Regulation of Human Epidermal Melanocyte Biology By β-Endorphin
2003; Elsevier BV; Volume: 120; Issue: 6 Linguagem: Inglês
10.1046/j.1523-1747.2003.12242.x
ISSN1523-1747
AutoresSöbia Kauser, Karin U. Schallreuter, A. J. Thody, Desmond J. Tobin, Christopher L. Gummer,
Tópico(s)Biochemical Analysis and Sensing Techniques
Resumoβ-Endorphin is an opioid peptide cleaved from the precursor pro-hormone pro-opiomelanocortin, from which other peptides such as adrenocorticotropic hormone, β-lipotropic hormone, and α-melanocyte-stimulating hormone are also derived. α-Melanocyte-stimulating hormone and adrenocorticotropic hormone are well documented to regulate human skin pigmentation via action at the melanocortin-1 receptor. Whereas plasma β-endorphin is reported to increase after exposure to ultraviolet radiation, to date a functional role for β-endorphin in the regulation of human epidermal melanocyte biology has not been demonstrated. This study was designed to examine the involvement of the β-endorphin/μ-opiate receptor system in human epidermal melanocytes. To address this question we employed reverse transcription–polymerase chain reaction, and immunohistochemistry/cytochemistry and immunoelectron microscopy using β-endorphin and μ-opiate receptor specific antibodies. A functional role for β-endorphin was assessed in epidermal melanocyte cultures by direct stimulation with the peptide. This study demonstrated the expression of μ-opiate receptor mRNA in cultured epidermal melanocytes, as well as mRNA for pro-opiomelanocortin. In addition, we have shown that β-endorphin and μ-opiate receptor are expressed at the protein level in situ in glycoprotein100-positive melanocytes. The expression of both β-endorphin and μ-opiate receptor correlated positively with their differentiation status in vitro. Furthermore, immunoelectron microscopy studies revealed an association of β-endorphin with melanosomes. Functional studies showed that β-endorphin has potent melanogenic, mitogenic, and dendritogenic effects in cultured epidermal melanocytes deprived of any exogenous supply of pro-opiomelanocortin peptides. Thus, we report that human epidermal melanocytes express a fully functioning β-endorphin/μ-opiate receptor system. In the absence of any data showing cross-talk between the μ-opiate receptor and the melanocortin-1 receptor, we conclude that the β-endorphin/μ-opiate receptor system participates in the regulation of skin pigmentation. β-Endorphin is an opioid peptide cleaved from the precursor pro-hormone pro-opiomelanocortin, from which other peptides such as adrenocorticotropic hormone, β-lipotropic hormone, and α-melanocyte-stimulating hormone are also derived. α-Melanocyte-stimulating hormone and adrenocorticotropic hormone are well documented to regulate human skin pigmentation via action at the melanocortin-1 receptor. Whereas plasma β-endorphin is reported to increase after exposure to ultraviolet radiation, to date a functional role for β-endorphin in the regulation of human epidermal melanocyte biology has not been demonstrated. This study was designed to examine the involvement of the β-endorphin/μ-opiate receptor system in human epidermal melanocytes. To address this question we employed reverse transcription–polymerase chain reaction, and immunohistochemistry/cytochemistry and immunoelectron microscopy using β-endorphin and μ-opiate receptor specific antibodies. A functional role for β-endorphin was assessed in epidermal melanocyte cultures by direct stimulation with the peptide. This study demonstrated the expression of μ-opiate receptor mRNA in cultured epidermal melanocytes, as well as mRNA for pro-opiomelanocortin. In addition, we have shown that β-endorphin and μ-opiate receptor are expressed at the protein