Dispersion Of Pigment Granules Research Articles

The regulation of melanocyte-stimulating hormone on the pigment granule dispersion in the xanthophores and melanophores of the large yellow croaker (Larimichthys crocea)

The large yellow croaker (Larimichthys crocea) is one of the most valuable economic mariculture fish in China, and its yellowness in skin is considered as the most important traits that determine the market value. The fish skin colors present naturally silvery white during the daytime and golden yellow during the night-time. In the present study, we found that the xanthosomes of xanthophores aggregated during the daytime and dispersed during the night-time, while the melanosomes of melanophores always dispersed all day long in vivo. We further cloned three proopiomelanocortin (POMC) genes of the large yellow croaker. All these POMC genes were expressed in the pituitary, but only POMC-C mRNA was expressed in the skin. The results of single-cell RT-PCR showed that the POMC-C mRNA was expressed in isolated xanthophores but not in melanophores. The xanthophores, but not melanophores, were found in the ventral skin, where only one subtype of melanocortin receptor (MCR), i.e. MC5R, was expressed. MC1R, MC4R and MC5R mRNAs were detected in the dorsal skin, where there existed both the xanthophores and melanophores. The results of single-cell RT-PCR showed that only MC5R mRNA was expressed in the xanthophores, while only MC1R mRNA was detected in the melanophores. Thirty min after in vivo and in vitro treatment with melanocyte-stimulating hormone (MSH) peptides (α-MSH, Des-Ac-α-MSH, α-MSH-C and β-MSH-C), dispersion degrees of the xanthosomes and melanosomes significantly increased, even under light exposure. Interestingly, when we immediately observed the dorsal scales that have been maintained under in vitro complete dark condition without MSH peptides treatment for at least 30 min, the melanosomes aggregated as expected, but the xanthosomes dispersed. Both α-MSH and α-MSH-C exhibited similar, but stronger effects than Des-Ac-α-MSH on dispersion of both the xanthosomes and melanosomes. Although the two MSH peptides (α-MSH-C and β-MSH-C) are generated from POMC-C, the former showed stronger effect on xanthosome dispersion than the latter. Forskolin, an Adenylyl cyclase activator, dispersed pigment granules in both the xanthophores and melanophores. H 89 2HCl, a specific inhibitor of protein kinase A blocked α-MSH-induced pigment granule dispersion of xanthophores and melanophores. Taken together, our results indicated that the dispersion of melanosomes is stimulated by MSH, possibly secreted from pituitary, through endocrine pathway. The yellowness of the large yellow croaker skin was mainly due to the dispersion of xanthosomes. The dispersion of xanthosomes was regulated by MSH that is possibly released both from the pituitary through endocrine pathway and from the xanthophores through autocrine pathway.

Interaction and developmental activation of two neuroendocrine systems that regulate light-mediated skin pigmentation.

Lower vertebrates use rapid light-regulated changes in skin colour for camouflage (background adaptation) or during circadian variation in irradiance levels. Two neuroendocrine systems, the eye/alpha-melanocyte-stimulating hormone (α-MSH) and the pineal complex/melatonin circuits, regulate the process through their respective dispersion and aggregation of pigment granules (melanosomes) in skin melanophores. During development, Xenopus laevis tadpoles raised on a black background or in the dark perceive less light sensed by the eye and darken in response to increased α-MSH secretion. As embryogenesis proceeds, the pineal complex/melatonin circuit becomes the dominant regulator in the dark and induces lightening of the skin of larvae. The eye/α-MSH circuit continues to mediate darkening of embryos on a black background, but we propose the circuit is shut down in complete darkness in part by melatonin acting on receptors expressed by pituitary cells to inhibit the expression of pomc, the precursor of α-MSH.

