Oxygenated polycyclic aromatic hydrocarbons from ambient particulate matter induce electrophysiological instability in cardiomyocytes.

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.

Effects of Ca2+-Activated Cl- Channel ANO1inhibitors on Pacemaker Activity in Interstitial Cells of Cajal.

BACKGROUND/AIMS: Anoctamin1 (Ca2+-activated Cl- channel, ANO1) is a specific marker of the interstitial cells of Cajal (ICC) in the gastrointestinal tract, and are candidate proteins that can function as pacemaker channels. Recently, novel selective ANO1 inhibitors were discovered and used to study Ca2+-activated Cl- channels. Therefore, to investigate whether ANO1 channels function as pacemaker channels, selective ANO1 inhibitors were tested with respect to the pacemaker potentials in ICC. METHODS: Whole-cell patch-clamp recording, RT-PCR, and intracellular Ca2+ ([Ca2+]i) imaging were performed in cultured ICC obtained from mice. RESULTS: Though CaCCinh-A01 (5 µM), T16Ainh-A01 (5 µM), and MONNA (5 µM) (selective ANO1 inhibitors) blocked the generation of pacemaker potentials in colonic ICC, they did not do so in small intestinal ICC. Though nifulmic acid (10 µM) and DIDS (10 µM) (classical Ca2+-activated Cl- channel inhibitors) also had no effect in small intestinal ICC, they suppressed the generation of pacemaker potentials in colonic ICC. In addition, knockdown of ANO1 reduced the pacemaker potential frequency in colonic ICC alone. Though ANO1 inhibitors suppressed [Ca2+]i oscillations in colonic ICC, they did not do so in small intestinal ICC. T-type Ca2+ channels were expressed in the both the small intestinal and colonic ICC, but mibefradil (5 µM) and NiCl2 (30 µM) (T-type Ca2+ channel inhibitors) inhibited the generation of pacemaker potentials in colonic ICC alone. CONCLUSION: These results indicate that though ANO1 and T-type Ca2+ channels participate in generating pacemaker potentials in colonic ICC, they do not do so in small intestinal ICC. Therefore, the mechanisms underlying pacemaking in ICC might be different in the small intestine and the colon.

Regulatory Roles of Endogenous Mitogen-Activated Protein Kinases and Tyrosine Kinases in the Pacemaker Activity of Colonic Interstitial Cells of Cajal.

BACKGROUND AND PURPOSE: Mitogen-activated protein (MAP) and tyrosine kinases play an important role in regulating smooth muscle contraction of the gastrointestinal (GI) tract. Interstitial cells of Cajal (ICCs) are pacemaker cells that regulate GI smooth muscle activity. Thus, the role of MAP and tyrosine kinases on the pacemaker potentials of colonic ICCs was investigated. METHODS: Cultured ICCs were prepared from mice colons, and their pacemaker potentials were recorded using whole-cell patch clamping. RESULTS: In current-clamping mode, colonic ICCs displayed spontaneous pacemaker potentials. SB203580 (a p38 MAP kinase inhibitor), SP600125 (a c-jun NH2-terminal kinase (JNK) inhibitor), genistein and herbimycin A (tyrosine kinase inhibitors) blocked the generation of pacemaker potentials. However, PD98059 (a p42/44 MAP kinase inhibitor) had no effects on pacemaker potentials. LY-294002 (phosphoinositide 3-kinase inhibitor) also reduced the pacemaker potential frequency but calphostin C and chelerythrine (protein kinase C inhibitors) had no effects. However, PD98059, SB203589, SP600125, genistein, herbimycin A, LY-294002, and calphostin C had no effect on normal pacemaker activity in small intestinal ICCs. CONCLUSIONS: Endogenous p38 MAP kinases, JNKs, tyrosine kinases, and PI3-kinases participate in the generation of pacemaker potentials in colonic ICCs but not in ICCs of the small intestine.

Effects of lubiprostone on pacemaker activity of interstitial cells of cajal from the mouse colon.

