October 2006
Volume 47, Issue 10
Free
Physiology and Pharmacology  |   October 2006
Uveal Melanocytes Do Not Respond To or Express Receptors for α-Melanocyte-Stimulating Hormone
Author Affiliations
  • Li Li
    From the Department of Dermatology, University of Cincinnati College of Medicine, Cincinnati, Ohio; and the
  • Dan-Ning Hu
    Tissue Culture Center, New York Eye and Ear Infirmary, New York, New York.
  • Huiquan Zhao
    From the Department of Dermatology, University of Cincinnati College of Medicine, Cincinnati, Ohio; and the
  • Steven A. McCormick
    Tissue Culture Center, New York Eye and Ear Infirmary, New York, New York.
  • James J. Nordlund
    From the Department of Dermatology, University of Cincinnati College of Medicine, Cincinnati, Ohio; and the
  • Raymond E. Boissy
    From the Department of Dermatology, University of Cincinnati College of Medicine, Cincinnati, Ohio; and the
Investigative Ophthalmology & Visual Science October 2006, Vol.47, 4507-4512. doi:10.1167/iovs.06-0391
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      Li Li, Dan-Ning Hu, Huiquan Zhao, Steven A. McCormick, James J. Nordlund, Raymond E. Boissy; Uveal Melanocytes Do Not Respond To or Express Receptors for α-Melanocyte-Stimulating Hormone. Invest. Ophthalmol. Vis. Sci. 2006;47(10):4507-4512. doi: 10.1167/iovs.06-0391.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Whereas cutaneous pigmentation increases after exposure to ultraviolet (UV) irradiation, ocular pigmentation does not. This study was designed to examine the evidence that α-melanocyte-stimulating hormone (α-MSH), which is thought to be the mediator of UV response in the skin, has any role to play in uveal melanocytes.

methods. Human uveal melanocytes derived from the choroid and the iris were cultivated by using eyes harvested from adult cadaveric donors and were assessed by Northern blot analysis for growth and melanogenic response to α-MSH and expression of the receptor for α-MSH (MC1-R). In addition, expression of α-MSH was evaluated in ocular tissue by immunocytochemistry.

results. Uveal melanocytes, unlike cutaneous melanocytes in vitro, exhibited no stimulation of proliferation in response to α-MSH at dosages ranging from 0.1 to 100 μM. In addition, tyrosine hydroxylase, DOPA oxidase, and protein levels for tyrosinase, TRP-1, and TRP-2 were not influenced by α-MSH. Associated with the lack of α-MSH response in cultured uveal melanocytes was the absence of expression of the receptor for α-MSH (MC1-R), as assessed by Northern blot analysis. Also in contrast to the skin, pigmented ocular tissue lacked expression of the α-MSH ligand, as assessed by immunocytochemistry.

conclusions. In conclusion, ocular pigmentation does not appear to be regulated by melanocyte stimulating hormone.