Prostaglandin signaling in ciliogenesis during development

Prostaglandin signaling in ciliogenesis during development

5-HT receptor subtypes as key targets in mediating pigment dispersion within melanophores of teleost, Oreochromis mossambicus

The presence of distinct class of 5-HT receptors in the melanophores of tilapia (Oreochromis mossambicus) is reported. The cellular responses to 5-HT (5-hydroxytryptamine), 5-HT(1), and 5-HT(2), agonists on isolated scale melanophores were observed with regard to pigment translocation within the cells. It was found that 5-HT exerted rapid and strong concentration dependent pigment granule dispersion within the melanophores. The threshold pharmacological dose of 5-HT that could elicit a measurable response was as low as 4.7×10(-12) M/L. Selective 5-HT(1) and 5-HT(2) agonists, sumatriptan and myristicin were investigated and resulted in dose-dependent pigment dispersion. The dispersing effects were effectively antagonized by receptor specific antagonists. It is suggested that 5-HT-induced physiological effects are mediated via distinct classes of receptors that possibly participate in modulation of pigmentary responses of the fish.

Developmental expression of muscarinic receptors in the eyes of zebrafish

In previous work, we have shown that light-adaptive pigment granule dispersion can be induced in vitro by treating retinal pigment epithelium (RPE) isolated from bluegill retina with acetylcholine or its analog carbachol and that these agents act through muscarinic receptors to induce pigment granule dispersion. RPE is a monolayer of tissue found between the neural retina and the choroid. In fish, RPE has long apical projections enmeshed with the distal part of photoreceptors, reaching down to the level of their nuclei. The RPE disperses melanin pigment granules into the apical projections to shield light-sensitive photoreceptor outer segments from photobleaching when fish are under bright-light conditions. During development, RPE begin to respond to light at 5days post-fertilization, raising the question of whether responsiveness is correlated to receptor expression. Here, we isolate, clone and sequence chrm-odd receptor genes in zebrafish, characterize them phylogenetically and observe their expression in the eyes of the zebrafish at different developmental stages using RT-PCR and immunofluorescence microscopy. We find that zebrafish express six unique chrm-odd receptor subtypes: chrm1a, chrm1b, chrm3a, chrm3b, chrm5a and chrm5b — and these receptors are differentially expressed during development. Our phylogenetic analysis confirms the assignments of chrm1b and chrm5b, isolated here, as well as other muscarinic receptor genes and their duplicates and suggests previously described muscarinic receptors may need to be reclassified. Differences between the expression patterns of ostensibly duplicated genes raise the possibility that subtle differences between the duplicates may enable refined regulation of specific developmental events.

Diurnal rhythms of muscarinic receptor expression in the eye of zebrafish

In teleosts, pigment granules disperse into the apical projections of the RPE to protect photoreceptors from photobleaching in ambient daylight. Activation of muscarinic receptors induces light‐adaptive pigment granule dispersion in isolated RPE. In order to determine whether muscarinic receptors could be involved in acetylcholine‐mediated pigment granule dispersion in vivo, we looked at muscarinic receptor expression in zebrafish eyes at time points in which the pigment granules of the RPE are fully dispersed or fully aggregated, midday and midnight, respectively. We used qRT‐PCR to study the differential expression of muscarinic receptors. Our data indicate that M3 muscarinic receptor RNA is expressed at higher levels at midnight than midday. Studies are underway to determine whether other subtypes of muscarinic receptors are also differentially expressed and to localize expression of the receptors in retinal sections using in situ hybridization and immunofluorescence microscopy. This work was supported by NSF IOB 0615762.

Conservation and Convergence of Colour Genetics: MC1R Mutations in brown Cavefish

Conservation and Convergence of Colour Genetics: MC1R Mutations in brown Cavefish

The Protein Kinase A‐Anchoring Protein Moesin is Bound to Pigment Granules in Melanophores

Major signaling cascades have been shown to play a role in the regulation of intracellular transport of organelles. In Xenopus melanophores, aggregation and dispersion of pigment granules are regulated by the second messenger cyclic AMP through the protein kinase A (PKA) signaling pathway. PKA is bound to pigment granules where it forms complexes with molecular motors involved in pigment transport. Association of PKA with pigment granules occurs through binding to A-kinase-anchoring proteins (AKAPs), whose identity remains largely unknown. In this study, we used mass spectrometry to examine an 80 kDa AKAP detected in preparations of purified pigment granules. We found that tryptic digests of granule protein fractions enriched in the 80 kDa AKAP contained peptides that corresponded to the actin-binding protein moesin, which has been shown to function as an AKAP in mammalian cells. We also found that recombinant Xenopus moesin interacted with PKA in vitro, copurified with pigment granules and bound to pigment granules in cells. Overexpression in melanophores of a mutant moesin lacking conserved PKA-binding domain did not affect aggregation of pigment granules but partially inhibited their dispersion. We conclude that Xenopus moesin is an AKAP whose PKA-scaffolding activity plays a role in the regulation of pigment dispersion in Xenopus melanophores.

