MSH stimulates adenylate cyclase and tyrosinase in cultivated melanoma cells in the presence of cytochalasin B

MSH and cholera toxin increase intracellular levels of cyclic AMP (cAMP) and tyrosinase activity in cultivated mouse melanoma cells in the presence of cytochalasin B (CB) at a concentration sufficient to prevent pinocytosis and cytokinesis. The data suggest that MSH and cholera toxin exert their effects without entering the cell.     

The effect of cytochalasin B on pigment dispersion and aggregation in perfused Xenopus laevis tailfin melanophores

A perfusion technique is described for the study of melanosome response in ventral tailfin melanophores of Xenopus laevis tadpoles. The melanosomes remain aggregated (punctate melanophores) in Ringer's. Theophylline (15 mM) and caffeine (30 mM) cause a reversible dispersion (stellate melanophores) of melanosomes which is partly blocked by cytochalasin B (10 μg/ml). When added with theophylline or caffeine to stellate cells, cytochalasin B causes a disrupted distribution of pigment granules, characterized by a melanosome free central region. C-AMP (20 mM) and dibutyryl c-AMP (1 mM) cause a reversible dispersion of melanosomes which is partly inhibited by cytochalasin. When cytochalasin plus a nucleotide are added to stellate cells, some show the disrupted distribution of melanosomes. Colchicine (5 mM) causes irreversible, while griseofulvin (0.2 mM) causes a slight, but reversible dispersion of melanosomes, and cytochalasin has little effect on these reactions. Perfused tailfin melanophores remain capable of responding to reversible reagents for at least 12 hours and are unresponsive to changes in illumination.     

Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase

Adenosie, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phophodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell suface receptor. Like theophylline, the addition of adenine alone caused no accumulation of cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides has no effect on the accumulation of cyclic AMP. Among other adenine nucleotides was tested, adenosine 5′-monophosphoramidate, but not adenosine 5′-monosulfate, significantly increased cyclic AMP especially with the addition of papaverine. Neither 2′- nor 3′-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.     

Heterochromatin dispersion and blebbing of the nuclear membrane induced by melanophore stimulating hormone

Stimulation (in vivo and in vitro) of dermal melanophores of the leaf frog, Agalychnis dacnicolor, by melanophore stimulating hormone (MSH) elicits two responses in addition to the dispersion of melanosomes: (1) dispersion of heterochromatin; and (2) blebbing of the outer membrane of the nuclear envelope. The latter leads to the formation of dilated rough endoplasmic reticulum with the involvement of 80–100 Å microfilaments. N⁶,O²-dibutyryl adenosine 3′,5′-monophosphate (db-cAMP) elicits a similar response. Actinomycin D prevents both heterochromatin dispersion and membrane blebbing while cytochalasin B (CB) prevents only the latter.     

In vitro response of goldfish (Carassius auratus L.) dermal melanophores to cyclic 3',5'-nucleotides, nucleoside 5'-phosphates and methylxanthines

cAMP, dbcAMP, cCMP, cGMP, theophylline and caffeine caused reversible melanosome dispersion within 5 minutes at 10 mM in the dermal melanophores of the black goldfish, Carassius auratus L. cTMP, cUMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-TMP, and 5′-UMP did not produce melanosome dispersion or aggregation in this melanophore system. cAMP was the most effective nucleotide in the induction of melanosome dispersion; at 10 mM, cGMP and at 5 mM, dbcAMP were the least effective of those nucleotides inducing melanosome dispersion. At the 10 mM level dbcAMP required 30 minutes to evoke the same degree of melanosome dispersion as 5 minutes cAMP treatment. Theophylline was more effective than caffeine in eliciting melanosome dispersion. At 1 mM, theophylline and caffeine first induced melanosome dispersion which was followed by aggregation in the course of the 30 minute test period. These reactions suggest both a high melanophore phosphodiesterase activity and competitive inhibition of phosphodiesterase by theophylline and caffeine. Induction of melanosome dispersion by several cyclic 3′,5′-nucleotides suggest multi-nucleotide control of melanosome dispersion. These findings also support a proposed mechanism of prostaglandin induced melanosome dispersion as well as the “second messenger” hypothesis.     