level in situ in glycoprotein100-positive melanocytes. The expression of both β-endorphin and μ-opiate receptor correlated positively with their differentiation status in vitro. Furthermore, immunoelectron microscopy studies revealed an association of β-endorphin with melanosomes. Functional studies showed that β-endorphin has potent melanogenic, mitogenic, and dendritogenic effects in cultured epidermal melanocytes deprived of any exogenous supply of pro-opiomelanocortin peptides. Thus, we report that human epidermal melanocytes express a fully functioning β-endorphin/μ-opiate receptor system. In the absence of any data showing cross-talk between the μ-opiate receptor and the melanocortin-1 receptor, we conclude that the β-endorphin/μ-opiate receptor system participates in the regulation of skin pigmentation. adrenocorticotropic hormone α-melanocyte-stimulating hormone β-endorphin β-lipotropic hormone epidermal keratinocytes epidermal melanocytes melanocortin-1 receptor pro-opiomelanocortin β-Endorphin (β-END) is a cleavage product of the precursor pro-hormone pro-opiomelanocortin (POMC). Other peptides, including adrenocorticotropic hormone (ACTH), α-melanocyte-stimulating hormone (α-MSH), and β-lipotropic hormone (β-LPH) are also produced from POMC by the actions of the pro-hormone convertases 1 and 2 (Benjannet et al., 1991Benjannet S. Rondeau N. Day R. Chretien M. Seidah N.G. PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues.Proc Natl Acad Sci USA. 1991; 88: 3564-3568Crossref PubMed Scopus (521) Google Scholar;Seidah et al., 1994Seidah N.G. Chretien M. Day R. The family of subtilisin/kexin like pro-protein and pro-hormone convertases: divergent or shared functions.Biochimie. 1994; 76: 197-209Crossref PubMed Scopus (374) Google Scholar). It is now well established that the skin is also a local source and target for POMC-derived peptides (Thody et al., 1983Thody A.J. Ridley K. Penny R.J. Chalmers R. Fisher C. Shuster S. MSH peptides are present in mammalian skin.Peptides. 1983; 4: 813-816Crossref PubMed Scopus (159) Google Scholar;Slominski et al., 1993Slominski A. Wortsman J. Mazurkiewicz J.E. Detection of proopiomelanocortin-derived antigens in normal and pathologic human skin.J Lab Clin Med. 1993; 122: 658-666PubMed Google Scholar;Wintzen and Gilchrest, 1996Wintzen M. Gilchrest B.A. Proopiomelanocortin, its derived peptides, and the skin.J Invest Dermatol. 1996; 106: 3-10Abstract Full Text PDF PubMed Scopus (123) Google Scholar). α-MSH and ACTH have been well documented to regulate human skin pigmentation via action at the melanocortin-1 receptor (MC-1R) (Lerner and McGuire, 1964Lerner A.B. McGuire J.S. Melanocyte-stimulating hormone and adrenocorticotrophic hormone: Their relation to pigmentation.N Engl J Med. 1964; 270: 539-546Crossref PubMed Scopus (102) Google Scholar;Hunt et al., 1994aHunt G. Todd C. Cresswell J.E. Thody A.J. Alpha-melanocyte stimulating hormone and its analogue Nle4DPhe7 alpha-MSH affect morphology, tyrosinase activity and melanogenesis in cultured human melanocytes.J Cell Sci. 1994; 107: 205-211PubMed Google Scholar;Suzuki et al., 1996Suzuki I. Cone R.D. Im S. Nordlund J. Abdel-Malek Z.A. Binding of melanotropic hormones to the melanocortin receptor MC1R on human melanocytes stimulates proliferation and melanogenesis.Endocrinology. 1996; 137: 1627-1633Crossref PubMed Scopus (238) Google Scholar;Wakamatsu et al., 1997Wakamatsu K. Graham A. Cook D. Thody A.J. Characterisation of ACTH peptides in human skin and their activation of the melanocortin-1 receptor.Pigment Cell Res. 1997; 10: 288-297Crossref PubMed Scopus (167) Google Scholar). β-END is an opioid peptide representing the 61–91 amino acid sequence of β-LPH (Dalayeun et al., 1993Dalayeun J.F. Nores J.M. Bergal S. Physiology of Beta-endorphins: A close-up view and a review of the literature.Biomed Pharmacother. 