In both cutaneous and uveal melanocytes, the variation in coloration is primarily determined by the type and amount of melanin produced. 1 2 3 However, uveal melanocytes differ from epidermal melanocytes in certain aspects. Epidermal melanocytes respond to UV radiation by darkening the skin color, whereas iris color does not darken after exposure to UV radiation. The methodologies for cultivation and in vitro study of epidermal and uveal melanocytes have been well established. Therefore, it is possible to compare the modulation of growth and melanogenesis of cultured uveal melanocytes to that of epidermal melanocytes. 
It has been reported that both mouse and human epidermal melanocytes in culture respond to α-melanocyte-stimulating hormone (α -MSH) with increased proliferation and melanogenesis. 4 5 Response by epidermal melanocytes to α-MSH is documented to be attributable to the expression of a specific surface receptor, melanocortin 1 receptor (MC1R). 6 7 8 9 10 11 On the basis of the mitogenic effect of α-MSH on human epidermal melanocytes, development of melanocyte growth medium used α-MSH as a specific mitogen. 12  
In the skin, α-MSH is synthesized mainly by epidermal keratinocytes, especially in response to ultraviolet light exposure, to regulate, via MC1R, the melanin content of the melanocytes. Epidermal melanocytes and keratinocytes respond to UV radiation by increasing their expression of α-MSH, which upregulates the expression of MC1R and consequently enhances the response to α-MSH. 13 14 The gene encoding MC1R is one of the key genes that regulate human skin pigmentation and is the only gene known to affect variance of skin and hair pigmentation within the normal human population. 15  
Very little was known concerning MC1R in human uveal melanocytes and the effect of α-MSH on the growth and melanogenesis of human uveal melanocytes. 16 17 18 The purpose of this study was to investigate the effect of α-MSH on growth and melanin content in 10 cell lines of uveal melanocytes from human donor eyes with various iris colors and to test the effect of α-MSH on the activity and expression of tyrosinase, tyrosinase-related protein (TRP)-1 and -2 in cultured uveal melanocytes. The expression of MC1R in uveal melanocytes was studied by Northern blot analysis. Epidermal melanocytes and fibroblasts were used as positive and negative controls, respectively. The presence of α-MSH in the iris and skin in vivo was determined by immunohistochemical studies. 
Methods
Cell Culture
Human uveal melanocytes derived from the choroid and iris were established in culture, as previously described. 19 Briefly, human donor eyes were obtained from the Cincinnati Eye Bank or New York Eye Bank for Sight Restoration Eye Bank, in accordance with the guidelines of the Declaration of Helsinki. The eyes were opened at the pars plana and separated into anterior and posterior segments. The vitreous and the retina of the posterior portion were excised. The retinal pigment epithelium (RPE) was separated from the choroid after treatment with trypsin-EDTA solution (Invitrogen-Gibco, Carlsbad, CA). The choroid was then separated from the sclera. The iris from the anterior portion was excised, and the iris pigment epithelium was separated after treatment with trypsin (Invitrogen-Gibco), leaving the iris stroma which contains predominantly the melanocytes. The iris stroma and the choroid were treated with trypsin solution, refrigerated overnight, and then incubated for 1 hour at 37°C. The cells released were collected. The remaining tissue was treated with collagenase (Sigma-Aldrich, St. Louis, MO) for 1 hour, and the treatment was repeated two to three times to release all the melanocytes. 
Cell suspensions of uveal melanocytes were collected and established as primary cultures by using FIC medium (Ham’s F12; Invitrogen-Gibco) containing 25 ng/mL basic fibroblast growth factor (bFGF; PeproTech, Rocky Hills, NJ), 0.1 mM isobutyl methylxanthine (IBMX), 10 ng/mL cholera toxin (Sigma-Aldrich), and 10% fetal bovine serum (FBS; Invitrogen-Gibco) or TISH medium (F12 medium containing 0.1 μM 12-O-tetradecanoyl phorbol acetate [TPA]; Sigma-Aldrich), 0.1 mM IBMX and 15% FBS]. Geneticin (Invitrogen-Gibco), a cytotoxic agent, was added at a concentration of 100 μg/mL for 3 days when necessary, to eliminate contaminating RPE cells and fibroblasts. After reaching confluence, the uveal melanocytes were detached using trypsin-EDTA solution, diluted 1:3 to 1:6, and subcultured. 19  
Human ocular fibroblasts were established in culture by taking a portion of the cell suspension from the iridal tissue and developing primary cultures using Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen-Gibco), containing 6% FBS. 
Human epidermal melanocytes were established in culture as previously described. 20 In short, neonatal foreskins were treated with 0.25% trypsin for 2 hours at 37°C. The tissue was vortexed for 30 seconds. Epidermal cell suspensions were collected and established as primary cultures using MCDB-153 medium containing 0.6 ng/mL bFGF, 8 nM TPA, 5 μg/mL insulin, 5 μg/mL transferrin, 1 μg/mL α-tocopherol, 30 μg/mL crude pituitary extract, 0.5 μg/mL hydrocortisone (all from Clonetics Corp., San Diego, CA), and 5% heat-inactivated FBS (Sigma-Aldrich). 
Human keratinocytes were established in culture, as previously described. 21 In short, an epidermal cell suspension was cultured in M154 basal medium (Cascade Biologicals, Portland, OR) supplemented with human keratinocyte growth supplements (Cascade Biologicals). 
Human cutaneous fibroblasts were established in culture by taking the dermis after trypsinization of the foreskins and developing primary cultures with DMEM containing 6% FBS. 
Effect of α-MSH on the Growth and Melanin Content of Uveal Melanocytes
Ten uveal melanocytes cell lines isolated from five donors with different iris colors (blue, green, hazel, brown, and dark brown) were used in the study (five from the iris and five from the choroid). These cells had been in culture no longer than 1 month and had been passaged three to four times at a dilution of 1:3 to 1:4. The purity of the cell lines was demonstrated by immunocytochemical methods. Uveal melanocytes display S-100 antigen but not cytokeratin, whereas pigment epithelial cells display both proteins, and fibroblasts display neither of these proteins. 19  
