Background Hyperpolarization-activated cyclic nucleotide (HCN) channels are pacemaker channels that regulate heart rate and neuronal rhythm in spontaneously active cardiac and neuronal cells. Interstitial cells of Cajal (ICCs) are also spontaneously active pacemaker cells in the gastrointestinal tract. Here, we investigated the existence of HCN channel and its role on pacemaker activity in colonic ICCs. Methods We performed whole-cell patch clamp, RT-PCR, and Ca²⁺-imaging in cultured ICCs from mouse mid colon. Results SQ-22536 and dideoxyadenosine (adenylate cyclase inhibitors) decreased the frequency of pacemaker potentials, whereas both rolipram (cAMP-specific phosphodiesterase inhibitor) and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. CsCl, ZD7288, zatebradine, clonidine (HCN channel blockers), and genistein (a tyrosine kinase inhibitor) suppressed the pacemaker activity. RT-PCR revealed expression of HCN1 and HCN3 channels in c-kit and Ano1 positive colonic ICCs. In recordings of spontaneous intracellular Ca²⁺ [Ca²⁺]_i oscillations, rolipram and 8-bromo-cAMP increased [Ca²⁺]_i oscillations, whereas SQ-22536, CsCl, ZD7288, and genistein decreased [Ca²⁺]_i oscillations. Conclusions HCN channels in colonic ICCs are tonically activated by basal cAMP production and participate in regulation of pacemaking activity.

Parajuli SP, et al. Korean J Physiol Pharmacol. 2010 Apr;14(2):83-89. http://dx.doi.org/10.4196/kjpp.2010.14.2.83

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 M₃ receptor antagonist, but not by methotramine, a muscarinic M₂ receptor antagonist. Intracellular GDP-β-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 M₃ receptors by a G-protein dependent intracellular Ca²⁺ release mechanism.

Background: Epidemiologic studies have suggested that elevated concentrations of particulate matter (PM) are strongly associated with an increased risk of developing cardiovascular diseases, including arrhythmia. However, the cellular and molecular mechanisms by which PM exposure causes arrhythmia and the component that is mainly responsible for this adverse effect remains to be established. In this study, the arrhythmogenicity of mobilized organic matter from two different types of PM collected during summer (SPM) and winter (WPM) seasons in the Seoul metropolitan area was evaluated. In addition, differential effects between polycyclic aromatic hydrocarbons (PAHs) and oxygenated PAHs (oxy-PAHs) on the induction of electrophysiological instability were examined. Results: We extracted the bioavailable organic contents of ambient PM, measuring 10 μm or less in diameter, collected from the Seoul metropolitan area using a high-volume air sampler. Significant alterations in all factors tested for association with electrophysiological instability, such as intracellular Ca²⁺ levels, reactive oxygen species (ROS) generation, and mRNA levels of the Ca²⁺-regulating proteins, sarcoplasmic reticulum Ca²⁺ATPase (SERCA2a), Ca²⁺/calmodulin-dependent protein kinase II (CaMK II), and ryanodine receptor 2 (RyR2) were observed in cardiomyocytes treated with PM. Moreover, the alterations were higher in WPM-treated cardiomyocytes than in SPM-treated cardiomyocytes. Three-fold more oxy-PAH concentrations were observed in WPM than SPM. As expected, electrophysiological instability was induced higher in oxy-PAHs (9,10-anthraquinone, AQ or 7,12-benz(a) anthraquinone, BAQ)-treated cardiomyocytes than in PAHs (anthracene, ANT or benz(a) anthracene, BaA)-treated cardiomyocytes; oxy-PAHs infusion of cells mediated by aryl hydrocarbon receptor (AhR) was faster than PAHs infusion. In addition, ROS formation and expression of calcium-related genes were markedly more altered in cells treated with oxy-PAHs compared to those treated with PAHs. Conclusions: The concentrations of oxy-PAHs in PM were found to be higher in winter than in summer, which might lead to greater electrophysiological instability through the ROS generation and disruption of calcium regulation.

Shahi PK, et al. Korean J Physiol Pharmacol. 2011 Jun;15(3):129-135. http://dx.doi.org/10.4196/kjpp.2011.15.3.129

Tricyclic antidepressants (TCAs) have been used to treat depression and were recently approved for treating irritable bowel syndrome (IBS) patients with severe or refractory IBS symptoms. However, the molecular mechanism of TCA action in the gastrointestinal (GI) tract remains poorly understood. Transient receptor potential channel canonical type 4 (TRPC4), which is a Ca²⁺‐permeable nonselective cation channel, is a critical regulator of GI excitability. Herein, we investigated whether TCA modulates TRPC4 channel activity and which mechanism in colonic myocytes consequently causes constipation. To prove the clinical benefit in patients with diarrhoea caused by TCA treatment, we performed mechanical tension recording of repetitive motor pattern (RMP) in segment, electric field stimulation (EFS)‐induced and spontaneous contractions in isolated muscle strips. From these recordings, we observed that all TCA compounds significantly inhibited contractions of colonic motility in human. To determine the contribution of TRPC4 to colonic motility, we measured the electrical activity of heterologous or endogenous TRPC4 by TCAs using the patch clamp technique in HEK293 cells and murine colonic myocytes. In TRPC4‐overexpressed HEK cells, we observed TCA‐evoked direct inhibition of TRPC4. Compared with TRPC4‐knockout mice, we identified that muscarinic cationic current (mI_cat) was suppressed through TRPC4 inhibition by TCA in isolated murine colonic myocytes. Collectively, we suggest that TCA action is responsible for the inhibition of TRPC4 channels in colonic myocytes, ultimately causing constipation. These findings provide clinical insights into abnormal intestinal motility and medical interventions aimed at IBS therapy.