1993; 47: 311-320Crossref PubMed Scopus (46) Google Scholar). Opioids exert their biologic effects by activating membrane-bound receptors, which consist of three major classes mu (μ), delta (δ), and kappa (κ) (Pasternak, 1993Pasternak G.W. Pharmacological mechanisms of opioid analgesics.Clin Neuropharmacol. 1993; 16: 1-18Crossref PubMed Scopus (486) Google Scholar). β-END binds with high affinity to μ and δ opiate receptors (Gilmore and Weiner, 1989Gilmore W. Weiner L.P. The opioid specificity of beta-endorphin enhancement of murine lymphocyte proliferation.Immunopharmacology. 1989; 17: 19-30Crossref PubMed Scopus (49) Google Scholar). β-END and other opioids, such as the enkephalins and dynorphins, mediate their biologic actions by inhibition of the adenylate cyclase pathway and a decrease in the formation of intracellular cyclic adenosine monophosphate (Kieffer, 1995Kieffer B.L. Recent advances in molecular recognition and signal transduction of active peptides: receptors for opioid peptides.Cell Mol Neurobiol. 1995; 15: 615-635Crossref PubMed Scopus (344) Google Scholar). Elevated plasma β-END levels have been reported in dermatoses, including psoriasis (Glinski et al., 1994Glinski W. Brodecka H. Glinska-Ferenz M. Kowalski D. Increased concentration of beta-endorphin in sera of patients with psoriasis and other inflammatory dermatoses.Br J Dermatol. 1994; 131: 260-264Crossref PubMed Scopus (34) Google Scholar), atopic dermatitis (Glinski et al., 1995Glinski W. Brodecka H. Glinska-Ferenz M. Kowalski D. Increased concentration of beta-endorphin in the sera of patients with severe atopic dermatitis.Acta Derm Venereol. 1995; 75: 9-11PubMed Google Scholar), and the pigmentation disorder vitiligo (Mozzanica et al., 1992Mozzanica N. Villa M.L. Foppa S. Vignati G. Cattaneo A. Diotti R. Finzi A.F. Plasma alpha-melanocyte-stimulating hormone, beta-endorphin, met-enkephalin, and natural killer cell activity in vitiligo.J Am Acad Dermatol. 1992; 26: 693-700Abstract Full Text PDF PubMed Scopus (42) Google Scholar;Caixia et al., 2001Caixia T. Daming Z. Xiran L. Levels of beta-endorphin in the plasma and skin tissue fluids of patients with vitiligo.J Dermatol Sci. 2001; 26: 62-66Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). The onset of these conditions can be triggered or their course can be aggravated by psychologic stress (Seville, 1989Seville R.H. Stress and psoriasis: The importance of insight and empathy in prognosis.J Am Acad Dermatol. 1989; 20: 97-100Abstract Full Text PDF PubMed Scopus (40) Google Scholar), thus implicating the involvement of classical stress hormones such as the endorphins in these disorders. Moreover, increased plasma β-END and β-LPH levels post-ultraviolet (UV)A exposure have been associated with the observed increase in skin pigmentation in humans (Levins et al., 1983Levins P.C. Carr D.B. Fisher J.E. Momtaz K. Parrish J.A. Plasma beta-endorphin and beta-lipoprotein response to ultraviolet radiation.Lancet. 1983; ii: 166Abstract Scopus (60) Google Scholar); however, more recent studies have reported that plasma β-END levels do not rise after single or repeated exposure to UVA, UVA-1, or UVB radiation (Wintzen et al., 2001Wintzen M. Ostijn D.M. Polderman M.C. Le Cessie S. Burbach J.P. Vermeer B.J. Total body exposure to ultraviolet radiation does not influence plasma levels of immunoreactive beta-endorphin in man.Photodermatol Photoimmunol Photomed. 2001; 17: 256-260Crossref PubMed Scopus (36) Google Scholar). ACTH, α-MSH (Schauer et al., 1994Schauer E. Trautinger F. Kock A. Proopiomelanocortin-derived peptides are synthesized and released by human keratinocytes.J Clin Invest. 