UV light activates a Gαq/11-coupled phototransduction pathway in human melanocytes

While short exposure to solar ultraviolet radiation (UVR) can elicit increased skin pigmentation, a protective response mediated by epidermal melanocytes, chronic exposure can lead to skin cancer and photoaging. However, the molecular mechanisms that allow human skin to detect and respond to UVR remain incompletely understood. UVR stimulates a retinal-dependent signaling cascade in human melanocytes that requires GTP hydrolysis and phospholipase C β (PLCβ) activity. This pathway involves the activation of transient receptor potential A1 (TRPA1) ion channels, an increase in intracellular Ca²⁺, and an increase in cellular melanin content. Here, we investigated the identity of the G protein and downstream elements of the signaling cascade and found that UVR phototransduction is Gα_q/11 dependent. Activation of Gα_q/11/PLCβ signaling leads to hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP₂) to generate diacylglycerol (DAG) and inositol 1, 4, 5-trisphosphate (IP₃). We found that PIP₂ regulated TRPA1-mediated photocurrents, and IP₃ stimulated intracellular Ca²⁺ release. The UVR-elicited Ca²⁺ response appears to involve both IP₃-mediated release from intracellular stores and Ca²⁺ influx through TRPA1 channels, showing the fast rising phase of the former and the slow decay of the latter. We propose that melanocytes use a UVR phototransduction mechanism that involves the activation of a Gα_q/11-dependent phosphoinositide cascade, and resembles light phototransduction cascades of the eye.     

Metabotropic glutamate receptor 6 signaling enhances TRPM1 calcium channel function and increases melanin content in human melanocytes

Mutations in TRPM1, a calcium channel expressed in retinal bipolar cells and epidermal melanocytes, cause complete congenital stationary night blindness with no discernible skin phenotype. In the retina, TRPM1 activity is negatively coupled to metabotropic glutamate receptor 6 (mGluR6) signaling through Gα_o and TRPM1 mutations result in the loss of responsiveness of TRPM1 to mGluR6 signaling. Here, we show that human melanocytes express mGluR6, and treatment of melanocytes with L‐AP4, a type III mGluR‐selective agonist, enhances Ca²⁺ uptake. Knockdown of TRPM1 or mGluR6 by shRNA abolished L‐AP4‐induced Ca²⁺ influx and TRPM1 currents, showing that TRPM1 activity in melanocytes is positively coupled to mGluR6 signaling. Gα_o protein is absent in melanocytes. However, forced expression of Gα_o restored negative coupling of TRPM1 to mGluR6 signaling, but treatment with pertussis toxin, an inhibitor of G_i/G_o proteins, did not affect basal or mGluR6‐induced Ca²⁺ uptake. Additionally, chronic stimulation of mGluR6 altered melanocyte morphology and increased melanin content. These data suggest differences in coupling of TRPM1 function to mGluR6 signaling explain different cellular responses to glutamate in the retina and the skin.     

Melanin content and MC1R function independently affect UVR‐induced DNA damage in cultured human melanocytes

Malignant transformation of melanocytes leads to melanoma, the most fatal form of skin cancer. Ultraviolet radiation (UVR)‐induced DNA photoproducts play an important role in melanomagenesis. Cutaneous melanin content represents a major photoprotective mechanism against UVR‐induced DNA damage, and generally correlates inversely with the risk of skin cancer, including melanoma. Melanoma risk is also determined by susceptibility genes, one of which is the melanocortin 1 receptor (MC1R) gene. Certain MC1R alleles are strongly associated with melanoma. We hereby present experimental evidence for the role of two melanoma risk factors, constitutive pigmentation, as assessed by total melanin, eumelanin and pheomelanin contents, and MC1R genotype and function, in determining the induction and repair of DNA photoproducts in cultured human melanocytes after irradiation with increasing doses of UVR. We found that total melanin and eumelanin contents (MC and EC) correlated inversely with the extent of UVR‐induced growth arrest, apoptosis and induction of cyclobutane pyrimidine dimers (CPD), but not with hydrogen peroxide release in melanocytes expressing functional MC1R. In comparison, melanocytes with loss‐of‐function MC1R, regardless of their MC or EC, sustained more UVR‐induced apoptosis and CPD, and exhibited reduced CPD repair. Therefore, MC, mainly EC, and MC1R function are independent determinants of UVR‐induced DNA damage in melanocytes.     