Expression of Modd muscarinic acetylcholine receptors in the retinas of bluegill and zebra fish

Light adaptation in most vertebrates occurs through adjustments in pupil diameter. Teleosts, however, have a fixed pupil size, so they rely on retinomotor movements to regulate the amount of light incident on the photoreceptors. Retinomotor movements include photoreceptor contraction and elongation as well as the aggregation and dispersion of membrane bound pigment granules located in the retinal pigment epithelium. Previous work has shown that pigment granule dispersion can be induced using the neurotransmitter acetylcholine acting via muscarinic receptors and that dispersion can be inhibited by Modd receptor specific antagonists. In this study we demonstrate Modd receptor expression using immunolabeling with an anti M3 antibody and with in situ hybridization using probes that target the M5 receptor. Labeling with the M3 antibody was seen multiple layers of the bluegill retina and was supported by Western blot analysis using bluegill and zebrafish tissue. In situ hybridization of the M5 sequence indicated a similar distribution of the receptor as that seen with the antibody. These results suggest that multiple muscarinic receptor subtypes are expressed in the fish retina, and lay the foundation for future studies to elucidate the role of muscarinic signaling pathways in the retina, especially as they pertain to light adaptation.

Carbachol-mediated pigment granule dispersion in retinal pigment epithelium requires Ca2+ and calcineurin

BackgroundInside bluegill (Lepomis macrochirus) retinal pigment epithelial cells, pigment granules move in response to extracellular signals. During the process of aggregation, pigment motility is directed toward the cell nucleus; in dispersion, pigment is directed away from the nucleus and into long apical processes. A number of different chemicals have been found to initiate dispersion, and carbachol (an acetylcholine analog) is one example. Previous research indicates that the carbachol-receptor interaction activates a Gq-mediated pathway which is commonly linked to Ca2+ mobilization. The purpose of the present study was to test for involvement of calcium and to probe calcium-dependent mediators to reveal their role in carbachol-mediated dispersion.ResultsCarbachol-induced pigment granule dispersion was blocked by the calcium chelator BAPTA. In contrast, the calcium channel antagonist verapamil, and incubation in Ca2+-free medium failed to block carbachol-induced dispersion. The calcineurin inhibitor cypermethrin blocked carbachol-induced dispersion; whereas, two protein kinase C inhibitors (staurosporine and bisindolylmaleimide II) failed to block carbachol-induced dispersion, and the protein kinase C activator phorbol 12-myristate 13-acetate failed to elicit dispersion.ConclusionA rise in intracellular calcium is necessary for carbachol-induced dispersion; however, the Ca2+ requirement is not dependent on extracellular sources, implying that intracellular stores are sufficient to enable pigment granule dispersion to occur. Calcineurin is a likely Ca2+-dependent mediator involved in the signal cascade. Although the pathway leads to the generation of diacylglycerol and calcium (both required for the activation of certain PKC isoforms), our evidence does not support a significant role for PKC.

Melanopsin in chicken melanocytes and retina

The vertebrate pigment cell, with the exception of mammals and birds, is able to provide the animal with rapid colour changes, which involve dispersion and aggregation of pigment granules in response to hormonal and neuronal agents, and in some cases as a direct response to light. The search for the mechanisms through which Xenopus leavis melanophores respond to light led to the discovery of a new photopigment, melanopsin, with a different spectral sensitivity to that of rhodopsin. This photopigment was also found in mammalian retinal ganglion cells that project to the suprachiasmatic nucleus and other non-visual retinorecipient areas. Herein we demonstrate (by RT-PCR, cloning and sequencing) for the first time that chick melanocytes express melanopsin, and confirmed the presence of the protein by immunocytochemistry. In the chicken retina, we revealed by immunocytochemistry that ganglion cells express melanopsin, but the highest density of immunopositive cells was found in the inner nuclear layer. Quantitative PCR showed that the retina of animals kept in 6 h light: 18 h dark possessed three-fold higher melanopsin mRNA content than animals kept in longer photoperiod, thus demonstrating that light modulates melanopsin expression in chickens.

