Spontaneous oscillations in intracellular Ca2+ concentration ([Ca2+]i) and membrane potential were used to monitor rhythmicity in freshly dispersed and cultured interstitial cells (IC) from the canine colon. The frequency of oscillations and responses to a number of channel blockers, agonists, ionic substitutions, and temperature were similar in freshly dispersed and cultured cells. An increase in the amplitude of Ca2+ oscillations after 3-6 days in culture and an increase in the rate of decline of [Ca2+]i in cultured IC were two differences noted between freshly dispersed and cultured cells. The frequency and amplitude of oscillations were a function of extracellular Ca2+ concentrations, and oscillations were abolished when the transmembrane flux of Ca2+ was reduced by nicardipine, La3+, or removal of Ca2+ from the extracellular medium. Oscillations persisted in the presence of ryanodine and ouabain. Lowered temperatures or a reduction in the concentration of Ca2+ in the medium reduced the frequency of spontaneous oscillations. Carbachol and substance P caused a transient increase in [Ca2+]i. Substance P then abolished spontaneous events. ATP and calcitonin gene-related peptide increased the frequency of spontaneous activity. Vasoactive intestinal peptide caused a temporary delay in spontaneous oscillations when added to the medium. Results indicate that freshly dispersed and cultured IC may be useful in studies of the mechanisms of rhythmicity in the gastrointestinal system.
ruptions of networks of interstitial cells of Cajal (ICC), gastrointestinal pacemakers and mediators of neurotransmission, can lead to disordered phasic contractions and peristalsis by reducing and uncoupling electrical slow waves. However, detailed analysis of the ICC network behavior has been hampered by their scarcity, limited accessibility in intact tissues, and contamination with other cell types in culture. Our goal was to develop a simple technique to purify ICC from murine gastrointestinal muscles for functional studies. We identified ICC in live small intestinal muscles or primary cell cultures by Kit immunoreactivity using fluorescent antibodies. Because this technique also labels resident macrophages nonspecifically, parallel studies were performed in which nonfluorescent Kit antibodies and macrophages labeled with fluorescent dextran were used for subtractive analysis of ICC. In both groups, Kit-positive cells were tagged with superparamagnetic antibodies and sorted on magnetic columns. Efficacy was assessed by flow cytometry. ICC enrichment from primary cultures and freshly dissociated tissues was ϳ63-fold and ϳ8-fold, respectively. Unlike the cells derived directly from tissues, cells sorted from cultures frequently yielded extensive, nearly homogenous ICC networks on reseeding. Monitoring oscillations in mitochondrial Ca 2ϩ or membrane potential by imaging revealed spontaneous rhythmicity in these networks. Cells that did not bind to the columns yielded cultures that were depleted of ICC and dominated by smooth muscle cells. In conclusion, immunomagnetic sorting of primary cultures of ICC results in relatively homogenous, functional ICC networks. This technique is less suitable for obtaining ICC from freshly dispersed cells. mouse; Kit; macrophage; pacemaking; fluorescent imaging INTERSTITIAL CELLS OF CAJAL (ICC) are specialized mesenchymal cells located within the tunica muscularis of the gastrointestinal (GI) tract that play critical roles in GI motor functions and their regulation (32). Multipolar ICC, which form two-dimensional networks in the myenteric region, on the submucosal border of the circular smooth muscle layer, and within intermuscular septa of phasic GI smooth muscles, play an essential role in phasic contractile activity and peristalsis by generating and propagating electrical slow waves (4,6,12,13,24,26, 42). Elongated or bipolar ICC, which are more loosely distributed within bundles of smooth muscle cells (where they form close contacts with varicose processes of enteric motor neurons), mediate excitatory and inhibitory neural inputs to the smooth musculature (45), may amplify slow waves (5), and contribute to vagally mediated mechanoreception (9).The discovery that ICC can be identified by expression of the receptor tyrosine kinase Kit [stem cell factor (SCF) receptor, CD117] (13, 21, 39, 42) has led to several studies on the morphology of ICC networks in human GI disorders. Damages to ICC networks have been described in congenital and acquired GI disorders including anorectal ...
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