Melanosome Dispersion Research Articles

L‐NAME‐Induced Dispersion of Melanosomes in Melanophores Activates PKC, MEK and ERK1

Melanosome movement represents a good model of cytoskeleton-mediated transport of organelles in eukaryotic cells. We recently observed that inhibiting nitric oxide synthase (NOS) with Nomega-nitro-L-arginine methyl ester (L-NAME) induced dispersion in melanophores pre-aggregated with melatonin. Activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) or calcium-dependent protein kinase (PKC) is known to cause dispersion. Also, PKC and NO have been shown to regulate the mitogen/extracellular signal-regulated kinase (MEK)-ERK pathway. Accordingly, our objective was to further characterize the signaling pathway of L-NAME-induced dispersion. We found that the dispersion was decreased by staurosporine and PD98059, which respectively inhibit PKC and MEK, but not by the PKA inhibitor H89. Furthermore, Western blotting revealed that ERK1 kinase was phosphorylated in L-NAME-dispersed melanophores. L-NAME also caused dispersion in latrunculin-B-treated cells, suggesting that this effect is not due to inhibition of the melatonin signaling pathway. Summarizing, we observed that PKC and MEK inhibitors decreased the L-NAME-induced dispersion, which caused phosphorylation of ERK1. Our results also suggest that NO is a negative regulator of phosphorylations that leads to organelle transport.

Melanosome and erythrosome positioning regulates cAMP-induced movement in chromatophores from spotted triplefin, Grahamina capito.

This study investigated regulation of uniform positioning of melanosomes and erythrosomes in chromatophores from spotted triplefin Grahamina capito from New Zealand, by modulating levels of intracellular cAMP. Elevated cAMP levels, caused by forskolin treatment, inhibited aggregation and induced rapid dispersion of melanosomes and erythrosomes. The dispersing organelles moved to and accumulated at the cell periphery, leading to an abnormal hyperdispersed state with a melanosome- or erythrosome-depleted cell center. Minutes after hyperdispersion, these organelles reversed direction and moved towards the center again to finally distribute throughout the cells. When chromatophores with initially dispersed melanosomes or erythrosomes were treated with forskolin, no hyperdispersion was seen, but the erythrosomes aggregated slowly. Disassembly of actin by latrunculin resulted in a similar but constant hyperdispersed melanosome and erythrosome distribution. The results show that cAMP not only disperses but also aggregates melanosomes and erythrosomes, and that it is the intracellular position of these organelles that determine the directionality of the cAMP-induced movement. To ascertain the even distribution in the dispersed state, regulatory components associated with the actin cytoskeleton in the cell periphery might modify activity of cytoplasmic dynein or kinesin upon contact with dispersing melanosomes or erythrosomes.

Use of a cell-based, lawn format assay to rapidly screen a 442,368 bead-based peptide library

A cell-based, lawn format assay utilizing an in situ photocleavage method has been developed that allows the rapid examination of large bead-based compound libraries as discrete molecules. The format uses frog melanophore cells in a contiguous, adherent, confluent layer in small petri dishes covered with a 0.5–1-mm layer of agarose containing 130 micron diameter TentaGel beads at a density of 2–20 beads/mm 2. Employing this technique a 9-mer, 442,368-member peptide library (designed around the 13 amino acid α-MSH peptide sequence) made up of 12 separate pools of 36,864 peptides/pool was assayed. Initially, a fraction (∼10%) of each pool was scanned (∼3700 beads from each pool) in 60-mm petri dishes to identify the most active pools. Upon direct photocleavage of the beads with UV light (365 nm), each petri dish was photographed over a 60-min period with a CCD camera to record changes in light intensity as an index of melanosome dispersion. Active beads were those that were surrounded by a localized decrease in light transmittance indicating melanosome dispersed cells. Upon examination with a dissecting microscope, single beads centrally located to a circular array of dispersed cells were identified and removed from the agarose and sequenced by Edman degradation to determine the peptide sequence. Re-synthesized peptides were re-examined against α-MSH receptor to confirm and quantify the activity. Several 9-mer peptides were identified with potencies similar to the natural 13-mer peptide. This method allows for the rapid screening of large bead-based photo-cleavable peptide libraries with the advantage that each compound is screened as a discrete molecule in a well-less format.

Identification of microtubule-organizing centers in interphase melanophores of Xenopus laevis larvae in vivo.