Molecular Characterization of G‐proteins in bluegill

The focus of research in our laboratory is directed towards understanding pigment granule dispersion of bluegill (Lepomis macrochirus) retinal pigment epithelium (RPE) and the role of muscarinic receptors in the signaling pathways of pigment granule dispersion. Muscarinic receptors belong to the G-protein coupled receptor (GPCR) superfamily. Binding of muscarinic receptors by acetylcholine or carbachol activates the G-protein coupled to it, causing a cascade of events that lead to dispersion of pigment granules in RPE. To better understand this signaling pathway, an attempt was made to molecularly characterize G-proteins from cDNA extracted from brain and heart of bluegill. Database screening yielded Gαq, Gα11 and Gαi sequences from different organisms. Primers were designed based on highly conserved domains of these genes in all organisms. PCR was used to amplify the cDNAs and the products were sequenced. So far, we have successfully amplified 893bp and 692bp of the expected 1080bp of bluegill Gαq and Gα11 respectively, and 793bp of the expected 1065bp of bluegill Gαi. Future studies will be directed towards finding the expression of these G-proteins in different tissues and their function in carbachol-induced pigment granule dispersion in bluegill RPE.

Molecular and pharmacological characterization of muscarinic receptors in retinal pigment epithelium: role in light‐adaptive pigment movements

Muscarinic receptors are the predominant cholinergic receptors in the central and peripheral nervous systems. Recently, activation of muscarinic receptors was found to elicit pigment granule dispersion in retinal pigment epithelium isolated from bluegill fish. Pigment granule movement in retinal pigment epithelium is a light-adaptive mechanism in fish. In the present study, we used pharmacological and molecular approaches to identify the muscarinic receptor subtype and the intracellular signaling pathway involved in the pigment granule dispersion in retinal pigment epithelium. Of the muscarinic receptor subtype-specific antagonists used, only antagonists specific for M1 and M3 muscarinic receptors were found to block carbamyl choline (carbachol)-induced pigment granule dispersion. A phospholipase C inhibitor also blocked carbachol-induced pigment granule dispersion, and a similar result was obtained when retinal pigment epithelium was incubated with an inositol trisphosphate receptor inhibitor. We isolated M2 and M5 receptor genes from bluegill and studied their expression. Only M5 was found to be expressed in retinal pigment epithelium. Taken together, pharmacological and molecular evidence suggest that activation of an odd subtype of muscarinic receptor, possibly M5, on fish retinal pigment epithelium induces pigment granule dispersion.

Protein Kinase A, which Regulates Intracellular Transport, Forms Complexes with Molecular Motors on Organelles

Major signaling cascades have been shown to play a role in the regulation of intracellular organelle transport . Aggregation and dispersion of pigment granules in melanophores are regulated by the second messenger cAMP through the protein kinase A (PKA) signaling pathway ; however, the exact mechanisms of this regulation are poorly understood. To study the role of signaling molecules in the regulation of pigment transport in melanophores, we have asked the question whether the components of the cAMP-signaling pathway are bound to pigment granules and whether they interact with molecular motors to regulate the granule movement throughout the cytoplasm. We found that purified pigment granules contain PKA and scaffolding proteins and that PKA associates with pigment granules in cells. Furthermore, we found that the PKA regulatory subunit forms two separate complexes, one with cytoplasmic dynein ("aggregation complex") and one with kinesin II and myosin V ("dispersion complex"), and that the removal of PKA from granules causes dissociation of dynein and disruption of dynein-dependent pigment aggregation. We conclude that cytoplasmic organelles contain protein complexes that include motor proteins and signaling molecules involved in different components of intracellular transport. We propose to call such complexes 'regulated motor units' (RMU).

Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill.

BackgroundIn fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents.ResultsThe general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl) carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion.ConclusionsOur results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion.