The uveal melanocytes were plated into 12-well plates (Corning Costar, Corning, NY) with FIC or TISH medium at a density of 5 × 104 cells per well. These media are referred to as the positive control. After 24 hours, the FIC or TISH medium was replaced with 1.0 mL of the test medium, which was cAMP-deleted medium (FIC medium without the c-AMP elevating agents IBMX and cholera toxin, or TISH medium without IBMX). α-MSH (Sigma-Aldrich) at various concentrations (0.01–10 μM) were added to test their effects. The cAMP-deleted media are referred to as the negative control. Each concentration was tested in triplicate in each cell line. The cAMP-deleted medium without α-MSH was used in three wells as the negative control. Cells cultured with FIC or TISH medium were used as the positive control. Human epidermal melanocytes cultured with FIC or TISH medium were tested as mentioned earlier, for comparison. The media were replaced every 3 days. After 6 days, the cells were detached with trypsin-EDTA solution for cell counting and melanin measurement. Cells were counted in a hemocytometer. Cell suspensions were centrifuged, and the pellet was dissolved in 1 N NaOH. Melanin concentration was determined by measurement of optical density at 475 nm and compared with a standard curve determined using synthetic melanin. Melanin content was expressed as micrograms per culture. 22 Student’s t-test was used to assess statistical significance. 
Effects of α-MSH on the Activity of Tyrosinase in Uveal Melanocytes
To investigate the influence of α-MSH on tyrosinase activity, 6 × 105 uveal or epidermal melanocytes were seeded, in triplicate, into 100-mm culture dishes and subcultured in their respective growth medium for 4 days with medium being renewed every other day. IBMX and BPE were then removed from the uveal and epidermal melanocyte growth medium, respectively, and the cells were grown for three additional days. After this, the depleted growth media containing various concentrations (0.1–100 μM) of α-MSH were applied to the cells and renewed every other day. After 7 days’ exposure of melanocytes to α-MSH treatment, the cells were harvested and assayed for cell number, tyrosine hydroxylase activity, DOPA oxidase activity, and melanogenic proteins. 
Tyrosine hydroxylase activity was determined in cells treated with α-MSH for 7 days. Cells were harvested, counted, and the tyrosinase activity within NP-40 cell lysates was determined by using a modification of the charcoal absorption method of Pomerantz as described in Zhao et al. 23 24 Tyrosine hydroxylase activity was expressed as disintegrations per minute (DPM) per cell per time. 
DOPA oxidase activity of uveal melanocytes was assayed using the SDS/PAGE/DOPA staining procedure previously described. 25 Cell lysates were centrifuged (10,000g) at 4°C for 10 minutes, and the resultant supernatants were used as the sample. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) at a 10% concentration. After electrophoresis at 50 mA for approximately 100 minutes, the gels were rinsed with a few changes of phosphate buffer (pH 6.8) and stained with 0.2% l-DOPA until bands appeared. The gels were then photographed. 
Effect of α-MSH on the Expression of Tyrosinase, TPR-1, and TRP-2 by Immunoblot Analysis
Cell proteins from cultured uveal melanocytes were solubilized on 0.5% Nonidet P40 (NP40) and nonreduced sample buffer, and equal amounts of protein per lane was separated by SDS-PAGE. The nonreduced sample buffer consisted of 62.5 mM of Tris/hydrochloric acid (pH 6.8), 1% SDS, and 10% glycerol. After electrophoresis, the proteins were transferred electrophoretically onto nitrocellulose membranes (Trans-Blot Transfer Medium; BioRad, Richmond, CA). Nonspecific sites were blocked by incubation in 5% (wt/vol) nonfat dry milk in 50 mM Tris/hydrochloric acid [pH 7.5] for 1 hour. The primary antibodies used at a dilution of 1:2000 with an overnight incubation included αTy-SP for tyrosinase (Seymore Pomerantz, University of Maryland, Baltimore, MD), TA99 for TRP-1 (Vijayasaradhi Setaluri, University of Wisconsin, Madison, WI), and α-PEP8 for TRP-2 (Vincent Hearing, NIH, Bethesda, MD). After extensive washing, alkaline phosphatase–conjugated secondary antibody (diluted 1:1000) was incubated with membrane for 2 hours. After extensive washing with Tris buffer, the membrane was developed with BCIP/NBT (5-bromo-4-chloro-3-indoyl phosphate–nitroblue tetrazolium) substrate solution (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). Prestained, low-range, molecular weight markers (BioRad) were run as molecular weight standards throughout the entire experiment. 24  
Northern Blot Analysis for the Expression of MC1R and Tyrosinase
Total RNA was isolated from pure, established cultures of uveal and epidermal melanocytes, ocular and cutaneous fibroblasts, and cutaneous keratinocytes using the technique of Chomczynski and Saachi. 26 The RNA was separated on 1.2% agarose-ethidium bromide formaldehyde gels in 1 × 3-[N-morpholino] propanesulfonic acid. After separation, the RNA was transferred to nylon membranes and stored at −85°C. Full-length cDNA fragments of human MCR-1 (a gift from Zalfa Abdel-Malek, University of Cincinnati, OH) and the human tyrosinase were labeled by random priming with [32P]ATP by aDNA labeling system (Megaprime; GE Healthcare, Arlington Heights, IL). The membranes were prehybridized for 3 to 10 hours in the hybridization buffer consisting of 10% dextran sulfate sodium salt, 40% formamide, 5× SSC, 5× Denhardt’s solution, 100 μg/mL DNA sodium salt-type XIV, herring testes, and 0.1% SDS. The entire probe solution was added directly to the hybridization solution and incubated for 12 to 14 hours at 42°C. The membranes were rinsed twice with 2× SSC buffer at 42°C followed by several rinses with 0.5× SSC at 55°C. Several rinses with 0.1 SSC buffer at 55°C were included when necessary to reduce background. Images were obtained with autoradiographic film. 
Immunohistochemistry for α-MSH in Skin and Iris Tissues
Transverse sections, 6 μm in thickness, were cut from snap-frozen skin and iridal biopsy specimens on a cryostat at −25°C, placed on poly-l-lysine–coated glass microslides, and stored at −70°C before use. Frozen sections were allowed to warm to room temperature for 30 minutes. Three washes with PBS were performed between each staining step. All incubations were performed in a humidified chamber and at room temperature. The tissues on the slides were fixed with freshly made 4% paraformaldehyde in PBS solution for 5 minutes. Tissues were treated with three changes of 0.5 mg/mL sodium borohydride in PBS. The tissues were overlaid with blocking solution (10% vol/vol FBS, 1% wt/vol BSA in PBS) for 45 minutes and then incubated with or without anti-human α-MSH rabbit serum (Accurate Chemical, Westbury, NY) at a dilution of 1:400 overnight at 4°C and rinsed three times with PBS. Fluorochrome-conjugated secondary antibody was layered on the tissue for 45 minutes. Tissue sections were rinsed, mounted, and observed by using a fluorescence microscope (Dialux 20; Leitz, Wetzlar, Germany) with an appropriate filter. 
Results
Effects of α-MSH on Growth and Melanin Content of Cultured Uveal Melanocytes