Ultraviolet radiation directly induces pigment production by cultured human melanocytes

In humans the major stimulus for cutaneous pigmentation is ultraviolet radiation (UVR). Little is known about the mechanism underlying this response, in part because of the complexity of interactions in whole epidermis. Using a recently developed culture system, human melanocytes were exposed daily to a physiologic range of UVR doses from a solar simulator. Responses were determined 24 hours after the last exposure. There was a dose-related increase in melanin content per cell and uptake of 14C-DOPA, accompanied by growth inhibition. Cells from donors of different racial origin gave proportionately similar increases in melanin, although there were approximately tenfold differences in basal values. Light and electron microscopy revealed UVR-stimulated increases in dendricity as well as melanosome number and degree of melanization, analogous to the well-recognized melanocyte changes following sun exposure of intact skin. Similar responses were seen with Cloudman S91 melanoma cells, although this murine cell line required lower UVR dosages and fewer exposures for maximal stimulation. These data establish that UVR is capable of directly stimulating melanogenesis. Because cyclic AMP elevation has been associated in some settings with increased pigment production by cultured melanocytes, preliminary experiments were conducted to see if the effects of UVR were mediated by cAMP. Both alpha-MSH and isobutylmethylxanthine (IBMX), as positive controls, caused a fourfold increase in cAMP level in human melanocytes and/or S91 cells, but following a dose of UVR sufficient to stimulate pigment production there was no change in cAMP level up to 4 hours after exposure. Thus it appears that the UVR-induced melanogenesis is mediated by cAMP-independent mechanisms.     

Melanin content and MC1R function independently affect UVR-induced DNA damage in cultured human melanocytes

Malignant transformation of melanocytes leads to melanoma, the most fatal form of skin cancer. Ultraviolet radiation (UVR)-induced DNA photoproducts play an important role in melanomagenesis. Cutaneous melanin content represents a major photoprotective mechanism against UVR-induced DNA damage, and generally correlates inversely with the risk of skin cancer, including melanoma. Melanoma risk is also determined by susceptibility genes, one of which is the melanocortin 1 receptor (MC1R) gene. Certain MC1R alleles are strongly associated with melanoma. We hereby present experimental evidence for the role of two melanoma risk factors, constitutive pigmentation, as assessed by total melanin, eumelanin and pheomelanin contents, and MC1R genotype and function, in determining the induction and repair of DNA photoproducts in cultured human melanocytes after irradiation with increasing doses of UVR. We found that total melanin and eumelanin contents (MC and EC) correlated inversely with the extent of UVR-induced growth arrest, apoptosis and induction of cyclobutane pyrimidine dimers (CPD), but not with hydrogen peroxide release in melanocytes expressing functional MC1R. In comparison, melanocytes with loss-of-function MC1R, regardless of their MC or EC, sustained more UVR-induced apoptosis and CPD, and exhibited reduced CPD repair. Therefore, MC, mainly EC, and MC1R function are independent determinants of UVR-induced DNA damage in melanocytes.     

Premature senescence in human melanocytes after exposure to solar UVR: An exosome and UV‐miRNA connection

Ultraviolet radiation (UVR) can play two roles: induce cellular senescence and convert skin melanocytes into melanoma. To assess whether this conversion might rely on melanocytes having to first acquire a senescent phenotype, we studied the effects of physiological doses of UVR (UVA + UVB) on quiescent melanocytes in vitro. Repeated doses of UVR induced these melanocytes into a senescent‐like state. Additionally, these cells secrete exosomes with specific miRNAs that differ in quantity from those of the un‐irradiated melanocytes. Many of the exosomal miRNAs that were differentially enriched regulated genes comprising a “senescence core signature” and encoding factors of the senescence‐messaging secretome (SASP), while a subset of the differentially reduced miRNAs targeted DNA repair genes that have been experimentally shown to be repressed in senescent melanocytes. Thus, the selection of specific miRNAs by exosomes and their release from melanocytes after exposure to UVR have activities in inducing these cells into premature senescence.     