The morphological characteristics of microtubule-organizing centers (MTOCs) in dermal interphase melanophores of Xenopus laevis larvae in vivo at 51-53 stages of development has been studied using immunostained semi-thick sections by fluorescent microscopy combined with computer image analysis. Computer image analysis of melanophores with aggregated and dispersed pigment granules, stained with the antibodies against the centrosome-specific component (CTR210) and tubulin, has revealed the presence of one main focus of microtubule convergence in the cell body, which coincides with the localization of the centrosome-specific antigen. An electron microscopy of those melanophores has shown that aggregation or dispersion of melanosomes is accompanied by changes in the morphological arrangement of the MTOC/centrosome. The centrosome in melanophores with dispersed pigment exhibits a conventional organization, and their melanosomes are situated in an immediate vicinity of the centrioles. In melanophores with aggregated pigment, MTOC is characterized by a three-zonal organization: the centrosome with centrioles, the centrosphere, and an outlying radial arrangement of microtubules and their associated inclusions. The centrosome in interphase melanophores is presumed to contain a pair of centrioles or numerous centrioles. Because of an inability of detecting additional MTOCs, it has been considered that an active MTOC in interphase melanophores of X. laevis is the centrosome. We assume that remaining intact microtubules in the cytoplasmic processes of mitotic melanophores (Rubina et al., 1999) derive either from the aster or the centrosome active at the interphase.

Functional characterization of a receptor for vasoactive-intestinal-peptide-related peptides in cultured dermal melanophores from Xenopus laevis.

A receptor for vasoactive-intestinal-peptide (VIP)-related peptides was functionally characterized in a cell line derived from Xenopus melanophores using a recently described microtiter-plate-based bioassay. Activation of the melanophore VIP receptor by VIP or the peptides pituitary-adenylate-cyclase-activating polypeptide (PACAP 38), PACAP 27, and helodermin stimulated intracellular 3'-5' cyclic adenosine monophosphate (cAMP) accumulation and pigment dispersion in the cells. Helodermin, with an EC50 (concentration of peptide inducing half-maximal melanosome dispersion) of 46.5 pM, was the most potent activator of pigment dispersion, followed by PACAP 38 > VIP > PACAP 27. A similar order of potencies was observed for the peptides to induce cAMP accumulation. The responses to VIP agonists were selectively inhibited by the VIP antagonists PACAP-(6-27) and (N-Ac-Tyr(1)-D-Phe2)-growth-hormone-releasing factor[GRF](1-29)-NH2. Taken together, the results suggest that the melanophores express a VIP receptor that shares certain characteristics of, but also differs significantly from, other previously identified VIP receptors.

Effects of lithium on pigmentation in the embryonic zebrafish ( Brachydanio rerio)

Pigment cell precursors of the embryonic zebrafish give rise to melanophores, xanthophores and/or iridophores. Cell signaling mechanisms related to the development of pigmentation remain obscure. In order to examine the mechanisms involved in pigment cell signaling, we treated zebrafish embryos with various activators and inhibitors of signaling pathways. Among those chemicals tested, LiCl and LiCl/forskolin had a stimulatory effect on pigmentation, most notable in the melanophore population. We propose that the inositol phosphate (IP) pathway, is involved in pigment pattern formation in zebrafish through its involvement in the: (1) differentiation/proliferation of melanophores; (2) dispersion of melanosomes; and/or (3) synthesis/deposition of melanin. To discern at what level pigmentation was being effected we: (1) counted the number of melanophores in control and experimental animals 5 days after treatment; (2) measured tyrosinase activity and melanin content; and (3) employed immunoblotting techniques with anti-tyrosine-related protein-2 and anti-melanocyte-specific gene-1 as melanophore-specific markers. Although gross pigmentation increased dramatically in LiCl- and LiCl/forskolin treated embryos, the effect on pigmentation was not due to an increase in the proliferation of melanophores, but was possibly through an increase in melanin synthesis and/or deposition. Collectively, results from these studies suggest the involvement of an IP-signaling pathway in the stimulation of pigmentation in embryonic zebrafish through the synthesis/deposition of melanin within the neural crest-derived melanophores.

