Human skin fibroblasts express m2, m4, and m5 subtypes of muscarinic acetylcholine receptors

Previous studies have demonstrated that muscarinic acetylcholine receptors (mAChRs) are expressed by human skin fibroblasts (HSF). We have identified the molecular subtypes of these receptors by reverse transcription-polymera se chain reaction (RT-PCR), using m1-m5 subtype-specific primers. These exp eriments showed that only mRNAs for m2, m4, and m5 mAChR subtypes are prese nt in HSF. The RT-PCR products were characterized by restriction analysis a nd Southern blotting. Northern blot analysis showed the presence of m2 and m4 mAChR RNA. Rabbit antibodies were raised using a synthetic peptide as im munogen corresponding to the C-terminus of the m2 protein and were used to visualize fibroblast mAChRs. Cell membranes of HSF in cell culture and spec imens of normal human skin had a unique staining pattern specific for anti- m2 antibody, as well as for antibodies against m4 and m5. In Western blots of fibroblast proteins, the antibodies visualized the m2 receptor at 65 kDa , m4 at 70 kDa, and m5 at 95 kDa. The function of fibroblast mAChRs was exa mined by measuring muscarinic effects on intracellular free Ca2+ concentrat ion ([Ca2+](i)). Muscarine increased transiently [Ca2+](i) in cultured HSF. This effect could be abolished by the muscarinic antagonist atropine; Thus , the results of this study showed that HSF express m2, m4, and m5 mAChR su btypes, and that fibroblast mAChRs are coupled to the regulation of [Ca2+]( i). J. Cell. Biochem. 74:264-277, 1999. (C) 1999 Wiley-Liss, Inc.