Uveal melanocytes seeded in 12-well plates attached and spread well within 24 hours. Cells grew well in positive control media (FIC or TISH medium), showed multiple dendrites and demonstrated an increase in melanin content per culture time. Uveal melanocytes cultured with negative control medium (i.e., medium without c-AMP-elevating agents) grew much more slowly, with lower melanin content per culture. α-MSH did not show any stimulatory effects on growth or melanogenesis of uveal melanocytes. In all uveal melanocyte cell lines, the number of cells (Fig. 1)and the amount of melanin (Fig. 2)per culture of cells incubated with α-MSH at all concentrations showed no significant difference compared with negative controls (P > 0.05). 
The epidermal melanocytes cultured with cAMP-deleted medium without α-MSH also grew slowly and showed less pigmentation. α-MSH had a dose-dependent stimulative effect on cell growth (Fig. 1)and melanin content (Fig. 2) . In cells tested in cAMP-deleted FIC medium, the difference in number of cells between the negative control and α-MSH-treated cultures at all concentrations was statistically significant (P < 0.01). The difference in amount of melanin per well between the negative control and α-MSH-treated cultures was statistically significant (0.05 > P > 0.01, between the negative control and α-MSH at 0.1 μM and P < 0.01 between the negative control and α-MSH at 1.0–100 μM). 
Figures 1 and 2show the effects of α-MSH on growth (Fig. 1)and melanin content (Fig. 2)of iridal and choroidal melanocytes from an eye with green iris and epidermal melanocytes cultured in FIC medium. The results of tests of cells cultured with TISH medium or uveal melanocytes from eyes with blue, hazel, brown, and dark brown irides (in FIC medium) were the same as those of tests of cells from green eyes cultured with FIC medium (data not shown). 
Effects of α-MSH on the Expression and Activity of Tyrosinase and Related Proteins
α-MSH showed no effect on tyrosine hydroxylase activity in uveal melanocytes (Fig. 3) , whereas the epidermal melanocytes showed a significant and dose-dependent increase of tyrosine hydroxylase activity after stimulation by α-MSH. In addition, the DOPA oxidase activity of tyrosinase was not influenced in cultured uveal melanocytes exposed to α-MSH (Fig. 4) . Concomitantly, there was no increase in expression of the gene products of the tyrosinase gene family—that is, tyrosinase (Fig. 5) , TRP-1 (Fig. 5) , and TRP-2 (not shown) by uveal melanocytes cultured with α-MSH. 
Expression of the Receptor for α-MSH in Cultured Uveal Melanocytes
Expression of the receptor for α-MSH (MC1-R) in cultured uveal and epidermal melanocytes, ocular and cutaneous fibroblasts, and keratinocytes was assessed by Northern blot analysis (Fig. 6) . Uveal melanocytes, cutaneous fibroblasts and keratinocytes did not express MC1R. MC1R transcripts were expressed only in cultured epidermal melanocytes. In addition, both epidermal and uveal melanocytes cell lines expressed transcripts for tyrosinase. 
Expression of α-MSH in Ocular and Cutaneous Tissues
Expression of α-MSH hormone in ocular (iridal) and cutaneous tissues was assessed by immunocytochemistry (Fig. 7) . α-MSH was present within keratinocytes throughout the epidermis of human skin. In contrast, α-MSH was not present in the stroma or in the melanocytes of the iris. 
Discussion
The melanotropic hormone α-MSH has been studied for several decades and has become one of the best-described physiological regulators of human melanization. The darkening effects on the skin of human subjects injected with α-MSH were observed several decades ago. 27 Early reports on the influence of α-MSH on the function of human epidermal melanocytes were conflicting 28 —a consequence of the medium used for testing the effects of α-MSH on the melanocytes. By using a medium without cAMP-elevating agents, α-MSH definitely stimulates the proliferation and melanogenesis of human epidermal melanocytes. 10 With the demonstration that epidermal melanocytes respond to α-MSH (with increase of proliferation and melanogenesis), combined with the cloning and characterization of the human MC1R 29 and the demonstration of functional MC1R in human epidermal melanocytes, the long-standing controversy about the role of α-MSH in regulating human cutaneous pigmentation was finally ended. 4 8 9 10 11 α-MSH at physiologic concentrations is mitogenic as well as melanogenic for cultured murine melanocytes 5 as well as normal human melanocytes derived from different skin types, 4 9 indicating the biological importance of α-MSH in melanogenesis. 
The MC1R regulates cutaneous melanin synthesis by epidermal melanocytes qualitatively and quantitatively. The MC1R is modulated by the physiological agonists α-MSH and ACTH, and the antagonist agouti signaling protein. Activation of MC1R by binding of α-MSH stimulates the proliferation and melanogenesis by increasing cAMP formation. UV radiation stimulates the production of α-MSH and upregulates the MC1R; both effects result in an increase in cutaneous pigmentation. 14 30 31 Therefore, MC1R is a key regulator of human cutaneous pigmentation. 30  
MC1R gene is highly polymorphic in human populations, more than 65 human MC1R alleles have been identified, and current evidence suggests that many of them vary in their physiological activity. 15 32 33 Allelic variation at this locus accounts, to a large extent, for the variation of skin and hair color in normal human populations, and MC1R is the only gene known to account for substantial variation in human skin and hair color. 14 32 33  
In the present study, our findings indicate that regulation of melanin content, expression of melanogenic enzymes, and cell proliferation in 10 cell lines of cultured iridal or choroidal melanocytes from human donor eyes with various colors of iris did not respond to α-MSH treatment. Hedley et al. 34 reported that the response of cultured human epidermal melanocytes to MSH is dependent on the culture medium used for treating cells. α-MSH induced an increase of melanin content and DOPA oxidase activity in only one culture medium, where bFGF was the sole mitogen. In the present studies, uveal melanocytes cultured with FIC medium and tested with cAMP-deleted FIC medium (the tested medium contains only bFGF) or TISH medium (tested medium contains TPA and without cAMP-elevating agents) showed identical results: None of these cells responded to α-MSH, suggesting that there is no significant contribution of α-MSH on uveal growth and melanogenesis in vitro. Expression of tyrosinase and TRP-1 and -2 in uveal melanocytes did not increase after the addition of α-MSH. Tyrosine hydroxylase and DOPA oxidase activity in uveal melanocytes did not respond to α-MSH. This lack of responsiveness of uveal melanocytes to α-MSH was further defined by the observation that uveal melanocytes did not express a detectable amount of MC1 receptors and thus presented no apparent response capacity. The difference in response to α-MSH by uveal melanocytes and epidermal melanocytes may be one of the factors that determine the difference in in vivo behavior between uveal and epidermal melanocytes, including the absence of changes of iris color after exposure to solar radiation. 
 