1994; 93: 2258-2262Crossref PubMed Scopus (309) Google Scholar), and β-END (Wintzen et al., 1996Wintzen M. Yaar M. Burbach J.P. Gilchrest B.A. Proopiomelanocortin gene product regulation in keratinocytes.J Invest Dermatol. 1996; 106: 673-678Abstract Full Text PDF PubMed Scopus (122) Google Scholar) are produced by human epidermal keratinocytes (EK) in vitro and their production is upregulated in response to UVB radiation. In addition, the presence of β-END, α-MSH, and ACTH has also been identified in normal and malignant human melanocytes in vitro (Slominski, 1998Slominski A. Identification of beta-endorphin, alpha-MSH and ACTH peptides in cultured human melanocytes, melanoma and squamous cell carcinoma cells by RP-HPLC.Exp Dermatol. 1998; 7: 213-216Crossref PubMed Scopus (43) Google Scholar), as well as in human skin (Slominski et al., 2000bSlominski A. Szczesniewski A. Wortsman J. Liquid chromatography-mass spectrometry detection of corticotropin-releasing hormone and proopiomelanocortin-derived peptides in human skin.J Clin Endocrinol Metab. 2000; 85: 3582-3588PubMed Google Scholar). The expression of β-END and other POMC peptides has been demonstrated in normal and pathologic skin, and was elevated under pathologic conditions but also during healing and regeneration (Slominski et al., 1993Slominski A. Wortsman J. Mazurkiewicz J.E. Detection of proopiomelanocortin-derived antigens in normal and pathologic human skin.J Lab Clin Med. 1993; 122: 658-666PubMed Google Scholar;Nagahama et al., 1998Nagahama M. Funasaka Y. Fernandez-Frez M.L. Ohashi A. Chakraborty A.K. Ueda M. Ichihashi M. Immunoreactivity of alpha-melanocyte-stimulating hormone, adrenocorticotrophic hormone and beta-endorphin in cutaneous malignant melanoma and benign melanocytic naevi.Br J Dermatol. 1998; 138: 981-985Crossref PubMed Scopus (50) Google Scholar). Recent data indicate that the human epidermis expresses a functionally active β-END receptor. Both μ-opiate receptor mRNA and protein expression have been found in human epidermis (Bigliardi et al., 1998Bigliardi P.L. Bigliardi-Qi M. Buechner S. Rufli T. Expression of mu-opiate receptor in human epidermis and keratinocytes.J Invest Dermatol. 1998; 111: 297-301Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). β-END is a specific agonist at the μ-opiate receptor in human skin; receptor expression was downregulated by β-END and this was reversed with an anti-μ-opiate receptor antibody or the antagonist naloxone. Notably, β-END was also shown to upregulate the expression of cytokeratin 16, a marker for keratinocyte differentiation (Bigliardi-Qi et al., 2000Bigliardi-Qi M. Bigliardi P.L. Eberle A.N. Buchner S. Rufli T. beta-endorphin stimulates cytokeratin 16 expression and downregulates mu-opiate receptor expression in human epidermis.J Invest Dermatol. 2000; 114: 527-532Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Whereas α-MSH and ACTH has been shown to regulate human skin pigmentation (Lerner and McGuire, 1964Lerner A.B. McGuire J.S. Melanocyte-stimulating hormone and adrenocorticotrophic hormone: Their relation to pigmentation.N Engl J Med. 1964; 270: 539-546Crossref PubMed Scopus (102) Google Scholar;Hunt et al., 1994aHunt G. Todd C. Cresswell J.E. Thody A.J. Alpha-melanocyte stimulating hormone and its analogue Nle4DPhe7 alpha-MSH affect morphology, tyrosinase activity and melanogenesis in cultured human melanocytes.J Cell Sci. 1994; 107: 205-211PubMed Google Scholar;Suzuki et al., 1996Suzuki I. Cone R.D. Im S. Nordlund J. Abdel-Malek Z.A. Binding of melanotropic hormones to the melanocortin receptor MC1R on human melanocytes stimulates proliferation and melanogenesis.Endocrinology. 