Molecular and Cellular Biochemistry

, Volume 228, Issue 1–2, pp 57–72 | Cite as

Identification and characterization of muscarinic acetylcholine receptor subtypes expressed in human skin melanocytes

  • Rico Buchli
  • Assane Ndoye
  • Juan Arredondo
  • Robert J. Webber
  • Sergei A. Grando
Article

Abstract

The present study was designed to identify and characterize muscarinic acetylcholine receptors in normal human melanocytes. We used subtype-specific oligonucleotide primers to localize the five genetically defined mAChR mRNAs (m1 through m5) by reverse transcription-polymerase chain reaction. These experiments showed that all five mAChR subtype mRNAs are expressed in melanocytes. The PCR products were verified by restriction analysis and Southern blotting. Receptors were visualized in cultures of normal human melanocytes and specimens of normal human skin by subtype-specific rabbit anti-receptor polyclonal antibodies. Radioligand binding assays with the lipophilic drug [3H]quinuclidinyl benzilate demonstrated approximately 9000 high affinity binding sites/cell. Micromolar concentrations of muscarine or carbachol transiently increased intracellular Ca2+, which could be attenuated by atropine, demonstrating coupling of the receptors to mobilization of intracellular free Ca2+. Lower concentrations of muscarine induced spontaneous repetitive spike-like increases of intracellular Ca2+ which is characteristic for the activation of muscarinic receptors. These results indicate that normal human skin melanocytes express the m1, m2, m3, m4, and m5 subtypes of classic muscarinic acetylcholine receptors on their cell membrane and that these receptors regulate the concentration of intracellular free Ca2+, which may play an important physiologic role in melanocyte behavior and skin pigmentation.

