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RECEPTORS AND SIGNAL TRANSDUCTION

Selective labeling and isolation of functional classes of interstitial cells of Cajal of human and murine small intestine

¹Department of Physiology and Cell Biology and ²Cytometry Center, University of Nevada, and ³Sierra Cytometry, Reno, Nevada

Submitted 31 March 2006 ; accepted in final form 16 August 2006

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, M₂ and M₃ muscarinic receptors, P2Y₁ and P2Y₄ 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 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.

Kit; substance P; neurokinin-1 receptor; flow cytometry; RT-PCR

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