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EP receptor activation by PGE2 regulates gastrointestinal motility by modulating smooth muscle contractility. Interstitial cells of Cajal (ICCs) are pacemaker cells that regulate smooth muscle activity. We aimed to determine effects of the EP3 receptor agonist sulprostone on pacemaker potentials in colonic ICCs. We performed a whole cell patch clamp, RT-PCR, and Ca2+ imaging in cultured ICCs from mouse colon. Sulprostone depolarized the membrane and increased pacemaker frequency. EP3 receptor antagonist blocked these sulprostone-induced effects. EP3 receptors were expressed in ANO1-positive ICCs. Phospholipase C inhibitor or Ca2+-ATPase inhibitor from the endoplasmic reticulum blocked the sulprostone-induced effects and sulprostone increased intracellular Ca2+ ([Ca2+]i) oscillations. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blockers also suppressed the sulprostone-induced effects. Sulprostone enhanced pacemaker activity through EP3 receptors by activating HCN channels via the [Ca2+]i release pathway. Therefore, EP3 receptor activation in ICCs may modulate colonic motility and could be a therapeutic target for enhancing colonic GI motility.

Cyclic guanosine 3',5'-monophosphate (cGMP) inhibited the generation of pacemaker activity in interstitial cells of Cajal (ICCs) from the small intestine. However, cGMP role on pacemaker activity in colonic ICCs has not been reported yet. Thus, we investigated the role of cGMP in pacemaker activity regulation by colonic ICCs. We performed a whole-cell patch-clamp and Ca(2+) imaging in cultured ICCs from mouse colon. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) increased the pacemaker potential frequency, whereas zaprinast (an inhibitor of phosphodiesterase) and cell-permeable 8-bromo-cGMP decreased the pacemaker potential frequency. KT-5823 (an inhibitor of protein kinase G [PKG]) did not affect the pacemaker potential. L-N(G)-nitroarginine methyl ester (L-NAME, an inhibitor of nitric oxide [NO] synthase) increased the pacemaker potential frequency, whereas (±)-S-nitroso-N-acetylpenicillamine (SNAP, a NO donor) decreased the pacemaker potential frequency. Glibenclamide (an ATP-sensitive K(+) channel blocker) did not block the effects of cell-permeable 8-bromo-cGMP and SNAP. Recordings of spontaneous intracellular Ca(2+) ([Ca(2+)]i) oscillations revealed that ODQ and L-NAME increased [Ca(2+)]i oscillations. In contrast, zaprinast, 8-bromo cGMP, and SNAP decreased the [Ca(2+)]i oscillations. Basal cGMP levels regulate the resting pacemaker potential frequency by the alteration on Ca(2+) release via a PKG-independent pathway. Additionally, the endogenous release of NO seems to be responsible maintaining basal cGMP levels in colonic ICCs.

The aim of this study was to clarify if phentolamine has proven effects on the pacemaker activities of interstitial cells of Cajal (ICC) from the mouse small intestine involving the ATPsensitive K(+) channels and adrenergic receptor. The actions of phentolamine on pacemaker activities were investigated using whole-cell patch-clamp technique and intracellular Ca(2+) analysis at 30 degrees C in cultured mouse intestinal ICC. ICC generated spontaneous pacemaker currents at a holding potential of -70 mV. Treatment with phentolamine reduced the frequency and amplitude of the pacemaker currents and increased the resting outward currents. Moreover, under current clamping (I = 0), phentolamine hyperpolarized the ICC membrane and decreased the amplitude of the pacemaker potentials. We also observed that phentolamine inhibited spontaneous [Ca(2+)](i) oscillations in ICC. The alpha-adrenergic drugs prazosin, yohimbine, methoxamine, and clonidine had no effect on ICC intestinal pacemaker activity and did not block phentolamine-induced effects. Phentolamine-induced effects on the pacemaker currents and the pacemaker potentials were significantly inhibited by ATP sensitive K(+) channel blocker glibenclamide, but not by TEA, apamin, or 4-aminopyridine. In addition, the NO synthase inhibitor, L-NAME and the guanylate cyclase inhibitor, ODQ were incapable of blocking the phentolamine-induced effects. These results demonstrate that phentolamine regulates the pacemaker activity of ICC via ATP-sensitive K(+) channel activation. Phentolamine could act through an adrenergic receptor- and also through NO-independent mechanism that involves intracellular Ca(2+) signaling.