Regulation of the human melanocortin 1 receptor expression in epidermal melanocytes by paracrine and endocrine factors and by ultraviolet radiation

The aim of this study is to investigate the regulation of the human melanocortin 1 receptor (MC1R) expression in cultured normal human melanocytes (NHM) by specific paracrine and endocrine factors, and by ultraviolet radiation (UVR). Treatment of NHM with alpha-melanotropin [alpha-melanocyte stimulating hormone (alpha-MSH)] increased MC1R mRNA level; the response was often more pronounced in NHM with a low (NHM-c) than in NHM with a high melanin content (NHM-b). Endothelin-1 increased MC1R mRNA level in NHM regardless of their melanin content. Basic fibroblast growth factor consistently up regulated MC1R mRNA level in NHM-b but not in NHM-c. Activation of protein kinase C by 12-0-tetradecanoylphorbol-13-acetate slightly increased, while stimulation of adenylate cyclase by forskolin markedly up-regulated the MC1R mRNA level. beta-Estradiol increased, and combined treatment with beta-estradiol and alpha-MSH further elevated, MC1R mRNA level in NHM-c and NHM-b. Testosterone reduced, while progesterone had no effect on, MC1R mRNA level. Agouti signaling protein reduced, and UVR down regulated dose-dependently MC1R mRNA level in NHM-b and NHM-c. This effect was reversed 24 h after irradiation with the lower doses of 7 or 14 mJ/cm2, but not after exposure to a higher, more cytotoxic dose of UVR. We conclude that the MC1R is regulated by paracrine factors, including its own ligands, by specific endocrine sex hormones, and by UVR. Differences in the responses of NHM to some of these factors suggest differential regulation of MC1R gene expression, which may contribute to the variation in constitutive and UV-induced cutaneous pigmentation in humans.     

MC1R and the response of melanocytes to ultraviolet radiation

The constitutive color of our skin plays a dramatic role in our photoprotection from solar ultraviolet radiation (UVR) that reaches the Earth and in minimizing DNA damage that gives rise to skin cancer. More than 120 genes have been identified and shown to regulate pigmentation, one of the key genes being melanocortin 1 receptor (MC1R) that encodes the melanocortin 1 receptor (MC1R), a seven-transmembrane G protein-coupled receptor expressed on the surface of melanocytes. Modulation of MC1R function regulates melanin synthesis by melanocytes qualitatively and quantitatively. The MC1R is regulated by the physiological agonists alpha-melanocyte-stimulating hormone (alphaMSH) and adrenocorticotropic hormone (ACTH), and antagonist agouti signaling protein (ASP). Activation of the MC1R by binding of an agonist stimulates the synthesis of eumelanin primarily via activation of adenylate cyclase. The significance of cutaneous pigmentation lies in the photoprotective effect of melanin, particularly eumelanin, against sun-induced carcinogenesis. Epidermal melanocytes and keratinocytes respond to UVR by increasing their expression of alphaMSH and ACTH, which up-regulate the expression of MC1R, and consequently enhance the response of melanocytes to melanocortins. Constitutive skin pigmentation dramatically affects the incidence of skin cancer. The pigmentary phenotype characterized by red hair, fair complexion, inability to tan and tendency to freckle is an independent risk factor for all skin cancers, including melanoma. The MC1R gene is highly polymorphic in human populations, and allelic variation at this locus accounts, to a large extent, for the variation in pigmentary phenotypes and skin phototypes (SPT) in humans. Several allelic variants of the MC1R gene are associated with the red hair and fair skin (RHC) phenotype, and carrying one of these variants is thought to diminish the ability of the epidermis to respond to DNA damage elicited by UVR. The MC1R gene is considered a melanoma susceptibility gene, and its significance in determining the risk for skin cancer is of tremendous interest.     

MC1R,the cAMP pathway,and the response to solar UV: extending the horizon beyond pigmentation

The melanocortin 1 receptor (MC1R) is a G protein‐coupled receptor crucial for the regulation of melanocyte proliferation and function. Upon binding melanocortins, MC1R activates several signaling cascades, notably the cAMP pathway leading to synthesis of photoprotective eumelanin. Polymorphisms in the MC1R gene are a major source of normal variation of human hair color and skin pigmentation, response to ultraviolet radiation (UVR), and skin cancer susceptibility. The identification of a surprisingly high number of MC1R natural variants strongly associated with pigmentary phenotypes and increased skin cancer risk has prompted research on the functional properties of the wild‐type receptor and frequent mutant alleles. We summarize current knowledge on MC1R structural and functional properties, as well as on its intracellular trafficking and signaling. We also review the current knowledge about the function of MC1R as a skin cancer, particularly melanoma, susceptibility gene and how it modulates the response of melanocytes to UVR.     

