Spontaneous electrical rhythmicity in cultured interstitial cells of cajal from the murine small intestine

Abstract

1. Interstitial cells of Cajal (ICC) are pacemaker cells in the small bowel, and therefore this cell type must express the mechanism responsible for slow wave activity. Isolated ICC were cultured for 1-3 days from the murine small intestine and identified with c-Kit-like immunoreactivity (c-Kit-LI). 2. Electrical recordings were obtained from cultured ICC with the whole-cell patch clamp technique. ICC were rhythmically active, producing regular slow wave depolarizations with waveforms and properties similar to slow waves in intact tissues. 3. Spontaneous activity of c-Kit-LI cells was inhibited by reduced extracellular Na+, gadolinium, and reduced extracellular Ca2+. The activity was not affected by nisoldipine. Voltage clamp studies showed rhythmic inward currents that were probably responsible for the slow wave activity. The current-voltage relationship showed that the spontaneous currents reversed at about +17 mV. These observations are consistent with the involvement of a non-selective cation current in the generation of slow waves, but do not rule out contributions from other conductances or transporters. 4. A Ba2+-sensitive inwardly rectifying K+ current in c-Kit-LI cells that may be involved in slow wave repolarization and maintenance of a negative potential between slow waves was also found. Similar pharmacology was observed in studies of intact murine intestinal muscles. 5. Cultured ICC may be a useful model for studying the properties and pharmacology of some of the ionic conductances involved in spontaneous rhythmicity in the gastrointestinal tract.

Figure 1. Phase contrast and fluorescence micrographs of cultured ICC (2 days)

A, single, multi-processed cell (arrow) that expressed strong c-Kit-like immunoreactivity (c-Kit-LI; B, arrow). Occasionally, we observed apparent connections between cells with c-Kit-LI and non-c-Kit positive cells (see phase image of cell noted with arrowhead in A, and lack of immunoreactivity in B). C, the networks formed by cells with c-Kit-LI. Multiple cells of similar morphology (arrows) formed distinctive network structures via long thin processes. D shows that these cells possessed c-Kit-LI. The cells that formed networks had similar properties and morphology to ICC in situ. Other cells within the field of view did not possess c-Kit-LI (arrowheads).

Figure 2. Spontaneous membrane potential and current oscillations from ICC in murine small intestine

A, typical slow wave oscillations in membrane potential recorded from ICC under current clamp. B, spontaneous inward currents recorded under voltage clamp. Currents were obtained at holding potentials from −60 to +30 mV. C, summary current-voltage relationship for 4 cells.

Figure 3. Effects of Na⁺ replacement on spontaneous slow wave activity of ICC

A, normal membrane potential oscillations before and after switching to a Na⁺-free solution (indicated by bar). After a brief period of waxing and waning, slow waves were greatly reduced in amplitude to events of only a couple of millivolts (B). A break of 5 min exists between the end of the record in A and the beginning of B. C begins 15 min after the start of washout of Na⁺-free conditions, and shows restoration of normal frequency and amplitude of slow waves.

Figure 4. Pharmacology of slow wave oscillations in cultured ICC

A, lack of effect of nisoldipine (1 μm) on slow waves. B, inhibition of slow waves when cells were exposed to a bathing solution in which Ca²⁺ was replaced by Mn²⁺ (in physiological saline solution, MnPSS). C, effects of cyclopiazonic acid (CPA). CPA increased frequency and produced a small hyperpolarization in MDP. D, gadolinium (Gd³⁺) blocked slow wave activity. E, DIDS reduced the amplitude of slow waves, caused a small hyperpolarization in MDP, but did not block spontaneous rhythmicity.

Figure 5. Evidence that a Ba²⁺-sensitive inward rectifier current participates in the regulation of membrane potential in cultured ICC

A, effect of adding Ba²⁺ (50 μm) to a spontaneously active network of ICC under current clamp conditions. Ba²⁺ caused depolarization of the MDP. B, ramp clamp protocol utilized to show inward rectification in isolated ICC. C, current responses of an isolated cell to voltage ramps before (○) and in the presence (*) of Ba²⁺ (10 μm). D, difference current obtained by subtracting the response during the exposure to Ba²⁺ from the control response. The difference current demonstrates the presence of a Ba²⁺-sensitive inwardly rectifying current.

Figure 6. Effects of reduced Na⁺ on slow waves from intact small intestinal muscles

A-E, similar to the cultured ICC, there was a reduction in slow wave amplitude when extracellular Na⁺ was reduced from 140 to 0 mm. Distinct oscillations in membrane potential were still present in 1 mm Na⁺. F, summary of the relationship between extracellular [Na⁺] and slow wave amplitude. G, relationship between extracellular [Na⁺] and slow wave frequency. Data in F and G are means ± s.e.m. (n = 5).