We studied the effect of carbachol on pacemaker currents in cultured interstitial cells of Cajal (ICC) from the mouse small intestine by muscarinic stimulation using a whole cell patch clamp technique and Ca2+-imaging. ICC generated periodic pacemaker potentials in the current-clamp mode and generated spontaneous inward pacemaker currents at a holding potential of-70 mV. Exposure to carbachol depolarized the membrane and produced tonic inward pacemaker currents with a decrease in the frequency and amplitude of the pacemaker currents. The effects of carbachol were blocked by 1-dimethyl-4-diphenylacetoxypiperidinium, a muscarinic M(3) receptor antagonist, but not by methotramine, a muscarinic M(2) receptor antagonist. Intracellular GDP-beta-S suppressed the carbachol-induced effects. Carbachol-induced effects were blocked by external Na+-free solution and by flufenamic acid, a non-selective cation channel blocker, and in the presence of thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum. However, carbachol still produced tonic inward pacemaker currents with the removal of external Ca2+. In recording of intracellular Ca2+ concentrations using fluo 3-AM dye, carbachol increased intracellular Ca2+ concentrations with increasing of Ca2+ oscillations. These results suggest that carbachol modulates the pacemaker activity of ICC through the activation of non-selective cation channels via muscarinic M(3) receptors by a G-protein dependent intracellular Ca2+ release mechanism.

The interstitial cells of Cajal (ICC) are pacemaking cells required for gastrointestinal motility. The possibility of whether DA-9701, a novel prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber, modulates pacemaker activities in the ICC was tested using the whole cell patch clamp technique. DA-9701 produced membrane depolarization and increased tonic inward pacemaker currents in the voltage-clamp mode. The application of flufenamic acid, a non-selective cation channel blocker, but not niflumic acid, abolished the generation of pacemaker currents induced by DA-9701. Pretreatment with a Ca2+-free solution and thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum, abolished the generation of pacemaker currents. In addition, the tonic inward currents were inhibited by U-73122, an active phospholipase C inhibitor, but not by GDP-beta-S, which permanently binds G-binding proteins. Furthermore, the protein kinase C inhibitors, chelerythrine and calphostin C, did not block the DA-9701-induced pacemaker currents. These results suggest that DA-9701 might affect gastrointestinal motility by the modulation of pacemaker activity in the ICC, and the activation is associated with the non-selective cationic channels via external Ca2+ influx, phospholipase C activation, and Ca2+ release from internal storage in a G protein-independent and protein kinase C-independent manner.

The effects of imipramine on A-type delayed rectifier K+ currents and ATP-sensitive K+ (KATP) currents were studied in isolated murine proximal colonic myocytes using the whole-cell patch-clamp technique. Depolarizing test pulses between -80 mV and +30 mV with 10 mV increments from the holding potential of -80 mV activated voltage-dependent outward K+ currents that peaked within 50 ms followed by slow decreasing sustained currents. Early peak currents were inhibited by the application of 4-aminopyridine, whereas sustained currents were inhibited by the application of TEA. The peak amplitude of A-type delayed rectifier K+ currents was reduced by external application of imipramine. The half-inactivation potential and the half-recovery time of A-type delayed rectifier K+ currents were not changed by imipramine. With 0.1 mM ATP and 140 mM K+ in the pipette and 90 mM K+ in the bath solution and a holding potential of -80 mV, pinacidil activated inward currents; this effect was blocked by glibenclamide. Imipramine also inhibited KATP currents. The inhibitory effects of imipramine in A-type delayed rectifier K+ currents and KATP currents were not changed by guanosine 5-O-(2-thiodiphosphate) (GDPbetaS) and chelerythrine, a protein kinase C inhibitor. These results suggest that imipramine inhibits A-type delayed rectifier K+ currents and KATP currents in a manner independent of G-protein and protein kinase C.