THE MELANOCYTE MODEL : Colchicine-like Effects of Other Antimitotic Agents

The effect of various agents that cause metaphase arrest in dividing cells was studied on the rapid reversible darkening of frog skin under the influence of melanocyte-stimulating hormone (MSH). Darkening is due to dispersion of melanin granules in melanocytes and is thought to be accompanied by a gel-to-sol cytoplasmic transformation. After subsequent washing the skin lightens, with aggregation of melanin granules and cytoplasmic gelation. As previously shown with colchicine, preincubation of frog skin with vinblastine, vincristine, or colcemid produced an increase in darkening induced by MSH, as compared to control skins, and a dosage-dependent inhibition of subsequent lightening. Preincubation with each drug, without subsequent MSH, produced a gradual, irreversible, dosage-dependent darkening over several hours. On a molar basis, the relative strength of the various agents was vinblastine > vincristine > colcemid > colchicine; vinblastine was about 100 times stronger than colchicine. Preincubation of frog skin with griseofulvin, followed by washing, had no subsequent effects on darkening or lightening. However, effects similar to those of the Colchicum and Vinca alkaloids were seen if griseofulvin was kept in the ambient media. These effects were rapidly reversible on removal of the drug from the media. These findings support the melanocyte model originally proposed for the action of colchicine, and emphasize certain facts that models of melanin granule movement will have to accommodate.     

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. α-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.     

Ionic requirements for melanin concentrating hormone (MCH) actions on teleost Poecilia reticulata melanophores

Melanin concentrating hormone (MCH) is a cyclic heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, synthesized in the hypothalamus and released by the neurohypophysis of teleost fish. This hormone is a potent lightening agent of fish skin. This lightening results from the stimulation of a centripetal melanosome (melanin granule) migration to a perinuclear position within integumental melanophores. MCH and related fragment analogues, MCH5-17 and MCH1-14 were used to investigate the ionic requirements for receptor activation by MCH on dermal melanophores of the fish Poecilia reticulata. In calcium-free saline, the sensitivity of the melanophores to MCH and MCH1-14 increased, whereas the sensitivity of the cells to MCH5-17 decreased. Verapamil diminished the sensitivity to MCH5-17, but did not affect melanophore responses to MCH or MCH1-14. The melanosome aggregating response to MCH was not affected in the presence of tetrodotoxin or in sodium- or potassium-free (choline-substituted) saline. These results suggest that neither TTX-sensitive sodium channels nor extracellular sodium or potassium ions play a role in MCH-induced melanosome aggregation. It is known that MCH and MCH1-14 also exhibit MSH-like melanosome dispersion within melanophores, skin darkening activity on fish melanophores whereas MCH5-17 lacks this characteristic. Since the darkening activity of MCH and MCH1-14 requires calcium, these analogues exhibited a diminished lightening (MCH-like) activity in the presence of the divalent cation. In the absence of the N-terminal tetrapeptide sequence (necessary for the expression of MSH-like activity), a role for calcium on melanosome aggregation became evident. These results demonstrate a bifunctional role of calcium on melanosome movements.     

Effects of purine compounds and forskolin on melanophores of the medaka,oryzias latipes: Evidence for an A2-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 P₁-purinoceptor that displays characteristics of the A₂-subtype and that adenine nucleotides directly activate the A₂-receptor without conversion to adenosine.     

The effect of prostaglandins on ox pituitary content of adenosine 3′:5′-cyclic monophosphate and the release of growth hormone

1. An assay, based on competition between adenosine 3':5'-cyclic monophosphate (cyclic AMP) and cyclic [(3)H]AMP for binding to a rabbit skeletal muscle protein, has been used to measure tissue contents of cyclic AMP. The assay has a sensitivity of 0.05pmol of cyclic AMP. Cyclic GMP and cyclic CMP have 0.5%, and cyclic IMP 6.5%, of the ability of cyclic AMP to displace cyclic [(3)H]AMP from binding protein; AMP, ADP and ATP have no effect. 2. By using this method, the cyclic AMP content of ox pituitary slices exposed to prostaglandin was determined; release of growth hormone was measured by radioimmunoassay. 3. Release of growth hormone was increased by 45min incubation in 1mum-prostaglandin E(2) in the absence of theophylline, or in 10nm-prostaglandin E(2), 0.1mum-prostaglandin A(1) or 1mum-prostaglandin B(1) in the presence of 0.5mm-theophylline. 4. Pituitary cyclic AMP content was increased by 10min incubation in 1mum-prostaglandin E(2) in the absence of theophylline, or in 0.1mum-prostaglandin E(2) in the presence of 0.5mm-theophylline. 5. The maximum increase in cyclic AMP content was observed 10min, and significant changes in growth hormone release 30min, after introduction of prostaglandin E(2). 6. The increase in pituitary cyclic AMP content, but not in the rate of release of growth hormone, was observed in the absence of external Ca(2+). 7. The stimulation of release of growth hormone by prostaglandin was decreased by preincubation of tissue for 2h in colchicine (100mum) or cytochalasin B (10mug/ml). 8. These results support the suggestion that increased release of growth hormone after treatment with prostaglandin is the result of increased tissue cyclic AMP content, and possibly involves a microfilamentous or microtubular protein.     