1996; 137: 1627-1633Crossref PubMed Scopus (238) Google Scholar;Wakamatsu et al., 1997Wakamatsu K. Graham A. Cook D. Thody A.J. Characterisation of ACTH peptides in human skin and their activation of the melanocortin-1 receptor.Pigment Cell Res. 1997; 10: 288-297Crossref PubMed Scopus (167) Google Scholar). These studies have led to an MC-1R-centric view of the regulation of mammalian melanogenesis; however, β-LPH, the immediate precursor of β-END, stimulates melanogenesis in amphibians and in sheep (Lohmar and Li, 1968Lohmar P. Li C.H. Biological properties of ovine beta-lipotropic hormone.Endocrinology. 1968; 82: 898-904Crossref PubMed Scopus (32) Google Scholar). Moreover, elevated serum levels of β-LPH have been associated with generalized hyperpigmentation in humans (Al Rustom et al., 1986Al Rustom K. Gerard J. Pierard G.E. Extrapituitary neuroendocrine melanoderma. Unique association of extensive melanoderma with macromelanosomes and extrapituitary secretion of a high molecular weight neuropeptide related to pro-opiomelanocortin.Dermatologica. 1986; 173: 157-162Crossref PubMed Scopus (7) Google Scholar). Despite these tentative clues, however, a functional role for β-END in the regulation of human skin pigmentation has not yet been demonstrated. This study examines the β-END/μ-opiate receptor system in epidermal melanocyte (EM) biology and presents evidence that β-END is a potent melanocyte modifier by upregulating melanocyte proliferation, dendricity, and melanogenesis. EM cultures were established from normal human haired scalp tissue, obtained with informed consent after elective plastic surgery, from eight females (age range 43–62 y, mean age 52 y, photo skin type III–V, Fitzpatrick classification;Fitzpatrick et al., 1974Fitzpatrick T.B. Pathak M.A. Parrish J.A. Protection of human skin against the effects of the sunburn ultraviolet (290–320 nm).in: Fitzpatrick T.B. Sunlight and Man—Normal and Abnormal Photobiological Responses. University of Tokyo Press, Tokyo1974: 751-765Google Scholar). This study was conducted with Local Human Ethics Committee approval. All cell culture reagents were obtained from Invitrogen Ltd (Paisley, U.K.) unless otherwise stated. The skin samples were collected in RPMI 1640 medium supplemented with 10% fetal bovine serum, 12.5 μg fungizone per ml, 500 units per ml penicillin, and 500 μg streptomycin per ml, and were processed within 5 h of surgery. Briefly, tissue specimens were washed with 0.1 M phosphate-buffered saline (PBS) pH 7.4 containing 12.5 μg fungizone per ml, 500 units per ml penicillin, and 500 μg streptomycin per ml. Epidermal sheets were separated from the underlying dermis after an 18 h incubation in 0.25% trypsin solution at 4°C and placed in T25 cell culture flasks (Corning Costar Corporation, Cambridge, MA) containing Eagle's minimal essential medium (EMEM) supplemented 2% FBS, 1 × concentrated nonessential amino acid mixture, antibiotics, 2 mM L-glutamine, 5 ng basic fibroblast growth factor per ml, and 5 ng endothelin-1 per ml (Sigma, Poole, Dorset, U.K.). Residual epidermal material was carefully removed and the medium was replenished after 48 h. Cells were incubated at 37°C in a 5% CO2 atmosphere and fed every third day. These were established by selectively trypsinizing EM from the coculture at the primary culture stage, using 0.05% trypsin and 0.53 mM ethylenediamine tetraacetic acid solution. The detached EM were transferred into a separate culture dish, the remaining EK were then switched to keratinocyte serum free medium. EM and EK were maintained in culture media without FBS or bovine pituitary extract (BPE) 48 h prior to all experiments. This treatment removes all exogenous sources of POMC peptides, as the half-lives of α-MSH, ACTH (Eberle, 1988Eberle A.N. The Melanotropins. Chemistry, Physiology and Mechanisms of Action. Basel, Karger1988Google Scholar), and β-END (Morch and Pedersen, 1995Morch H. Pedersen B.K. Beta-endorphin and the immune system-possible role in autoimmune diseases.Autoimmunity. 