Figure 1.
 
Effect of α-MSH on growth of iridal and choroidal melanocytes from eye with green iris and epidermal melanocytes from a white donor. Cultured iridal melanocytes (iris M), choroidal melanocytes (choroidal M), and epidermal melanocytes (epidermal M) were seeded into 12-well plates and cultured with cAMP-deleted medium (FIC medium without cAMP-elevating agents, IBMX, and cholera toxin) without or with α-MSH at various concentrations (0.1–100 μM). Cells cultured with cAMP-deleted medium alone and FIC medium were used as negative control (Negative) and positive (Positive) controls, respectively. Six days later, the cells were detached and counted. The number of cells was expressed as the percentages of the positive control (average of three wells in each group, mean ± SD).
Figure 1.
 
Effect of α-MSH on growth of iridal and choroidal melanocytes from eye with green iris and epidermal melanocytes from a white donor. Cultured iridal melanocytes (iris M), choroidal melanocytes (choroidal M), and epidermal melanocytes (epidermal M) were seeded into 12-well plates and cultured with cAMP-deleted medium (FIC medium without cAMP-elevating agents, IBMX, and cholera toxin) without or with α-MSH at various concentrations (0.1–100 μM). Cells cultured with cAMP-deleted medium alone and FIC medium were used as negative control (Negative) and positive (Positive) controls, respectively. Six days later, the cells were detached and counted. The number of cells was expressed as the percentages of the positive control (average of three wells in each group, mean ± SD).
Figure 2.
 