Effect of modulators of cytoskeletal function on desensitization and recovery of PGE2-responsive ovarian adenylate cyclase

Exposure of cultured Graafian follicles to PGE₂ for 20 h resulted in a loss of the cyclic AMP response to fresh hormone. This desensitization was prevented by addition to the medium of D₂O (25–50%) or Li⁺ (0.6 – 6 mM), agents believed to stabilize microtubules, as well as by phalloidin (1.0 – 10 μM), believed to stabilize the polymerized state of actin, in a dose-dependent manner.The spontaneous recovery of responsiveness to PGE₂ upon incubation of refractory follicles for 6 h in hormone-free medium was prevented by addition to the medium of cytochalasin B (CB; 3 μg/ml) or of the actin-binding myosin subfragment HMM S-1 (80 μg/ml) or of anti-actin serum; viz. by agents likely to interfere with microfilament function. D₂O (50%) caused morphological damage to the inner layer of the membrana granulosa and severe depression of protein synthesis. The other drugs used (phalloidin, LiCl and cytochalasin B) had no such effects. Resensitization of refractory follicles was also prevented by cycloheximide (10 μg/ml) and by actinomycin D (10 μg/ml). It is speculated that the recovery process may involve the insertion of a newly synthesized protein, such as PG-receptor, into the membrane by a mechanism dependent on microfilament action.     

Effect of cyclic AMP and cytochalasin B on tissue cultured melanophores of Xenopus laevis

The effects of cytochalasin B or low concentrations of adenosine 3′,5′-monophosphate (cyclic AMP) were tested on melanophores in hanging drop preparations of neural fold explants from Xenopus laevis embryos in Barths' solution. After one week in culture, the melanophores were punctate in this medium. Cyclic AMP at 5 mM consistently caused reversible morphological transformation of these cells to the stellate state, whether they were situated within an epithelial outgrowth or isolated on the surface of the coverglass. Only the isolated melanophores consistently responded to 1 mM cyclic AMP. Cytochalasin B at 1–10 μg/ml caused aggregation of melanin granules in stellate cells, but left long, narrow cell branches containing some melanosomes. Its effect was at least partially reversible and appeared to be dose dependent. At 1% concentration, dimethyl sulfoxide caused melanin dispersion.     

Iontophoretic release of cyclic AMP and dispersion of melanosomes within a single melanophore

Selective dispersion of melanosomes was often observed after iontophoretic injection of cyclic adenosine monophosphate (AMP) from a glass microelectrode positioned in a target melanophore in frog skin (as viewed from above through a microscope), with other melanophores in the field serving as controls. Because the skin has orderly arrays of several types of closely spaced cells, it is probable that at times the microelectrode also impales cells other than melanophores. When cyclic AMP injection inside a cell resulted in dispersion of melanosomes from a perinuclear position into dendritic processes, the onset of dispersion was relatively rapid, in many cases less than 4 min (mean time of onset, 5.3 +/- 2.9 [SD] min). A much slower dispersion (mean time of onset, 19.0 +/- 5.0 min) of melanosomes was observed when the microelectrode was positioned adjacent to a melanophore, and much larger quantities of cyclic AMP were released. In addition, no changes were observed for injections of 5'-AMP or cyclic guanosine monophosphate (GMP) through electrodes positioned inside or adjacent to melanophores. Potential measurements showed that after impaling a clell, a constant transmembrane potential could often be recorded over many minutes, indicating that the membrane tends to seal around the microelectrode. The results indicate that cyclic AMP acts more rapidly on the inside of a cell than when applied outside a cell and allowed to diffuse through the plasma membrane. This study introduces a model system whereby the properties of the plasma membrane and melanocyte-stimulating hormone (MSH) receptors can be studies within a single target cell.     