cholinergic drugs indirect immunofluorescence intracellular free calcium reverse transcription-polymerase chain reaction radioligand binding 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Buchli R, Ndoye A, Rodriguez JG, Zia S, Webber RJ, Grando SA: Human skin fibroblasts express m2, m4, and m5 subtypes of muscarinic acetylcholine receptors. J Cell Biochem 74: 264–277, 1999Google Scholar
  2. 2.
    Grando SA, Horton RM: The keratinocyte cholinergic system: Acetylcholine as an epidermal cytotransmitter. Curr Opin Dermatol 4: 262–268, 1997Google Scholar
  3. 3.
    Grando SA: Biological functions of keratinocyte cholinergic receptors. J Invest Dermatol Symp Proc 2: 41–48, 1997Google Scholar
  4. 4.
    Grando SA, Kist DA, Qi M, Dahl MV: Human keratinocytes synthesize, secrete, and degrade acetylcholine. J Invest Dermatol 101: 32–36, 1993Google Scholar
  5. 5.
    Allard WJ, Sigal IS, Dixon RA: Sequence of the gene encoding the human M1 muscarinic acetylcholine receptor. Nucleic Acids Res 15: 10604, 1987Google Scholar
  6. 6.
    Bonner TI, Buckley NJ, Young AC, Brann MR: Identification of a family of muscarinic acetylcholine receptor genes. Science 237: 527–532, 1987Google Scholar
  7. 7.
    Bonner TI, Young AC, Brann MR, Buckley NJ: Cloning and expression of the human and rat m5 muscarinic acetylcholine receptor genes. Neuron 1: 403–410, 1988Google Scholar
  8. 8.
    Peralta EG, Winslow JW, Peterson GL, Smith DH, Ashkenazi A, Ramachandran J, Schimerlik MI, Capon DJ: Primary structure and biochemical properties of an M2 muscarinic receptor. Science 236: 600–605, 1987Google Scholar
  9. 9.
    Peralta EG, Ashkenazi A, Winslow JW, Smith DH, Ramachandran J, Capon DJ: Distinct primary structures, ligand-binding properties and tissue-specific expression of four human muscarinic acetylcholine receptors. EMBO J 6: 3923–3929, 1987Google Scholar
  10. 10.
    Hulme EC, Birdsall NJ, Buckley NJ: Muscarinic receptor subtypes. Annu Rev Pharmacol Toxicol 30: 633–673, 1990Google Scholar
  11. 11.
    Hosey MM: Diversity of structure, signaling and regulation within the family of muscarinic cholinergic receptors. FASEB J 6: 845–852, 1992Google Scholar
  12. 12.
    Iyengar B: Modulation of melanocytic activity by acetylcholine. Acta Anat 136: 139–141, 1989Google Scholar
  13. 13.
    Grando SA, Zelickson BD, Kist DA, Weinshenker D, Bigliardi PL, Wendelschafer Crabb G, Kennedy WR, Dahl MV: Keratinocyte muscarinic acetylcholine receptors: Immunolocalization and partial characterization. J Invest Dermatol 104: 95–100, 1995Google Scholar
  14. 14.
    Ndoye A, Buchli R, Greenberg B, Nguyen VT, Zia S, Rodriguez JG, Webber RJ, Lawry MA, Grando SA: Identification and mapping of keratinocyte muscarinic acetylcholine receptor subtypes in human epidermis. J Invest Dermatol 111: 101–108, 1998Google Scholar
  15. 15.
    Vestling M, Cowburn RF, Venizelos N, Lannfelt L, Winblad B, Adem A: Characterization of muscarinic acetylcholine receptors in cultured adult skin fibroblasts: Effects of the Swedish Alzheimer's disease APP 670/671 mutation on binding levels. J Neural Transm Park Dis Dement Sect 10: 1–10, 1995Google Scholar
  16. 16.
    Gardner-Medwin JM, Taylor JY, Macdonald IA, Powell RJ: An investigation into variability in microvascular skin blood flow and the responses to transdermal delivery of acetylcholine at different sites in the forearm and hand. Br J Clin Pharmacol 43: 391–397, 1997Google Scholar
  17. 17.
    Abello J, Roche C, Cuber JC, Bernard C, Philippe J, Chayvialle JA: Characterization of muscarinic acetylcholine receptors on the rat pancreatic gastrin-producing cell line B6 RIN. FEBS Lett 270: 37–40, 1990Google Scholar
  18. 18.
    Mak JC, Barnes PJ: Muscarinic receptor subtypes in human and guinea pig lung. Eur J Pharmacol 164: 223–230, 1989Google Scholar
  19. 19.
    Grynkiewicz G, Poenie M, Tsien RY: A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985Google Scholar