1995; 21: 161-171Crossref PubMed Scopus (27) Google Scholar) are less than 60 min. Total RNA was isolated from detached EM and EK by the guanidinium thiocyanate–phenol–chloroform-based method, using Tri-Reagent™ (Sigma, Dorset, U.K.) according to the manufacturer's instructions. Total RNA was isolated from the aqueous phase and the resulting RNA pellet was dissolved in nuclease-free water (Sigma, Dorset, U.K.). Total RNA isolated from EM was then purified using a Dynabeads mRNA direct kit (Dynal AS, Oslo, Norway) according to the manufacturer's instructions. This additional purification step was necessary to remove traces of melanin, which can be inhibitory to the polymerase chain reaction (PCR). (Eckhart et al., 2000Eckhart L. Bach J. Ban J. Tschachler E. Melanin binds reversibly to thermostable DNA polymerase and inhibits its activity.Biochem Biophys Res Commun. 2000; 271: 726-730Crossref PubMed Scopus (145) Google Scholar). To avoid possible contamination of genomic DNA, extracted total RNA samples were additionally treated with deoxyribonuclease I, amplification grade (Invitrogen Ltd) according to the manufacturer's instructions. The synthesis of cDNA was performed using RevertAidTM M-MuLV Reverse Transcriptase (MBI Fermentas, Vilnius, Lithuania) according to the manufacturer's instructions using 2 μg of total RNA or 0.2 μg of mRNA and oligo(dT)18 and random hexamer primers (Sigma Genosys, Pampisford, Cambridgeshire, U.K.). All PCR reagents were obtained from MBI Fermentas, unless otherwise stated. One microliter of cDNA was used in the PCR amplification in a 50 μl reaction mixture containing the following components: 1×PCR buffer [75 mM Tris–HCl pH 8.8, 20 mM (NH4)2SO4, 0.01% Tween 20], 1.0 or 2.5 mM MgCl2 for POMC and μ-opiate receptor-specific primers, respectively, 20 pmol of each primer (Sigma Genosys), 0.2 mM deoxyribonucleoside triphosphate mix, 1.0 unit of Taq DNA polymerase, and, finally, nuclease-free water (Sigma, Dorset, U.K.) was added to make a final reaction volume of 50 μl. POMC was amplified using primer sets as previously described bySlominski et al., 1995Slominski A. Ermak G. Hwang J. Chakraborty A. Mazurkiewicz J.E. Mihm M. Proopiomelanocortin, corticotropin releasing hormone and corticotropin releasing hormone receptor genes are expressed in human skin.FEBS Lett. 1995; 374: 113-116Crossref PubMed Scopus (152) Google Scholar and a modified protocol with one cycle at 94°C for 5 min, 35 cycles at 94°C for 1 min, 67°C for 1 min, 72°C for 1 min, and a final cycle at 94°C for 1 min, 67°C for 1 min, 72°C for 10 min. Plasmid containing POMC gene (gift from Dr J. Ancans, University of Riga, Latvia) was used as a positive control for POMC mRNA expression. μ-Opiate receptor was amplified using primers sets and PCR parameters as described byBigliardi et al., 1998Bigliardi P.L. Bigliardi-Qi M. Buechner S. Rufli T. Expression of mu-opiate receptor in human epidermis and keratinocytes.J Invest Dermatol. 1998; 111: 297-301Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar. EK were used as a positive control cell line for μ-opiate receptor mRNA expression. RNA samples that were not reverse transcribed and the omission of cDNA from the reaction mixture served as negative controls. Amplifications were performed using the Hybaid PCR sprint temperature cycling system (Hybaid, Ashford, Middlesex, U.K.). After amplification, 10 μl of the reaction mixture was mixed with 4 μl gel loading solution (MBI Fermentas) and loaded directly on to 1.5% agarose gel (Sigma, Dorset, UK) containing 1 μg per ml of ethidium bromide (Sigma, Dorset, UK). A 100 bp DNA