Differential Actions of Melatonin on Melanophores of the Threeline Pencilfish, Nannostomus trifasciatus

Abstract The longitudinally striped pattern of the threeline pencilfish, Nannostomus trifasciatus, is observed only in the daytime, and changes into a different pattern with three dark spots at night. In this study, microscopic examinations and photoelectric measurements revealed that melanophores in the greater part of the integument respond to melatonin by the aggregation of melanosomes. By contrast, larger melanophores existing in dark spots respond to the amine by the dispersal of pigment. Physiological tests indicated that the nervous mechanisms controlling these melanophores are practically identical to each other, and also with those existing in many teleost species known hitherto. It is further shown that membrane-permeating analogues of 3′,5′-cyclic nucleotides effectively disperse melanosomes in all these melanophores. We conclude that melanophores in the spots possess melatonin receptors that mediate melanosome dispersion, which we have recently described as “β-melatonin receptors” in some mela...

Heterotrimeric kinesin II is the microtubule motor protein responsible for pigment dispersion in Xenopus melanophores.

Melanophores move pigment organelles (melanosomes) from the cell center to the periphery and vice-versa. These bidirectional movements require cytoplasmic microtubules and microfilaments and depend on the function of microtubule motors and a myosin. Earlier we found that melanosomes purified from Xenopus melanophores contain the plus end microtubule motor kinesin II, indicating that it may be involved in dispersion (Rogers, S.L., I.S. Tint, P.C. Fanapour, and V.I. Gelfand. 1997. Proc. Natl. Acad. Sci. USA. 94: 3720-3725). Here, we generated a dominant-negative construct encoding green fluorescent protein fused to the stalk-tail region of Xenopus kinesin-like protein 3 (Xklp3), the 95-kD motor subunit of Xenopus kinesin II, and introduced it into melanophores. Overexpression of the fusion protein inhibited pigment dispersion but had no effect on aggregation. To control for the specificity of this effect, we studied the kinesin-dependent movement of lysosomes. Neither dispersion of lysosomes in acidic conditions nor their clustering under alkaline conditions was affected by the mutant Xklp3. Furthermore, microinjection of melanophores with SUK4, a function-blocking kinesin antibody, inhibited dispersion of lysosomes but had no effect on melanosome transport. We conclude that melanosome dispersion is powered by kinesin II and not by conventional kinesin. This paper demonstrates that kinesin II moves membrane-bound organelles.

Regulation of organelle movement in melanophores by protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2A (PP2A).

We used melanophores, cells specialized for regulated organelle transport, to study signaling pathways involved in the regulation of transport. We transfected immortalized Xenopus melanophores with plasmids encoding epitope-tagged inhibitors of protein phosphatases and protein kinases or control plasmids encoding inactive analogues of these inhibitors. Expression of a recombinant inhibitor of protein kinase A (PKA) results in spontaneous pigment aggregation. alpha-Melanocyte-stimulating hormone (MSH), a stimulus which increases intracellular cAMP, cannot disperse pigment in these cells. However, melanosomes in these cells can be partially dispersed by PMA, an activator of protein kinase C (PKC). When a recombinant inhibitor of PKC is expressed in melanophores, PMA-induced pigment dispersion is inhibited, but not dispersion induced by MSH. We conclude that PKA and PKC activate two different pathways for melanosome dispersion. When melanophores express the small t antigen of SV-40 virus, a specific inhibitor of protein phosphatase 2A (PP2A), aggregation is completely prevented. Conversely, overexpression of PP2A inhibits pigment dispersion by MSH. Inhibitors of protein phosphatase 1 and protein phosphatase 2B (PP2B) do not affect pigment movement. Therefore, melanosome aggregation is mediated by PP2A.

Beta2-adrenoceptors mediate melanosome dispersion in winter flounder (Pleuronectes americanus))

Although the melanosome-aggregating mediation of catecholamines through alpha -adrenoceptors is well established for teleost melanophores, the regulation of the dispersive process is not as clearly understood. The melanosome-aggregating effect of high concentrations of catecholamines in vitro is reversed at low concentrations with melanophores of the winter flounder, Pleuronectes americanus. In vitro incubation in low concentrations of isoproterenol ( <<= 10-7M) and noradrenaline ( <<= 10-8M) enhances Na+-induced melanosome dispersion in balanced salt solution, which can be depressed by propranolol (10-4M), indicating beta -adrenoceptor mediation in pigment dispersion. The subtype of this adrenoceptor appears to be the beta2conformation, since the beta2-adrenoceptor agonist terbutaline (>=>3.15 times 10-6M) reverses the melanosome-aggregating effect associated with higher concentrations of noradrenaline and with electrical stimulation. It is concluded that in this species there is adrenergic neuronal control of melanosome aggregation through alpha -adrenoceptors on release of noradrenaline and of dispersion through beta2-adrenoceptors during a subsequent decrease in concentration.

