Related Experiment Videos
Rhythmic Cl- current and physiological roles of the intestinal c-kit-positive cells
N Tokutomi1, H Maeda, Y Tokutomi
1Department of Pharmacology, Kumamoto University School of Medicine, Japan.
Abstract:
Chronic injection of an anti-c-KIT receptor tyrosine kinase monoclonal antibody (ACK2) results in the disruption of the normal motility patterns of young BALB/c mice intestine. This effect is accompanied by a drastic decrease in the number of intestinal c-kit-expressing (c-kit+) cells when studied immunohistochemically with the fluorescence-labelled antibody. In order to clarify the mechanism underlying the ACK2 action and the physiological roles of intestinal c-kit+ cells, we studied the excitability of intestinal c-kit+ cells in primary culture by use of the nystatin perforated-patch-clamp technique. Under voltage-clamp at -40 mV, the majority of c-kit+ cells tested (59/70) elicited rhythmic current waves with an amplitude and frequency of 263 +/- 24 pA and 2.30 +/- 0.25 cycles/min (mean +/- SEM), respectively. Intracellular perfusion of the c-kit+ cells with ethylenebis (okonitrilo) tetraacetate (EGTA) as well as a nominally Ca(2+)-free external solution or low holding voltage (< -60 mV) prevented the rhythmic current. The reversal potential of the rhythmic current was close to the equilibrium potential for Cl-(ECl). Moreover the rhythmic current was depressed by a Cl- channel blocker, 4-acetoamido-4-isothiocyanat-ostilbene-2,2'-disulphoni c acid (SITS). The smooth muscle cells freshly dissociated from the same intestinal specimen revealed a Ca(2+)-activated K+ current, as has been described in a variety of smooth muscle cells. Cultured smooth muscle cells from the ileum preparation lacked neither the Ca(2+)-activated K+ nor rhythmic Cl- currents. Smooth muscle cells freshly dissociated from the same ileum preparation and those in culture showed no immunoreactivity with the labelled ACK2, which was consistent with our previous in situ study. Results provided direct evidence that the intestinal c-kit+ cells, but not the smooth muscle cells, possess a rhythmic Cl- current oscillation, suggesting their participation in pacemaker activity for the peristaltic gut movement.
Insights
Intestinal c-kit+ cells exhibit rhythmic chloride currents, suggesting their role in gut motility. Blocking c-KIT receptor tyrosine kinase disrupts normal intestinal movement and reduces these crucial cells.
Area of Science:
- Gastroenterology
- Cell Physiology
- Neuroscience
Background:
- Intestinal motility is crucial for nutrient absorption and waste elimination.
- c-KIT receptor tyrosine kinase expressing cells (c-kit+) are present in the intestine.
- Antibody targeting c-KIT disrupts intestinal motility in mice.
Purpose of the Study:
- To investigate the physiological roles of intestinal c-kit+ cells.
- To clarify the mechanism of action of anti-c-KIT antibody (ACK2).
- To study the excitability of intestinal c-kit+ cells.
Main Methods:
- Primary culture of intestinal c-kit+ cells.
- Nystatin perforated-patch-clamp technique.
- Immunohistochemistry and Western blotting.
Main Results:
- Intestinal c-kit+ cells exhibited rhythmic chloride current oscillations.
- These currents were dependent on calcium and chloride ions.
- Smooth muscle cells did not display these rhythmic currents or ACK2 immunoreactivity.
Conclusions:
- Intestinal c-kit+ cells, not smooth muscle cells, possess rhythmic chloride currents.
- These oscillations suggest a pacemaker role for c-kit+ cells in gut peristalsis.
- ACK2 disrupts intestinal motility by affecting c-kit+ cell function.