ladder (New England Biolabs, Hitchin, Hertfordshire, U.K.) was also loaded followed by electrophoresis at a constant voltage of 100 V using 0.5×Tris-borate buffer. Gels were photo-documented using the UVitec gel documentation system (UVitec Limited, Cambridge, U.K.). Normal human haired scalp tissue was obtained after elective plastic surgery (from five females, age range 43–60 y, mean age 50 y) or from occipital scalp tissue of two normal healthy males (age 23 and 36 y). The tissue was cut into approximately 0.4 cm2 segments and was mounted using OCT™ embedding medium (Raymond A. Lamb, Eastbourne, East Sussex, U.K.). Seven micrometer thick cryosections were cut on to poly L-lysine coated slides. The slides were air-dried at room temperature for 1 h and then fixed in ice cold acetone for 10 min at -20°C and subsequently rehydrated in PBS for 5 min. Sections were blocked in 10% normal donkey serum in PBS for 90 min at room temperature and then rinsed briefly in PBS. This was followed by incubation with the first primary monoclonal antibody β-END 1:10 (Biogenesis, Poole, Dorset, UK) or μ-opiate receptor 1:100 (Gramsch Laboratories, Schwabhausen, Germany) in 1% normal donkey serum in PBS at 4°C for 18 h. After incubation the antibodies were carefully aspirated and the sections were washed four times in PBS for 20 min followed by a final rinse in distilled water. The sections were then incubated with a rhodamine-conjugated donkey anti-rabbit or anti-mouse secondary antibody (Jackson Immunoresearch Laboratories, Inc., West Grave, Pennsylvania, USA). The secondary antibodies were used at a 1:50 dilution and incubated for 60 min at room temperature, followed by four washes in PBS. For the detection of the second marker the sections were further blocked with 10% normal donkey serum, covered and incubated for 90 min at room temperature. After a brief rinse in PBS they were incubated with the second primary antibody, NKI/beteb 1:30 (Monosan, Uden, the Netherlands), a melanocyte lineage-specific marker against glycoprotein100 for 2 h at room temperature, followed by subsequent washing steps. The sections were then incubated with a fluorescein-conjugated donkey anti-mouse secondary antibody (Jackson Immunoresearch Laboratories, Inc.) for 60 min at room temperature. The sections were washed in PBS as previously described, rinsed in distilled water, carefully blotted dry and then mounted in Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories Ltd, Peterborough, U.K.). The omission of both primary antibodies, but the inclusion of secondary antibodies, served as negative controls. The resulting staining was visualized with a Leica DMIRB/E fluorescence microscope (Leica, Wetzlar, Ossett, Germany) and photo-documented with the aid of a computer assisted 3-CCD color video camera (Optivision, Ossett, West Yorkshire, U.K.) and the Image Grabber PCI graphics program (Neotech Ltd, Easleeigh, Hampshire, U.K.). The images produced with the two different fluorochromes rhodamine (red) and fluorescein (green) were subsequently merged together using the Paint Shop Pro™ 7 graphics program (JascSoftware, Banbury, Oxon, U.K.). Co-localization of β-END and μ-opiate receptor with gp100-positive EM was indicated by the production of a yellow color. For staining of cultured EM and EK (passages 2–5), cells were seeded into eight-well Laboratory-Tek® chamber slides (ICN Biomedicals, Inc., Aurora, OH, USA) at a seeding density of 5000 cells per well and were grown for at least of 2–3 d. FBS and BPE were omitted from the culture media 48 h prior to immunostaining. Cells were rinsed briefly in PBS for 5 min and then fixed in ice cold methanol for 10 min at – 20°C and subsequently rehydrated in PBS for 5 min. Briefly, cells were blocked in 10% normal