Effect of antioxidant supplementation on the adaptive response of human skin fibroblasts to UV-induced oxidative stress

Abstract

The effect of supplementation with substances having antioxidant properties on the adaptive responses of human skin fibroblasts to UV-induced oxidative stress was studied in vitro. UVR was found to induce a substantial oxidative stress in fibroblasts, resulting in an increased release of superoxide anions and an increase in lipid peroxidation (shown by an elevated malonaldehyde content). Sub-lethal doses of UVR were also found to induce adaptive responses in the fibroblast antioxidant defences, with a transient rise in catalase and superoxide dismutase activities followed by a slower, large increase in cellular glutathione content. Supplementation of the fibroblasts with the antioxidants, Trolox (a water soluble analogue of α-tocopherol), ascorbic acid or β-carotene, had differential effects on these responses. Trolox supplementation reduced the UVR-induced cellular oxidative stress and adaptive response in a predictable concentration-dependant manner. This was in contrast to ascorbic acid which increased superoxide release from fibroblasts. At low doses, ascorbate supplements also reduced the magnitude of the adaptive increases in catalase and superoxide dismutase activities and increase in glutathione content. β-Carotene had a similar effect to ascorbic acid, reducing the extent of the adaptations to UVR at lower doses while simultaneously increasing superoxide release and malonaldehyde content. These in vitro data indicate that only the vitamin E analogue suppressed UVR-induced oxidative stress in a predictable manner and suggest that common dietary antioxidants may not be equally effective in reducing the potential deleterious effects of UVR-induced oxidative stress in skin.     

The impact of skin colour on human photobiological responses

Terrestrial solar ultraviolet radiation (UVR) exerts both beneficial and adverse effects on human skin. Epidemiological studies show a lower incidence of skin cancer in people with pigmented skins compared to fair skins. This is attributed to photoprotection by epidermal melanin, as is the poorer vitamin D status of those with darker skins. We summarize a wide range of photobiological responses across different skin colours including DNA damage and immunosuppression. Some studies show the generally modest photoprotective properties of melanin, but others show little or no effect. DNA photodamage initiates non‐melanoma skin cancer and is reduced by a factor of about 3 in pigmented skin compared with white skin. This suggests that if such a modest reduction in DNA damage can result in the significantly lower skin cancer incidence in black skin, the use of sunscreen protection might be extremely beneficial for susceptible population. Many contradictory results may be explained by protocol differences, including differences in UVR spectra and exposure protocols. We recommend that skin type comparisons be done with solar‐simulated radiation and standard erythema doses or physical doses (J/m²) rather than those based solely on clinical endpoints such as minimal erythema dose (MED).     

Kaempferol promotes melanogenesis and reduces oxidative stress in PIG1 normal human skin melanocytes

Vitiligo is an autoimmune disease characterized by depigmentation. Kaempferol is a flavonoid compound with broad anti-inflammatory and antioxidant properties. The purpose of this study was to investigate the effect of kaempferol on melanogenesis in PIG1 normal human skin melanocytes and its response to oxidative stress. The effect of kaempferol on melanin synthesis in PIG1 normal human skin melanocytes was explored by measuring tyrosinase activity, melanin content, mRNA and protein expression of key enzymes and expression of related pathway proteins. The effects of kaempferol pretreatment on cell viability, apoptosis, ROS level and HO-1 protein level under H₂O₂ stimulation were explored. When treated with kaempferol, the tyrosinase activity and melanin content of PIG1 cells increased, the mRNA and protein expressions of TYR, TRP1, TRP2 and MITF increased, and the phosphorylation level of ERK1/2 increased. Upon the stimulation of H₂O₂, kaempferol reduced the production of ROS, decreased apoptosis and increased the protein expression of HO-1 in PIG1 cells. In addition, kaempferol inhibited oxidative stress-induced melanin reduction and promoted melanin synthesis in PIG1 cells and protected against H₂O₂-induced oxidative stress damage.     