  20. 20.
    Fisher SK: Recognition of muscarinic cholinergic receptors in human SK-N-SH neuroblastoma cells by quaternary and tertiary ligands is dependent upon temperature, cell integrity, and the presence of agonists. Mol Pharmacol 33: 414–422, 1988Google Scholar
  21. 21.
    Baumgartner MK, Wei J, Aronstam RS: Retinoic acid-induced differentiation of a human neuroblastoma cell line alters muscarinic receptor expression. Brain Res Dev Brain Res 72: 305–308, 1993Google Scholar
  22. 22.
    Klapproth H, Reinheimer T, Metzen J, Munch M, Bittinger F, Kirkpatrick CJ, Hohle KD, Schemann M, Racke K, Wessler I: Non-neuronal acetylcholine, a signalling molecule synthezised by surface cells of rat and man. Naunyn-Schmiedebergs Arch Pharmacol 355: 515–523, 1997Google Scholar
  23. 23.
    Sailer M, Oppitz M, Drews U: Induction of cellular contractions in the human melanoma cell line SK-mel 28 after muscarinic cholinergic stimulation. Anat Embryol 201: 27–37, 2000Google Scholar
  24. 24.
    Warren JB: Nitric oxide and human skin blood flow responses to acetylcholine and ultraviolet light. FASEB J 8: 247–251, 1994Google Scholar
  25. 25.
    Buckley NJ, Bonner TI, Brann MR: Localization of a family of muscarinic receptor mRNAs in rat brain. J Neurosci 8: 4646–4652, 1988Google Scholar
  26. 26.
    Maeda A, Kubo T, Mishina M, Numa S: Tissue distribution of mRNAs encoding muscarinic acetylcholine receptor subtypes. FEBS Lett 239: 339–342, 1988Google Scholar
  27. 27.
    Weiner DM, Levey AI, Brann MR: Expression of muscarinic acetylcholine and dopamine receptor mRNAs in rat basal ganglia. Proc Natl Acad Sci USA 87: 7050–7054, 1990Google Scholar
  28. 28.
    Ndoye A, Buchli R, Greenberg B, Nguyen VT, Zia S, Rodriguez JG, Webber RJ, Lawry MA, Grando SA: Identification and mapping of keratinocyte muscarinic acetylcholine receptor subtypes in human epidermis. J Invest Dermatol 111: 100–106, 1998Google Scholar
  29. 29.
    Ishizaka N, Noda M, Yokoyama S, Kawasaki K, Yamamoto M, Higashida H: Muscarinic acetylcholine receptor subtypes in the human iris. Brain Res 787: 344–347, 1998Google Scholar
  30. 30.
    Janossy A, Li JY, Saez JM: Characterisation of the muscarinic receptor subtype M3 in the bovine zona fasciculata-reticularis cells by receptor binding, mRNA and functional studies. J Endocrinol 163: 329–336, 1999Google Scholar
  31. 31.
    Mei L, Roeske WR, Yamamura HI: Molecular pharmacology of muscarinic receptor heterogeneity. Life Sci 45: 1831–1852, 1989Google Scholar
  32. 32.
    Lammerding-Koppel M, Noda S, Blum A, Schaumburg-Lever G, Rassner G, Drews U: Immunohistochemical localization of muscarinic acetylcholine receptors in primary and metastatic malignant melanomas. J Cutan Pathol 24: 137–144, 1997Google Scholar
  33. 33.
    Noda S, Lammerding-Koppel M, Oettling G, Drews U: Characterization of muscarinic receptors in the human melanoma cell line SK-Mel-28 via calcium mobilization. Cancer Lett 133: 107–114, 1998Google Scholar
  34. 34.
    Kohn EC, Alessandro R, Probst J, Jacobs W, Brilley E, Felder CC: Identification and molecular characterization of a m5 muscarinic receptor in A2058 human melanoma cells. Coupling to inhibition of adenylyl cyclase and stimulation of phospholipase A2. J Biol Chem 271: 17476–17484, 1996Google Scholar
  35. 35.
    Sage SO, Adams DJ, van Breemen C: Synchronized oscillations in cytoplasmic free calcium concentration in confluent bradykinin-stimulated bovine pulmonary artery endothelial cell monolayers. J Biol Chem 264: 6–9, 1989Google Scholar
  36. 36.
    Berridge MJ: Calcium oscillations. J Biol Chem 265: 9583–9586, 1990Google Scholar
  37. 37.
    Jacob R: Calcium oscillations in electrically non-excitable cells. Biochim Biophys Acta 1052: 427–438, 1990Google Scholar
  38. 38.
    Rink TJ, Merritt JE: Calcium signalling. Curr Opin Cell Biol 2: 198–205, 1990Google Scholar
  39. 39.
