scholarly journals Expression of the Regulated Isoform of the Electrogenic Na+/HCO3- Cotransporter, NBCe1, is Enriched in Pacemaker Interstitial Cells of Cajal

2020 ◽  
Author(s):  
Maria-Gabriela Colmenares Aguilar ◽  
Amelia Mazzone ◽  
Seth T Eisenman ◽  
Peter R Strege ◽  
Cheryl E Bernard ◽  
...  
Keyword(s):  
Small Intestine ◽  
Slow Wave ◽  
Interstitial Cells Of Cajal ◽  
Interstitial Cells ◽  
Transcriptional Analysis ◽  
Pacemaker Activity ◽  
Ip3 Receptor ◽  
Gene Transcripts ◽  
Tunica Muscularis ◽  
Cells Of Cajal

Interstitial cells of Cajal (ICC) generate electrical slow waves, which are required for normal gastrointestinal motility. The mechanisms for generation of normal pacemaking are not fully understood. Normal gastrointestinal contractility and electrical slow wave activity depend on the presence of extracellular HCO3-. Previous transcriptional analysis identified enrichment of mRNA encoding the electrogenic Na+/HCO3- cotransporter (NBCe1) gene (Slc4a4) in pacemaker myenteric ICC in mouse small intestine. We aimed to determine the distribution of NBCe1 protein in ICC of the mouse gastrointestinal tract, and to identify the transcripts of the Slc4a4 gene in mouse and human small intestinal tunica muscularis. We determined the distribution of NBCe1-immunoreactivity (NBCe1-IR) by immunofluorescent labeling in mouse and human tissues. In mice, NBCe1-IR was restricted to Kit-positive myenteric ICC of the stomach and small intestine and sub-muscular ICC of the large intestine; that is the slow wave generating subset of ICC. Other sub-types of ICC were NBCe1-negative. Quantitative real time PCR identified >500 fold enrichment of Slc4a4‑207 and Slc4a4‑208 transcripts (IP3-receptor binding protein released by IP3" (IRBIT) regulated isoforms) in Kit expressing cells isolated from KitcreERT2/+, Rpl22tm1.1Psam/Sj mice and from single GFP-positive ICC from Kittm1Rosay mice. Human jejunal tunica muscularis ICC were also NBCe1-positive and SLC4A4‑201 and SLC4A4‑204 RNAs were >300 fold enriched relative to SLC4A4‑202. In summary, NBCe1 protein expressed in ICC with electrical pacemaker function is encoded by Slc4a4 gene transcripts that generate IRBIT regulated isoforms of NBCe1. In conclusion Na+/HCO3- cotransport through NBCe1 contributes to the generation of pacemaker activity in subsets of ICC.

scholarly journals Effects of ATP on Pacemaker Activity of Interstitial Cells of Cajal from the Mouse Small Intestine

2018 ◽  
Vol 54 (1) ◽  
pp. 63
Author(s):  
Il Koo Park ◽  
Jin Ho Kim ◽  
Chan Guk Park ◽  
Man Yoo Kim ◽  
Shankar Prasad Parajuli ◽  
...  
Keyword(s):  
Small Intestine ◽  
Interstitial Cells Of Cajal ◽  
Interstitial Cells ◽  
Pacemaker Activity ◽  
Mouse Small Intestine ◽  
Cells Of Cajal

Action potential generation in the small intestine of W mutant mice that lack interstitial cells of Cajal

1996 ◽  
Vol 271 (3) ◽  
pp. G387-G399 ◽  
Author(s):  
J. Malysz ◽  
L. Thuneberg ◽  
H. B. Mikkelsen ◽  
J. D. Huizinga
Keyword(s):  
Small Intestine ◽  
Action Potentials ◽  
Interstitial Cells Of Cajal ◽  
Interstitial Cells ◽  
K Channel ◽  
Pacemaker Activity ◽  
Channel Blockade ◽  
Auerbach’S Plexus ◽  
Cells Of Cajal ◽  
Auerbach's Plexus