Ultraviolet stimulated melanogenesis by human melanocytes is augmented by di-acyl glycerol but not TPA

Epidermal melanocytes (MC) synthesise melanin in response to ultraviolet radiation (UVR). The mechanisms mediating the UV-induced activation of melano-genesis are unknown but since UVR induces turnover of membrane phospholipids generating prostaglandins (PGs) and other products, it is possible that one of these might provide the activating signal. We have examined the effects of prostaglandins (PGs) E₁, E₂, D₂, F_2α, and di-acyl glycerol upon the UV-induced responses of cultured human MC and the Cloudman S91 melanoma cell line. The PGs had little effect on unirradliated cells and did not alter the response to UVR in either human MC or S91 melanoma cells. However, a synthetic analogue of di-acyl glycerol, 1-oleyl 2-acetyl glycerol (OAG), caused a significant (P<0.0001), dose-related augmentation of melanin content both in human MC (seven-fold) and S91 cells (three-fold). UVR caused a significant augmentation of the OAG-induced melanognesis of both human MC and S91 cells. Since OAG is known to activate protein kinase C, it was possible that the observed modulation of the UVR signal could be via that pathway. Di-octanoyl glycerol, another di-acyl glycerol, which activates kinase C, caused a small (70%) increase in melanogenesis in MC which was not altered by UVR. However, 12-0 tetradecanoyl phorbol 13-acetate (TPA), a potent activator of protein kinase C, had no significant effect on either basal or UV-induced melanin synthesis in either cell type. These data suggest that the UV-induced signal activating melanogenesis could be mediated by di-acyl glycerol. Furthermore, they imply that the signal is transduced via an alternative, pathway that might be independent of protein kinase C.     

Wnt inhibitory factor (WIF)‐1 promotes melanogenesis in normal human melanocytes

Wnt signaling plays a role in the differentiation as well as the development of melanocytes. Using a microarray analysis, hyperpigmentary skin of melasma expressed high levels of Wnt inhibitory factor‐1 (WIF‐1) compared with perilesional normal skin. In this study, the expression and functional roles of WIF‐1 on melanocytes were investigated. WIF‐1 was expressed both in the melanocytes of normal human skin and in cultured melanocytes. The upregulation of WIF‐1 on cultured normal human melanocytes significantly induced expressions of MITF and tyrosinase, which were associated with increased melanin content and tyrosinase activity. Consistent with the stimulatory effect of WIF‐1, WIF‐1 siRNA reduced melanogenesis in the cells. Moreover, WIF‐1 increases pigmentation in melanocytes co‐cultured with WIF‐1‐overexpressed fibroblasts and of organ‐cultured human skin. These findings suggest that melanocytes express WIF‐1 constitutively in vivo and in vitro and that WIF‐1 promotes melanogenesis in normal human melanocytes.     

Paclitaxel-induced increase in mitochondrial volume mediates dysregulation of intracellular Ca2+ in putative nociceptive glabrous skin neurons from the rat

We have recently demonstrated that in a rat model of chemotherapy-induced peripheral neuropathy (CIPN), there is a significant decrease in the duration of the depolarization-evoked Ca²⁺ transient in small diameter, IB4+, and capsaicin-responsive neurons innervating the glabrous skin of the hindpaw. This change was specific to the transient duration and significantly smaller if not undetectable in neurons innervating the dorsal skin of the hindpaw or the skin of the inner thigh. Given the importance of mitochondria in intracellular Ca²⁺ regulation and the findings of chemotherapy-associated increase in mitotoxicity along the sensory neuron axons, we hypothesized that CIPN is due to both increases and decreases in mitochondria function, with changes manifest in distinct subpopulations of afferents. To begin to test this hypothesis, we used confocal microscopy and Ca²⁺ imaging in combination with pharmacological manipulations to study paclitaxel-induced changes in retrograde tracer-labeled neurons from naïve, vehicle-treated, and paclitaxel-treated rats. Paclitaxel treatment was not associated with decreased mitochondrial membrane potential or increased superoxide levels in the somata of putative nociceptive glabrous skin neurons. However, it was associated with significant increases in the relative contribution of mitochondria to the control of the evoked Ca²⁺ transient duration in putative nociceptive glabrous skin neurons, as well as increases in mitotracker and Tom20 staining which reflected an increase in mitochondrial volume. Furthermore, the relative contribution of the sarco-endoplasmic reticulum Ca²⁺ ATPase to the regulation of the duration of the depolarization evoked Ca²⁺ transient was also increased in this subpopulation of neurons from paclitaxel treated rats. Our results indicate that the paclitaxel-induced decrease in the duration of the evoked Ca²⁺ transient is due to both direct and indirect influences of mitochondria. It remains to be determined if and how these changes contribute to the manifestation of CIPN.     

Molecular Cascades in UV Induced Melanogenesis: A Central Role for Melanotropins?

