title: Selective labeling and isolation of functional classes of interstitial cells of Cajal of human and murine small intestine.
abstract: Specific functions of interstitial cells of Cajal (ICC) have been linked to distinct classes that differ by morphology and distribution. In the small intestine, slow wave-generating ICC are located in the myenteric region (ICC-MY), whereas ICC that mediate neuromuscular neurotransmission occur either throughout the circular muscle layer (intramuscular ICC, ICC-IM) or in association with the deep muscular plexus (ICC-DMP). Selective isolation of ICC to characterize specific properties has been difficult. Recently, neurokinin-1 receptors have been detected in murine ICC-DMP and neurons but not in ICC-MY. Here we identified and isolated ICC-DMP/IM by receptor-mediated internalization of fluorescent substance P and Kit immunofluorescence. Specificity of labeling was verified by confocal microscopy. Mouse and human ICC-DMP/IM were detected in suspension by fluorescent microscopy and harvested for RT-PCR with micropipettes. The isolated cells expressed Kit but not markers for neurons, smooth muscle, or antigen-presenting cells. ICC-DMP expressed neurokinin-1 receptor, M(2) and M(3) muscarinic receptors, P2Y(1) and P2Y(4) purinergic receptors, VIP receptor 2, soluble guanylate cyclase-1 subunits, and protein kinase G. L- or T-type Ca(2+) channels were not detected in these cells. ICC-MY and ICC-DMP were simultaneously detected and enumerated by flow cytometry and sorted to purity by fluorescence-activated cell sorting. In summary, functional classes of ICC have distinct molecular identities that can be used to selectively identify and harvest these cells with, for example, receptor-mediated uptake of substance P and Kit immunofluorescence. ICC-DMP express neurotransmitter receptors and signaling intermediate molecules that are consistent with their role in neuromuscular neurotransmission.
authors.affiliation: Department of Physiology and Cell Biology, School of Medicine, University of Nevada-Reno, Reno, NV 89557, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.forename: Doug
authors.initials: D
authors.lastname: Redelman
authors.forename: Seungil
authors.initials: S
authors.lastname: Ro
authors.forename: Sean M
authors.initials: SM
authors.lastname: Ward
authors.forename: Tamás
authors.initials: T
authors.lastname: Ordög
authors.forename: Kenton M
authors.initials: KM
authors.lastname: Sanders
mesh_terms: D000818:Animals; D002469:Cell Separation; D002477:Cells / Q000145:classification; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007421:Intestine, Small / Q000166:cytology* / Q000378:metabolism / Q000502:physiology*; D051379:Mice / Q000033:anatomy & histology*; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000378:metabolism; D017981:Receptors, Neurotransmitter / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013373:Substance P / Q000378:metabolism
chemical_list: D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter; D013373:Substance P; D019009:Proto-Oncogene Proteins c-kit