Identification of protein kinase C (PKC) isoforms in teleostean, amphibian and avian pigment cells.

The beta isoform of protein kinase C (PKC) has been described as the main isoform involved in the stimulation of melanogenesis in mammalian skin melanocytes. Little is known about PKC isoforms in non-mammalian pigment cells. In neopterigian fish (holostei and teleostei), PKC is associated with pigment granule aggregation within the pigment cells (skin lightening), whereas in elasmobranchs and tetrapods, the activation of PKC leads to pigment granule dispersion (skin darkening). In an attempt to a better understanding of this distinct functional behavior upon PKC activation, we decided to investigate the PKC isoforms expressed in pigment cell lines of teleost fish, amphibians and birds, using RT-PCR followed by cloning and sequencing. Our results demonstrate the presence of messenger RNA (mRNA) for the following PKC isoforms: beta 1, lambda and iota in GEM-81 cells (Carassius auratus erythrophoroma), beta 1, beta 2 and zeta in Xenopus laevis (amphibian) melanophores; beta 1 and lambda in Gallus gallus (chicken) primary melanocytes. Beta 1 PKC seems to be conserved throughout phylogeny, but the diversity of the other isoforms in the different groups may account for the functional differences after PKC activation, which are observed between teleost and tetrapod pigment cells.

Corazonin promotes tegumentary pigment migration in the crayfish Procambarus clarkii

The undecapeptide corazonin (pGlu-Thr-Phe-Gln-Tyr-Ser-His-Gly-Trp-Thr-AsnNH 2) elicits a retraction of erythrophore pigment granules and dispersion of leucophore pigment granules in the crayfish Procambarus clarkii. The effects are dose-dependent from 10 −10 to 10 −5 M. Influence on erythrophores is lower than that of Red Pigment Concentrating Hormone (RPCH), which is inactive on leucophores. Corazonin effects are partly blocked by an anti-corazonin antibody, and even less by an anti-RPCH antibody. Corazonin effects are completely suppressed by the calcium chelator BAPTA. Immunoreactive somata and fibers were identified in various regions of the eyestalk (medulla terminalis, medulla interna and medulla externa) with the anti-corazonin antibody. These results suggest the possible existence of a corazonin-like peptide in crustaceans.

Daily morphological variations in the viscacha (Lagostomus maximus maximus) retina. Probable local modulatory action of melatonin.

Given that the local melatonin levels exhibit rhythmic daily changes in the retina of the viscacha, we considered it important to study the likely daily variations in morphology and specific 2-[(125)I]-iodomelatonin binding in retinas from this rodent and to correlate these putative changes with local indole levels. Adult animals of both sexes were captured in their habitat and were kept under a natural photoperiod. For light and electron microscopic studies the viscachas were sacrificed by decapitation at 08:00, 16:00, and 24:00 hr. A computer-assisted image analysis system was used to measure the thickness of the complete retina, the photoreceptor layer, the rod outer and inner segments, and the outer nuclear layer. The daily variation in 2-[(125)I]-iodomelatonin binding sites was followed during a 24-hr light-dark cycle, the animals being sacrificed at six time points. The parameters studied showed significant variations throughout the 24-hr period. Maximal specific binding, lysosomal content in the pigment epithelium, and photoreceptor layer outer segment thicknesses were observed at 24:00 hr. Close contact between photoreceptor membranes and microvilli of the pigment epithelium was observed at 08:00 and 16:00 hr. Moreover, the minimal outer segment thickness at 16:00 hr was accompanied by a scarcity of dense bodies, such as lysosomes, a maximum dispersion of melanin pigment granules, and a minimum density of radioligand binding sites. Therefore, in the retina of the viscacha, we suggest that the interaction between melatonin and specific sites could be one of the factors or causes that participate in the regulation of the daily morphological changes observed in viscacha.

