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1 Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada, United States
2 Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada, United States; Reno, Nevada, United States
3 Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada, United States; Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada, United States
4 University of Nevada; Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada, United States; University of Nevada, Reno, Nevada, United States
5 Dept. of Physiology & Cell Biology/352, University of Nevada School of Medicine, Reno, Nevada, United States
* To whom correspondence should be addressed. E-mail: kent{at}unr.edu.
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 using micropipettes. The isolated cells expressed Kit but not markers for neurons, smooth muscle or antigen-presenting cells. ICC-DMP expressed neurokinin-1 receptor, M2 and M3 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 Ca2+ 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 using, for example, receptor-mediated uptake of substance P and Kit immunofluorescence. ICC-DMP express neurotransmitter receptors and signaling intermediate molecules that is consistent with their role in neuromuscular neurotransmission.
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