Effect of α-MSH on melanogenesis of iridal and choroidal melanocytes from an eye with a green iris and epidermal melanocytes from a white donor. Cultured iridal melanocytes (iris M), choroidal melanocytes (choroidal M), and epidermal melanocytes (epidermal M) were seeded into 12-well plates and cultured with cAMP-deleted medium (FIC medium without cAMP-elevating agents, IBMX, and cholera toxin), without or with α-MSH at various concentrations (0.1–100 μM). Cells cultured with cAMP-deleted medium alone and FIC medium were used as negative control (Negative) and positive (Positive) controls, respectively. Six days later, the cells were detached and melanin content per well was measured. Melanin content was expressed as the percentages of the positive control (average of three wells in each group, mean ± SD).
Figure 2.
 
Effect of α-MSH on melanogenesis of iridal and choroidal melanocytes from an eye with a green iris and epidermal melanocytes from a white donor. Cultured iridal melanocytes (iris M), choroidal melanocytes (choroidal M), and epidermal melanocytes (epidermal M) were seeded into 12-well plates and cultured with cAMP-deleted medium (FIC medium without cAMP-elevating agents, IBMX, and cholera toxin), without or with α-MSH at various concentrations (0.1–100 μM). Cells cultured with cAMP-deleted medium alone and FIC medium were used as negative control (Negative) and positive (Positive) controls, respectively. Six days later, the cells were detached and melanin content per well was measured. Melanin content was expressed as the percentages of the positive control (average of three wells in each group, mean ± SD).
Figure 3.
 
Effect of α-MSH on tyrosine hydroxylase activity of iridal and choroidal melanocytes from an eye with a green iris and epidermal melanocytes from a white donor. Human uveal melanocytes were maintained in TISH medium and subcultured for 3 days in the absence of IBMX. Human epidermal melanocytes were maintained in growth medium and subcultured for 3 days in the absence of BPE. The cells were then exposed to α-MSH at concentrations from 0 to 100 μM for 7 days. The treated cells were assayed for tyrosine hydroxylase. Data demonstrate that uveal melanocytes did not respond to α-MSH in contrast to epidermal melanocytes that exhibited an increase in tyrosine hydroxylase activity after α-MSH exposure.
Figure 3.
 
Effect of α-MSH on tyrosine hydroxylase activity of iridal and choroidal melanocytes from an eye with a green iris and epidermal melanocytes from a white donor. Human uveal melanocytes were maintained in TISH medium and subcultured for 3 days in the absence of IBMX. Human epidermal melanocytes were maintained in growth medium and subcultured for 3 days in the absence of BPE. The cells were then exposed to α-MSH at concentrations from 0 to 100 μM for 7 days. The treated cells were assayed for tyrosine hydroxylase. Data demonstrate that uveal melanocytes did not respond to α-MSH in contrast to epidermal melanocytes that exhibited an increase in tyrosine hydroxylase activity after α-MSH exposure.
Figure 4.
 
Effect of α-MSH on DOPA oxidase activity of choroidal melanocytes. Human choroidal melanocytes were treated with or without various concentrations of α-MSH and processed for DOPA oxidase activity by SDS/PAGE/DOPA staining. Data demonstrate that DOPA oxidase activity was not altered by α-MSH.
Figure 4.
 
Effect of α-MSH on DOPA oxidase activity of choroidal melanocytes. Human choroidal melanocytes were treated with or without various concentrations of α-MSH and processed for DOPA oxidase activity by SDS/PAGE/DOPA staining. Data demonstrate that DOPA oxidase activity was not altered by α-MSH.
Figure 5.
 
Effect of α-MSH on tyrosinase and TRP-1 expression of choroidal melanocytes. Human choroidal melanocytes were treated without or with various concentrations of α-MSH and processed for expression of tyrosinase and TRP-1 by immunoblot analysis. Data demonstrate that neither tyrosinase nor TRP-1 expression levels were significantly altered by α-MSH.
Figure 5.
 
Effect of α-MSH on tyrosinase and TRP-1 expression of choroidal melanocytes. Human choroidal melanocytes were treated without or with various concentrations of α-MSH and processed for expression of tyrosinase and TRP-1 by immunoblot analysis. Data demonstrate that neither tyrosinase nor TRP-1 expression levels were significantly altered by α-MSH.
Figure 6.
 
Expression of mRNA for the α-MSH receptor (MC1-R) by iridal and choroidal melanocytes from an eye with a green iris and epidermal cells from a white donor. Northern blot analysis was performed on RNA samples isolated from cultured choroidal (lanes 1, 2) and iridal (lanes 3, 4) melanocytes, ocular fibroblasts (lane 5), cutaneous fibroblasts (lane 6), cutaneous keratinocytes (lane 7), and epidermal melanocytes (lane 8) and probed with 32P-labeled human melanotropin receptor MC1R (middle), human tyrosinase (top), and G3PDH (bottom) cDNA. The data demonstrate that MC1R mRNA was expressed only by the epidermal melanocytes and that tyrosinase mRNA was expressed by all lines of melanocytes.
Figure 6.
 
