scholarly journals Ano1 is a better marker than c-Kit for transcript analysis of single interstitial cells of Cajal in culture

2014 ◽  
Vol 19 (4) ◽  
Author(s):  
Raúl Loera-Valencia ◽  
Xuan-Yu Wang ◽  
George Wright ◽  
Carlos Barajas-López ◽  
Jan Huizinga
Keyword(s):  
Single Cell ◽  
Interstitial Cells Of Cajal ◽  
Expression Patterns ◽  
Mrna Level ◽  
Primary Cultures ◽  
Interstitial Cells ◽  
Mrna Analysis ◽  
Cells Of Cajal ◽  
Cell Expression ◽  
The Relationship

AbstractThe interstitial cells of Cajal (ICC) drive the slow wave-associated contractions in the small intestine. A commonly used marker for these cells is c-Kit, but another marker named Ano1 was recently described. This study uses single-cell RT-PCR, qPCR and immunohistochemistry to determine if Ano1 could be reliably used as a molecular marker for ICC in single-cell mRNA analysis. Here, we report on the relationship between the expression of c-Kit and Ano1 in single ICC in culture. We observed that Ano1 is expressed in more than 60% of the collected cells, whereas c-Kit is found only in 22% of the cells (n = 18). When we stained ICC primary cultures for c-KIT and ANO1 protein, we found complete co-localization in all the preparations. We propose that this difference is due to the regulation of c-Kit mRNA in culture. This regulation gives rise to low levels of its transcript, while Ano1 is expressed more prominently in culture on day 4. We also propose that Ano1 is more suitable for single-cell expression analysis as a marker for cell identity than c-Kit at the mRNA level. We hope this evidence will help to validate and increase the success of future studies characterizing single ICC expression patterns.

scholarly journals Ca2+ signaling driving pacemaker activity in submucosal interstitial cells of Cajal in the murine colon

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Salah A Baker ◽  
Wesley A Leigh ◽  
Guillermo Del Valle ◽  
Inigo F De Yturriaga ◽  
Sean M Ward ◽  
...  
Keyword(s):  
Interstitial Cells Of Cajal ◽  
Colonic Motility ◽  
External Solution ◽  
Interstitial Cells ◽  
Pacemaker Activity ◽  
Voltage Dependent ◽  
Rhythmic Firing ◽  
Complex Patterns ◽  
Cells Of Cajal ◽  
The Relationship

Interstitial cells of Cajal (ICC) generate pacemaker activity responsible for phasic contractions in colonic segmentation and peristalsis. ICC along the submucosal border (ICC-SM) contribute to mixing and more complex patterns of colonic motility. We show the complex patterns of Ca2+ signaling in ICC-SM and the relationship between ICC-SM Ca2+ transients and activation of SMCs using optogenetic tools. ICC-SM displayed rhythmic firing of Ca2+ transients ~15 cpm and paced adjacent SMCs. The majority of spontaneous activity occurred in regular Ca2+ transients clusters (CTCs) that propagated through the network. CTCs were organized and dependent upon Ca2+ entry through voltage-dependent Ca2+ conductances, L- and T-type Ca2+ channels. Removal of Ca2+ from the external solution abolished CTCs. Ca2+ release mechanisms reduced the duration and amplitude of Ca2+ transients but did not block CTCs. These data reveal how colonic pacemaker ICC-SM exhibit complex Ca2+ firing patterns and drive smooth muscle activity and overall colonic contractions.

Immunomagnetic enrichment of interstitial cells of Cajal

2004 ◽  
Vol 286 (2) ◽  
pp. G351-G360 ◽  
Author(s):  
Tamás Ördög ◽  
Doug Redelman ◽  
Nancy N. Horowitz ◽  
Kenton M. Sanders
Keyword(s):  
Interstitial Cells Of Cajal ◽  
Primary Cultures ◽  
Cell Types ◽  
Interstitial Cells ◽  
Functional Studies ◽  
Immunomagnetic Enrichment ◽  
Small Intestinal ◽  
Cells Of Cajal ◽  
Fluorescent Dextran ◽  
Spontaneous Rhythmicity

Disruptions 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 ∼63-fold and ∼8-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 Ca2+ 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.

Sodium current in human intestinal interstitial cells of Cajal

2003 ◽  
Vol 285 (6) ◽  
pp. G1111-G1121 ◽  
Author(s):  
Peter R. Strege ◽  
Yijun Ou ◽  
Lei Sha ◽  
Adam Rich ◽  
Simon J. Gibbons ◽  
...  
Keyword(s):  
Single Cell ◽  
Slow Wave ◽  
Interstitial Cells Of Cajal ◽  
Sodium Current ◽  
Interstitial Cells ◽  
Wave Frequency ◽  
Rt Pcr ◽  
Α Subunit ◽  
Channel Current ◽  
Cells Of Cajal

Interstitial cells of Cajal (ICC) generate the electrical slow wave required for normal gastrointestinal motility. The ionic conductances expressed in human intestinal ICC are unknown. The aim of this study was to determine expression of a Na+ current in human intestinal ICC and to determine the effects of the Na+ current on the slow wave. Visually identified, freshly dissociated, single ICC were verified by the presence of c- kit mRNA by using single-cell RT-PCR. Standard whole cell currents were recorded from patch-clamped ICC held at -100 mV between pulse protocols. A Na+ current was identified in human intestinal ICC. The current activated at -55 mV and peaked at -30 mV. Extracellular N-methyl-d-glucamine abolished and QX-314 (500 μM) blocked the Na+ current, but nifedipine and Ni2+ did not. The Na+ current was activated by shear stress. Single-cell RT-PCR detected mRNA for the Na+ α-subunit SCN5A in single human intestinal ICC. Lidocaine (200 μm) and QX-314 (500 μM) decreased slow wave frequency, and stretch increased slow wave frequency. A mechanosensitive Na+ channel current is present in human intestinal ICC and appears to play a role in the control of intestinal motor function.

Sodium current in human small intestinal interstitial cells of cajal

2001 ◽  
Vol 120 (5) ◽  
pp. A201-A201 ◽  
Author(s):  
P STREGE ◽  
A RICH ◽  
Y OU ◽  
S GIBBONS ◽  
M SARR ◽  
...  
Keyword(s):  
Interstitial Cells Of Cajal ◽  
Sodium Current ◽  
Interstitial Cells ◽  
Small Intestinal ◽  
Cells Of Cajal
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