Human Skin Fibroblasts Express m2, m4, and m5 Subtypes of M uscarinic Acetylcholine Receptors Rico Buchli, 1 Assane Ndoye, 1 John G. Rodriguez, 2 Shaheen Zia, 1 Robert J. Webber, 2 and Sergei A. Grando 1 * 1 Department of Dermatology, University of California, Davis, California 95816 2 Research and Diagnostic Antibodies, Richmond, California 94806 Abstract Previousstudieshave 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- polymerase chain reaction (RT-PCR), using m1-m5 subtype-specific primers. These experiments showed that only mRNAsfor m2, m4, and m5 mAChRsubtypesare present in HSF. The RT-PCRproductswere characterized by restriction analysis and Southern blotting. Northern blot analysis showed the presence of m2 and m4 mAChR RNA. Rabbit antibodies were raised using a synthetic peptide as immunogen corresponding to the C-terminus of the m2 protein and were used to visualize fibroblast mAChRs. Cell membranesof HSFin cell culture and specimensof normal human skin had a unique staining pattern specific for anti-m2 antibody, aswell asfor antibodiesagainst m4 and m5. In Western blots of fibroblast proteins, the antibodiesvisualized the m2 receptor at 65 kDa, m4 at 70 kDa, and m5 at 95 kDa. The function of fibroblast mAChRs was examined by measuring muscarinic effects on intracellular free Ca 2+ concentration ([Ca 2+ ] i ). Muscarine increased transiently [Ca 2+ ] i in cultured HSF. This effect could be abolished by the muscarinic antagonist atropine. Thus, the resultsof thisstudy showed that HSFexpressm2, m4, and m5 mAChRsubtypes, and that fibroblast mAChRsare coupled to the regulation of [Ca 2+ ] i . J. Cell. Biochem. 74:264–277, 1999.  1999 Wiley-Liss, Inc. Key words: human skin fibroblasts; muscarinic acetylcholine receptors; reverse transcriptase-polymerase chain reac- tion; Northern blot; anti-peptide antibody production; Western blot; indirect immunofluorescence; intracellular free calcium concentration Muscarinic acetylcholine receptors (mAChRs) are present in a wide variety of tissues in which they modulate important physiological re- sponses. The mAChRs are members of a large family of receptors that mediate signal trans- duction by coupling with heterotrimeric gua- nine nucleotide-binding proteins, or G-pro- teins, which modulate the activity of a number of effector enzymes in addition to regulating ion channels directly, leading to increases or de- creases in second messengers or changes in ion concentrations [reviewed by Nathanson, 1987]. Each mAChR subtype is a single glycoprotein molecule that spans the plasma membrane seven times, resulting in seven hydrophobic transmembrane domains (TM1-TM7) connected by three extracellular (e1-e3) and three intracel- lular (i1-i3) hydrophilic loops. Through molecu- lar cloning, five human subtypes of mAChRs (m1-m5) have been identified to date [Allard et al., 1987; Bonner et al., 1987, 1988; Peralta et al., 1987a,b], all of which lack introns in their coding sequences. The amino acid sequences of these receptors are highly conserved, particu- larly within transmembrane domains. The re- gion of the least homology occurs in the cytoplas- mic loop i3 located between TM5 and TM6. These mAChR subtypes are generally grouped according to their functional coupling either to the mobilization of intracellular calcium (m1, m3, and m5) through the activation of phospho- lipase C, which results in the release of the second messenger inositol 1,4,5-trisphosphate (IP 3 ), or by the inhibition of adenylyl cyclase (m2 and m4), which results in the reduction of the intracellular levels of cyclic adenosine mono- phosphate [reviewed by Hulme et al., 1990; and Hosey, 1992]. Grant sponsor: Unilever Research-USA; Grant sponsor: NIH; Grant number: R29 AR42955. *Correspondence to: Sergei A. Grando MD, PhD, DSci, Department of Dermatology, University of California, Davis, 4860 Y Street, #3400, Sacramento, CA 95817. E-mail: sagrando@ ucdavis.edu Received 16 November 1998; Accepted 16 February 1999 Journal of Cellular Biochemistry 74:264–277 (1999)  1999 Wiley-Liss, Inc.