Light-sensitive response in melanophores of Xenopus laevis: I. Spectral characteristics of melanophore response in isolated tail fin of Xenopus tadpole.

Melanophores in the isolated tail from the amphibian larvae Xenopus laevis, Hyla japonicus, Rana pirica, and Hynobius retardatus aggregated melanin granules in response to light and dispersed them when placed in darkness. The spectral characteristics for the melanin-aggregation response were examined by irradiating the Xenopus tail-fin locally (diameter, 2.1 mm) with monochromatic light (380-1,020 nm). The spectral region of wave length which induced melanosome aggregation depended on the light intensity but was limited to the visible spectrum. At low light intensity (1.59 microW/cm2, delta lambda = 5 nm), the aggregation response occurred in the spectral region between 400 and 600 nm and the maximum response was observed at 500 nm. This range is very close to the absorption spectrum of rhodopsin in the visual rod cell. Hypodermic injection of cGMP into isolated tail-fin induced a marked melanin-dispersion in spite of light-stimuli. When the tail-fin was treated with isobutylmethylxanthine (IBMX; phosophodiesterase inhibitor) in darkness and then was re-exposed to light, the aggregation response was inhibited. The photo-sensitive melanin aggregation was independent of a requirement for Ca2+ ions but melanosome dispersion in darkness was Ca(2+)-dependent. K(+)-rich Hanks' solution, ouabain (inhibitor of Na(+)-K(+)-ATPase) or nonactin (cation ionophore), which induced a change of the membrane potential of melanophores, inhibited the aggregation response when the melanophores were re-exposed to light after a period in darkness. These results suggest that the molecular mechanism of photoreception in melanophores of amphibian tadpoles is similar to that in visual cells.

Light‐sensitive response in melanophores of Xenopus laevis: I. Spectral characteristics of melanophore response in isolated tail fin of Xenopus tadpole

Melanophores in the isolated tail from the amphibian larvae Xenopus laevis, Hyla japonicus, Rana pirica, and Hynobius retardatus aggregated melanin granules in response to light and dispersed them when placed in darkness. The spectral characteristics for the melanin-aggregation response were examined by irradiating the Xenopus tail-fin locally (diameter, 2.1 mm) with monochromatic light (380–1,020 nm). The spectral region of wave length which induced melanosome aggregation depended on the light intensity but was limited to the visible spectrum. At low light intensity (1.59 μW/cm2, Δλ = 5 nm), the aggregation response occurred in the spectral region between 400 and 600 nm and the maximum response was observed at 500 nm. This range is very close to the absorption spectrum of rhodopsin in the visual rod cell. Hypodermic injection of cGMP into isolated tail-fin induced a marked melanin-dispersion in spite of light-stimuli. When the tail-fin was treated with isobutylmethylxanthine (IBMX; phosophodiesterase inhibitor) in darkness and then was re-exposed to light, the aggregation response was inhibited. The photo-sensitive melanin aggregation was independent of a requirement for Ca2+ ions but melanosome dispersion in darkness was Ca2+-dependent. K+-rich Hanks' solution, ouabain (inhibitor of Na+-K+-ATPase) or nonactin (cation ionophore), which induced a change of the membrane potential of melanophores, inhibited the aggregation response when the melanophores were re-exposed to light after a period in darkness. These results suggest that the molecular mechanism of photoreception in melanophores of amphibian tadpoles is similar to that in visual cells. © 1996 Wiley-Liss, Inc.

Adrenoceptors in cryptic patterning of a flatfish, Pleuronectes americanus

Cryptic patterns of winter flounder, Pleuronectes americanus , possess general background, dark band and white spot components that display differential chromatic responsiveness which has been studied pharmacologically in the current work. In vitro experiments with catecholamines and phentolamine demonstrate that α-adrenoceptors mediate melanosome aggregation in each pattern component. There was pattern-related differential responsiveness to noradrenaline, with EC 50 values between 5.2 x 10 -7 M for dark bands and 2.6 x 10 -6 M for white spots. Phentolamine (10 -4 M) completely dispersed melanosomes, previously aggregated by noradrenaline (10 -5 M), in 9 min for the white spots compared with approximately 82.5% dispersion in 15 min for the other two components. High degrees of melanosome aggregation induced in vitro by isoproterenol (3.15 x 10 -5 M) were reversed differentially by propranolol (10 -4 M) indicating β-adrenoceptor involvement, white spots displaying complete melanosome dispersion in 15 min with partial or no dispersion in the other two components. Phentolamine (10 -4 M) also reversed the effect of isoproterenol, so that the apparent β-adrenoceptor synergism can be explained in terms of pattern-related presynaptic neuromodulation.