Molecular cloning and functional characterization of murine cysteinyl-leukotriene 1 (CysLT 1) receptors

We sought to clone and characterize the murine cysteinyl-leukotriene D 4 receptor (mCysLT 1R) to complement our studies with leukotriene-deficient mice. A cDNA, cloned from trachea mRNA by reverse transcriptase-polymerase chain reaction, has two potential initiator ATG codons that would encode for polypeptides of 352 and 339 amino acids, respectively. These two potential forms, predicted to be seven transmembrane-spanning domain proteins, have 87% amino acid identity with the human CysLT 1 receptor (hCysLT 1R). Membrane fractions of Cos-7 cells transiently expressing the short mCysLT 1R demonstrated high affinity and specific binding for leukotriene D 4 (LTD 4, K d = 0.25 ± 0.04 nM). In competition binding experiments, LTD 4 was the most potent competitor ( K i = 0.8 ± 0.2 nM) followed by LTE 4 and LTC 4 ( K i = 86.6 ± 24.5 and 100.1 ± 17.1 nM, respectively) and LTB 4 ( K i > 1.5 μM). Binding of LTD 4 was competitively inhibited by the specific CysLT 1 receptor antagonists MK-571 [(+)-3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-(dimethylamino)-3-oxopropyl)thio)methyl)thio)propanoic acid], pranlukast (Onon™), and zafirlukast (Accolate™), while the CysLT 1/CysLT 2 receptor antagonist BAY-u9773 [6( R)-(4′-carboxyphenylthio)-5( S)-hydroxy-7(E),9(E),11(Z),14(Z)-eicosatetrenoic acid] was 1000 times less potent than LTD 4. In transiently transfected HEK293-T cells expressing either the long or short form of mCysLT 1R, LTD 4 induced an increase of intracellular calcium. In Xenopus laevis melanophores transiently expressing either isoform, LTD 4 induced the dispersion of pigment granules, consistent with the activation by LTD 4 of a G αq (calcium) pathway. Functional elucidation of mCysLT 1R properties as described here will enable further experiments to clarify the selective role of LTD 4 in murine models of inflammation and asthma.

An endogenous 5‐HT7 receptor mediates pigment granule dispersion in Xenopus laevis melanophores

Melatonin (5-methoxy N-acetyltryptamine) and serotonin (5-HT) exert rapid, but opposite effects on pigment granule distribution in Xenopus laevis melanophores. Low concentrations of melatonin (10(-11) - 10(-9) M) cause a dramatic perinuclear aggregation of the melanin-containing granules, while 5-HT (10(-8) - 10(-5) M) disperses pigment granules throughout the cell. The present study found that pharmacological doses of melatonin (> or =10(-6) M) induced a time- and concentration-dependent pigment granule dispersion, which was mediated by an endogenous melanophore 5-HT receptor. 5-HT produced a concentration-dependent elevation of melanophore cyclic AMP, and 5-HT-induced dispersion was blocked by H89 (10(-4) M), an inhibitor of protein kinase A (PKA), but not by a PKC inhibitor (Ro 31-8220, 10(-5) M), indicating a vital role for cyclic AMP in 5-HT-induced dispersion. 5-HT-mediated dispersion was not blocked by antagonists selective for G(s)-coupled 5-HT(4) (GR113808) or 5-HT(6) (Ro 04-6790, Ro 63-0563, olanzepine) receptors, nor by 5-HT(1 - 3) (pindolol, ketanserine, metoclopramide, MDL72222, tropisetron) receptor antagonists, but was inhibited by a selective 5-HT(7) receptor antagonist, DR4004, and other antagonists with a high affinity for 5-HT(7) receptors. The rank order of antagonist potency was: risperidone (mean pK(B) 7.82)>methiothepin (7.43)>DR4004 (6.92)>mesulergine (6.83)>methysergide (6.60)>[+/-]-sulpiride (5.81)>spiperone (5.52). The agonist potency order [mean pEC(50), 5-CT (8.68)>5-HT (7.13)>5-MT (6.94)>8-OH-DPAT (4.79)>sumatriptan (<4)] was also consistent with an action on 5-HT(7) receptors. RT - PCR confirmed that melanophores express 5-HT(7) receptor mRNA. The pigment dispersing effect of high melatonin concentrations in melanophores is most likely mediated by activation of 5-HT(7) receptors. Conceivably some of the effects attributed to pharmacological doses of melatonin in mammals may be mediated by activation of 5-HT(7) receptors.