The small intestine of W/Wv mice lacks both the network of interstitial cells of Cajal (ICC), associated with Auerbach's plexus, and pacemaker activity, i.e., it does not generate slow-wave-type action potentials. The W/Wv muscle preparations showed a wide variety of electrical activities, ranging from total quiescence to generation of action potentials at regular or irregular frequency with or without periods of quiescence. The action potentials consisted of a slow component with superimposed spikes, preceded by a slowly developing depolarization and followed by a transient hyperpolarization. The action potentials were completely abolished by L-type Ca2+ channel blockers. W/Wv mice responded to K+ channel blockade (0.5 mM Ba2+ or 10 mM tetraethylammonium chloride) with effects on amplitude, frequency, rate of rise, and duration of the action potentials. In quiescent tissues from W/Wv mice, K+ channel blockade evoked the typical spikelike action potentials. Electron microscopy identified few methylene blue-positive cells in the W/Wv small intestine associated with Auerbach's plexus as individual ICC. Numbers of resident macrophage-like cells (MLC) and fibroblast-like cells (FLC) were significantly changed. Neither FLC nor MLC were part of a network nor did they form specialized junctions with neighboring cells as ICC do. Hence no cell type had replaced ICC at their normal morphological position associated with Auerbach's plexus. ICC were present in W/Wv mice at the deep muscular plexus in normal organization and numbers, indicating that they are not dependent on the Kit protein and do not take part in generation of pacemaker activity.

scholarly journals Molecular markers expressed in cultured and freshly isolated interstitial cells of Cajal

2000 ◽  
Vol 279 (2) ◽  
pp. C529-C539 ◽  
Author(s):  
Anne Epperson ◽  
William J. Hatton ◽  
Brid Callaghan ◽  
Philip Doherty ◽  
Rebecca L. Walker ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Small Intestine ◽  
Interstitial Cells Of Cajal ◽  
Expression Profiles ◽  
Interstitial Cells ◽  
Gastric Fundus ◽  
Soluble Form ◽  
Pacemaker Activity ◽  
Murine Small Intestine ◽  
Cells Of Cajal

Located within the tunica muscularis of the gastrointestinal (GI) tract are networks of cells known as interstitial cells of Cajal (ICC). ICC are critical for important basic functions of GI motility such as generation and propagation of slow-wave pacemaker activity and reception of regulatory inputs from the enteric nervous system. We have developed a novel procedure to identify and isolate individual ICC from freshly dispersed cell preparations of the murine small intestine and gastric fundus and to determine differential transcriptional expression We have compared the expression profiles of pacemaker ICC isolated from the murine small intestine (IC-MY) and ICC involved in neurotransmission from the gastric fundus (IC-IM). We have also compared expression profiles between ICC and smooth muscle cells (SMC) and between freshly isolated ICC and cultured ICC. Cultured ICC express smooth muscle myosin, whereas freshly dispersed ICC do not. All cell types express muscarinic receptor types M2and M3, neurokinin receptors NK1and NK3, and inhibitory receptor VIP-1, whereas only cultured ICC and SMC express VIP-2. Both cultured and freshly dispersed IC-IM and IC-MY express the soluble form of stem cell factor, whereas SMC from the gastric fundus express only the membrane-bound form.

scholarly journals Capsaicin Inhibits the Spontaneous Pacemaker Activity in Interstitial Cells of Cajal From the Small Intestine of Mouse

2010 ◽  
Vol 16 (3) ◽  
pp. 265-273 ◽  
Author(s):  
Seok Choi ◽  
Jae Myeong Sun ◽  
Pawan Kumar Shahi ◽  
Dong Chuan Zuo ◽  
Hyun Il Kim ◽  
...  
Keyword(s):  
Small Intestine ◽  
Interstitial Cells Of Cajal ◽  
Interstitial Cells ◽  
Pacemaker Activity ◽  
Cells Of Cajal

scholarly journals Intracellular Ca2+ release from endoplasmic reticulum regulates slow wave currents and pacemaker activity of interstitial cells of Cajal