Expression of mRNA for the α-MSH receptor (MC1-R) by iridal and choroidal melanocytes from an eye with a green iris and epidermal cells from a white donor. Northern blot analysis was performed on RNA samples isolated from cultured choroidal (lanes 1, 2) and iridal (lanes 3, 4) melanocytes, ocular fibroblasts (lane 5), cutaneous fibroblasts (lane 6), cutaneous keratinocytes (lane 7), and epidermal melanocytes (lane 8) and probed with 32P-labeled human melanotropin receptor MC1R (middle), human tyrosinase (top), and G3PDH (bottom) cDNA. The data demonstrate that MC1R mRNA was expressed only by the epidermal melanocytes and that tyrosinase mRNA was expressed by all lines of melanocytes.
Figure 7.
 
Expression of α-MSH in situ. Cryosections of the iris and skin were immunolabeled for α-MSH expression. Expression of α-MSH was absent from the anterior area of the iris (I) and the more posterior stroma of the uveal tract (S). White arrow: melanocytes in the stroma of the iris. In contrast, expression of α-MSH was detected throughout the viable epidermis (black arrow, top middle) and was prominent in the basal epithelial layer (white arrows, top middle). PC, posterior chamber; E, epidermis; D, dermis.
Figure 7.
 
Expression of α-MSH in situ. Cryosections of the iris and skin were immunolabeled for α-MSH expression. Expression of α-MSH was absent from the anterior area of the iris (I) and the more posterior stroma of the uveal tract (S). White arrow: melanocytes in the stroma of the iris. In contrast, expression of α-MSH was detected throughout the viable epidermis (black arrow, top middle) and was prominent in the basal epithelial layer (white arrows, top middle). PC, posterior chamber; E, epidermis; D, dermis.
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Figure 1.
 
Effect of α-MSH on growth of iridal and choroidal melanocytes from eye with green iris and epidermal melanocytes from a white donor. Cultured iridal melanocytes (iris M), choroidal melanocytes (choroidal M), and epidermal melanocytes (epidermal M) were seeded into 12-well plates and cultured with cAMP-deleted medium (FIC medium without cAMP-elevating agents, IBMX, and cholera toxin) without or with α-MSH at various concentrations (0.1–100 μM). Cells cultured with cAMP-deleted medium alone and FIC medium were used as negative control (Negative) and positive (Positive) controls, respectively. Six days later, the cells were detached and counted. The number of cells was expressed as the percentages of the positive control (average of three wells in each group, mean ± SD).
Figure 1.
 
Effect of α-MSH on growth of iridal and choroidal melanocytes from eye with green iris and epidermal melanocytes from a white donor. Cultured iridal melanocytes (iris M), choroidal melanocytes (choroidal M), and epidermal melanocytes (epidermal M) were seeded into 12-well plates and cultured with cAMP-deleted medium (FIC medium without cAMP-elevating agents, IBMX, and cholera toxin) without or with α-MSH at various concentrations (0.1–100 μM). Cells cultured with cAMP-deleted medium alone and FIC medium were used as negative control (Negative) and positive (Positive) controls, respectively. Six days later, the cells were detached and counted. The number of cells was expressed as the percentages of the positive control (average of three wells in each group, mean ± SD).
Figure 2.
 
Effect of α-MSH on melanogenesis of iridal and choroidal melanocytes from an eye with a green iris and epidermal melanocytes from a white donor. Cultured iridal melanocytes (iris M), choroidal melanocytes (choroidal M), and epidermal melanocytes (epidermal M) were seeded into 12-well plates and cultured with cAMP-deleted medium (FIC medium without cAMP-elevating agents, IBMX, and cholera toxin), without or with α-MSH at various concentrations (0.1–100 μM). Cells cultured with cAMP-deleted medium alone and FIC medium were used as negative control (Negative) and positive (Positive) controls, respectively. Six days later, the cells were detached and melanin content per well was measured. Melanin content was expressed as the percentages of the positive control (average of three wells in each group, mean ± SD).
Figure 2.
 
Effect of α-MSH on melanogenesis of iridal and choroidal melanocytes from an eye with a green iris and epidermal melanocytes from a white donor. Cultured iridal melanocytes (iris M), choroidal melanocytes (choroidal M), and epidermal melanocytes (epidermal M) were seeded into 12-well plates and cultured with cAMP-deleted medium (FIC medium without cAMP-elevating agents, IBMX, and cholera toxin), without or with α-MSH at various concentrations (0.1–100 μM). Cells cultured with cAMP-deleted medium alone and FIC medium were used as negative control (Negative) and positive (Positive) controls, respectively. Six days later, the cells were detached and melanin content per well was measured. Melanin content was expressed as the percentages of the positive control (average of three wells in each group, mean ± SD).
Figure 3.
 