The effects of background colouration and ?-MSH treatment on melanophore frequency in winter flounder, Pleuronectes americanus

Winter flounder, Pleuronectes americanus, adapting to black or white backgrounds display significant increase and decline respectively in the number of visible epidermal melanophores over periods up to 8 weeks or longer. This contrasts with a stability in the number of visible dermal melanophores during the same periods of exposure to each background. Flounders treated with α-melanophore stimulating hormone exhibited an enhanced rate of increase in number of visible epidermal melanophores when the background was changed from white to black, whereas white-adapted flounder treated with α-melanophore stimulating hormone without background change did not manifest any such increase in number of epidermal melanophores. Flounder treated with α-melanophore stimulating hormone after transfer from black to white displayed a similar initial decline in visible epidermal melanophore number as in control fish, but the final decline was significantly attenuated. Thus α-melanophore stimulating hormone, which has no apparent influence on melanosome dispersion in this species, may have a limited morphological melanophore regulatory role which is discussed in relation to possible antagonistic and synergistic factors that could influence melanogenesis and visible melanophore numbers.

Functional Expression of Recombinant G-Protein-Coupled Receptors Monitored by Video Imaging of Pigment Movement in Melanophores

Xenopus laevis melanophores are capable of functionally expressing recombinant receptors which couple via G-proteins to adenylate cyclase or phospholipase C (PLC). Receptor-mediated stimulation of either of these enzymes causes dispersion of melanosomes while receptor stimulation leading to inhibition of adenylate cyclase induces their aggregation. Translocation of melanosomes within thousands of individual pigment cells was simultaneously tracked by capturing gray scale video images before and after receptor activation. Digital subtraction of poststimulation from prestimulation images was performed on a microcomputer, generating bitplane images containing pixels with nonzero gray scale values wherever melanosome movement had occurred. Movement in both centripetal and centrifugal directions was detectable. The assay was tested using four receptors: a human β2-adrenergic receptor which stimulates adenylate cyclase, murine substance P and bombesin receptors which stimulate PLC, and a human D2 dopamine receptor which inhibits adenylate cyclase. Based on melanosome translocation following application of ligands, expression of functional receptors could be consistently detected in melanophores which received only single copies of a plasmid encoding any of the four receptors. By imaging fields containing up to 11,000 melanophores, the presence of a plasmid coding for a receptor could be detected when its frequency was one per 10,000 plasmids transfected. Combining receptor-mediated pigment translocation in melanophores with the rapid data-handling ability of video technology should provide a bioassay useful for investigating the function of G-protein-coupled receptors and for screening cDNA libraries for clones encoding new receptors.

Effects of purine compounds and forskolin on melanophores of the medaka, oryzias latipes: Evidence for an A 2-adenosine receptor

1. The effects of purines on denervated melanophores of the medaka were studied under experimental conditions in which melanosomes were aggregated by norepinephrine or lithium ion beforehand. 2. Adenosine and its derivatives caused melanosome dispersion; the order of potency for the series was; NECA > adenosine > ATP > 2-chloroadenosine > PIA > CHA > cyclic AMP. 3. 8-Phenyltheophylline, a potent purinoceptor antagonist, blocked the effect of purines and caused a rightward shift of the adenosine and analog concentration-response curves. 4. 8-Br cyclic AMP also caused melanosome dispersion but its action was not blocked by 8-phenyladenosine. Dibutyryl cyclic AMP, cyclic GMP, dibutyryl cyclic GMP, and 8-br cyclic GMP were all ineffective. 5. The effect of adenosine was immediately eliminated by adenosine deaminase but, actions of NECA, AMP, ADP, ATP, and cyclic AMP were not. 6. Forskolin, a potent activator of adenylate cyclase, mimicked the action of adenosine. 7. It is concluded that adenosine and its derivatives mediate their melanosome-dispersing effect via a P 1-purinoceptor that displays characteristics of the A 2-subtype and that adenine nucleotides directly activate the A 2-receptor without conversion to adenosine.

The effects of spinal nerve section on responses of melanophores in the minnow, Phoxinus phoxinus(L.)