Lubiprostone is a chloride (Cl(-)) channel activator derived from prostaglandin E1 and used for managing constipation. In addition, lubiprostone affects the activity of gastrointestinal smooth muscles. Interstitial cells of Cajal (ICCs) are pacemaker cells that generate slow-wave activity in smooth muscles. We studied the effects of lubiprostone on the pacemaker potentials of colonic ICCs. We used the whole-cell patch-clamp technique to determine the pacemaker activity in cultured colonic ICCs obtained from mice. Lubiprostone hyperpolarized the membrane and inhibited the generation of pacemaker potentials. Prostanoid EP1, EP2, EP3, and EP4 antagonists (SC-19220, PF-04418948, 6-methoxypyridine-2-boronc acid N-phenyldiethanolamine ester, and GW627368, respectively) did not block the response to lubiprostone. L-NG-nitroarginine methyl ester (L-NAME, an inhibitor of nitric oxide synthase) and 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) did not block the response to lubiprostone. In addition, tetraethylammonium (TEA, a voltage-dependent potassium [K(+)] channel blocker) and apamin (a calcium [Ca(2+)]-dependent K(+) channel blocker) did not block the response to lubiprostone. However, glibenclamide (an ATP-sensitive K(+) channel blocker) blocked the response to lubiprostone. Similar to lubiprostone, pinacidil (an opener of ATP-sensitive K(+) channel) hyperpolarized the membrane and inhibited the generation of pacemaker potentials, and these effects were inhibited by glibenclamide. These results suggest that lubiprostone can modulate the pacemaker potentials of colonic ICCs via activation of ATP-sensitive K(+) channel through a prostanoid EP receptor-independent mechanism.

Spontaneous electrical activity of cultured interstitial cells of cajal from mouse urinary bladder.

Interstitial cells of Cajal (ICCs) from the urinary bladder regulate detrusor smooth muscle activities. We cultured ICCs from the urinary bladder of mice and performed patch clamp and intracellular Ca(2+) ([Ca(2+)]i) imaging to investigate whether cultured ICCs can be a valuable tool for cellular functional studies. The cultured ICCs displayed two types of spontaneous electrical activities which are similar to those recorded in intact bladder tissues. Spontaneous electrical activities of cultured ICCs were nifedipine-sensitive. Carbachol and ATP, both excitatory neurotransmitters in the urinary bladder, depolarized the membrane and increased the frequency of spike potentials. Carbachol increased [Ca(2+)]i oscillations and basal Ca(2+) levels, which were blocked by atropine. These results suggest that cultured ICCs from the urinary bladder retain rhythmic phenotypes similar to the spontaneous electrical activities recorded from the intact urinary bladder. Therefore, we suggest that cultured ICCs from the urinary bladder may be useful for cellular and molecular studies of ICCs.

Decreased Expression of Urethral Caveolin-1, -2, and -3 in the Rat Model of Overactive Bladder: Potential Mediator of Functional Interaction of Urethra and Urinary Bladder.

PURPOSE: To investigate the effect of detrusor overactivity (DO) on the urethral expression of caveolin (CAV)-1, -2, and -3 of urethra in an animal model of cyclophosphamide (CYP)-induced cystitis rat. METHODS: Female Sprague-Dawley rats were divided into the control group (n=20) and the cystitis group (n=20). Cystitis was induced by intraperitoneal injection of CYP (200 mg/kg). An urodynamic study was done 3 days after the CYP injection to measure functional change of the urinary bladder and urethra. Cellular localization and expression of CAV-1, -2, and -3 in the rat urethra were determined by immunohistochemistry (IHC) and Western blot. RESULTS: Urodynamic experiments demonstrated a decreased contraction interval in the cystitis group compared to the control (3.9±1.0 minutes vs. 6.6±1.2 minutes, P<0.05). Conversely, contraction pressure increased significantly in the cystitis group compared to the control (22.4±0.7 mmHg vs. 11.5±0.4 mmHg, P<0.05). The urethral pressure was decreased in the cystitis group compared to the control (4.05 ±2.5 mmHg vs. 5.8 ±2.8 mmHg, P <0.05). The IHC and Western blot data showed that CAV-1, -2, and -3 expression decreased significantly in the cystitis group compared control group (P<0.05). CONCLUSION: The decreased urethral CAV-1, -2, and -3 in the DO rats suggests that CAVs might be related with the functional change of urethra in association with DO of urinay bladder.

Using the modified Delphi method to research the influencing factors of long-term health-related quality of life in patients with unruptured intracranial aneurysms after endovascular treatment.