2015 ◽  
Vol 308 (8) ◽  
pp. C608-C620 ◽  
Author(s):  
Mei Hong Zhu ◽  
Tae Sik Sung ◽  
Kate O'Driscoll ◽  
Sang Don Koh ◽  
Kenton M. Sanders
Keyword(s):  
Slow Wave ◽  
Interstitial Cells Of Cajal ◽  
Ryanodine Receptors ◽  
Interstitial Cells ◽  
Pacemaker Activity ◽  
Current Clamp ◽  
Anoctamin 1 ◽  
Intracellular Ca ◽  
Inward Currents ◽  
Cells Of Cajal

Interstitial cells of Cajal (ICC) provide pacemaker activity in gastrointestinal muscles that underlies segmental and peristaltic contractions. ICC generate electrical slow waves that are due to large-amplitude inward currents resulting from anoctamin 1 (ANO1) channels, which are Ca2+-activated Cl− channels. We investigated the hypothesis that the Ca2+ responsible for the stochastic activation of ANO1 channels during spontaneous transient inward currents (STICs) and synchronized activation of ANO1 channels during slow wave currents comes from intracellular Ca2+ stores. ICC, obtained from the small intestine of Kit +/copGFP mice, were studied under voltage and current clamp to determine the effects of blocking Ca2+ uptake into stores and release of Ca2+ via inositol 1,4,5-trisphosphate (IP3)-dependent and ryanodine-sensitive channels. Cyclocpiazonic acid, thapsigargin, 2-APB, and xestospongin C inhibited STICs and slow wave currents. Ryanodine and tetracaine also inhibited STICs and slow wave currents. Store-active compounds had no direct effects on ANO1 channels expressed in human embryonic kidney-293 cells. Under current clamp, store-active drugs caused significant depolarization of ICC and reduced spontaneous transient depolarizations (STDs). After block of ryanodine receptors with ryanodine and tetracaine, repolarization did not restore STDs. ANO1 expressed in ICC has limited access to cytoplasmic Ca2+ concentration, suggesting that pacemaker activity depends on Ca2+ dynamics in restricted microdomains. Our data from studies of isolated ICC differ somewhat from studies on intact muscles and suggest that release of Ca2+ from both IP3 and ryanodine receptors is important in generating pacemaker activity in ICC.

scholarly journals Conditional genetic deletion of Ano1 in interstitial cells of Cajal impairs Ca2+ transients and slow waves in adult mouse small intestine

2017 ◽  
Vol 312 (3) ◽  
pp. G228-G245 ◽  
Author(s):  
John Malysz ◽  
Simon J. Gibbons ◽  
Siva A. Saravanaperumal ◽  
Peng Du ◽  
Seth T. Eisenman ◽  
...  
Keyword(s):  
Small Intestine ◽  
Slow Wave ◽  
Interstitial Cells Of Cajal ◽  
Adult Mouse ◽  
Rank Order ◽  
Interstitial Cells ◽  
Slow Waves ◽  
Functional Reserve ◽  
Mouse Small Intestine ◽  
Cells Of Cajal