A continuous observation apparatus was used to study the responses of Phoxinus phoxinus melanophores to illuminated black/white backgrounds and their reversal. The fish. Although confined, showed maximum melanosome dispersion (MI 5) and maximum melanosome aggregation (MI 1) when exposed to illuminated black and white backgrounds respectively. Melanophores affected by spinal nerve section showed full melanosome dispersion and the affected area appeared as a black band. The affected melanophores marginally and gradually aggregated their melanosomes if the fish was exposed to an illuminated white background for about a week. The responses of these melanophores to illuminated black and white backgrounds and their reversal indicates that the dispersal of their melanosomes in response to a black background is much faster than their aggregation in response to a white background. It is concluded that an active mechanism is involved and possible factors controlling it are discussed.

Identification of a Single Melanin‐Concentrating Hormone Messenger Ribonucleic Acid in Coho Salmon: Structural Relatedness with 7SL Ribonucleic Acid

Abstract Melanin-concentrating hormone (MCH) is a cyclic neuropeptide possessing antagonistic function to alpha-melanocyte-stimulating hormone and corticotropin-releasing factor in the control of melanosome dispersion within melanophores and adrenocorticotropin release in fish. We have isolated and characterized MCH cDNAs from coho salmon (Oncorhyncus kisutch). The precursor protein predicted by the longest cDNA consists of 132 amino-acids with a characteristic signal peptide at the N-terminus and the biologically active salmon MCH (sMCH) peptide at the C-terminus. The coho sMCH mRNA and protein sequences are very similar but not identical to the previously reported chum or chinook salmon counterparts, suggesting the existence of species polymorphism. Sequence similarities were revealed between alpha-melanocyte-stimulating hormone and part of the C-terminal domain of sMCH precursor. Two sMCH genes were found in coho salmon. By contrast to other salmon species, only one major sMCH mRNA was detected in coho species suggesting that differential MCH gene expression might occur in salmon. In addition, under low stringency oligoprobes complementary to the sMCH RNA recognize a 0.3 kb RNA which was identified as the 7SL RNA. The regions conserved between those RNAs fold in a similar secondary structure. These similarities might reflect common ancestry which may have functional significance.

Melanophore response enhancement during in vitro K + and Na + stimulus repetition in Pseudopleuronectes americanus

1. 1. An effect of repeated in vitro K + and Na + stimulation on respective melanosome aggregation and dispersion responsiveness in winter flounder, Pseudopleuronectes americanus, is described. 2. 2. There is pronounced enhancement of in vitro melanosome aggregation between a first and second response to K +, and of melanosome dispersion during three consecutive responses to Na +, in melanophores from flounders adapted to both black and white backgrounds. 3. 3. Persistence of the enhanced melanophore responsiveness was observed 6 hr after a single ionic stimulation. 4. 4. It is proposed that this response enhancement probably involves a melanophore intracellular priming process.

Synthesis and Actions of a Melanotropin Conjugate, Ac-[Nle4, Glu(gamma-4′-hydroxyanilide)5, D-Phe7]α-MSH4–10-NH2, on Melanocytes and Melanoma Cells In Vitro

L-Glutamic acid (γ-4′-hydroxyanilide) (GHB) is oxidized by tyrosinase to a quinone which inhibits DNA polymerase, RNA polymerase, and mitochondrial energy production within mushrooms. It was previously shown that GHB can kill B16 melanoma cells in culture, but lacks cytotoxicity for nontyrosinase-containing cells. We have conjugated this drug to a superpotent melanotropic peptide and examined the bioactivity of this conjugate to melanoma cells. 4′-Hydroxyaniline was attached to glutamic acid at position 5 in the superpotent melanotropin fragment analogue, Ac-[Nle4, d-Phe7]α-MSH4–10-NH2. The melanotropin:anilide conjugate, Ac-(Nle4, Glu(γ-4′-hydroxyanilide)5, d-Phe7]α-MSH4–10-NH2, was not cytotoxic to B16 or Cloudman S91 mouse melanoma cells in culture, as determined by cell counts and protein assays. Interestingly, we also found that GHB stimulated melanoma cell tyrosinase above control levels in both melanoma cell lines. In our study, GHB itself also was found not to be cytotoxic to B16 or S91 melanoma cells in culture. In the frog skin bioassay, the melanotropin conjugate was more potent than α-MSH or Ac-[Nle4, d-Phe7]α-MSH4–10 in stimulating melanosome dispersion. These results demonstrate that putative chemotherapeutic ligands can be incorporated into active-site fragment analogues of MSH without loss of biological activity.