Molecular and Cellular Biochemistry

, Volume 228, Issue 1, pp 57–72

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

DOI: 10.1023/A:1013368509855

Cite this article as:
Buchli, R., Ndoye, A., Arredondo, J. et al. Mol Cell Biochem (2001) 228: 57. doi:10.1023/A:1013368509855

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 drugsindirect immunofluorescenceintracellular free calciumreverse transcription-polymerase chain reactionradioligand binding

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