Myenteric plexus interstitial cells of Cajal (ICC-MY) in the small intestine are Kit+ electrical pacemakers that express the Ano1/TMEM16A Ca2+-activated Cl– channel, whose functions in the gastrointestinal tract remain incompletely understood. In this study, an inducible Cre-LoxP-based approach was used to advance the understanding of Ano1 in ICC-MY of adult mouse small intestine. KitCreERT2/+;Ano1Fl/Fl mice were treated with tamoxifen or vehicle, and small intestines (mucosa free) were examined. Quantitative RT-PCR demonstrated ~50% reduction in Ano1 mRNA in intestines of conditional knockouts (cKOs) compared with vehicle-treated controls. Whole mount immunohistochemistry showed a mosaic/patchy pattern loss of Ano1 protein in ICC networks. Ca2+ transients in ICC-MY network of cKOs displayed reduced duration compared with highly synchronized controls and showed synchronized and desynchronized profiles. When matched, the rank order for Ano1 expression in Ca2+ signal imaged fields of view was as follows: vehicle controls>>>cKO(synchronized)>cKO(desynchronized). Maintenance of Ca2+ transients’ synchronicity despite high loss of Ano1 indicates a large functional reserve of Ano1 in the ICC-MY network. Slow waves in cKOs displayed reduced duration and increased inter-slow-wave interval and occurred in regular- and irregular-amplitude oscillating patterns. The latter activity suggested ongoing interaction by independent interacting oscillators. Lack of slow waves and depolarization, previously reported for neonatal constitutive knockouts, were also seen. In summary, Ano1 in adults regulates gastrointestinal function by determining Ca2+ transients and electrical activity depending on the level of Ano1 expression. Partial Ano1 loss results in Ca2+ transients and slow waves displaying reduced duration, while complete and widespread absence of Ano1 in ICC-MY causes lack of slow wave and desynchronized Ca2+ transients. NEW & NOTEWORTHY The Ca2+-activated Cl− channel, Ano1, in interstitial cells of Cajal (ICC) is necessary for normal gastrointestinal motility. We knocked out Ano1 to varying degrees in ICC of adult mice. Partial knockout of Ano1 shortened the widths of electrical slow waves and Ca2+ transients in myenteric ICC but Ca2+ transient synchronicity was preserved. Near-complete knockout was necessary for transient desynchronization and loss of slow waves, indicating a large functional reserve of Ano1 in ICC. View this article's corresponding video summary at https://youtu.be/cyPtDP0KLY4 .

scholarly journals Na+-K+-Cl− cotransporter (NKCC) maintains the chloride gradient to sustain pacemaker activity in interstitial cells of Cajal

2016 ◽  
Vol 311 (6) ◽  
pp. G1037-G1046 ◽  
Author(s):  
Mei Hong Zhu ◽  
Tae Sik Sung ◽  
Masaaki Kurahashi ◽  
Lauren E. O'Kane ◽  
Kate O'Driscoll ◽  
...  
Keyword(s):  
Slow Wave ◽  
Interstitial Cells Of Cajal ◽  
High Current Density ◽  
Reversal Potential ◽  
Interstitial Cells ◽  
Slow Waves ◽  
Hek293 Cells ◽  
Pacemaker Activity ◽  
Inward Currents ◽  
Cells Of Cajal

Interstitial cells of Cajal (ICC) generate electrical slow waves by coordinated openings of ANO1 channels, a Ca2+-activated Cl− (CaCC) conductance. Efflux of Cl− during slow waves must be significant, as there is high current density during slow-wave currents and slow waves are of sufficient magnitude to depolarize the syncytium of smooth muscle cells and PDGFRα+ cells to which they are electrically coupled. We investigated how the driving force for Cl− current is maintained in ICC. We found robust expression of Slc12a2 (which encodes an Na+-K+-Cl− cotransporter, NKCC1) and immunohistochemical confirmation that NKCC1 is expressed in ICC. With the use of the gramicidin permeabilized-patch technique, which is reported to not disturb [Cl−]i, the reversal potential for spontaneous transient inward currents ( ESTICs) was −10.5 mV. This value corresponds to the peak of slow waves when they are recorded directly from ICC in situ. Inhibition of NKCC1 with bumetanide shifted ESTICs to more negative potentials within a few minutes and reduced pacemaker activity. Bumetanide had no direct effects on ANO1 or CaV3.2 channels expressed in HEK293 cells or L-type Ca2+ currents. Reducing extracellular Cl− to 10 mM shifted ESTICs to positive potentials as predicted by the Nernst equation. The relatively rapid shift in ESTICs when NKCC1 was blocked suggests that significant changes in the transmembrane Cl− gradient occur during the slow-wave cycle, possibly within microdomains formed between endoplasmic reticulum and the plasma membrane in ICC. Recovery of Cl− via NKCC1 might have additional consequences on shaping the waveforms of slow waves via Na+ entry into microdomains.

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