When human skin is exposed to ultraviolet (UV) light, a highly complex cascade of events ensues that culminates, among other things, in increased skin melanin content. From analyses at the tissue and cellular level, it has been shown that following exposure to UV light there is an increase in the number of active melanocytes in the basal layer of the epidermis, and individual melanocytes are stimulated to produce more melanin. In addition, the rate of transfer of melanosomes from melanocytes to keratinocytes is apparently increased, although the role of UV light in this process remains to be demonstrated. Recent biochemical evidence is reviewed on factors that regulate these processes. A plausible explanation for the effects of UV on pigmentation is that there are mechanisms in the skin for the orderly, regulated reception of UV signals that are then transduced to initiate the cascade. The signals involve both melanocytes and keratinocytes, and avail-able evidence supports a model in which melanotropins and their receptors play a central role in the process.     

Quantification of Ca2+ binding to melanin supports the hypothesis that melanosomes serve a functional role in regulating calcium homeostasis

Calcium regulation in melanocytes affects numerous biological pathways including protecting the redox balance in the cell and regulating the supply of substrate, l ‐tyrosine, for melanogenesis. The pigment contained in the melanocytes, melanin, has been implicated in maintaining calcium homeostasis in the cell and is known to be involved with calcium ion regulation in the inner ear. Herein, the association constant for Ca²⁺ binding to Sepia melanin is determined by isothermal titration calorimetry to be 3.3 (±0.2) × 10³/M. This value is comparable with other well‐established intracellular calcium‐binding proteins that serve to buffer calcium concentrations, lending further support to the hypothesis that melanosomes serve as intracellular mediators of calcium homeostasis in melanocytes. Using this binding constant and the data from a fluorescent Ca²⁺ displacement assay, the pK_a of the carboxyl group coordinated to Ca²⁺ is determined to be 3.1 ± 0.1.     

mTOR-Controlled Autophagy Requires Intracellular Ca2+ Signaling

Autophagy is a lysosomal degradation pathway important for cellular homeostasis and survival. Inhibition of the mammalian target of rapamycin (mTOR) is the best known trigger for autophagy stimulation. In addition, intracellular Ca²⁺ regulates autophagy, but its exact role remains ambiguous. Here, we report that the mTOR inhibitor rapamycin, while enhancing autophagy, also remodeled the intracellular Ca²⁺-signaling machinery. These alterations include a) an increase in the endoplasmic-reticulum (ER) Ca²⁺-store content, b) a decrease in the ER Ca²⁺-leak rate, and c) an increased Ca²⁺ release through the inositol 1,4,5-trisphosphate receptors (IP₃Rs), the main ER-resident Ca²⁺-release channels. Importantly, buffering cytosolic Ca²⁺ with BAPTA impeded rapamycin-induced autophagy. These results reveal intracellular Ca²⁺ signaling as a crucial component in the canonical mTOR-dependent autophagy pathway.     

Nitric oxide enhances the sensitivity of alpaca melanocytes to respond to α-melanocyte-stimulating hormone by up-regulating melanocortin-1 receptor

Nitric oxide (NO) and α-melanocyte-stimulating hormone (α-MSH) have been correlated with the synthesis of melanin. The NO-dependent signaling of cellular response to activate the hypothalamopituitary proopiomelanocortin system, thereby enhances the hypophysial secretion of α-MSH to stimulate α-MSH-receptor responsive cells. In this study we investigated whether an NO-induced pathway can enhance the ability of the melanocyte to respond to α-MSH on melanogenesis in alpaca skin melanocytes in vitro. It is important for us to know how to enhance the coat color of alpaca. We set up three groups for experiments using the third passage number of alpaca melanocytes: the control cultures were allowed a total of 5 days growth; the UV group cultures like the control group but the melanocytes were then irradiated everyday (once) with 312 mJ/cm² of UVB; the UV + L-NAME group is the same as group UV but has the addition of 300 μM L-NAME (every 6 h). To determine the inhibited effect of NO produce, NO produces were measured. To determine the effect of the NO to the key protein and gene of α-MSH pathway on melanogenesis, the key gene and protein of the α-MSH pathway were measured by quantitative real-time PCR and Western immunoblotting. The results provide exciting new evidence that NO can enhance α-MSH pathway in alpaca skin melanocytes by elevated MC1R. And we suggest that the NO pathway may more rapidly cause the synthesis of melanin in alpaca skin under UV, which at that time elevates the expression of MC1R and stimulates the keratinocytes to secrete α-MSH to enhance the α-MSH pathway on melanogenesis. This process will be of considerable interest in future studies.