Receptor-mediated gonadotropin action in the ovary. Action of cytoskeletal element-disrupting agents on gonadotropin-induced steroidogenesis in rat luteal cells.

The role of the cellular cytoskeletal system of microtubules and microfilaments on gonadotropin-stimulated progesterone production by isolated rat luteal cells has been investigated. Exposure of luteal cells to human choriogonadotropin resulted in a stimulation of cyclic AMP (4-7-fold) and progesterone (3-4-fold) responses.l Incubation of cells with the microfilament modifier cytochalasin B inhibited the gonadotropin-induced steroidogenesis in a dose- and time-dependent manner. The effect of cytochalasin B on basal production of steroid was less pronounced. Cytochalasin B also inhibited the accumulation of progesterone in response to lutropin, cholera enterotoxin, dibutyryl cyclic AMP and 8-bromo cyclic AMP. The inhibition of steroidogenesis by cytochalasin B was not due to (a) inhibition of 125I-labelled human choriogonadotropin binding to luteal cells, (b) inhibition of gonadotropin-stimulated cyclic AMP formation or (c) a general cytotoxic effect and/or inhibition of protein biosynthesis. Cytochalasin D, like cytochalasin B, inhibited gonadotropin- and 8-bromo cyclic AMP-stimulated steroidogenesis. Although cytochalasin B also blocked the transport of 3-O-methyl-glucose into luteal cells, cytochalasin D was without such an effect. Increasing glucose concentration in the medium, or using pyruvate as an alternative energy source, failed to reverse the inhibitory effect of cytochalasin B. The anti-microtubular agent colchicine failed to modulate synthesis and release of progesterone by luteal cells in response to human choriogonadotropin. These studies suggest that the cellular microfilaments may be involved in the regulation of gonadotropin-induced steroidogenesis. In contrast, microtubules appear to be not directly involved in this process.     

Effects of dopamine on prolactin secretion and cyclic AMP accumulation in the rat anterior pituitary gland

The effects of dopamine on pituitary prolactin secretion and pituitary cyclic AMP accumulation were studied by using anterior pituitary glands from adult female rats, incubated in vitro. During 2h incubations, significant inhibition of prolactin secretion was achieved at concentrations between 1 and 10nm-dopamine. However, 0.1–1μm-dopamine was required before a significant decrease in pituitary cyclic AMP content was observed. In the presence of 1μm-dopamine, pituitary cyclic AMP content decreased rapidly to reach about 75% of the control value within 20min and there was no further decrease for at least 2h. Incubation with the phosphodiesterase inhibitors theophylline (8mm) or isobutylmethylxanthine (2mm) increased pituitary cyclic AMP concentrations 3- and 6-fold respectively. Dopamine (1μm) had no effect on the cyclic AMP accumulation measured in the presence of theophylline, but inhibited the isobutylmethylxanthine-induced increase by 50%. The dopamine inhibition of prolactin secretion was not affected by either inhibitor. Two derivatives of cyclic AMP (dibutyryl cyclic AMP and 8-bromo cyclic AMP) were unable to block the dopamine (1μm) inhibition of prolactin secretion, although 8-bromo cyclic AMP (2mm) significantly stimulated prolactin secretion and both compounds increased somatotropin (growth hormone) release. Cholera toxin (3μg/ml for 4h) increased pituitary cyclic AMP concentrations 4–5-fold, but had no effect on prolactin secretion. The inhibition of prolactin secretion by dopamine was unaffected by cholera toxin, despite the fact that dopamine had no effect on the raised pituitary cyclic AMP concentration caused by this factor. Dopamine had no significant effect on either basal or stimulated somatotropin secretion under any of the conditions tested. We conclude that the inhibitory effects of dopamine on prolactin secretion are probably not mediated by lowering of cyclic AMP concentration, although modulation of the concentration of this nucleotide in some other circumstances may alter the secretion of the hormone.     

The role of RNA and protein synthesis in mediating the action of MSH on mouse melanoma cells.