    Tsien RW, Tsien RY: Calcium channels, stores, and oscillations. Annu Rev Cell Biol 6: 715–760, 1990Google Scholar
  40. 40.
    de Roos AD, Willems PH, Peters PH, van Zoelen EJ, Theuvenet AP: Synchronized calcium spiking resulting from spontaneous calcium action potentials in monolayers of NRK fibroblasts. Cell Calcium 22: 195–207, 1997Google Scholar
  41. 41.
    Ishizaka N, Noda M, Kimura Y, Hashii M, Fukuda K, Katayama M, Brown DA, Higashida H: Inositol 1,4,5-trisphosphate formation and ryanodine-sensitive oscillations of cytosolic free Ca2+ concentrations in neuroblastoma × fibroblast hybrid NL308 cells expressing m2 and m4 muscarinic acetylcholine receptor subtypes. Eur J Physiol 429: 426–433, 1995Google Scholar
  42. 42.
    Putney JW Jr: Excitement about calcium signaling in inexcitable cells. Science 262: 676–678, 1993Google Scholar
  43. 43.
    Harootunian AT, Kao JP, Paranjape S, Tsien RY: Generation of calcium oscillations in fibroblasts by positive feedback between calcium and IP3. Science 251: 75–78, 1991Google Scholar
  44. 44.
    Cuthbertson KS, Cobbold PH: Phorbol ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca2+. Nature 316: 541–542, 1985Google Scholar
  45. 45.
    Fujii R, Miyashita Y: Receptor mechanisms in fish chromatophores - III. Neurally controlled melanosome aggregation in a siluroid (Parasilurus asotus) is strangely mediated by cholinoceptors. Comp Biochem Physiol C 55: 43–49, 1976Google Scholar
  46. 46.
    Fujii R, Miyashita Y, Fujii Y: Muscarinic cholinoceptors mediate neurally evoked pigment aggregation in glass catfish melanophores. J Neural Transm 54: 29–39, 1982Google Scholar
  47. 47.
    Hayashi H, Fujii R: Muscarinic cholinoceptors that mediate pigment aggregation are present in the melanophores of cyprinids (Zacco spp.). Pigment Cell Res 6: 37–44, 1993Google Scholar
  48. 48.
    Hayashi H, Fujii R: Pharmacological profiles of the subtypes of muscarinic cholinoceptors that mediate aggregation of pigment in the melanophores of two species of catfish. Pigment Cell Res 7: 175–183, 1994Google Scholar
  49. 49.
    Ovais M: Control of melanosome movements in isolated skin melanophores of a catfish Clarias batrachus (Linn.). Indian J Physiol Pharmacol 38: 185–188, 1994Google Scholar
  50. 50.
    Ali AS, Peter J, Ali SA: Role of cholinergic receptors in melanophore responses of amphibians. Acta Biol Hung 46: 61–73, 1995Google Scholar
  51. 51.
    Moller H, Lerner AB: Melanocyte stimulating hormone inhibition by acetylcholine and noradrenaline in the frog skin bioassay. Acta Endocrinol 51: 149–160, 1966Google Scholar
  52. 52.
    Lamacz M, Tonon MC, Louiset E, Cazin L, Strosberg D, Vaudry H: Acetylcholine stimulates alpha-melanocyte-stimulating hormone release from frog pituitary melanotrophs through activation of muscarinic and nicotinic receptors. Endocrinology 125: 707–714, 1989Google Scholar
  53. 53.
    Zhao H, Boissy RE, Nordlung JJ: Down-regulation of human melanogenesis by acetylcholine in culture. J Invest Dermatol 106: 910, 1996Google Scholar
  54. 54.
    Schallreuter KU, Wood JM, Pittelkow MR, Buttner G, Swanson N, Korner C, Ehrke C: Increased monoamine oxidase A activity in the epidermis of patients with vitiligo. Arch Dermatol Res 288: 14–18, 1996Google Scholar
  55. 55.
    Schallreuter KU, Pittelkow MR: Defective calcium uptake in keratinocyte cell cultures from vitiliginous skin. Arc Dermatol Res 280: 137–139, 1988Google Scholar
  56. 56.
    Buchli R, Ndoye A, Slominski A, Grando SA: Cholinergic control of melanogenesis. J Invest Dermatol 114: 859, 2000Google Scholar

Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • Rico Buchli
    • 1
  • Assane Ndoye
    • 1
  • Juan Arredondo
    • 1
  • Robert J. Webber
    • 2
  • Sergei A. Grando
    • 1
  1. 1.Department of DermatologyUniversity of CaliforniaDavisUSA
  2. 2.Research and Diagnostic AntibodiesRichmondUSA

Personalised recommendations