pH-mediated Regulation of Pacemaker Activity in Cultured Interstitial Cells of Cajal

Interstitial cells of Cajal (ICCs) are pacemakers in gastrointestinal tracts, regulating rhythmicity by activating nonselective cation channels (NSCCs). In the present study, we investigated the general characteristics and pH-mediated regulation of pacemaker activity in cultured interstitial cells of Cajal. Under voltage clamp mode and at the holding potential of -60 mV, the I-V relationships and difference current showed that there was no reversal potential and voltage-independent inward current. Also, when the holding potentials were changed from +20 mV to -80 mV with intervals of 20 mV, there was little difference in inward current. In pacemaker activity, the resting membrane potential (RMP) was depolarized (In pH 5.5, 23+/-1.5 mV depolarized) and the amplitude was decreased by a decrease of the extracellular pH. However, in case of increase of extracellular pH, the RMP was slightly hyperpolarized and the amplitude was decreased a little. The melastatin type transient receptor potential (TRPM) channel 7 has been suggested to be required for intestinal pacemaking activity. TRPM7 produced large outward currents and small inward currents by voltage ramps, ranging from +100 to -100 mV from a holding potential of -60 mV. The inward current of TRPM7 was dramatically increased by a decrease in the extracellular pH. At pH 4.0, the average inward current amplitude measured at -100 mV was increased by about 7 fold, compared with the current amplitude at pH 7.4. Changes in the outward current (measured at +100 mV) were much smaller than those of the inward current. These results indicate that the resting membrane potential of pacemaking activity might be depolarized by external acidic pH through TRPM7 that is required for intestinal pacemaking activity.

The Role of Mitochondrial ATP-sensitive Potassium Channel on Intestinal Pacemaking Activity

Interstitial cells of Cajal (ICCs) are the pacemaker cells that generate slow waves in the gastrointestinal (GI) tract. In the present study, we investigated the effect of mitochondrial ATP-sensitive potassium (mitoKATP) channel on pacemaking activity in cultured ICCs from murine small intestine by using whole-cell patch clamp techniques. Under current clamp mode, at 10microM glibenclamide, there was no change in pacemaking activity of ICCs. At 30microM glibenclamide, an inhibitor of the ATP sensitive K+ channels, we could find two examples. If pacemaking activity of ICCs was irregulating, pacemaking activity of ICCs was changed into regulating and if in normal conditions, membrane potential amplitude was increased. At 50microM glibenclamide, the resting membrane potential was depolarized. At 3mM 5-HDA, an inhibitor of the mitoKATP channels, inhibited the pacemaking activity of ICCs. Both the amplitude and the frequency were decreased. At 5 mM 5-HDA, both the amplitude and the frequency were completely abolished. Diazoxide, an opener of the mitoKATP channels, was applied to examine its effect on pacemaking activity of ICCs. At 50microM concentration, the pacemaking activity of ICCs was inhibited. Both the amplitude and the frequency were decreased. At 1 mM concentration, both the amplitude and the frequency were completely abolished and the resting membrane potential was shaked. These results indicate that mitoKATP channel has an important role in pacemaking activity of ICCs.

PGE2 Regulates Pacemaker Currents through EP2-Receptor in Cultured Interstitial Cells of Cajal from Murine Small Intestine

The interstitial cells of Cajal (ICCs) are the pacemaker cells in gastrointestinal tract and generate electrical rhythmicity in gastrointestinal muscles. Therefore, ICC may be modulated by endogenous agents such as neurotransmitter, hormones, and prostaglandins (PGs). In the present study, we investigated the effects of prostaglandins, especially PGE2, on pacemaker currents in cultured ICCs from murine small intestine by using whole-cell patch clamp techniques. ICCs generated spontaneous slow waves under voltage-clamp conditions and showed a mean amplitude of -452+/-39 pA and frequency of 18+/-2 cycles/min (n=6). Treatments of the cells with PGE2 (1muM) decreased both the frequency and amplitude of the pacemaker currents and increased the resting currents in the outward direction. PGE2 had only inhibitory effects on pacemaker currents and this inhibitory effect was dose-dependent. For characterization of specific membrane EP receptor subtypes, involved in the effects of PGE2 on pacemaker currents in ICCs, EP receptor agonists were used: Butaprost (1muM), EP2 receptor agonist, reduced the spontaneous inward current frequency and amplitude in cultured ICCs (n=5). However sulprostone (1muM), a mixed EP1 and EP3 agonist, had no effects on the frequency, amplitude and resting currents of pacemaker currents (n=5). SQ-22536 (an inhibitor of adenylate cyclase; 100muM) and ODQ (an inhibitor of guanylate cyclase; 100muM) had no effects on PGE2 actions of pacemaker currents. These observations indicate that PGE2 alter directly the pacemaker currents in ICCs, and that the PGE2 receptor subtypes involved are the EP2 receptor, independent of cyclic AMP- and GMP-dependent pathway.