Exposure of mouse melanoma cells in culture to MSH (melanocyte stimulating hormone) results in a marked increase in tyrosinase (O-diphenyl: O₂ oxidoreductase) activity following a lag period of 6–9 hours. Within 20 minutes after exposure of cells to MSH, the intracellular levels of cyclic AMP rise to levels which are ten times those of controls but fall to concentrations twice control values by 60 minutes. Transient increases in both protein and RNA synthetic rates also occur following MSH administration correlating in time with the dramatic but rapidly decaying increase in cellular cyclic AMP. The increase in tyrosinase activity observed in response to either MSH, dibutyryl cAMP, or theophylline, is completely suppressed by the addition of either cycloheximide (0.28 μg/ml) or actinomycin D (0.05 μg/ml) as is the basal activity of the enzyme. Results from ¹⁴C/³H leucine studies suggest that MSH may cause increased $\text{de}$$\text{novo}$ synthesis of tyrosinase.     

Melanocyte-stimulating hormone affects melanogenic differentiation of quail neural crest cells in vitro

Quail neural crest cells were treated in vitro with alpha-melanocyte-stimulating hormone (alpha-MSH) or dibutyryl cyclic AMP (dbcAMP) plus theophylline. These treatments increased the proportion of melanocytes to total cells in crest cell outgrowth cultures. Pigmentation of neural crest cell clusters proceeded more rapidly when cultures were treated with alpha-MSH or dbcAMP plus theophylline than when untreated. In clonal cell cultures, the proportion of pigmented colonies to total colonies was increased by MSH treatment. From these results, MSH seems not only to accelerate melanogenic differentiation but also to affect the state of commitment of neural crest cells to melanogenic differentiation in vitro, and this action of MSH appears to be mediated by cAMP.     

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.     

Cyclic 3′,5′-AMP phosphodiesterase in Physarum polycephalum: II. Kinetic properties

The effect of several inhibitors of the enzyme cyclic 3′,5′-AMP phosphodiesterase as chemoattractants in Physarum polycephalum was examined. Of the compounds tested, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Roche 20-1724/001) and 1-ethyl-4-(isopropylidinehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid ethyl ester, hydrochloride (Squibb 20009) were the most potent attractants. 3-Isobutyl-1-methyl xanthine, theophylline, and morin (a flavanoid) were moderate attractants and sometimes gave negative chemotaxis at high concentrations. Cyclic 3′,5′-AMP was an effective, but not potent attractant. A repellent effect following the positive chemotactic action was sometimes observed with cyclic 3′,5′-AMP at concentrations as high as 1 · 10⁻² M. Dibutyryl cyclic AMP appeared to be a somewhat more potent attractant than cyclic 3′,5′-AMP. The 8-thiomethyl and 8-bromoderivatives of cyclic AMP, which are poorly hydrolyzed by the phosphodiesterase, were not attractants in Physarum. Possible participation of cyclic 3′,5′-AMP in the directional movement in P. polycephalum is discussed.     

Manassantin B inhibits melanosome transport in melanocytes by disrupting the melanophilin–myosin Va interaction

Human skin hyperpigmentation disorders occur when the synthesis and/or distribution of melanin increases. The distribution of melanin in the skin is achieved by melanosome transport and transfer. The transport of melanosomes, the organelles where melanin is made, in a melanocyte precedes the transfer of the melanosomes to a keratinocyte. Therefore, hyperpigmentation can be regulated by decreasing melanosome transport. In this study, we found that an extract of Saururus chinensis Baill (ESCB) and one of its components, manassantin B, inhibited melanosome transport in Melan‐a melanocytes and normal human melanocytes (NHMs). Manassantin B disturbed melanosome transport by disrupting the interaction between melanophilin and myosin Va. Manassantin B is neither a direct nor an indirect inhibitor of tyrosinase. The total melanin content was not reduced when melanosome transport was inhibited in a Melan‐a melanocyte monoculture by manassantin B. Manassantin B decreased melanin content only when Melan‐a melanocytes were co‐cultured with SP‐1 keratinocytes or stimulated by α‐MSH. Therefore, we propose that specific inhibitors of melanosome transport, such as manassantin B, are potential candidate or lead compounds for the development of agents to treat undesirable hyperpigmentation of the skin.