Effect of α-MSH on tyrosine hydroxylase activity of iridal and choroidal melanocytes from an eye with a green iris and epidermal melanocytes from a white donor. Human uveal melanocytes were maintained in TISH medium and subcultured for 3 days in the absence of IBMX. Human epidermal melanocytes were maintained in growth medium and subcultured for 3 days in the absence of BPE. The cells were then exposed to α-MSH at concentrations from 0 to 100 μM for 7 days. The treated cells were assayed for tyrosine hydroxylase. Data demonstrate that uveal melanocytes did not respond to α-MSH in contrast to epidermal melanocytes that exhibited an increase in tyrosine hydroxylase activity after α-MSH exposure.
Figure 3.
 
Effect of α-MSH on tyrosine hydroxylase activity of iridal and choroidal melanocytes from an eye with a green iris and epidermal melanocytes from a white donor. Human uveal melanocytes were maintained in TISH medium and subcultured for 3 days in the absence of IBMX. Human epidermal melanocytes were maintained in growth medium and subcultured for 3 days in the absence of BPE. The cells were then exposed to α-MSH at concentrations from 0 to 100 μM for 7 days. The treated cells were assayed for tyrosine hydroxylase. Data demonstrate that uveal melanocytes did not respond to α-MSH in contrast to epidermal melanocytes that exhibited an increase in tyrosine hydroxylase activity after α-MSH exposure.
Figure 4.
 
Effect of α-MSH on DOPA oxidase activity of choroidal melanocytes. Human choroidal melanocytes were treated with or without various concentrations of α-MSH and processed for DOPA oxidase activity by SDS/PAGE/DOPA staining. Data demonstrate that DOPA oxidase activity was not altered by α-MSH.
Figure 4.
 
Effect of α-MSH on DOPA oxidase activity of choroidal melanocytes. Human choroidal melanocytes were treated with or without various concentrations of α-MSH and processed for DOPA oxidase activity by SDS/PAGE/DOPA staining. Data demonstrate that DOPA oxidase activity was not altered by α-MSH.
Figure 5.
 
Effect of α-MSH on tyrosinase and TRP-1 expression of choroidal melanocytes. Human choroidal melanocytes were treated without or with various concentrations of α-MSH and processed for expression of tyrosinase and TRP-1 by immunoblot analysis. Data demonstrate that neither tyrosinase nor TRP-1 expression levels were significantly altered by α-MSH.
Figure 5.
 
Effect of α-MSH on tyrosinase and TRP-1 expression of choroidal melanocytes. Human choroidal melanocytes were treated without or with various concentrations of α-MSH and processed for expression of tyrosinase and TRP-1 by immunoblot analysis. Data demonstrate that neither tyrosinase nor TRP-1 expression levels were significantly altered by α-MSH.
Figure 6.
 
Expression of mRNA for the α-MSH receptor (MC1-R) by iridal and choroidal melanocytes from an eye with a green iris and epidermal cells from a white donor. Northern blot analysis was performed on RNA samples isolated from cultured choroidal (lanes 1, 2) and iridal (lanes 3, 4) melanocytes, ocular fibroblasts (lane 5), cutaneous fibroblasts (lane 6), cutaneous keratinocytes (lane 7), and epidermal melanocytes (lane 8) and probed with 32P-labeled human melanotropin receptor MC1R (middle), human tyrosinase (top), and G3PDH (bottom) cDNA. The data demonstrate that MC1R mRNA was expressed only by the epidermal melanocytes and that tyrosinase mRNA was expressed by all lines of melanocytes.
Figure 6.
 
Expression of mRNA for the α-MSH receptor (MC1-R) by iridal and choroidal melanocytes from an eye with a green iris and epidermal cells from a white donor. Northern blot analysis was performed on RNA samples isolated from cultured choroidal (lanes 1, 2) and iridal (lanes 3, 4) melanocytes, ocular fibroblasts (lane 5), cutaneous fibroblasts (lane 6), cutaneous keratinocytes (lane 7), and epidermal melanocytes (lane 8) and probed with 32P-labeled human melanotropin receptor MC1R (middle), human tyrosinase (top), and G3PDH (bottom) cDNA. The data demonstrate that MC1R mRNA was expressed only by the epidermal melanocytes and that tyrosinase mRNA was expressed by all lines of melanocytes.
Figure 7.
 
Expression of α-MSH in situ. Cryosections of the iris and skin were immunolabeled for α-MSH expression. Expression of α-MSH was absent from the anterior area of the iris (I) and the more posterior stroma of the uveal tract (S). White arrow: melanocytes in the stroma of the iris. In contrast, expression of α-MSH was detected throughout the viable epidermis (black arrow, top middle) and was prominent in the basal epithelial layer (white arrows, top middle). PC, posterior chamber; E, epidermis; D, dermis.
Figure 7.
 
Expression of α-MSH in situ. Cryosections of the iris and skin were immunolabeled for α-MSH expression. Expression of α-MSH was absent from the anterior area of the iris (I) and the more posterior stroma of the uveal tract (S). White arrow: melanocytes in the stroma of the iris. In contrast, expression of α-MSH was detected throughout the viable epidermis (black arrow, top middle) and was prominent in the basal epithelial layer (white arrows, top middle). PC, posterior chamber; E, epidermis; D, dermis.
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