BACKGROUND: The purpose of this study was to use the modified Delphi method to identify the influencing factors of health-related quality of life (HRQoL) in patients with unruptured intracranial aneurysms (UIAs) after endovascular treatment. METHODS: A modified Delphi method to obtain expert consensus on the content of potential influencing factors of HRQoL in patients with UIAs treated by endovascular intervention was employed. The research team consists of three neuroradiologists and one epidemiologist from Xuanwu Hospital of Capital Medical University. They randomly selected 21 well-known experts in cerebrovascular disease diagnosis and treatment as participating experts. The importance of the indicator is based on the 5-Likert scale. The standard deviation (SD), coefficient of variation (CV), mean ( x ¯ ), and minimum and maximum scores of each indicator were calculated. The consistency was described by Kendall coefficient of concordance with a p value < 0.05 indicating that the expert consistency was high. RESULT: Twenty-one and 18 questionnaires were responded in 2 rounds, with effective response rates of 85.7% and 100.0%, respectively. The average authoritative coefficient (Cr) of all 21 experts was 0.88, familiarity with the indicators (Cs) was 0.82, and the judgment basis of the indicators (Ca) was 0.94. Eventually, the x ¯ values of arterial puncture hematoma, hyperlipidemia, gender, marital status, and hospitalization for other diseases were lower than 3.5; CV for marital status and gender was higher than 0.35. The Kendall coefficient of concordance in the first round was 0.19 (p < 0.001), and the second round was 0.15 (p < 0.001). CONCLUSION: In this study, the factors affecting the recovery of HRQoL after endovascular treatment in patients with UIAs were analyzed by the modified Delphi method, which provided a valuable evidence for the clinical management and daily life guidance for UIAs patients.

Regulation of Intracellular Calcium by Endoplasmic Reticulum Proteins in Small Intestinal Interstitial Cells of Cajal.

BACKGROUND/AIMS: We investigated the role of representative endoplasmic reticulum proteins, stromal interaction molecule 1 (STIM1), and store-operated calcium entry-associated regulatory factor (SARAF) in pacemaker activity in cultured interstitial cells of Cajal (ICCs) isolated from mouse small intestine. METHODS: The whole-cell patch clamp technique applied for intracellular calcium ions ([Ca2+]i) analysis with STIM1 or SARAF overexpressed cultured ICCs from mouse small intestine. RESULTS: In the current-clamping mode, cultured ICCs displayed spontaneous pacemaker potentials. External carbachol exposure produced tonic membrane depolarization in the current-clamp mode, which recovered within a few seconds into normal pacemaker potentials. In STIM1-overexpressing cultured ICCs pacemaker potential frequency was increased, and in SARAF-overexpressing ICCs pacemaker potential frequency was strongly inhibited. The application of gadolinium (a non-selective cation channel inhibitor) or a Ca2+-free solution to understand Orai channel involvement abolished the generation of pacemaker potentials. When recording intracellular Ca2+ concentration with Fluo 3-AM, STIM1-overexpressing ICCs showed an increased number of spontaneous intracellular Ca2+ oscillations. However, SARAF-overexpressing ICCs showed fewer spontaneous intracellular Ca2+ oscillations. CONCLUSION: Endoplasmic reticulum proteins modulated the frequency of pacemaker activity in ICCs, and levels of STIM1 and SARAF may determine slow wave patterns in the gastrointestinal tract.

ATP-sensitive K+ channels maintain resting membrane potential in interstitial cells of Cajal from the mouse colon.

To investigate the role of ATP-sensitive K+(KATP) channels on pacemaker activity in interstitial cells of Cajal (ICC), whole-cell patch clamping, RT-PCR, and intracellular Ca2+([Ca2+]i) imaging were performed in cultured colonic ICC. Pinacidil (a K+ channel opener) hyperpolarized the membrane and inhibited the generation of pacemaker potential, and this effect was reversed by glibenclamide (a KATP channel blocker). RT-PCR showed that Kir 6.1 and SUR2B were expressed in Ano-1 positive colonic ICC. Glibenclamide depolarized the membrane and increased pacemaker potential frequency. However, 5-hydroxydecanoic acid (a mitochondrial KATP channel blocker) had no effects on pacemaker potentials. Phorbol 12-myristate 13-acetate (PMA; a protein kinase C activator) blocked the pinacidil-induced effects, and PMA alone depolarized the membrane and increased pacemaker potential frequency. Cell-permeable 8-bromo-cyclic AMP also increased pacemaker potential frequency. Recordings of spontaneous intracellular Ca2+([Ca2+]i) oscillations showed that glibenclamide increased the frequency of [Ca2+]i oscillations. In small intestinal ICC, glibenclamide alone did not alter the generation of pacemaker potentials, and Kir 6.2 and SUR2B were expressed in Ano-1 positive ICC. Therefore, KATP channels in colonic ICC are activated in resting state and play an important role in maintaining resting membrane potential.

Prostanoid EP3 receptor agonist sulprostone enhances pacemaker activity of colonic interstitial cells of Cajal.

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.