goat serum for 90 min at room temperature, rinsed briefly in PBS and incubated with either β-END 1:10 or μ-opiate receptor 1:100 antibodies in 1% normal goat serum in PBS at 4°C for 18 h. Subsequent steps in immunostaining were performed using the DAKO LSAB®2 HRP kit and DAKO AEC substrate system (DAKO, Carpinteria, CA) according to the manufacturer's instructions. EM (passages 2–5) were seeded at a density of 1 × 105 cells per T25 cell culture flask (Corning Costar Corporation, Cambridge, MA) and allowed to attach overnight. Melanocyte cultures were grown without FBS and BPE for 48 h prior to stimulation with 10–8Mβ-END (Sigma, Dorset, U.K.) for 72 h. Melanocytes from the same donor were maintained in parallel in the absence of the peptide and served as a negative control. β-END (10–8M) was chosen for stimulation, as the greatest effects on melanogenesis, dendricity, and proliferation were seen at this concentration during preliminary experiments compared with 10–6M and 10–10M. The testing of a broader range of β-END concentrations was difficult given the limitations of melanocyte number at low passage. For the assessment of melanocyte dendricity, cells were photographed 24 and 72 h after β-END stimulation. Representative photographs were taken from seven to eight random and different fields incorporating approximately 10 cells for each cell line. Changes in EM dendricity were assessed and compared with controls. The cells were subsequently trypsinized and counted using a Neubauer counting chamber. Melanocytes were then pelleted by centrifugation and solubilized in up 0.5 ml of 1 M sodium hydroxide, followed by boiling for 10 min. Melanin content was measured spectrophotometrically at 475 nm. A standard curve of synthetic melanin (Sigma, Dorset, U.K.) 0.05–100 μg per ml was used as a basis for the determination of melanin content. Results were determined as picograms of melanin per cell and expressed as percentage increase in melanin content above control unstimulated cells. The increase in cell number was expressed as percentage increase in cell number above control unstimulated levels. EM were established from normal human scalp tissue. FBS and BPE were removed from the culture medium 48 h prior to experiments. Melanocyte cultures (passages 3–5) were processed for immunoelectron microscopy as follows: cells were trypsinized and then pelleted by gentle centrifugation. Cells were subsequently fixed in 0.5% glutaraldehyde (Agar Scientific, Stanstead, Essex, U.K.) and 2% paraformaldehyde (Sigma, Dorset, U.K.) in 0.1 M sodium cacodylate buffer (Sigma, Dorset, U.K.) containing 0.027 mM CaCl2 (Sigma, Dorset, U.K.) buffered to pH 7.4 for 1 h at room temperature. After fixation cells were washed in PBS over 25 min, rinsed in 0.1 M glycine (Sigma, Dorset, U.K.) in PBS for 5 min and then pelleted through 1% low melting point agarose (Bio-Rad Laboratories, Hercules, CA) in PBS. The cell/agar blocks were dehydrated in graded series of ethanol. Blocks were infiltrated with hydrophillic Unicryl resin (British BioCell International, Cardiff, Wales, U.K.) followed by polymerization using UV light (360 nm) of 2 × 8 W for 3 d at 4°C. Ultrathin sections (90 nm) were cut using a Reichert-Jung ultramicrotome (Vienna, Austria), mounted on 200 mesh nickel grids coated with a carbon film (Agar Scientific) and blocked in 10% normal goat serum and 2% bovine serum albumin in PBS (pH 8.2) for 1 h. One percent normal goat serum in the above blocking buffer was used to dilute both the primary and secondary antibodies. The sections were then washed twice in PBS containing 2% bovine serum albumin (pH 8.2) followed by incubation with the primary antibodies β-END neat or μ-opiate rec
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