America 92: 8734–8738. Scher, J., Wankiewicz, E., Brown, G. M., and Fujieda, H. (2002). MT1 melatonin receptor in the human retina: Expression and localization. Investigative Ophthalmology and Visual Science 43: 889–897.
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Wiechmann, A. F. and Smith, A. R. (2001). Melatonin receptor RNA is expressed in photoreceptors and displays a cyclic rhythm in Xenopus retina. Molecular Brain Research 91: 104–111. Wiechmann, A. F. and Summers, J. A. (2008). Circadian rhythms in the eye: The physiological significance of melatonin receptors in ocular tissues. Progress in Retinal and Eye Research 27: 137–160. Wiechmann, A. F., Vrieze, M. J., Dighe, R. K., and Hu, Y. (2003). Direct modulation of rod photoreceptor responsiveness through a Mel1c melatonin receptor in transgenic Xenopus laevis retina. Investigative Ophthalmology and Visual Science 44: 4522–4531. Wiechmann, A. F., Udin, S. B., and Summers Rada, J. A. (2004). Localization of Mel1b melatonin receptor protein expression in ocular tissues of Xenopus laevis. Experimental Eye Research 79: 585–594. Young, R. W. and Bok, D. (1969). Participation of the retinal pigment epithelium in the rod outer segment renewal process. Journal of Cell Biology 42: 392–403.